DNA PALINDROME METABOLISM IN MAMMALIAN CELLS

By

Tara Anne Belsito

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Molecular Genetics University of Toronto

©Copyright by Tara Anne Belsito, 2009

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DNA Palindrome Metabolism in Mammalian Cells Master of Science 2009 Tara Anne Belsito Department of Molecular Genetics University of Toronto

Abstract

A DNA palindrome is a sequence of DNA followed by an exact inverted copy of itself.

Palindromes are associated with gross chromosomal instability in mammalian cells. This may be

related to their ability to extrude a double-stranded cruciform structure. In mammalian cells,

palindromes have been shown to undergo centre-directed rearrangements resulting in a central

region of asymmetry. This process occurs via a mechanism termed „centre break palindrome

revision‟. In this thesis, I have investigated palindrome revision in mammalian cells using two

existing assays. In the first, performed by transfection of an extrachromosomal palindromic

dimer, I have shown that joining of palindrome-mediated double-strand breaks does not

depend solely on NHEJ and instead relies heavily on an alternate end-joining pathway. Using

the second assay, the Line78 mouse model which contains a 15.4kb transgenic palindrome, I

have shown that small modifications near the centre of the palindrome prevent these centre-

directed rearrangements possibly by inhibiting cruciform formation.

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Acknowledgements

I would like to thank my supervisor, Dr. Susanna Lewis, for her advice, support, and

encouragement. I would also like to thank my committee members, Dr. Bri Lavoie and Dr.

Steve Meyn for their comments and suggestions. Finally, I would like to thank the members of

the Lewis Lab, past and present, especially Atina Côté for her technical assistance and good

company.

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Table of Contents

DNA PALINDROME METABOLISM IN MAMMALIAN CELLS I

ABSTRACT II

ACKNOWLEDGEMENTS III

TABLE OF CONTENTS IV

LIST OF FIGURES VII

LIST OF TABLES VIII

LIST OF ABBREVIATIONS IX

CHAPTER 1: INTRODUCTION TO DNA PALINDROMES 1

Disease-Causing Palindromes in Humans 2 Translocations 2 Deletions 5 Amplifications 5

Evidence for Cruciform Formation 7

Palindrome Metabolism in E. coli 11 SbcCD-dependent breakage 12 Replication slipped mispairing 14

Palindrome Metabolism in Yeast 14

Palindrome Metabolism in Mammalian Cells: Insight from Model Systems 18 Revision of extrachromosomal palindromes 18 Revision of integrated chromosomal palindromes – the Line78 transgenic mouse line 19

Centre Break Palindrome Revision: A Model For Preventative Palindrome Modification in Mammals 21

NHEJ and its Potential Involvement in Centre Break Palindrome Revision 21

Thesis Rationale 25

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CHAPTER 2: MATERIALS AND METHODS 26

Note to Reader 27

Fetal Liver Harvest 30

Cloning and Subcloning by Limiting Dilution 31

Genomic DNA Preparation 31

Line 78 PCR conditions 31

Southern Analysis 35

Rate Analysis 36 Maximum likelihood estimate sample calculations 37 Likelihood ratio test 38

Palindromic Dimer DNA preparation 40

Transfection Procedure 42

Transformation Procedure 42

Revision Product Analysis 42

CHAPTER 3: PALINDROME REVISION IS A FORM OF NON-CANONICAL END-JOINING 44

Note to Reader 45

Introduction 46 Experimental approach 46

Results 51 Preliminary gene screen 51 Joint analysis reveals common features of palindrome revision between NHEJ-proficient and NHEJ-deficient cell lines 51 There is no difference in the quantity of palindrome revision joints between NHEJ-proficient and NHEJ-deficient cells 56

Discussion 61 Extrachromosomal mammalian palindrome revision occurs in the absence of NHEJ 61 A subset of palindrome revision joints are formed via microhomology 62 Palindrome revision may be a function of non-canonical end-joining 64 Inserted DNA is a common feature in both extrachromosomal and intrachromosomal palindrome revision 66 Future Directions 67

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CHAPTER 4: THE STABILIZING EFFECTS OF ∆4BP ON THE LINE78 PALINDROME 72

Note to Reader 73

Introduction 74 Experimental approach 77

Results 81

Discussion 85 Line78 Rearrangements do not occur via replication slipped mispairing 87 Future Directions 88

REFERENCES 90

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List of Figures

Chapter 1: Figure 1.1: DNA palindromes and inverted repeats can form secondary DNA structures 3 Figure 1.2: Model of palindrome-mediated amplification 6 Figure 1.3: Mechanism of cruciform formation 9 Figure 1.4: Model of SbcCD mediated palindrome deletions in E. coli 13 Figure 1.5: Palindrome deletion by replication slipped mispairing 15 Figure 1.6: Model of cruciform resolution 17 Figure 1.7: Centre break model of palindrome revision 22 Figure 1.8: Basic mechanism of the NHEJ pathway 24 Chapter 2: Figure 2.1: Maximum likelihood estimate 39 Figure 2.2: Schematic of plasmids used in this study 41 Chapter 3: Figure 3.1a: Schematic of the qualitative extrachromosomal palindromic dimer assay 48 Figure 3.1b: Schematic of the quantitative extrachromosomal palindromic dimer assay 49 Figure 3.1c: Schematic of the extrachromosomal V(D)J assay 50 Figure 3.2: Nucleotides retained in axis-inclusive joints at the SalI and BamHI axes 57 Figure 3.3: The number of nucleotides used at junctions formed via microhomology 58 Figure 3.4: Palindrome revision efficiency in NHEJ-deficient and NHEJ-proficient cells 60 Chapter 4: Figure 4.1: Schematic of Line78 variants relevant to this study 76 Figure 4.2: Schematic of the Line78 rate of rearrangement assay 78 Figure 4.3: Schematic of the Line78 transgene and outlying areas 79 Figure 4.4: Southern blot analysis of Line78 4bp rearrangements in the germline 82

Figure 4.5: Southern blot analysis of Line78 4bp rearrangements in somatic cells 83

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List of Tables

Chapter 2: Table 2.1: Source of cell lines used in this study 29 Table 2.2: Results of subcloning of Line 78 4bp by limiting dilution 32

Table 2.3: Primers used in this study 33 Chapter 3: Table 3.1: Selected monomer junction sequences 52 Table 3.2: Summary of qualitative revision product analysis 53 Table 3.3: Quantitative analysis of palindrome revision in NHEJ-proficient and

NHEJ-deficient cell lines 59

Table 3.4: Sequence of palindrome revision junctions 68 Chapter 4:

Table 4.1: Rate analysis of the 4bp Line 78 palindrome 84

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List of Abbreviations

DSB - double-strand break

PATRR - palindromic AT-rich repeat

HPFH - hereditary persistence of fetal hemoglobin

NHEJ - non-homologous end-joining

MMEJ - microhomology mediated end-joining

DNA-PKcs - catalytic subunit of DNA-PK

ATM - ataxia telangiectasia mutated

MRN - Mre11/Rad50/Nbs1 (human homolog)

MRX - Mre11/Rad50/Xrs2 (yeast homolog)

DC-PCR - digestion circularization PCR

MLE - maximum likelihood estimate

Ab-MLV - Abelson murine leukemia virus

dsDNA - double-stranded DNA

ssDNA - single-stranded DNA

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Chapter 1: Introduction to DNA Palindromes

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The term “palindrome” is often used to describe a variety of sequences arrangements

which include perfect palindromes, near-palindromes, inverted repeats and spaced inverted

repeats (reviewed in (Lewis and Cote 2006)). In this study, the term “palindrome” is used to

describe a perfect DNA palindrome, which is a sequence of DNA followed immediately by an

exact inverted copy of itself. The term “near-palindrome” is used to describe sequences which

are similar to perfect palindromes but contain small central spacers and/or small sequence

inconsistencies between the two arms. Perfect palindromes and near-palindromes behave

similarly to each other, particularly in their secondary structure forming abilities. They both

have the ability to self-anneal while single-stranded to form a hairpin structure as well as while

double-stranded to form a cruciform structure (Fig. 1.1). The term “inverted repeat” is used to

describe inverted DNA sequences separated by central spacers longer than approximately 10

bp. Inverted repeats only have the ability to self-anneal while single-stranded to form hairpins;

they cannot extrude a cruciform structure.

Disease-Causing Palindromes in Humans

DNA palindromes are actually quite common in the human genome ((Lu, Jia et al.

2007) and S. Lewis, in preparation); there are at least 3,000 near-palindromes in the human

reference sequence. Palindromes are increasingly becoming better understood as their

involvement in human disease comes to light. This involvement is a result of a palindrome‟s

ability to cause various types of chromosomal instability.

Translocations

Emanuel syndrome is an inherited genetic disorder characterized by severe mental

retardation, preauricular tag or sinus, ear anomalies, cleft or high-arched palate, micrognathia,

microcephaly, kidney abnormalities, heart defects, and genital abnormalities in males (Zackai

and Emanuel 1980). It is a result of a supernumerary der(22) t(11;22 ) translocation. The

3

Figure 1.1: DNA palindromes and inverted repeats can form secondary DNA structures. A: A DNA Palindrome is a sequence of DNA followed immediately by an exact, inverted copy of

itself. B: DNA Palindromes have the ability to extrude a double-stranded cruciform structure. C: An inverted repeat is similar to a palindrome except that it contains a central region of

asymmetry. D: Inverted repeats are able to form single-stranded hairpin structures when single stranded,

such as on the lagging strand during replication. A palindrome is also able to form a single-stranded hairpin, however an inverted repeat cannot form a cruciform. Arrows indicate the direction of replication.

Okazaki Fragment

D

A C

B

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breakpoints on chromosomes 11 and 22 map to the centres of large Palindromic AT-Rich

Repeats (PATRRs) located at 11q23 and 22q11 ((Kurahashi and Emanuel 2001) and see

(Kurahashi, Inagaki et al. 2006; Kurahashi, Inagaki et al. 2006) for reviews). A different PATRR

located at 17q11 is responsible for another recurrent constitutional translocation with 22q11;

again, the breakpoints map the centres of these near-palindromes (Kurahashi, Shaikh et al.

2003). Analysis of t(11;22) indicates that it occurs de novo at a high frequency during meiosis

in sperm samples, but not in lymphoblasts or fibroblasts (Kurahashi and Emanuel 2001).

Although the translocation occurs during meiosis, cytogenetics studies have determined that the

breakpoints on chromosomes 11 and 22 are not associated with MLH1 foci; MLH1 is involved

with reciprocal cross-overs, indicating that the breakpoints are not associated with

recombination hotspots (Ashley, Gaeth et al. 2006). Analysis of breakpoint sequences of

various translocations reveals that the breaks occur at the centres of the near-palindromes and

that joining is accompanied by small central deletions; thus joining is not dependent on

homology (Kurahashi, Inagaki et al. 2007).

It seems likely that the three PATRRs (22q11, 11q23, and 17q11) become involved in

translocations because of their ability to extrude cruciform structures. Using 2-D gel

electrophoresis and S1 nuclease digestion (techniques commonly used to detect cruciforms), it

was shown that plasmids containing the 22q11, 11q23, and 17q11 PATRRs can extrude

cruciforms in vitro (Kurahashi, Inagaki et al. 2004; Kogo, Inagaki et al. 2007). The 22q11

PATRR is the longest of the three studied at approximately 600 bp (the PATRR at 11q23 is

approximately 500bp and the 17q11 is the smallest at 200 bp) and is the PATRR which exhibits

the most homology between the two arms (Kurahashi, Inagaki et al. 2006). It is also the

PATRR most likely to undergo cruciform formation in vitro, followed by 11q23 (Kogo, Inagaki et

al. 2007). A small (9bp) spacer introduced into the centre of the 22q11 PATRR greatly reduces

its cruciform extrusion ability. The ability of the PATRRs to extrude in vitro compels one to

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further examine the relationship between secondary structure formation and chromosomal

aberrations.

Deletions

A subset of thalassemia, hereditary persistence of fetal haemoglobin or HPFH, is caused

by deletions in the beta globin gene cluster; these deletions are all associated with a 160bp

palindrome. In a Belgian family, the 3‟ breakpoint is located at the centre of the palindrome

(Fodde, Losekoot et al. 1990). In an Indian family, the breakpoint is 4 bp downstream of the

centre (Henthorn, Mager et al. 1986) and in a Chilean family the breakpoint is 10 bp upstream

(Game, Bergounioux et al. 2003).

These palindrome-associated deletions are centre-directed (Henthorn, Mager et al.

1986; Fodde, Losekoot et al. 1990; Game, Bergounioux et al. 2003) as are the PATRR-mediated

translocations (Kurahashi, Inagaki et al. 2006; Kurahashi, Inagaki et al. 2006). It may be that

they are both a result of the same initiation event, presumably secondary structure formation.

Amplifications

Gene amplification is a common form of genome instability and is often associated with

tumour progression in mammals (see (Albertson 2006) for a review). In many characterized

cases in human cancers, the amplified regions contain large inverted duplications ((Tanaka,

Bergstrom et al. 2005; Myllykangas, Himberg et al. 2006; Tanaka, Bergstrom et al. 2006). One

proposed mechanism involves introduction of a double-strand break (DSB) adjacent to a short

inverted repeat. After formation of the DSB, 5‟-3‟ resection occurs followed by intrastrand

annealing at the short inverted repeat to create a hairpin structure. A large palindrome is

formed after one round of replication (Tanaka, Tapscott et al. 2002; Tanaka, Cao et al. 2007)

(Fig. 1.2). This can produce a dicentric chromosome which can then go on to cause further

rearrangements or amplifications through breakage-fusion-bridge cycles (McClintock 1941).

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Figure 1.2: Model of palindrome-mediated amplification. Palindrome-mediated amplification occurs when a double-strand break occurs adjacent to a small inverted repeat (A). After the break occurs, 5‟-3‟ resection occurs, leaving a 3‟ overhang (B). The free end folds back on itself and intrastrand base-pairing occurs between the two arms of the inverted repeat (C). The gap is filled in, creating a hairpin end (D). After one round of replication, a large DNA palindrome is formed (E).

Inverted repeat

Double-strand break

5‟-3‟ resection

Foldback/ Intrastrand base-pairing

Gap fill-in

Replication

A

B

D B

C

E

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In this case, the resultant palindromes serve as an intermediate for further large-scale

chromosomal amplification. There are also cases where only the palindromic sequence is

amplified. The nucleolus organizer regions (NORs) contain tandem arrays with hundreds of

copies of rRNA genes. Normally, these arrays contain a transcribed region followed by a non-

transcribed spacer. Molecular combing revealed that in normal individuals one third of the

repeats were arranged in an atypical fashion resulting in inverted repeats and/or near or perfect

palindromes. In Werner‟s syndrome patients approximately 50% of the repeats are inverted or

palindromic and they are found in clusters, implying that they are a result of repeat expansion

(Caburet, Conti et al. 2005) via a mechanism distinct from that causing gross chromosomal

amplification.

Based on the previous examples, it is fair to stay that palindromes are troublesome in

the human genome; often their effects are quite disastrous. Palindrome-induced chromosomal

rearrangements such as those discussed above also occur in other organisms. These organisms

also seem to have evolved mechanisms for preventing such damage.

Evidence for Cruciform Formation

Although palindromes and near-palindromes do have the ability to form a cruciform

structure, they do not do so readily. Approximately 17 kcal/mol of free energy are required to

drive cruciform extrusion in DNA (Courey and Wang 1983; Gellert, O'Dea et al. 1983; Lilley

1985; Benham, Savitt et al. 2002). This free energy is derived from torsional stress created by

negative supercoiling in the DNA (Lilley 1980; Panayotatos and Wells 1981; Mizuuchi, Mizuuchi

et al. 1982). Once the DNA has reached what‟s called its “superhelicity threshold” (Courey and

Wang 1988; Benham, Savitt et al. 2002), the excess energy created by the tightly under-wound

DNA results in melting of the interstrand hydrogen bonds at the centre of symmetry of the

palindrome (Murchie and Lilley 1987; Courey and Wang 1988; Zheng and Sinden 1988).

Approximately 10 base pairs are initially unwound, followed by intrastrand base-pairing and

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branch migration, finally resulting in a fully extruded cruciform (Fig. 1.3) ((Murchie and Lilley

1987) and references therein). The formation of the cruciform results in the relaxation of

negative supercoils and a loss of the excess free energy that drove the initial change.

Therefore, when the DNA is past its superhelicity threshold, cruciforms are energetically

favoured and the process of cruciform formation is essentially irreversible.

In bacteria, the negative supercoiling required to drive cruciform extrusion may be

provided by DNA gyrase activity (Gellert, Mizuuchi et al. 1976) however recent work has shown

that supercoiling in the E. coli genome is tightly regulated by DNA gyrase, Topoisomerase I and

gene expression processes (i.e. transcription, see below) (Snoep, van der Weijden et al. 2002).

Given that negative supercoils in eukaryotic DNA are constrained by nucleosome formation, it

was originally thought that cruciform extrusion was not possible (Sinden, Carlson et al. 1980;

Mizuuchi, Mizuuchi et al. 1982). Subsequent studies have demonstrated that negative

supercoiling may occur during transcription (Liu and Wang 1987). Supercoils are induced in

plasmid DNA in prokaryotes in vivo (Wu, Shyy et al. 1988; Dayn, Malkhosyan et al. 1992) and in

vitro (Leng and McMacken 2002). This occurs as a result of the transcriptional machinery

progression; the DNA double-helix unwinds, the DNA ahead of the transcription machinery

becomes positively supercoiled and the DNA behind the transcription machinery becomes

negatively supercoiled. This is termed the “transcription-induced twin-supercoiled domain”

model (Liu and Wang 1987). Studies involving computer models indicate that the twin-domain

model generates sufficient stress to drive secondary structure transitions in DNA under

physiological conditions (Mielke, Fink et al. 2004).

Early evidence for cruciform formation in vitro came from electron micrographs of

plasmid DNA extruding cruciform structures in response to negative supercoiling (Mizuuchi,

Mizuuchi et al. 1982). The same study also demonstrated cruciform formation by showing the

loss of a restriction site located at the centre of the palindrome. Other in vitro studies make use

of the unique behaviour of the single-stranded nucleotides at the tips of the cruciform arms.

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Figure 1.3: Mechanism of cruciform formation. In response to torsional strain, approximately 10 base pairs at the centre of the palindrome melt. Intrastrand base pairing occurs within the melted nucleotides. Branch migration occurs and the cruciform structure extrudes. Please see text for a more detailed description. Also see (Mizuuchi, Mizuuchi et al. 1982; Sullivan and Lilley 1986; Murchie and Lilley 1987) and references cited therein.

Lineform

Torsional strain and Local Melting

Intrastrand base-pairing and branch migration

Cruciform

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Digestion of palindrome-containing DNA with S1 nuclease, which detects single-stranded

nucleotides at the cruciform tips, or Endonuclease VII, which cleaves across the base of the 4-

way junction, followed by electrophoresis and analysis of band size can reveal if the DNA

contains a nuclease-sensitive site (Panayotatos and Wells 1981). Single-strand bases at the tips

of cruciforms have been detected by chemical modification by chloroacetaldehyde (Dayn,

Malkhosyan et al. 1991), osmium tetroxide (McClellan, Boublikova et al. 1990) and

diethylpyrocarbonate (Scholten and Nordheim 1986)(reviewed in (Sinden 1994)). More

recently, cruciforms in DNA have been detected using atomic force microscopy (AFM)

(Chasovskikh, Dimtchev et al. 2005). Finally, trimethylpsoralen, which causes intrastrand

crosslinks to form, can be used to fix DNA in either the lineform or cruciform formation (Sinden,

Carlson et al. 1980). This assay has been used to demonstrate the ability of DNA to extrude a

cruciform structure in response to negative supercoiling (Sinden, Carlson et al. 1980) and the

rate of cruciform formation at different temperatures and at different ionic strengths (Sinden

and Pettijohn 1984).

Several of the above methods can be used to identify cruciform formation in vivo;

psoralen cross-linking (Zheng, Kochel et al. 1991), chemical modification of single stranded

bases (Dayn, Malkhosyan et al. 1992) and digestion by structure-specific nucleases in

protoplasts (del Olmo and Perez-Ortin 1993) have been used to demonstrate cruciform

formation in E. coli. Other in vivo studies that present more direct evidence for cruciform

formation involve use of an antibody that recognizes the cruciform structure (Frappier, Price et

al. 1987). These studies show that the cruciforms antibody binds DNA in living cells in G1/S

phase (Ward, Shihab-el-Deen et al. 1991). There is other indirect evidence for the existence of

cruciforms in vivo, mostly involving the location of site-specific DNA breaks; this will be

discussed later on.

The DNA sequence located at the centre of the palindrome has a drastic effect on its

ability to extrude a cruciform. Psoralen crosslinking assays have shown that palindromes with

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AT stretches at their centres are more likely to form a cruciform in vitro (Zheng and Sinden

1988).

Studies involving plating efficiencies of phage containing palindromes show that those

palindromes with ATs at the centre have a decreased plating efficiency compared to those with

CGs at the centre (Davison and Leach 1994). Also, the plating efficiency increases if an

asymmetric spacer is inserted into the centre of the palindrome; the larger the spacer, the

greater the increase in plating efficiency (Chalker, Okely et al. 1993). Thus, one can say that

plating efficiency is correlated to cruciform formation; if the phage extrudes a cruciform, it is

not viable and the plating efficiency is decreased. Other studies examining imperfections in

palindrome-containing plasmids have shown that a 1-2 bp mutation in the centre 8-10 bp of

perfect palindromes can cause as much as a 2000-fold decrease in the kinetics of cruciform

formation (Murchie and Lilley 1987). Finally, studies on the energy needed to drive cruciform

formation show that for every unpaired base, the energy required increases substantially,

indicating that sequence imperfections in the near-palindrome have a major effect on cruciform

formation (Benham, Savitt et al. 2002). These effects decrease the further the imperfection is

from the centre of the near-palindrome.

Short DNA palindromes and inverted repeats are known to occur naturally, such as at

origins of DNA replication in plasmids and viruses in prokaryotes and eukaryotes (Jin and Novick

2001; Willwand, Moroianu et al. 2002). They have also been implicated in the regulation of

gene expression given the requirement for palindromes/inverted repeat symmetry for

transcription of some genes (Glucksmann, Markiewicz et al. 1992; Kim, Peng et al. 1998).

Palindrome Metabolism in E. coli

In E. coli, the first experiments regarding palindromic DNA involved palindrome-

containing plasmids in phage constructed in vitro and introduced in the cell (reviewed in

(Leach 1994)). Analysis of transformants revealed that palindromes any larger than 150-200 bp

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were inviable (transformants did not contain any introduced DNA presumably due to a block in

replication) and palindromes as small as 20 bp were subject to partial or complete deletion,

referred to as instability. This instability increases with the length of the palindrome (up to 150-

200 bp). This instability or inviability in E. coli makes palindromic sequences very difficult to

analyze by standard molecular genetic techniques (incidentally, palindromes are also resistant

to amplification by standard PCR because of their ability to self-anneal). This „cloning barrier‟

explains why palindromes are under-represented and/or present in an incomplete form in the

human genome database (Lewis, Chen et al. 2005).

SbcCD-dependent breakage

There are two theories regarding palindrome metabolism in E. coli. The first theory is

that palindrome inviability in E. coli is dependent on the SbcCD complex, homologs of

eukaryotic Mre11/Rad50 (Naom, Morton et al. 1989; Gibson, Leach et al. 1992; Connelly and

Leach 1996). As SbcCD can cleave hairpin DNA molecules in vitro (Connelly, Kirkham et al.

1998), it likely exerts its effects by cleaving secondary structures formed in the cell (Leach,

Okely et al. 1997). A recent study (Eykelenboom, Blackwood et al. 2008) introduced a 246bp

closely spaced inverted repeat (24bp spacer) into the E. coli chromosome. Pulsed-field gel

electrophoresis revealed that DSBs are introduced in the vicinity of the inverted repeat centre.

The generation of these DSBs is both SbcCD- and replication-dependent. The physical and

genetic evidence presented by this study supports a model whereby a hairpin forms during

replication on the lagging strand after passage of the replication fork (Fig. 1.4). SbcCD cleaves

the hairpin and a DSB is produced. Further evidence indicates that the DSB is repaired via the

RecBCD recombination pathway.

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Figure 1.4: Model of SbcCD mediated palindrome deletions in E. coli. During replication, palindromic DNA forms a hairpin structure on the lagging strand. Replication progresses past the hairpin, then the tips of the hairpin are cleaved by SbcCD. This results in one intact strand (formed from the leading strand) and a DSB in the other (formed from the lagging strand) (Eykelenboom, Blackwood et al. 2008).

SbcCD

Okazaki Fragment

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Replication slipped mispairing

The second theory regarding palindrome metabolism in E. coli is also replication-

dependent and results in deletions (reviewed in (Leach 1994)). One group examined the

deleterious effects of a direct repeat flanked palindrome in the ampicillin resistance gene in

pBR322; deletion of the palindrome resulted in ampicillin resistant colonies. Sequence analysis

revealed that the deletion occurred via hybridization between the flanking repeats (DasGupta,

Weston-Hafer et al. 1987; Weston-Hafer and Berg 1989; Weston-Hafer and Berg 1991). They

propose a replication slipped mispairing model to explain their findings (Fig. 1.5). Hairpins are

formed in the lagging strand during DNA synthesis. When the replication machinery encounters

the hairpin, it stalls. The nascent strand dissociates and hybridizes with nearby complementary

sequence. This model is consistent with a similar, more recent study. A plasmid containing a

palindrome flanked by direct repeats was introduced into a sbcD mutant strain and sequence

analysis revealed deletion breakpoints at the direct repeats (Bzymek and Lovett 2001) implying

that these deletions are SbcCD-independent. Replication slipped mispairing can also occur

between a direct repeat located just inside the palindrome and a flanking direct repeat. This

implies that once the replication machinery encounters the hairpin, it progresses a short

distance into the hairpin before dissociating (Weston-Hafer and Berg 1989; Bzymek and Lovett

2001). A distinct feature of replication slipped mispairing is that the deletion always spans the

symmetry centre of the palindrome or inverted repeat.

Palindrome Metabolism in Yeast

Palindromes in yeast have a more varied behaviour than those in E. coli. There is

evidence that palindromes, inverted repeats and other hairpin-forming structures are deleted

during replication in a manner similar to replication slipped mispairing in E. coli (Gordenin,

Lobachev et al. 1993; Henderson and Petes 1993; Ruskin and Fink 1993; Tran, Degtyareva et

al. 1995; Lobachev, Shor et al. 1998). Short palindromes and inverted repeats can undergo

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Figure 1.5: Palindrome deletion by replication slipped mispairing. A-B: A replication fork proceeds towards DNA containing a palindromic sequence. C: While single stranded, the palindromic DNA on the lagging strand forms a hairpin. The synthesizing lagging strand initially stalls at the hairpin, then dissociates from the template and re-anneals at a homologous sequence beyond the hairpin. D: Deletion of the palindrome can occur after a second round of replication. If only the central part of the palindrome takes place in hairpin formation, then the deletion will only include this central area. The deletion always includes the symmetry centre of the palindrome. Please see text for references.

microhomology

slippage

Okazaki

Fragment

A

B

C

D

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amplification to create larger palindromes. For instance, budding yeast can maintain their

chromosome ends in the absence of telomerase or telomeric recombination by forming large

DNA palindromes; as such, this has been termed the PAL-mechanism of chromosome

maintenance ((Maringele and Lydall 2004) and reviewed in (Maringele and Lydall 2005)). The

PAL-mechanism occurs only in cells that have lost Exo1 activity, allowing the cells to overcome

checkpoint arrest and continue to divide. This type of palindrome amplification may be the

same as that which occurs in response to a DSB in yeast. The authors propose that the

inverted repeats can fold back on themselves to form a short hairpin and prime new DNA

synthesis. Break-induced replication of the hairpin-capped DNA creates a longer palindromic

sequence ((Butler, Yasuda et al. 1996)and reviewed in (Haber and Debatisse 2006)) (Fig. 1.2).

Amplification leading to giant palindromes occuring in response to chromosomal fragility

is observed at the location of an Alu inverted repeat in yeast chromosome V (320bp arms

separated by a 12bp spacer). The researchers observed that the inverted Alu repeats

stimulated a DSB at their centre and that the ends consisted of covalently closed hairpins

(Lobachev, Gordenin et al. 2002). The group proposes that the hairpin-capped DSB arises as a

result of cruciform extrusion in the Alu repeats followed by resolution of the four-way branch

structure. This occurs by diagonal cleavage across the base of the cruciform by a putative

nuclease in a manner analogous to Holliday junction resolution, hence a „cruciform resolution‟

model (Fig. 1.6). The MRX complex (Mre11-Rad50-Xrs2) and Sae2 are required to open the

hairpins and initiate single-strand resection of the DNA ends and that this can lead to the

formation of intrachromosomal amplicons (Narayanan, Mieczkowski et al. 2006; Narayanan and

Lobachev 2007). They propose that replication of the broken ends results in palindromic

dicentric chromosomes which break during anaphase. This stimulates break-induced

replication and recombination of the open-ended chromosome. Break-induced recombination

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Figure 1.6: Model of cruciform resolution. When palindromic DNA extrudes a cruciform (A), a putative resolvase enzyme cleaves diagonally across the base of the cruciform (B). This leaves a hairpin-capped DSB (C) (Leach and Stahl 1983; Lobachev, Gordenin et al. 2002).

A

B

C

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can occur intrachromosomally (Tran, Degtyareva et al. 1995; Narayanan, Mieczkowski et al.

2006) as well as interchromosomally, leading to translocations and gene conversion (Gordenin,

Lobachev et al. 1993; Tran, Degtyareva et al. 1995; Lobachev, Shor et al. 1998; Narayanan,

Mieczkowski et al. 2006).

Thus palindromes and inverted repeats in yeast can undergo simple deletions, such as

those seen in E. coli, or they can potentially lead to more extensive chromosomal aberrations

such as DNA amplification, although the specific mechanisms by which this amplification occurs

are speculative at this point.

Palindrome Metabolism in Mammalian Cells: Insight from Model Systems

Our lab has developed two systems for examining palindrome metabolism in mammalian

cells. The first is an extrachromosomal palindromic dimer assay. The second is an

intrachromosomal 15.4kb perfect palindromic transgene integrated into the mouse genome.

Examination of palindrome metabolism in these two systems has revealed that palindromes do

not always cause gross chromosomal instability leading to the idea that mammalian cells have

developed a system that modifies palindromes before they are able to cause damage.

Revision of extrachromosomal palindromes

The extrachromosomal palindromic dimer assay was developed in the course of

investigating hairpin DNA metabolism in eukaryotic cells (Lewis 1999). The specific details of

the assay and dimer construction will be discussed in Chapter 3. A palindromic dimer does not

transform E. coli, consistent with previous reports on large palindromes and their inviability

(reviewed in (Leach 1994)). When transfected into rodent cells, the dimer underwent

modifications to produce monomer sized DNA molecules capable of transforming E. coli. These

monomer products were DpnI resistant, indicating that they were unmethylated and thus had

19

been formed in the rodent cells. Sequence analysis revealed that breaks were induced quite

near the two symmetry axes of the dimer followed by rejoining; often the symmetry axes

remained intact. Monomer products also contained insertions which can be divided into two

categories: small insertions of fewer than 25bp of apparently random sequence, as well as

larger insertions, some of which contained vector-derived sequence. The joints also exhibited

variable sized deletions as well as joining apparently mediated by microhomology.

Revision of integrated chromosomal palindromes – the Line78 transgenic mouse line

The Line78 transgenic mouse line was originally created in a study that intended to

measure recombination in the male germline (Akgun, Zahn et al. 1997). The introduced

fragment contained two defective copies of the lacZ allele. Recombination between the two

alleles would result in a complete copy of the lacZ+ allele. One of the founder mice, mouse 78,

acquired two copies integrated in a tail-to-tail manner, thereby regenerating a PstI site at the

centre. The structure of the transgene was examined by Southern analysis (Akgun, Zahn et al.

1997) and DNA sequence analysis (Cunningham, Cote et al. 2003) and determined to be a

perfect palindrome.

A thorough analysis of the Line78 palindrome behaviour was conducted in the male

germline (Akgun, Zahn et al. 1997). Breeding analysis revealed that the Line78 transgene was

inherited in a Mendelian ratio. However, the transgene was not inherited stably. Novel

arrangements of the transgene were frequently detected in progeny of mice containing the

input palindrome – 15 to 56% of progeny were rearranged. Based on Southern analysis, it

appeared that the central region of the palindrome was particularly susceptible to

rearrangement; usually one, if not both, of the palindrome arms remained intact. The

variability of the bands seen by Southern analysis implied that this was due to some type of

nonhomolgous or illegitimate rearrangement. Rearrangement of the transgene was stabilizing,

20

so that the transgene was not observed to undergo any further rearrangements (Akgun, Zahn

et al. 1997).

Extensive Southern analysis of tail tip DNA from mice containing variants of the

transgene was performed; three classes of rearrangements were observed (Akgun, Zahn et al.

1997). The two relevant to this study were comprised of small (>200 bp) and large (1-3kb)

central deletions. These deletions often left the symmetry centre intact, indicating that they

occurred by a different mechanism than deletions in E.coli and yeast caused by replication

slipped mispairing. Both classes of deletions were stably inherited. The third class involved

homologous recombination between lacZ alleles and will not be discussed further.

In order to quantify the rate of rearrangement of the intact Line78 transgene in somatic

cells, permanent cell lines were established via Abelson Murine Leukemia Virus (Ab-MLV)

transformation of Line78 fetal liver cells (Cunningham, Cote et al. 2003). The specific details of

the rate assay will be discussed in Chapter 4. It was determined that rearrangement of the

intact Line78 transgene occurs at a rate of about 0.55% per population doubling. As in the

germline, the majority of the rearrangements involved non-homology based deletions extending

from the centre of the palindrome out to one or both arms.

The studies on the Line78 palindrome demonstrate that it can exist in the mouse

genome, although the intact palindrome undergoes frequent stabilizing rearrangements

affecting the symmetry centre. Although the intact Line78 palindrome is far from stable, it does

not appear to be associated with any gross chromosomal instability. For instance, there was no

unbridled amplification of the palindrome, only incremental expansion of the repeats (Zhou,

Akgun et al. 2001) and complete loss of the transgene was never seen (Akgun, Zahn et al.

1997; Cunningham, Cote et al. 2003).

21

Centre Break Palindrome Revision: A Model For Preventative Palindrome

Modification in Mammals

Our lab has proposed a model, termed Centre Break Palindrome Revision, to explain the

asymmetrical deletions and insertions observed in the Line78 palindrome as well as in the

extrachromosomal palindromic dimer assay (Fig. 1.7) (Lewis 1999; Cunningham, Cote et al.

2003). The model proposes that DNA containing a palindromic sequence extrudes a cruciform

structure. A nick is introduced at the tips of the hairpins. Branch migration and resorption of

the cruciform occur, resulting in a double strand break (DSB). The ends are processed by

nucleases and polymerases and eventually sealed. This results in an inverted repeat with a

central region of asymmetry, which is no longer able to extrude a cruciform structure. The

centre break palindrome revision model accounts for the structure of the joints seen in both the

intra- and extrachromosomal palindrome systems. The deletions encompassing only one arm of

the palindrome can be explained as a result of DSB formation at the symmetry centre of the

palindrome followed by processing. Similarly, the retention of the symmetry axes and

nucleotides past the symmetry centre provides evidence for centre-directed breaks, which can

arise from cruciform formation. The variable sized insertions observed can result from a free 3‟

end which can participate in strand invasion and extension. Finally, palindrome revision would

result in the palindrome containing sequence being modified, preventing further cruciform

extrusion and any possible destructive effects that may occur as a result.

NHEJ and its Potential Involvement in Centre Break Palindrome Revision

DSBs are extremely toxic to the cell. Some known causes are ionizing radiation, reactive

oxygen species, replication fork stalling, mechanical shearing at anaphase bridges, and during

V(D)J recombination and class-switch recombination (reviewed in (Sonoda, Hochegger et al.

2006; Soulas-Sprauel, Rivera-Munoz et al. 2007)). If not repaired, the broken chromosomes

22

23

can lead to deletions or translocations of chromosome fragments, potentially resulting in

inactivation of tumour suppressors and/or activation of oncogenes.

There are two well-characterized pathways that repair a DSB, homologous

recombination or NHEJ (reviewed in (Sonoda, Hochegger et al. 2006)). Homologous

recombination is a precise form of repair where a homologous sequence of DNA on a sister

chromatid serves as a template; for this reason homologous recombination occurs only in late S

and G2 phase. NHEJ on the other hand, is an imprecise mechanism active throughout the cell

cycle (reviewed in (Lieber 2008; Weterings and Chen 2008)).

The basic mechanism of NHEJ is outlined below (Fig. 1.8). The first step in NHEJ

involves recognition and binding of the broken DNA ends by Ku. Ku70 and Ku80 bind to either

hairpin-capped or open DNA ends with high affinity (Arosio, Cui et al. 2002) and form a

heterodimeric ring at the DNA ends (Walker, Corpina et al. 2001). Once Ku has bound to the

DNA, it attracts the catalytic subunit of DNA-PK (DNA-PKcs); the three proteins together from

the DNA-PK holoenzyme. DNA-PKcs, a kinase, may be involved in synapsis of the DNA ends

(DeFazio, Stansel et al. 2002). Next, incompatible ends are processed by nucleases and gap-

filling polymerases (TdT, polymerases and ) (reviewed in (Lieber, Lu et al. 2008). It is this

processing, resulting in the loss or gain of sequence, that makes NHEJ an imprecise form of

repair. Finally, DNA LigaseIV seals the break in conjunction with XRCC4 and XLF/Cernunnos

(Grawunder, Wilm et al. 1997; Grawunder, Zimmer et al. 1998; Ahnesorg, Smith et al. 2006).

NHEJ is especially important during development of the mammalian immune system; it

is responsible for joining the DSBs formed during V(D)J (reviewed in (Lieber, Ma et al. 2004)

(Soulas-Sprauel, Rivera-Munoz et al. 2007)). In V(D)J recombination during B-cell

development, DSBs are induced by the RAG proteins. At the coding ends, the RAG proteins

generate hairpin-capped ends that are opened by Artemis. Alone, Artemis acts as a 5‟-3‟

exonuclease, however upon phosphorylation by DNA-Pkcs, it acquires hairpin endonuclease

24

Figure 1.8: Basic Mechanism of the NHEJ pathway. When a DSB is formed, the DNA ends are recognized and bound by the Ku70/Ku80 heterodimer, which in turn attracts the catalytic subunit of DNA-PK (DNA-PKcs). The DNA-PK holoenzyme tethers the broken DNA ends together where they are processed by nucleases and polymerases. Once the ends are compatible they are sealed by LigIV in conjunction with XRCC4 and XLF. Redrawn from (Lees-Miller and Meek 2003).

DSB break

KU70/80

DNA-PKcs

LigIV/XRCC4/XLF

Restored DNA

DNA-Pk holoenzyme

Synapsis and processing

25

activity (Ma, Pannicke et al. 2002). The variability created by NHEJ during V(D)J recombination

leads to a greater diversity of antigen receptors.

On the face of it, NHEJ provides a mechanism for achieving centre break palindrome

revision for many reasons. NHEJ is highly variable because of the nuclease and polymerase

activity; there are a vast number of ways that NHEJ can repair the same DSB. This is

consistent with the variability observed in Palindrome Revision joints. Moreover, NHEJ accounts

for the opening of the hairpins (Artemis) and the joining of the processed ends (DNA LigaseIV

complex).

Thesis Rationale

Based on their involvement in translocations, deletions and amplifications, we know that

DNA palindromes and their propensity to form secondary structures have the ability to wreak

havoc in our tightly controlled genomes. A more comprehensive understanding of how cells

prevent palindrome-induced damage will help us understand how and why these palindromes

are so harmful when not properly maintained. Ultimately, we expect this knowledge will help

illuminate some of the mechanisms behind oncogenesis and other genetic diseases.

The focus of this thesis will be on elucidating the mechanism of palindrome revision in

mammalian cells. In the first half I have addressed the question „what DNA DSB repair factors

are implicated in centre break palindrome revision?‟. Using the extrachromosomal palindromic

dimer assay, I will present evidence that, contrary to junction appearance, palindrome revision

is not solely dependent on NHEJ and that other alternative end-joining factors are responsible

for a significant portion of the end-joining observed. In the second half, I will demonstrate how

small modifications near the symmetry centre of the Line78 palindrome stabilize it from further

rearrangements. The effect of these small modifications on Palindrome Revision provides

continuing evidence in support of the centre break palindrome revision model.

26

Chapter 2: Materials and Methods

27

Note to Reader

Lesley Cunningham performed the fetal liver harvests, Ab-MLV infection and cloning of the ATM

+/- and ATM -/- cell lines. Julia Appleby performed the fetal liver harvests and Ab-MLV

infection of the Ku70 +/- and Ku70 -/- cell lines. Ming Li performed fetal liver harvests and Ab-

MLV infection of the Line78 ∆4bp cell lines.

28

Mice and Cell Lines

The Line 78 mouse strain originated in Dr. Maria Jasin‟s lab (Sloane Kettering, NYC). It

contains a 15.4kb perfect DNA palindrome, as described in (Akgun, Zahn et al. 1997). We

obtained the line as a heterozygote for Line 78 in a (C57BL/6 X CBA/CA)F1 back-cross

background. Our Line 78 colony was maintained by crossing heterozygous males to Balb/C

(purchased from Charles River Laboratories) mice (Appleby 2005).

The Line 78 4bp originated in a female mouse heterozygous for Line78 (mouse

#B5383). #B5383 was then crossed with a male Balb/C; 3 of the female offspring (mouse #

78D1306, #78D1307 and #78D1310) were back-crossed to a male Balb/C (Table 2.1). Fetal

livers from these crosses were harvested and cell lines were generated by Ab-MLV infection, as

described below (Table 3.2). The 4bp genotype was confirmed by southern blotting, DC-PCR

and sequencing, as described below.

Atm +/- mice were purchased from Jackson Laboratories (129/SvEv-Atmtm1Awb) and bred

to C57B6/CBA, Balb/C and C.B.-17/lcrCR/-scidBR mice (Table 2.1). Ku70 null mice were

obtained from Dr. Yansong Gu (University of Washington, Seattle) (Gu, Jin et al. 1997).

The human pre-B cell line Nalm-6 (wild type) and NII4P2 (DNA LigIV null) cell lines were

obtained from Dr. Michael Lieber (University of Southern California, Los Angeles) (Table 2.1).

Briefly, to create the NII4P2 cell line, the open reading frame of the DNA Ligase IV gene in

Nalm-6 cells was disrupted by insertion of a neomycin or puromycin resistance gene, eliminating

DNA LigIV catalytic activity as well as suppressing binding to XRCC4 (Grawunder, Zimmer et al.

1998).

All cell lines used in this study were maintained in 5% CO2 at 37 in RPMI (Wisent or

HyClone) containing 10% heat-inactivated fetal bovine serum (Wisent or HyClone), and 50M

-mercaptoethanol (Sigma). Ab-MLV transformed cells were split 1:50 every 3 days, Nalm-6

and NII4P2 cells were split 1:20 every 3 days.

29

Table 2.1: Source of cell lines used in this study.

Genotype Cell Line Source (reference)

Line 78 4bp 78D1307FL-6-1 78D1307FL-1-2 78D1310FL-1-2

Generated by Ab-MLV infection of fetal

liver of Line 78 4bp mice generated in our lab.

ATM +/- M243FL-3(2)-1 Generated by Ab-MLV infection of mice fetal livers (Cunningham, 2002). ATM -/- M243FL-4-1

Ku70 +/- K2016FL-2 Generated by Ab-MLV infection of mice fetal livers (Gao et al., 1998). Ku70 -/- K2016FL-3

DNA LigIV +/+ Nalm-6

Grawunder et al., 1998. DNA LigIV -/- NII4P2

30

Fetal Liver Harvest

Breeding pairs were placed together on day 0 and separated by day 3. Pregnant females

were sacrificed on day 18, ensuring that the fetuses were between 16 and 18 days of gestation,

as this is when cells are sensitive to Ab-MLV infection and transformation (Rosenberg and

Baltimore 1976).

Under sterile conditions, uterine horns from pregnant females were removed. Each

fetus was then removed from the amniotic sac and placed in a separate 10cm dish containing

3ml of media (RPMI containing 20% heat-inactivated FBS, 50 uM B-mercaptoethanol and

100U/ml penicillin and 100 mg/ml streptomycin (Gibco)). Livers were dissected from the

fetuses and placed in 3cm dish containing 1ml of media.

In order to prepare a cell suspension, the livers and media were passed through a nylon

mesh (Cell MicroSieve, 70 micron pore size, Biodesign, or Nitex Nylon, 100 micron pore size,

Genesee Scientific). Cell suspensions were diluted to 2x106 cells/ml, and 1 ml was plated in a

6-well dish. Each well was then seeded with 1ml of Abelson murine leukemia virus (stocks were

prepared from a p-160 Ab-MLV producer cell line as previously described (Cho, Webber et al.

1999) and 4ug/ml polybrene (Sigma). Following infection, cells were rocked every 30 minutes

for 2 hours and incubated at 37C in 5% CO2. Five days post-infection, cells were fed with 2ml

of fresh media. Uninfected fetal liver cells were collected for DNA extraction and genotyping

(see below).

For the following 45-60 days, the cells undergo "crisis"; split ratios were initially only 1:2

every 3 days but gradually increased as the cultures because more confluent. Crisis is

considered complete when the cultures can be split 1:50 every three days and no apoptotic

cells were evident under the light microscope. At this time, cultures were switched to media

containing RPMI, 10% heat-inactivated FBS and 50uM B-mercaptoethanol. Cloned cell lines

were isolated by limiting dilution (see below).

31

Cloning and Subcloning by Limiting Dilution

Cells were plated on 96-well dishes at concentrations of 0.06, 0.19 or 0.31 cells per well

for ATM +/- and ATM -/- and for Ku70+/- and Ku70 -/- cell lines. Line 78 4bp cell lines were

plated at concentrations of 0.06 and 0.63 cells per well. After 12 days, wells with growth were

scored and select wells were expanded to cultures split 1:50 every 3 days (Table 2.2).

Genomic DNA Preparation

Approximately 2x107 cells from cell lines or liver tissue were collected at 100xg. Cells

were washed with RPMI and resuspended in 425 l of digestion buffer (100mM EDTA, 50mM

Tris pH 8.0, 1mg/ml Proteinase K (Sigma) and 0.5% SDS). When isolating DNA from mice tails,

approximately 0.5cm of tail was used.

Samples were rotated at 55C overnight, followed by one extraction with an equal

volume of phenol/chloroform/isoamyl alcohol (25:24:1) (500l) and one extraction with

chloroform in Phase Lock Gel 2.0ml heavy tubes (Eppendorf). The DNA was then precipitated

with an equal volume of isopropanol then pelleted. Precipitated DNA was resuspended in 200

to 500l TE and incubated overnight at 37C to reconsitute.

DNA concentration was determined using a Nanodrop (ND-1000) spectrophotometer

(Thermo Fisher Scientific).

Line 78 PCR conditions

PCR reactions were carried out using a PTC-200 thermal cycler (MJ research). Standard

reactions were comprised of 16mM (NH4)2SO4, 67mM Tris-HCl pH 8.8, 0.01% Tween-20, 25mM

MgCl2, 0.2mM of each primer, 0.2mM dNTPs and 0.5U of Taq (Bioron) in a total volume of 50l.

Primer sequences are listed in Table 2.3.

32

Table 2.2: Results of subcloning of Line 78 4bp by limiting dilution.

Cell Line Generation # of clones analyzed

(# of wells picked)

# of wells with growth

# of wells seeded

78D1307FL-1-2

66.7 7 7 96

96 4 15 96

148 24 24 384

78D1307FL-6-1 136 11 11 384

78D1310FL-1-2

52 9 23 96

81.3 3 5 96

133.3 29 29 384

Cultures were seeded at concentrations of 0.06 cells/ml and 0.63 cells/ml in 96-well plates.

33

Table 2.3: Primers used in this study.

Target Primer Name Sequence 5’-3’

Line 78 Left-flank (left-flank PCR)

AC174 CCG TGG CAG TAA CCA TTA AGA CC

DC5 GTC CAA CAA TCC AGC TTC AGG

Line 78 (DC-PCR)

Les1 CCT GTG TAG GTT CCA AAA TAT C

Erc1 AGC ATT ATC CTT ATC CAA AAC AGC

ACLF1 CTT TCC AGA TAC GAC GCA GG

ACR1 GGT GAC ATC ACA GTC TAA GGA GG

pGG52 monomer (sequencing)

TB-Fwd GAA AAG TGC CAC CTG ACG

TB-Rev GAG CAA CTG ACT GAA ATG CC

TB-004 AGA TCC AGT TCG ATG TAA CC

TB-005 ATG GCG GGA TCA ATT CGA GC

pJH298 monomer (sequencing)

lacprime CTC ATT AGG CAC CCC AGG CT

TB-001 CTA CAC AAC GAA GAG GTC C

TB-002 GAA CAC TAT CCC ATA TCA CC

TB-003 CGA TTC ATT AAT GCA GCT GG

34

The PCR conditions for Line 78 analysis are as follows: 94C for 3:00, 25 cycles at 94C

for 30sec, 58C for 45sec and 72C for 60sec, followed by a 10:00 extension at 72C. PCR

products were gel electrophoresed in a 2% agarose gel.

Mice were screened for the presence of Line 78 by using primers that spanned the 'left'

integration site (Left flank PCR), AC174 and DC5; a 200 bp band was indicative of transgene

inheritance.

DC-PCR

DC-PCR was used in order to determine the structure of the transgene in the area

adjacent to the centre of symmetry (Cunningham, Cote et al. 2003). 3g of DNA was digested

with 1U of Pst1 (Fermentas) overnight at 37C. The digestion reaction was then heat-

inactivated at 80C for 20min, then ligated with 80U T4 DNA ligase (NEB) in an 800 l reaction

for 2 hours at room temperature. The ligation reaction was then ethanol precipitated and

resuspended in 100l TE.

The left and right side of the Line 78 palindromic transgene were amplified in separate

nested reactions. The left side was amplified using the primers Les1/ACLF1 followed by

Erc1/ACLF1; the right side by using Les1/ACRF1 followed by Erc1/ACRF1. An intact transgene

resulted in a PCR product of approximately 200 bp from each side.

DC-PCR products were cloned into the pCR2.1-TOPO vector using the TOPO TA Cloning

kit (Invitrogen) using 1/3 of the manufacturers' recommended reaction volumes. The cloning

reaction was transformed into OneShot Top10 competent E. coli (Invitrogen) and plated onto

agar plates containing 100 g/ml of ampicillin. Plasmid DNA was prepared using the GeneJET

plasmid mini-prep kit (Fermentas) and sequenced by Macrogen Inc. (Seoul, Korea). Sequences

were aligned and analysed using BLAST 2 sequences (bl2seq program,

www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi, (Tatusova and Madden 1999).

35

Southern Analysis

5g of genomic DNA was digested with 10U of the desired enzyme with the

recommended buffer in a total reaction volume of 30l, overnight at 37C. DNA samples were

electrophoresed in a 0.8% ultra-pure agarose gel (Invitrogen) with ethidium bromide in 1x TAE

at 57V for 4 hours.

After electrophoresis, the gel was rocked for 8min in 0.25M HCl to depurinate the DNA,

followed by 2 brief rinsings in H2O to remove residual HCl. The gel was then rocked in

denaturation solution 1 (0.5M NaOH, 1.5M NaCl) for 20 minutes, followed by 20 minutes in

denaturation solution 2 (0.25M NaOH, 1.5M NaCl).

Transfer was performed in denaturation solution 2 onto Genescreen transfer membrane

using upwards capillary transfer. After transfer, the membrane was rinsed with 2xSSC + 0.2M

Tris-HCL pH 8.0 and dried between 2 pieces of Whatmann paper.

Blots were probed with a labelled 3.5kb BamH1 fragment prepared from the Ercan 1.8

plasmid (Akgun, Zahn et al. 1997). Probe DNA was labelled using random primed synthesis;

50ng of probe DNA and 1 g of hexamers were boiled for 10min in 14l total volume. The

reaction was cooled on ice for 2min, then 5U of Klenow (NEB) with supplied buffer, 2.5mM

dNTP-dCTs and 50Ci -32P-dCTP were added to make a total volume of 25l. The reaction

was incubated at room temperature for 4 hours. The probe was purified using the ProbeQuant

G-50 Micro Columns (Amersham) according to the manufacturers' instructions.

Pre-hybridization was carried out for 4 hours at 42C in 25mL of Stark's buffer (5x SSC,

25mM NaPO4 pH 6.5, 5x Denhardt's, 50% formamide) supplemented with 6.25mg of boiled

torula RNA, 0.01% SDS and 125mg milk powder. The solution was first heated to 65C.

Hybridization was carried out in the same solution as above but with the addition of 1g

Dextran-Sufate Sodium Salt (Fisher Scientific). The probe was boiled for 10min and cooled on

ice for 2 min before addition to the hybridization solution. Hybridization was allowed to occur

36

overnight at 42C.

The hybridization solution was discarded and the membrane was rinsed briefly in 25mL

of Blot Wash Solution 1 (2x SSC, 0.1% SDS), then washed in same for 15min at 42C. Blot

Wash Solution 1 was discarded and the membrane was washed with Blot Wash Solution 2 (0.2x

SSC, 0.1% SDS) at 65C for 20 minutes, twice. Southern blots were then exposed on a

Phosphor screen (Molecular Dynamics) for 5-7 days and visualized using a PhosphorImager

Storm 860 (Molecular Dynamics) and ImageQuant computer program.

Rate Analysis

The rate of rearrangement of the Line 78 palindrome was calculated as in Cunningham

et al. (2003). Rates were based on the following equation:

x/y=(1-p)n

The fraction „x/y‟ is equal to the fraction of subclones with an input Line 78 transgene out of all

the subclones analyzed, and is determined by sampling subcultures via limiting dilution

subcloning. „n‟ is the number of population doublings since the subcloned line was seeded as a

single cell, and is calculated by the number of days that the cell line (since single cell) has been

in culture, given a population doubling time of 18 hours. Inputting these values into the above

equation allows us to solve for „p‟, which is the rate of rearrangement per population doubling.

Rate determinations from a culture at different generations, and/or from different

cultures of the same genotype (at different generations) can be pooled using the Maximum

Likelihood Estimate (MLE). MLE calculates the Likelihood (L) of a series of rates and determines

which rate best fits with the observed data. It also takes into consideration the limiting dilution

results (number of wells of growth/ total number of wells seeded, Table 4.3), as well as allows

calculation of 95% confidence intervals, even when the rate of rearrangement is 0 (Edwards

1972; Rosche and Foster 2000).

37

Maximum likelihood estimate sample calculations

Sample calculations to determine the Log Likelihood (LL) will be carried out using the

values for cell line 1307-1-2-1 at population doubling (n) 96, as in Table 4.4. The following

values were used: the estimate rate (R*) is equal to 0.003, x equals 3, y equals 4, the number

of wells seeded equals 96, and the number of wells with growth equals 15.

1. The fraction of intact Line78 transgenes (Fraction Intact, or FI) expected at a particular

doubling time (n) is calculated for an estimated rate (R*) according to the following

formula: FI = (1-R*)n, so FI=(1-0.003)96, and therefore FI=0.7494.

2. Next calculate the Revised Fraction (RF), which takes into account that a given well with

growth has a certain probability of being seeded by one cell, two cells, three cells etc.

The probability of each well being seeded by x number of cells follows a Poisson

distribution of rare events. It is calculated as follows: RF= (e-) [(e()(FI)-1)/( 1- e-

)], so RF=(0.844)[(e(0.1698)(0.7494)-1)/(1-0.844)], and therefore RF=0.7332.

The following was used to calculate in our sample: e-=1-(total # of wells with

growth/total # of wells seeded), so e-=1-(15/96)=0.844. Since =-ln(e-), then

=0.1698.

3. Log likelihood is then calculated using RF, x and y using the following equation:

LL=x(ln(RF/(1-RF)))+y(ln(1-RF)), so LL=3{ln[0.7332/(1-7332)]}+4[ln(1-0.7332)], and

therefore LL=(-2.25).

Steps 1 through 3 are then repeated for a series of estimated rates. The LLs are then

plotted against the estimated rates; the peak of the curve represents the rate most likely to fit

the data. The 95% confidence interval is bounded by the rates corresponding to the peak LL

+2 (LL=-4.25) (Rosche and Foster 2000).

Data from different experiments can be pooled by adding the LLs at each rate together

and then plotting. A sample curve is represented in Figure 2.1.

38

Likelihood ratio test

The likelihood ratio test can determine whether the data sets from different samplings

differ significantly from one another. The alternate hypothesis, that all experiments are

significantly different, is determined by summing the LL of the peak rate for each experiment

(LL1 + LL2 + LL3 +….). The null hypothesis, that the experiments are not significantly

different, is calculated as though all data were from one experiment (LL (1 + 2 + 3+…)) and

then determining the peak LL of this summation. The critical value is then determined as the

difference between the LL of the alternate and null hypothesis multiplied by two (2*([LL1 + LL2

+ LL3+…] – [LL(1 + 2 + 3 +…)])). Using the calculated critical value and setting the degrees

of freedom to the number of experiments being compared minus one, the p-value can be

determined by the 2 test. A p-value less than 0.05 allows for rejection of the null hypothesis.

39

Figure 2.1: Maximum likelihood estimate. The Log Likelihood (y) is calculated for a series of estimated rates (x). The peak rate represents the rate that best fits with the observed data. The 95% confidence interval is bounded at the rates corresponding to the peak LL+2 (see text).

40

Palindromic Dimer DNA preparation

The source plasmid for the palindromic dimer used to transfect human cell lines (Nalm-

6, NII4P2) was pGG52 (Gauss and Lieber 1996) (Fig. 2.2A). This plasmid was also used for

V(D)J assays in these cell lines. The source plasmid for the mouse (Ku70+/-, Ku70-/-)

palindromic dimer was pJH298 (Lieber, Hesse et al. 1988) (Fig. 2.2B). This plasmid does not

contain a functional RSS signal sequences, so p12x23 was used for V(D)J assays in these cell

lines. Plasmid DNA was purified from DH10B competent E. coli using a CsCl extraction.

The palindromic dimer was constructed in vitro in the following manner: 150g of DNA

was digested with 50U of BamH1 (Fermentas) overnight in 200l. The digestion was

precipitated and resuspended, then digested overnight with 50U of Sal1 (Fermentas), again, in

200l. A linear fragment was then purified from a 0.7% low-melting point agarose gel by

phenol/choloroform extraction. The recovered linearized DNA was ligated at 100g/ml with

approximately 20U of T4 DNA Ligase (NEB) per g of DNA in the suppliers‟s recommended

buffer at room temperature overnight. The ligation reaction was then electrophoresed in a

0.5% low-melting point agarose gel at 40V for approximately 24 hours at 4C and purified via

phenol/choloroform extraction. The purified dimer was transformed into E. coli to determine

the level of non-palindromic DNA (background levels). Only preps with less than 2000 ampicillin

resistant colonies per g of dimer prep were used.

41

Figure 2.2: Schematic of plasmids used in this study. Orange boxes represent the chloramphenicol resistance gene. Purple boxes represent the ampicillin resistance gene. Arrowheads indicate orientation. Black bars represents the bacterial origin of replication. Red bars represent the mammalian origin of replication. Elements are not to scale. Sizes of the linear fragments recovered and BamHI and SalI co-digestion are given. A: pGG52 is the source plasmid used for construction of the human palindromic dimer. It is derived from pBR328. It contains an SV40 ori for mammalian replication (Gauss and Lieber 1996). B: pJH298 is the source plasmid used for construction of the mouse palindromic dimer. It is derived from pUC13. It contains a polyoma ori for mammalian replication (Lieber, Hesse et al. 1988).

pJH298 8012kb

7722kb linear

SalI BamHI

pGG52

6277kb

5948kb linear

BamHI SalI

A

B

42

Transfection Procedure Transfection of pre-B cells was done by electroporation in the presence of DEAE-

Dextran. Transfection procedure was modified from the protocol described in Gauss and Lieber,

1992. Electroporations were performed using a BioRad Gene Pulser with 0.4cm electrode gap

cuvettes (BioRad or Cell Projects). 3.0x106 cells were pelleted, washed in RPMI then

resuspended in 800l RPMI, 10g/ml DEAE-Dextran (Pharmacia) and 100ng-1g DNA. The

suspension was then pulsed at 960FD and 0.3kV. Electroporated cells were then immediately

diluted with 4.2 ml of complete media (see above). Cells were incubated for 48 hours at 37C

in 5% CO2.

Transfected DNA was then harvested by a rapid alkaline lysis protocol as previously

described (Lewis 1994). In the case of control transfections (with source plasmids) or for V(D)J

assays, 3/5 of the harvested DNA was digested with 60U of Dpn1 (NEB) at 37C for 3 hours.

The digestion reactions were then precipitated and resuspended in TE.

Transformation Procedure

Transformation of recovered and/or Dpn1 digested DNA was performed by

electroporation of DH10B E. coli competent cells in a BioRad Gene Pulser at 200 , 25FD in

0.2cm electrode gap BioRad Gene Pulser cuvettes. Electroporated DH10B cells were incubated

with 500l of SOC in a 37C shaker for 1 hour at 220rpm then plated.

For V(D)J assays, cells were plated on LB-agar plates containing 100g/ml ampicillin, as

well as plates containing 100g/ml ampicillin and 10g/ml chloramphenicol. Chloramphenicol

resistant colonies were then streaked out on LB-agar plates containing 100g/ml ampicillin and

25g/ml chloramphenicol. For palindromic dimer assays, cells were plated on LB-agar plates

containing 100g/ml ampicillin.

Revision Product Analysis

43

DNA was isolated from ampicillin resistant colonies using a GeneJet Mini-Prep kit

(Fermentas). Monomer structure was then assessed by restriction enzyme digestion and a

suitable primer was chosen from those listed in Table 4.3. Sequencing was performed by

Macrogen Inc. (Seoul, Korea). Sequences were aligned and analyzed using BLAST 2 sequences

(bl2seq program, www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi, (Tatusova and Madden

1999).

44

Chapter 3: Palindrome Revision is a Form of Non-Canonical End-Joining

45

Note to Reader

Screening for palindrome revision function in DNA-PKcs (+/+, +/-, -/-) and Artemis -/- was

performed by Julia Appleby, a previous MSc. student in the lab.

46

Introduction

The Line 78 mouse colony is a valuable tool for studying the effects of a chromosomally

located palindrome on genome stability. Attempts have been made to determine the enzymatic

activities necessary for palindrome processing by breeding the Line78 palindrome into mice

deficient for DNA repair, although these attempts have so far been unsuccessful. Fortunately, it

is possible to study palindrome processing in eukaryotic cells using the extrachromosomal

palindrome revision assay (Lewis 1999). I utilized this assay in cell lines deficient for NHEJ

components to determine if NHEJ is responsible for palindrome revision in eukaryotic cells.

Experimental approach

In this assay, the source plasmid from which the palindromic dimer is derived (Fig. 2.2)

is digested with BamHI and SalI to liberate the vector backbone fragment. The linearized

fragment is then ligated to obtain a palindromic dimer with a BamHI site at one axis of

symmetry and a SalI site at the other. The palindromic dimer itself is incapable of transforming

E. coli (Lewis 1999), but there is always a small residual amount of source plasmid in any dimer

preparation. When the prep is transformed into E. coli, only preps with less than 2000

ampicillin resistant colonies per µg of DNA (corresponding to residual source plasmid) were

used.

The extrachromosomal palindrome revision assay is illustrated in Figure 3.1A and is

briefly described below: 100 ng of dimer DNA was transfected into a given cell line.

Transfected cells were cultured for approximately 48 hours, at which time plasmid DNA was

harvested from the bulk pool of cells and transformed directly into E. coli. The palindromic

dimer cannot transform E. coli, but it is processed in eukaryotic cells to create a non-repeat

monomer circle. When harvested DNA is transformed into E. coli, transformants either contain

monomer DNA or source plasmid DNA; these can easily be distinguished from each other by

47

restriction enzyme digestion. Transformants containing monomers are scored, then DNA is

isolated and analyzed to assess the quality of palindrome revision junctions formed in all of the

cell lines studied.

It is also possible to measure differences in palindrome revision quantitatively (Fig.

3.1B). In these cases, 100 ng of unmanipulated source plasmid DNA is transfected in parallel

with dimer DNA to normalize for transfection efficiency. Harvested source plasmid DNA is then

Dpn1 digested to eliminate unreplicated plasmid and transformed into E. coli; transformants are

scored and used as a measure of transfection efficiency. There is no need to DpnI digest

recovered monomer products as we know they were processed in the mammalian cells by

virtue of their existence.

48

Figure 3.1A: Schematic of the qualitative extrachromosomal palindromic dimer assay. The palindromic dimer cannot transform E. coli. Upon transfection into mammalian cells, it undergoes site-specific cleavage at the symmetry axes and forms two monomer circles, which can transform E. coli. DNA is the isolated from the bacterial clones and the sequence of the revision joint is analyzed.

Transform

E. coli

(no colonies)

(colonies)

Palindromic dimer

Mammalian cells

Harvest monomers

Transform E. coli

49

Figure 3.1B: Schematic of the quantitative extrachromosomal palindromic dimer assay. The palindromic dimer and source plasmid are transfected in parallel. After harvesting, the source plasmid molecules are digested with DpnI to eliminate any unreplicated molecules. The remaining replicated molecules are transformed into E. coli and colonies are scored as a measure of transfection efficiency. Harvest monomers are transformed directly as in Fig. 3.1A. Individual revision events are scored and compared to the transfection efficiency to obtain a measurement of palindrome revision efficiency.

Revision events

Palindromic dimer

Mammalian cells

Harvest

Transform E. coli

DNA substrate

Transfection Efficiency

Dpn1 cut

B B S S

Source plasmid

B S

50

Figure 3.1C: Schematic of the extrachromosomal V(D)J assay. The V(D)J substrates contains a stop codon between the chloramphenicol resistance gene (camr) and the promoter, as such this substrate does not confer camr upon transformation into E. coli. Upon transfection into mammalian cells, the substrate can undergo V(D)J recombination, which deletes the sequence between the recombination signal sequences (blue and green arrow heads). The promoter is now located directly upstream of the camr gene. Harvested DNA contains 3 species of extrachromosomal DNA: rearranged (A), unreplicated unrearranged (B), and replicated unrearranged (C). DpnI digestion eliminates the unreplicated unrearranged species (B). The remaining DNA is transformed into E. coli. Plating on cam measures the amount of V(D)J rearrangement (A) and plating on amp measures the transfection efficiency (A + C). Colonies are scored and the values are compared to give a measure of V(D)J recombination efficiency.

V(D)J Substrate

Mammalian Cells

Harvest

Dpn1 Cut

cam to measure

V(D)J rearrangement amp to measure

transfection efficiency

(no camr colonies)

A B C

AMP

51

Results

Preliminary gene screen

To find genes involved in palindrome revision, a screen for candidate DNA repair

proteins involved in palindrome revision in mammalian cells was performed. DNA-PKcs (+/+,

+/- and -/-), Ku70 (+/-, -/-) and LigIV (+/+, -/-) were chosen based on their involvement in the

NHEJ pathway. Artemis (-/-) was chosen based on its hairpin-opening ability. ATM (ataxia

telangiectasia mutated)(+/-, -/-), a PIKK signalling kinase, was chosen because it is involved in

activating the signalling cascade resulting in cell-cycle arrest which provides the cell with

enough time to repair DSBs (reviewed in (Lobrich and Jeggo 2005; Lobrich and Jeggo 2005)).

All of the cell lines studied were able to support dimer-to-monomer resolution. A

qualitative analysis of monomers isolated from these cell lines was executed by restriction

digests and sequencing where possible. In total, 17 joints were sequenced from the Artemis -/-

cell line, 10 from DNA-PKcs +/+, 11 from DNA-PKcs +/-, 8 from DNA-PKcs -/-, 12 from ATM +/-

and 21 from ATM -/-. A selection of these sequences is displayed in Table 3.1, as well as a

selection from the Ku70 +/- and -/- and LigIV +/- and -/- cell lines (a complete list of these

junctions can be found in Table 3.S1). All of the monomers studied were variable in terms of

deletion size, insertion size and microhomology usage at the joint and there was no difference

in the degree of variability in any of the cell lines.

Joint analysis reveals common features of palindrome revision between NHEJ-proficient and

NHEJ-deficient cell lines

A more extensive study was conducted in the Ku70+/-, Ku70-/-, LigIV+/+ and LigIV -/-

cell lines (Table 3.2, Table 3.S1). In total, there were 77 monomers analyzed from LigIV+/+

cells, 67 from LigIV-/-, 73 from Ku70+/- and 51 from Ku70-/- cells. The junctions isolated from

NHEJ-deficient and NHEJ-proficient cells were compared based on 6 different criteria. The first

three categories were (1) the length of deletion from the SalI axis (0, <0.1, 0.1-1 or >1kb), (2)

52

Table 3.1: Selected monomer junction sequences.

Genotype

Input Palindrome Sequence

Symmetry Axis LEFT

Symmetry Axis RIGHT

ID#

AGCTTGGGCTGCAGGTC GACCTGCAGCCCAAGC AAGCTGATCCCCGGGGA TCCCCGGGGATCAGCTTG

Artemis -/-

AGCTTGGG GATCCCCGGGGA TCCCCGGGGATCAGCTTG 1

GGAT (-59) CAGCTTG 2

(-210) GCTTAGA (-21) 3

DNA-PK

-/-

AGCTTGGGCAGCAGG AGCTAAGTTTCTGAACTTAGGAGCTAAAGTTCCTGAA AACGTTTCAGTTTTAGTTCCTGAAAATCATGAATTGCAGGGTAT

T-CCCGGGATCAGCTTG 4

(-231) AGCTTG 5

DNA-PK +/-

AGCTTGGGCTGCAGGTC CCCGGGGATCAGCTTG 6

AGC (-39) G/T (-36) 7

DNA-PK +/+

(-208) CCCA(37bp)CTC GGGA TCCCCGGGGATCAGCTTG 8

AGCTTGG (-19) 9

ATM +/-

G (-217) (-1585) G 10

AGCTTGGGCTGCA TAGTTAAGCTGATCG CCCGGGGATCAGCTTG 11

ATM -/-

AGCTTGGGCTGCAGGTC GACCTGC CCAATCTAAATCTAAAGTATATATGAGTAAACTTGGTC (-90) C 12

AGCTTGGGCTGCAGGTC GACC ATG TCCCCGGGGATCAGCTTG 13

Ku70

+/-

AGCTTGGGCTGCAGGTC CCCGGGGATCAGCTTG 14

G (-14) (403bp)(277bp)(100bp)(~200bp)(349bp)(32bp)(165bp) (-635) T 15

AGCTTGGGCTGCAGGTC GACCT CTGG (-1115) T 16

AGCT (95bp) (22bp)A(193bp) (-1018) A 17

AGCTTGGGCTGCAGGTC GAC CTTCGGAAAAAGAGTTGGTAGCTCTTGATCC CCGGGGA TCCCCGGGGATCAGCTTG 18

Ku70 -/-

(-82) T

ATATAATATAATATATTATATATATATATATAATATATATATTATATATATAATAATATATA

TAATAAATATATTTATTATATATATAAGCA (-212) T

19

(-408) T TACG GGA TCCCCGGGGATCAGCTTG 20

AGCTTGGGCTGCAGGTC GAC TCTTCCATATT(420bp)TAG GGA TCCCCGGGGATCAGCTTG 21

AGCTTGGGCTGCAGGTC GACCTGCAGC (62bp)(226bp)ATT (-113) T 22

AGCTCA (-128) (-1504) G 23

(-263) G (263bp)TCCTCG GGGATCAGCTTG 24

Lig4 +/+

AGCTTGGCTGCAGGTC GAC T(675bp) CTAGCTGATCCCCGG GGA TCCCCGGGGATCAGCTTG 25

G (-98) 1.7kb (-201) A 26

GATCC (-417) CCGGGGATCAGCTTG 27

T (-234) (277bp)CCCCGGT (-69) T 28

AGC/TTTG/TG A (-521) 29

Lig4 -/-

AGCTTGGC GG (164bp)C (84bp) GGATCAGCTTG 30

TCCCCG/TG (-51) GGATCAGCTTG 31

AGCTTGGCTGC (-922) T 32

T (-235) (-863) A 33

AGCTTGGCTGCAGG (48bp)C (-876) C 34

AGCTTGGCTGCAGGTC GACCTGCAGCCAA (-61) T 35

Junction sequences from all genotypes studied are highly variable in terms of deletion size, insertion size and microhomology usage. Mutations from the reference sequence are indicated in purple, where the first base is the mutated base and the second is the original sequence. Insertion DNA of unknown origin is indicated in bold while insertion DNA of plasmid origin is indicated in red and insertion DNA of genomic origin is indicated in upper case blue. Underlined nucleotides indicate instances where the joint was formed using microhomology. In instances where the junction wasn‟t sequenced, the size of the deletions and insertions was estimated using restriction analysis and the approximate number of base pairs is indicated in green.

53

Table 3.2: Summary of qualitative revision product analysis. The number of junctions in each category is listed, followed by the percentage in brackets. Data from NHEJ-proficient and NHEJ-deficient cell lines was compared using a Fisher‟s Exact Test. a: all instances of joining via microhomology. For instance, if the left side of a junction was jointed to the insert via microhomology, and the insert was joined to the right side of the junction with microhomology, it was scored twice (see Table 3.1 #18 for an example). The following were considered significantly different: b:p=0.0170 c:p=0.0111

Cell

Line

Total

Left Side Deletion (kb) Right Side Deletion (kb) Insertion (kb) Intact

Sal1

site

Intact

BamH1

site

HOM

usagea 0 <0.1 0.1-1 >1 0 kb <0.1 0.1-1 >1 0 <0.1 0.1-1 >1

Lig4

+/+ 77

14

(18)

49

(64)

14

(18)

0

(0)

9

(11)

23

(30)

39

(51)

6

(8)

24

(31)

35

(45)

13

(17)

5

(7)

10

(13)

6

(8)

15

(19)

Lig4

-/- 67

22

(33)

35

(52)

10

(15)

0

(0)

16

(24)

23

(34)

27

(40)

1

(2)

29

(43)

29

(43)

6

(9)

3

(5)

18

(27)

11

(16)

20

(30)

Ku 70

+/- 73

29

(40)

35b

(48)

9

(12)

0

(0)

14

(19)

24

(33)

20

(27)

15

(21)

36

(49)

21

(29)

8

(11)

8

(11)

18c

(25)

4

(6)

34

(47)

Ku 70

-/- 51

34

(67)

9b

(17)

8

(16)

0

(0)

12

(24)

11

(22)

22

(43)

6

(11)

19

(37)

15

(29)

11

(22)

6

(12)

31c

(61)

8

(16)

25

(49)

53

54

length of deletion from the BamHI axis (0, <0.1, 0.1-1 or >1kb) and (3) length of insertion (0,

<0.1, 0.1-1 or >1kb). These criteria were chosen to reflect the variability in the joints while at

the same time allowing a simple statistical analysis. The other categories were whether the (4)

SalI or (5) BamHI site remained intact as a measure of the centre-specificity of palindrome

revision, and lastly (6) whether the joints were formed using microhomology to determine how

common its use was in palindrome revision.

There was no significant difference in any of these criteria between the LigIV+/+ and

LigIV-/- cell lines (Table 3.2). Two related categories were significantly different between the

Ku70+/- and Ku70-/- cell lines; in the Ku70-/- cell line, more monomers retained the restriction

site at the SalI axis and there were fewer monomers that had a <0.1kb deletion from the SalI

axis (Table 3.2, Fisher‟s Exact Test, p=0.0111 and 0.0170 respectively).

The sequencing results in Table 3.1 and Table 3.S1 revealed several important features

of palindrome revision in mammalian cells. Deletions in both NHEJ-proficient and NHEJ-

deficient cell lines range in size from no deletion to deletions of just over 400 base pairs from

the SalI axis. From the BamHI axis, deletions, when present, could be as large as 1.5kb.

Deletions larger than 400 base pairs from the SalI axis are not observed since either the

bacterial origin of replication (in the mouse monomer – Fig. 2.2B) or the ampicillin resistance

gene (in the human monomer – Fig. 2.2A) lies upstream; presumably, larger deletions may

occur but are excluded from analysis by the nature of the assay.

Palindrome revision joints can be formed simply from the two ends of the halved dimer

or instead may contain inserted sequences. In all 4 cases studied, the majority of the

recovered joints contained inserted DNA; 69% of the LigIV+/+, 57% of the LigIV-/-, 51% of

the Ku70+/- and 63% of the Ku70-/-. When inserted DNA is present in a joint, it can be as

little as a 1, 2 or 3 nucleotides (Table 3.1, #13), or as large as approximately 2kb (Table 3.1,

#26). The composition of the inserted DNA from one monomer to another varies. Inserted

55

DNA can be plasmid-derived (Table 3.1, #18) or genome-derived (Table 3.1, #8), but joints can

also contain insertions that appear to be neither genome- nor plasmid-derived (Table 3.1, #4).

Most monomers contain inserted DNA of mixed origin, for instance, stretches of plasmid-derived

DNA will be interspersed with stretches of untemplated DNA (Table 3.1, #15), or an insert may

contain both plasmid-derived and genome-derived DNA (Table 3.1, #30). Insertions larger than

approximately 800bp could not be sequenced in their entirety and their sizes are estimated by

restriction analysis; in these cases, the origin of the inserted DNA is not known (Table 3.1,

#15,26).

A major feature of palindrome revision is “axis-inclusive” breaks, breaks that retain the

axis of symmetry, as well as nucleotides beyond it; this is indicative of centre-directed

breakage. This occurred in all four genotypes studied, at both the SalI (Table 3.1, #12,13) and

BamHI (Table 3.1, #20,21) axes (Fig 3.2, Table 3.S1). The number of retained nucleotides at

the joints beyond the symmetry axes in all 4 cell lines was tallied and is presented as a

percentage of total joints in Figure 3.2. Up to 19 retained nucleotides were observed in any

given instance, but in the majority of the joints there were fewer than 10. There was no

significant difference between the LigIV+/+ and LigIV-/- genotypes in terms of the number of

axis-inclusive joints. There is also no significant difference in terms of the number of

nucleotides retained beyond the axis. Conversely, in both the Ku70+/- and Ku70-/- genotypes,

there are significantly more axis-inclusive joints at the Sal1 axis as opposed to the BamHI axis

(Fisher‟s exact test, p=0.0029 and 0.0015 respectively). In addition, there are also a greater

number of Ku70 -/- retained nucleotides beyond the SalI axis than Ku70+/- joints (Fisher‟s

exact test, p=0.045).

These retained nucleotides in axis-inclusive joints are reminiscent of the palindromic

nucleotides, or P-nucleotides, that arise when hairpin terminated ends are joined, as in the case

of V(D)J recombination (Lewis 1994). Thus, these retained nucleotides may be the result of

56

nicking hairpin arms arising from cruciform structures during centre break revision. The

difference in retained nucleotides between the BamHI and SalI axes may be sequence specific.

In a study which used related DNA substrates, Meier and Lewis (Meier and Lewis 1993) found

that during extrachromosomal V(D)J recombination in Ab-MLV transformed mouse cells, there

were more P-nucleotides retained at the coding end adjacent to a SalI site than that adjacent to

a BamHI site.

Joints exhibiting microhomology are common in palindrome revision (Table 3.1, #31).

All four genotypes had joints with some degree of microhomology; 17% of LigIV+/+, 31% of

LigIV-/-, 44% of Ku70+/- and 45% of Ku70-/-(Table 3.2). In each instance, the number of

homologous nucleotides was scored (Fig 3.3, Table 3.S1); the majority of microhomology-

derived joints hade between 1 and 3 homologous nucleotides. Use of 6 (Ku70 +/- and -/-) or

even 7 bases (LigIV-/-) did occur, but was rare.

There is no difference in the quantity of palindrome revision joints between NHEJ-proficient and

NHEJ-deficient cells

The efficiency of palindrome revision can be measured by performing side-by-side

transfections of the palindromic dimer and the source plasmid (Fig. 3.1B). Palindrome revision

efficiency is measured as the average number of palindrome revision events divided by the

average number of control colonies (Table 3.3). A large transfection-to-transfection variation in

palindrome revision efficiency was observed in all 4 genotypes. Revision efficiencies were

compared using a Student‟s T-test. There was no significant difference in palindrome revision

efficiency between LigIV+/+ and LigIV-/- genotypes (p=0.2) or between Ku70+/- and Ku70-/-

genotypes (p=0.7) (Fig. 3.4).

57

Figure 3.2: Nucleotides retained in axis-inclusive joints at the SalI and BamHI axes. A subset of revision joints formed were axis-inclusive, in that the symmetry axis remained intact. These joints retained a variable number of nucleotides past the symmetry axis. The number of joins that retained 1-19 nucleotides was tallied and is presented as a percentage of total junctions. Joints formed in Ku70-/- cells appear to retain more nucleotides at the SalI axis.

0

2

4

6

8

10

12

14

16

18

1 2 3 4 5 6 7 8 9 10 12 13 19

Percen

t o

f total

ju

nctio

ns

# of nucleotides retained from Sal1 axis

LigIV+/+

LigIV-/-

Ku70+/-

Ku70-/-

0

2

4

6

8

10

12

14

16

18

1 2 3 4 5 6 7 8 9 11 12 15 18

Percen

t o

f total

jun

ctio

ns

# of nucleotides retained from BamH1 axis

LigIV+/+

LigIV-/-

Ku70+/-

Ku70-/-

A

B

58

0

5

10

15

20

25

1 2 3 4 5 6 7

Pe

rce

nt

of

tota

l ju

ncti

on

s

# of nucleotides

LigIV+/+

LigIV-/-

Ku70+/-

Ku70-/-

Figure 3.3: The number of nucleotides used at junctions formed via microhomology. The number of junctions formed using a particular number of nucleotides of microhomology was scored and plotted as a percentage of total junctions analyzed. The majority of junctions formed using microhomology used only 1 or 2 nucleotides and no junctions used more than 7.

59

Table 3.3: Quantitative analysis of palindrome revision in NHEJ-proficient and NHEJ-deficient cell lines.

Genotype Average # control

coloniesa Average # palindrome

revision eventsb Palindrome revision

efficiencyc

LigIV +/+

103.1 3.825 0.03710

114.0 15.08 0.1323

11.83 6.825 0.5769

135.6 2.850 0.02102

18.48 2.725 0.1475

LigIV -/-

263.7 1.890 0.007167

12.63 1.590 0.1259

22.21 1.180 0.05313

74.53 1.540 0.02066

14.08 1.025 0.07280

Ku70+/-

2.525 1.070 0.4238

2.125 0.8200 0.3859

5.013 2.113 0.4215

Ku70-/-

0.1925 0.3175 1.649

9.750 0.1400 0.01436

0.4500 0.1500 0.3333

Palindrome revision efficiency between Lig4 +/+ and Lig4 -/- and Ku70+/- and Ku70-/- was compared by a Student‟s t-test. There was no significant difference between the NHEJ-proficient and NHEJ-deficient cell lines (Lig4, p=0.2; Ku70, p=0.7). a: Control transfections were done in duplicate. The DNA was harvested, Dpn1 digested to get rid of any unreplicated molecules, and then transformed in duplicate, for a total of 4 transformations. The number of colonies per transformation was counted and the average was taken. b: Dimer transfections were done in duplicate. The DNA was harvested from the mammalian cells and then transformed into E. coli in duplicate, for a total of 4 transformations. The number of independent revision junctions (as determined by sequencing of the junctions) was scored and the average was taken. c: Palindrome revision efficiency is measured as the average # of palindrome revision events divided by the average # of control colonies.

60

Figure 3.4: Palindrome revision efficiency in NHEJ-deficient and NHEJ-proficient cells. Palindrome Revision efficiency was compared using a Student‟s t-test. There was no significant difference between the NHEJ-proficient and NHEJ-deficient cell lines (LigIV, p=0.2; Ku70, p=0.7). Error bars represent the standard error of the mean.

Ku70-/- Ku70+/-

LigIV +/+ LigIV -/-

A

B

61

Discussion

Extrachromosomal mammalian palindrome revision occurs in the absence of NHEJ

The purpose of this study was to determine the role of NHEJ in palindrome revision in

mammalian cells. The NHEJ machinery is comprised of 8 key factors: Ku70, Ku80, DNA-PKcs,

Artemis, Pol X polymerases, XLF, XRCC4 and DNA ligase IV (reviewed in (Lieber, Ma et al.

2003)). I chose to examine two key NHEJ factors in this study. Ku70 is known to have many

diverse functions in the cell (i.e. telomere maintenance) (reviewed in (Tuteja and Tuteja

2000)); its function in NHEJ, in complex with Ku80, is to bind to the free DNA ends resulting

from a DSB, protecting them from degradation by nucleases and preventing binding of the

homologous recombination machinery (Pierce, Hu et al. 2001). DNA LigIV forms a complex

with XRCC4 and XLF and is responsible for re-joining the DNA ends after processing (Ahnesorg,

Smith et al. 2006); it has no other known function in the cell.

V(D)J recombination, a process known to occur via NHEJ, is severely crippled in the

absence of NHEJ-factors (Hesse, Lieber et al. 1989). The extrachromosomal V(D)J assays

revealed that while the LigIV+/+ and Ku70+/- were capable of producing rearranged V(D)J

products, there were no detected V(D)J rearrangement products in the LigIV -/- and Ku70 -/-

cell lines (Table 3.S1, Fig. 3.1C). Identical V(D)J assays using rearrangement substrates were

performed in the same LigIV+/+ and LigIV-/- cell lines (Grawunder, Zimmer et al. 1998). This

study reported a decrease in signal and coding joint formation of 100-1000 fold in the LigIV-/-

cell line. Moreover, in vivo plasmid end-joining assays in NII4P2 (LigIV-/-) cells and in vitro

using NII4P2 (LigIV-/-) cell extracts demonstrate a marked decrease in end-joining frequency

(~14-100-fold) as well as sequence fidelity; the joints had approximately twice as many deleted

nucleotides as the Nalm-6 (LigIV+/+) cell extracts (Smith, Riballo et al. 2003). Finally, as LigIV

has only one known function in the cell, and that is during NHEJ, it follows that if palindrome

62

revision were reliant upon LigIV and the remaining NHEJ factors, that it would be severely

crippled in its absence.

The same can be said of Ku70‟s involvement in palindrome revision. Although no

studies on V(D)J recombination in the absence of Ku70 have been performed in Ab-MLV

transformed cells, studies in ES cells and fibroblasts have shown a decrease in coding joint

formation (10-100 fold) (Zhu, Bogue et al. 1996; Bogue, Wang et al. 1997; Gu, Jin et al. 1997;

Ouyang, Nussenzweig et al. 1997). Also, plasmid end-joining studies in vitro and in vivo

demonstrate a requirement for Ku – in its absence, joints are accompanied by large deletions, a

reliance upon microhomology usage and a decreased frequency (Gu, Jin et al. 1997; Verkaik,

Esveldt-van Lange et al. 2002; Kuhfittig-Kulle, Feldmann et al. 2007).

Attempts to quantify palindrome revision were complicated (Table 3.3, Fig. 3.4), by the

variable transfection efficiencies in all four cell lines. Statistical analysis of this data did not

reveal any quantitative differences in palindrome revision efficiency but the results are

questionable due to this variation.

I observed no qualitative difference in the joints formed by palindrome revision between

NHEJ-proficient and NHEJ-deficient cell lines. In fact, in all genotypes studied (Artemis -/-,

DNA-PKcs +/+, +/- and -/-, ATM +/- and -/- as well as Ku70 +/- and -/- and LigIV +/+ and -/-)

several key features of palindrome revision became apparent: a broad spectrum of deleted DNA

from both sides of the symmetry axis, inserted DNA of multiple origins and varying lengths, use

of microhomologies and axis-inclusive joints. Although we can‟t rule out an involvement of

NHEJ entirely, it is undoubtedly not the sole and/or principal mechanism for palindrome revision

in mammalian cells.

A subset of palindrome revision joints are formed via microhomology

A microhomology-mediated end-joining (MMEJ) pathway was first identified in budding

yeast (Boulton and Jackson 1996); in yeast deficient for Ku70, end-joining occurred and was

63

accompanied by large deletions and short stretches of microhomology. Subsequent work in

NHEJ-deficient cells (Ku70, Ku80, LigIV) has further characterized this MMEJ pathway in

budding yeast (Ma, Kim et al. 2003) (Yu, Marshall et al. 2004) as well as in fission yeast

(Decottignies 2005; Decottignies 2007), Arabidopsis (Heacock, Spangler et al. 2004) and

mammals (Bentley, Diggle et al. 2004), (Liang, Deng et al. 2005; Kuhfittig-Kulle, Feldmann et

al. 2007; Liang, Deng et al. 2008). Typically, MMEJ joints contain terminal deletions up to

several hundred base pairs and somewhere between 3 and 16 bp of microhomology depending

on the study. In all of the above systems, few to no joints exhibiting defined MMEJ

characteristics are observed if NHEJ is fully functional.

Factors implicated in MMEJ in yeast are Mre11 and Rad50, Rad22, Exo1 and Pol4 (a

polymerase with gap-filling activity) (Ma, Kim et al. 2003), (Decottignies 2007). These factors

are also involved in single-strand annealing (SSA) in yeast. In humans, MMEJ is known to rely

upon the MRN complex (Mre11/Rad50/Nbs1), Fen-1 and DNA LigI and LigIII (Liang, Deng et al.

2005; Liang, Deng et al. 2008). Based on their in vitro studies with human Mre11, Paull and

Gellert (Paull and Gellert 2000) propose the following model for the function of Mre11 during

MMEJ. The model states that when a DSB occurs, the DNA ends are aligned by the MRN

complex. Mismatched ends stimulate Mre11‟s exonuclease activity, which degrades the DNA

until homology is reached. The single strand overhangs then pair with each other, preventing

further nucleation while a ligase seals one strand. The other strand undergoes further

processing until it too can be sealed and repair finished. In this model, the MRN complex

tethers the ends, preventing strand invasion of any 3‟ overhangs thereby preventing the

opportunity for filler DNA generation. Indeed, the majority of palindrome revision joints joined

by microhomology do not contain any inserted DNA (73% of LigIV+/+, 95% of LigIV-/-, 81% of

Ku70 +/- and 61% of Ku70 -/-) (Table 3.S1). In addition to its involvement in joining via

microhomologies, other activities of the MRN complex point to its potential involvement in

64

centre break palindrome revision; it is known to not only bind to hairpin ends, it also possesses

hairpin endonuclease activity (Paull and Gellert 1999) (Paull and Gellert 2000) in conjunction

with Sae2 (Lengsfeld, Rattray et al. 2007).

The majority of palindrome revision joints I observed did not use any microhomology;

those joints that did were often joined by only 1 or 2 nucleotides and none more than 7 (Fig.

3.3, Table 3.S1). This is fewer nucleotides than the typical MMEJ product although this may

reflect a difference in the experimental system – other end-joining studies in mammalian

systems were performed in vitro using cell free extracts and substrates with a defined regions

of microhomology (Bentley, Diggle et al. 2004); (Liang, Deng et al. 2005; Liang, Deng et al.

2008). If these palindrome revision joints are indeed formed by MMEJ, that only accounts for

approximately 36% of revision joints analyzed in this study. The remaining 64% must be

formed via another pathway (Table 3.S1).

Palindrome revision may be a function of non-canonical end-joining

A very small number of palindrome revision joints (approximately 10%, Table 3.4) were

joined with minimal deletions from both axes with no insertions, or small insertions that can be

explained by terminal deoxynucleotidyl transferase (TdT) activity (Mahajan, Gangi-Peterson et

al. 1999). While we can‟t rule out that in the NHEJ-proficient strains, these joints were formed

via canonical NHEJ, it is obviously not the primary end-joining pathway responsible for

extrachromosomal palindrome revision in mammalian cells.

A non-canonical end-joining pathway has been shown to be Ku-independent (Wang,

Perrault et al. 2003; Guirouilh-Barbat, Huck et al. 2004), DNA-PKcs-independent (Cheong,

Perrault et al. 1999; DiBiase, Zeng et al. 2000), DNA Ligase IV/XRCC4-independent (Wang,

Zeng et al. 2001; Smith, Riballo et al. 2003), and XL-F-independent (Zha, Alt et al. 2007). This

non-canonical end-joining pathway can join DSBs in vitro (Cheong, Perrault et al. 1999; Wang,

65

Perrault et al. 2003), in vivo (Wang, Zeng et al. 2001; Verkaik, Esveldt-van Lange et al. 2002)

as well as play a role in telomere fusion (Heacock, Idol et al. 2007), chromosomal translocations

(Weinstock, Brunet et al. 2007), IgH class switching and translocations (Yan, Boboila et al.

2007) and V(D)J recombination (Brown, Franco et al. 2002; Weinstock and Jasin 2006; Corneo,

Wendland et al. 2007). Non-canonical end-joining occurs with slower kinetics in vivo (DiBiase,

Zeng et al. 2000) than NHEJ and it is known to occur with decreased precision, resulting in

deletions. There is some evidence that non-canonical end-joining can rely on microhomology

(Corneo, Wendland et al. 2007; Audebert, Salles et al. 2008) implying that non-canonical end-

joining and MMEJ may be one and the same. Finally, when DNA-PK is fully functional, it

prevents non-canonical end-joining pathways by binding to DSB ends and shunting them

towards NHEJ (Perrault, Wang et al. 2004). It may be that in terms of DSB repair, NHEJ is the

preferred pathway; non-canonical end-joining is a back-up pathway in case NHEJ is non-

functional. Various studies have pointed to DNA Ligase III (Audebert, Salles et al. 2004; Wang,

Rosidi et al. 2005) and Poly(ADP-ribose) Polymerase-1 (Parp-1) (Audebert, Salles et al. 2004;

Audebert, Salles et al. 2008) as being involved in non-canonical end-joining.

In terms of palindrome revision, my results indicate that non-canonical end-joining is

likely to play a more important role than NHEJ. If this were the case, we would expect to see a

quantitative and/or qualitative difference between NHEJ-proficient and NHEJ-deficient cell lines.

Because this is not the case, we propose that this non-canonical end-joining pathway is the

intended mechanism for palindrome revision in mammalian cells. Furthermore, we propose that

palindrome revision may be the reason for the existence of this non-canonical end-joining

pathway. Under no other circumstances is joining a DSB with such decreased fidelity beneficial,

whereas with palindrome revision, a greater asymmetry at the joint is only further assurance

that the palindrome will not go on to cause chromosomal aberrations.

66

Inserted DNA is a common feature in both extrachromosomal and intrachromosomal palindrome

revision

Inserted, or filler, DNA is a feature of DSB repair by NHEJ in plants (Gorbunova and Levy

1997; Kirik, Salomon et al. 2000), yeast (Yu and Gabriel 1999; Haviv-Chesner, Kobayashi et al.

2007) and mammalian cells (Merrihew, Marburger et al. 1996; Lin and Waldman 2001; Lin and

Waldman 2001). In their studies on genomic DSBs, Lin and Waldman have observed inserted

DNA of both vector, genomic and unknown origin. Vector-derived inserts were the most

prevalent, occurring in approximately 50% of joints analyzed; they were approximately 150 to

490 bp in length. Inserts of unknown origin could be as large as 1490 bp. These results are

very similar to the inserted DNA found in palindrome revision joints in this study, both in terms

of origin and size (Table 3.2, Table 3.S1). Vector-derived inserted DNA was previously seen in

extrachromosomal palindrome revision joints in rodent cells (Lewis 1999).

The inserted DNA could be copied from either the Watson or the Crick strand, or even

both strands in the same insert; it could also be templated from distant or adjacent sequences

(data not shown). This hints at inserted DNA as originating from a strand invasion event as

those previously proposed (Merrihew, Marburger et al. 1996);(Gorbunova and Levy 1997);

(Zhou, Akgun et al. 2001). In the case of palindrome revision, if the hairpins are nicked

sequentially and 5‟ resection of the open end occurs, it would leave a 3‟ single stranded

overhang without another DNA end with which to interact. The 3‟ singled stranded DNA region

could then go on to invade a region of DNA via a short region of microhomology. DNA

synthesis occurs and the newly synthesized strand is immediately displaced from the template.

This newly synthesized DNA can then either join with another free end via end-joining or go on

to invade other regions of DNA, again via short microhomologous stretches. This last scenario

results in joints with short stretches of templated DNA pieced together, as well as joints with

templated DNA interspersed with non-templated DNA (Table 3.1, #15).

67

Finally, inserted DNA has been observed at rearrangement joints of two transgenic mice

lines containing inverted repeats (Collick, Drew et al. 1996). This same study observed

asymmetric deletions of variable sizes in both transgenes. This, in addition to the extensive

deletions and resulting asymmetry observed in the mouse Line78 transgenic palindrome

(Cunningham, Cote et al. 2003), indicates that the extrachromosomal palindrome revision assay

does indeed reflect processing of genomic palindromes.

Future Directions

In this study, I have demonstrated that NHEJ is not the main pathway responsible for

formation of palindrome revision joints in mammalian cells. Future research will focus on

determining which enzymes are specifically required, with particular interest being on the MRN

complex (Mre11, Rad50, Nbs1), Parp-1, LigIII and Fen-1 based on their involvement in non-

canonical end-joining pathways.

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Table 3.S1: Sequence of palindrome revision junctions.

Genotype

Input Palindrome Sequence

Symmetry Centre LEFT

Symmetry Centre RIGHT

AGCTTGGGCTGCAGGTC GACCTGCAGCCCAAGC AAGCTGATCCCCGGGGA TCCCCGGGGATCAGCTTG

LigIV

+/+

C (-65) CT (-531) A

AGCTTGGCTGCAGGTC G G (-391) A

AGCTTGGCTGCAGG GG (-174) G

C (-46) (-28) C

AGCTTGGCTGCAGGTC GA G GCTGATCCCCGGGGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGTC GACCT GA TCCCCGGGGATCAGCTTG

C (-35) CCCCC (-36) A

AG ATCAGCTTG

C (-22) (331bp) A (-191) A

C (-81) CAAGTCTCA (-264) A

AGCTTGGCTGCA CGG (-104) A

AGCTTGGCTGCAGGTC GACC CCT (-78) G

AGCTTGGCTGCAGGTC GACCTGCAG GGGA TCCCCGGGGATCAGCTTG

C (-81) CAAGTCTC (-263) A

AGCTTGGCTGCAG (-150) C

AGCTTGGC GGGGATCAGCTTG

AGCTTGGCTGCAG T A TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAG TC (-446) C

AGCTTGGCTGCAGGT ATAAGCATATGTTCAGTAACAA GGGATCAGCTTG

AGCTTGGCTGCAGGTC GAC T(675bp) CTAGCTGATCCCCGGGGA TCCCCGGGGATCAGCTTG

C (-133) 400 bp (-1092bp)

T (-23) (-89) T

T (-14) AATTTTTTT (-719) A

G (-98) (154bp) 1.5kb (-201) A

AGCTTGGC GC (-427) A

AGCTTGGCTGCAGGTC GA (-177) G

(-150bp) 1kb (-131) T

T (-20) (-88) T

AGCTT (-206) A

AGCTTGGCTGCAG CC (-28) C

A (-24) GA (-185) G

T (-30) GGG (52bp) GGGAT (82bp) CCTGTTT (-149) C

T (-83) (-12) C

AGCTTGGCTGCAGGTC GACC GG (-283) G

G (-439) GA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGT GAC GGGGGAAG (-436) A

C (-15) CCAGCCCG GGGGATCAGCTTG

T (-22) GGCCCAAA (-183) A

AGCTTGGCTGCAGGTC G C (-15) C

AGCTTGGCTGCAGG GGGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGC (-163) T

C (-418) CACAGTGGTAGTACTCCACTGTCTGGCTGTACAAAAACCCTCG GGA TCCCCGGGGATCAGCTTG

T (-202) CCCCGGGGA TCCCCGGGGATCAGCTTG

GATCC (-417) CCGGGGATCAGCTTG

A (-19) ACTAGTTAACTAGTTATAGTTAACTATAACTA (-126) T

T (-234) (277bp) CCCCGGT (-69) T

C (-93) 200bp (-265bp)

A (-74) CTCCT (-95) G

A (-97) (-200) T

T (-123) 700 bp (-265bp)

AGCTT (131bp) A GCTT

AGCTTGGCTGCA CA CCCCGGGGATCAGCTT

T (-76) (-152) G

AGCTTGGCTG GGATCAGCTT

AGCTTGGCTGC AGCG CCGGGGATCAGCTT

AGCTT (204bp) 1.3kb (-1092bp)

AGCTTGGCTGCAGG CCCTGG (-825) G

AGCT G (-526)

AGCTTGG A (-516)

AGCTTGGCTGCAGGTC GACCT AA CCCGGGGATCAGCTTG

(-90) A GA T (-213)

AGCTT T AGCTTG

AGCTTGG 600bp (-400bp)

(-200bp) 200 bp (-81) G

AGCTTGGCTGCAGGTC GACC 1kb (-1.2kb)

(-92) C T (-286) G

AGCTTGGCTGCAGGTC GACCTGCAGCCAAGCTGCG (-514) T

AGCTTGGCTGCAGGTC GACCTGCA TTGGTAACTGTCATACCACTTTTACACATATATACT(193bp)(312bp)1.5kb (-1.2kb)

69

G (-153) (-379) A

T (-267) (112bp)(104bp)(42bp)(233bp) TA (-1263) C

AGCTTGGCTGCAGGTC AT (-771) A

T (-83) CG (-515) T

T (-256) (361bp)(122bp) TACTT (-1263) C

C (-18) T (-347) A

G (-186) TTCAC (-315) C

AGCTTGGCTGCAGG (-371) A

G (-183) TTCAC (-315) C

LigIV

-/-

AGCTTGGC GG(164bp)C(84bp) GGATCAGCTTG

AGCTTGGCTGCA CGA (-108) A

AGCTTGGCTGCA CGG (-104) A

AGCTTGGCTGCAGGTC GACCTGCAGCCAA C (-14) C

A (-209) (-127) C

AGCTTGGCTGCAGG ACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTC (-876) C

AGCTT CGG (-687) G

AGCTTGGCTG (-779) A

AGCTTGGCTGCAGGT ATCAGCTTG

AGCTTGGCTGCAGGTC GAC GCTTG

AGCTTGGCTGCAGGTC GACCTG GGA (-97) A

AGCTTGGCTGC (-922) T

AGCTTGGCTGCAGGTC GACCTG GCATACCTCAAAATGTTCTTTA (-19) C

TCCCC(G/T)G (-51) GGATCAGCTTG

(-200bp) 200bp TCCCCGGGGATCAGCTTG

G (-17) GGGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGC GTATAGTTCCGCACA (-671) A

A (-104) (-401) C

T (-235) (-863) A

AGCTTGGCTGCAGGTC GACCT TTT (-38) A

G (-332) TTTTT (-240) C

AGCTTGGCTGC (-860) C

AGCTTGGCTGCAGGTC 2.3 kb (-200bp)

C (-142) CTG CCGGGGATCAGCTTG

A (-25) TCCA CCGGGGATCAGCTTG

C (-23) GCCA (-540) C

G (-180) CGGAA (-280) A

TCCCCG(G/A) (-160) GGATCAGCTTG

AGCTTGG (-40) A

AGCTTG TGACCCT CGGGGA(T/C)CAGCTTG

C (-18) (-361) T

AGCTTGGCTGCAGGTC GAC (-591) A

AGCTTGGCTGCAGGTC GACCTGCAGCCAA (-61) T

AGCTTGGCTGCAGGTC GACCTGC TCCTGAAAATCTCGCCCCG GGGGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGTC GAC (-583) A

AGCTTGGCTGCAGGTC GACCTG G(204bp)CCCCAGAGATCCGG(212bp) 2 kb (-875bp)

GT (-27) (-500) G

AGCTTGGCTGCAGGTC GACCTGCAG GTCGACCCCG GGGGA TCCCCGGGGATCAGCTTG

T (-50) A (-441) G

C (-18) CCTATACCATGGC GGGATCAGCTTG

C (-18) (157bp) 1.3kb (-1092bp)

AGCTTGGCTG GGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGTC GACC ACAGTGCTACAGACTGGAACAAAAACCCTGCAGTCGACTCTCATCGATG GGGGATCAGCTTG

AGCTTGGCTGCAGGTC GACC CGGGGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGTC GACCT AGGCCAAGGTTAAAAAAATGCTTTGCATTTTTTACATGCTTAAT G (-704)

AGCTTGGCTGCAG CA GA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGT A T (-35)

AGCTTGGCTGCAGGT T (-557)

AGCTTGGCTGCAGGTC GAC A (-148)

(-62) T GAGATAATTGACATACTGTTATGTCAATATA CCCGGGGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGG AAA ATCAGCTTG

AGCTTG A TCCCCGGGGATCAGCTTG

AGCTTGG A TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGTC G T (-526)

AGCTTGGCTGCAGGTC G GGGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGT GGGATCAGCTTG

AGCTTGGCTGCAGG GA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGTC GAC CCGGGGA TCCCCGGGGATCAGCTTG

(-100bp) 500 bp GGGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGTC GAC CGGGGA TCCCCGGGGATCAGCTTG

AGCTTGGCT GGGG (-581) G

70

C (-94) CC (542bp)… …GGATCAGCTTG

A (-25) GGGC (-61) T

G (-183) TTCACCCTTGTT (-315) C

AGCTTGGCTGCAGGTC GACCT A(155bp)TTGGTCGAGG (-955) T

AGCTTGGC G GATCAGCTTG

Ku70+/-

A (-40) (778bp) (-939) T

AGCTTGGGCTGCAG 1.3kb (-489) A

AGCTTGGGCTGCA (-207) C

G (-14) (403bp)(277bp)(100bp)200bp(349bp)(32bp)(165bp) (-635) T

AGCTTGGGCTGC 4kb (-265bp)

(-50bp) 500bp (-265bp)

AGCTTGGGCTGCAGG GGATCAGCTTG

AGCTTGGGCTGCAGG GGGATCAGCTTG

AGCTTGGGCTGCAGGTC CCCGGGGATCAGCTTG

A(-133) ATTAATTTGAGTTAATGTGAGTTAGCATA CCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GA TCCCCGGGGATCAGCTTG

A(-112) CTCAACCCCCGGGTTGTTTGTTATCCATACCA A TCCCCGGGGATCAGCTTG

(-435bp) TGTCAGACATCTCAAGATGAGTGTAG(363bp) 1.2bp CGGG(G/A)ATCAGC(G/T)T

G

(-435bp) 1275bp (-1169bp)

G (-111) 500bp (-265bp)

CA (-38) (-65) C

T(-33) (-29)C

AGCTTGGGCTGCAGGTC GAC (-20) C

AGCTTGGGCTGCAGGTC GACCTGCA (96bp)T GA TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GACCTGCA TACAAGCTGTATGCTT CCGGGGATCAGCTTG

AGCTTGGGCTGCAGG CAAGATTTTCAAGATTTTGGG (-20) C

ATTA (-25) (-578) T

AGCTTGGGCTGCAGGTC GACCTG (670bp) 1.4kb (-265bp)

G (-11) A (-87) T

AGCTTGGGCTGCAG (672bp) (350bp) (-265bp)

AGCTTGGGC A (-180) T

AGCTTGGGCTGCAGGTC GACCT CTGG (-1115) T

AGCTTGGGCTGCAGGTC TACTGGGTTGCAATTTCGATCTGCTACCATG (-339) G

AGCTTGGGCTGCAGGTC GACCT CTGG T (-107)

TCC (-91) (-793) (G/C)

AGCTTGGGCTGCAGGTC GACC (27bp)(27bp)(36bp) GGA TCCCCGGGGATCAGCTTG

C (-18) TGCCTTACTAATGCCTTACTAATGACCGT A TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GAC (728bp) 1.27kb (-265bp)

AGCTTGGGCTGCAGGTC T (-811)

A T (-27)

AGCTTGGGCTGCAGGTC GACCTGC TGGGCTGT (-1187) G

AGCTTGGGCTGCAGGTC GACC CGGGGATCAGCTTG

AGCTTGGGCTGCAGG CGCTTAAT G (-1445)

(-37) G A (-48)

AGCTTGGGC GTAATCAGATGGTAAGCACTGGGGCCAGATGGTAAGCCC GGGGA TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAG A (-1125)

(-83) A CTG A (-1600)

(-89) TTC(A/C)GG (-32) A

AGCTTGGGCTGCAGGTC GA TCCCCGGGGATCAGCTTG

AGCT (94bp)CTGGTGATATGGGATAGTGTTCA(193bp) (-1018) A

AGCTTGGGCTGCAGGTC GA TGTAATTTATGTAATTGA T (-1676)

AGCTTGGGCTGCAGGTC GACCTG A TCCCCGGGGATCAGCTTG

AGCTT T (-1807)

AGCTTGGGCTGCAGGTC GACCT GA TCCCCGGGGATCAGCTTG

(-97) T CCCGGGGATCAGCTTG

TGA (-31) (-759) A

G (-334) 500bp (-1.5kb)

AGCTTGG (-69) A

AGCTTGGGCTGCAGGTC G GGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GACCT TTTTTTT (-716) A

AGCTTGGGCTGCAGGT G(109bp)TTACGCAAATCTCCCCGGCT GA TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GACCTGC (40bp)TG C (-1510)

(-465bp) CCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GACCT GA TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GAC T (-1193)

G (-20) (778bp) 1kb (-265bp)

AGCTTGGGCTGCAGGTC GAC CTTCGGAAAAAGAGTTGGTAGCTCTTGATCC CCGGGGA TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC G (253bp) 1.5kb (-265bp)

AGCTTGGGCTGCAGGTC G GGGA TCCCCGGGGATCAGCTTG

(-25) A (-766) A

71

AGCTTGGGCTGCAGGTC GA T (-1480)

AGCTTGGGCTGCAGGTC CCCGGGGATCAGCTTG

(-54) CAA (56bp)AT CCCCGGGGATCAGCTTG

(-406) G (-500) T

(-349) A A (-1117)

AGCTTGG TTG

AGCTTGGGCTGCAGG CA(571bp)(51bp) C (-1675)

AGCTTGGGCTGCAGG A (-40)

AGCTTGGGCTGCAGGTC CGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GAC CCCCGGGGATCAGCTTG

Ku70-/-

AGCTTGGGCTGCAGGTC GACCTGCAGCCA TCGCAGCCTGAATGG(55bp)G GATCCCCGGGGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGTC GACCTG AAAATCTCGCCAAGCTGATCCCC CCCGGGGATCAGCTTG

AGCTTGGCTGCAGGTC GACCTGC (-476) T

(-82) T ATATAATATAATATATTATATATATATATATAATATATATATTATATATATAATAATATATATAAT

AAATATATTTATTATATATATAAGCA

(-212) T

AGCTTGGCTGCAGGTC GAC AAGCT GA TCCCCGGGGATCAGCTTG

(-408) T TACG GGA TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GACCTGC (-53) A

AGCTTGGGCTGCAGGTC GAC TCTTCCATAT(421bp)TAG GGA TCCCCGGGGATCAGCTTG

AGCTTGGCTGCAGGTC GACCTGCA (103bp)CTTAGCTCCTGAAAATCTCGCCAAGCTAATTTTTAACCAA (-37) A

AGCTTGGCTGCAGGTC GACCTGCA 100bp (-100bp)

AGCTTGGCTGCAGGTC GACCTGCAGC 800bp (-200bp)

AGCTTGGCTGCAGGTC GACCTG TA (-142) A

AGCTTGGCTGCAGGTC GAC (-510) C

(-405) GG ATCAGCTTG

AGCTTGGCTGCAGGTC GACCTGCAGCCA TCGCCAAGCTGATCCCCGGGGATC1.3kb (-265bp)

AGCTTGGGCTGCAGGTC GAC C (-564)

AGCTTGGCTGCAGGTC GACCTGCAGCCA TCGCCAAGCTGATCCCCGGGGATCC CCCGGGGA TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GAC C (-110)

AGCTTGGCTGCAGGTC GAC C(856bp)650bp (-265bp)

AGCTTGGCTGCAGGTC GACCTGCA (686bp)900bp (-265bp)

AGCTTGGG (84bp)(219bp)GCTGAA (-475) T

AGCTTGGGCTGCAGGTC GACCTGCAGC (62bp)(226bp) ATT (-113) T

AGCTTGGGCTGCAGGTC GAC

(737bp)

750bp

(-265bp)

AGCTTGGG (159bp)G (-32) G

AGCTTGGGCTGCAGGTC GACCTG ACTATTC GGA TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC T (-45)

(-100bp) 1.5bp TTG

(-45) GGA T (-168)

AGCTTGGGCTGCAGGTC 600bp (-100bp)

A (-236) C (-1619) A

AGCTTGGGCTGCAGGTC GA (-573) A

AGCTCA (-128) (-1504) G

T (-93) (-85) A

AGCTTGGGCTGCAGGTC GACCTGCA TCGCCAAGCTGATCCCCGGGGATCC CCGGGGA TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GACC 1.5kb (-265bp)

CC (-18) (-6) G

AGCTTGGGCTG 300bp GGGA TCCCCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GACCTGC A (-141)

AGCTTGGGCTGCAGGTC GACCTGCAGCCCAAGCTG (123bp)AGAAAA (-983) G

(-278) A ATCCCCGGGGA TCCCCGGGGATCAGCTTG

AGC CCGGGGATCAGCTTG

AGCTTGGGCTGCAGGTC GACCTGCA 89bp A TCCCCGGGGATCAGCTTG

(-324) ACC C (-1441)

(-263) G (263bp)TCCTCG GGGATCAGCTTG

AGCTTGGGCTGCAGGTC GACCTGC CAGT G (-689)

AGCTTGGGCTG G (-67)

AGCTTGGGCTGCAGGTC GACCTGCAG TAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTG C (-1510)

AGCTTGGGCTGCAGGTC GACCTGCA ACAACAACA A (-671)

AGCTTGGGCTGCAGGTC GACCTGCAG (38bp) CTTAGGAAATAGTAGA G (-1514)

AGCTTGGGCTGCAGGTC GAC C (-1759)

AGCTTGGGCTGCAGGTC GAC C (-438)

Mutations from the reference sequence are indicated in purple, where the first base is the mutated base and the second is the original sequence. Insertion DNA of unknown origin is indicated in bold while insertion DNA of plasmid origin is indicated in red and insertion DNA of genomic origin is indicated in upper case blue. Underlined nucleotides indicate instances where the joint was formed using microhomology. In instances where the junction wasn‟t sequenced, the size of the deletions and insertions was estimated using restriction analysis and the approximate number of base pairs is indicated in green.

72

Chapter 4: The Stabilizing Effects of ∆4bp on the Line78 Palindrome

73

Note to Reader

Line 78 ∆4bp germline analysis, fetal liver harvests and Ab-MLV infections were performed by

Ming Li, a technician in the lab.

74

Introduction

The Line78 transgenic mouse contains a 15.4kb perfect palindrome (Akgun, Zahn et al.

1997). The intact transgene undergoes centre-directed rearrangements, predominantly

deletional, in the germline (Akgun, Zahn et al. 1997) as well as in somatic cells, where it

rearranges at a rate of 0.55% per population doubling (Cunningham, Cote et al. 2003).

The centre break palindrome revision model, as described in Chapter 1, proposes that

DNA containing a palindromic sequence extrudes a cruciform structure and, in the case of the

Line78 palindrome, leads to transgene rearrangements. A nick is introduced at the tips of the

hairpins. Branch migration and resorption of the cruciform occur, resulting in a double strand

break (DSB). The ends are processed by nucleases and polymerases and eventually sealed

(Fig. 1.7). As we learned in Chapter 3, end-processing and joining during palindrome revision

occur via a non-homology based end-joining mechanism, either NHEJ or an alternate pathway.

However, the formation of a cruciform, a crucial step in centre break palindrome revision,

remained unconfirmed. A series of spontaneously arising mutations near the symmetry centre

provided an opportunity to further examine the centre break palindrome revision model in

particular, as well as palindrome metabolism in general.

The first spontaneously arising mutation occurred during attempts to determine the

enzymes responsible for rearrangements of the Line78 palindrome. Line78 mice were bred into

a WRN/hel background (Lebel and Leder 1998) with hopes of obtaining a mouse with an input

version of the Line78 transgene in a hel/hel background. A cell line thought to contain an

intact Line78 transgene in a WRN/hel background aroused suspicion because it was

anomalously stable. DNA sequence analysis was performed and a „tcc‟ mismatch near the

symmetry centre was identified (Fig. 4.1B) (Appleby 2005). This led towards a search for small

stabilizing modifications of the Line78 palindrome.

75

In the course of examining the origin of the „tcc‟ mismatch, it was determined that the

mutation arose from another spontaneous mutation of the Line78 perfect palindrome. The „g‟

insertion is between three and six base pairs away from the symmetry centre (Fig. 4.1C). It

originated in the germline of the wild type Line78 mouse colony and went undetected through

several generations. A cell line was created to analyze the rate of rearrangement of the „g‟

insertion palindrome in somatic cells; this cell line was also WRN/hel. The „g‟ insertion

palindrome was found to rearrange at a rate of 0.00189 events per population doubling

(95%CI:0.0012-0.0028). None of the „tcc‟ mismatch palindrome samples were rearranged, and

so are assigned a rate of 0.0 events per population doubling (95%CI: 0-0.00015) (Appleby

2005).

The purpose of this study is to determine how effective palindrome revision is at

preventing further Line78 rearrangements, or more specifically, the minimum size of asymmetry

that will stabilize Line78. In order to determine this, I examined the effects of a third small

mutation, a 4bp deletion in the Line78 palindrome 3 bp away from the symmetry centre on the

right side; this results in a 10 bp spacer (Fig. 4.1D). These three palindromes have grossly

different effects on palindrome revision, revealing new features of palindrome metabolism in

the mammalian system.

76

Figure 4.1: Schematic of Line78 variants relevant to this study. A. Intact Line78 transgene. B. The „g‟ insertion mutation. In this case, the inserted „g‟ is depicted as

the fourth in a short stretch of 4 „g‟s; in reality, it is not clear which „g‟ would bulge out of the hairpin stem. C. The „tcc‟ mismatch mutation. D. The ∆4bp mutation.

The left panel represents the central 18 nucleotides of the Line78 palindrome. Nucleotides surrounded by

the black box represent the central PstI site, which is conserved in all four mutations. The right panel the hypothetical structure that would form upon cruciform extrusion; for simplicity, only the top strand is

represented.

CAGGGTGTG CACACCCTG

G C

T A CG

CG CG

AT

CG AT

CG

G C

T A CG

CG CG

AT CG

AT

CG

>G

G C

T A

C G C G

C T A C

C C

AT CG

G C

T A C G

G

G T

G AT

CG

CAGGGTGTG CA - - - -CTG

CAGGTCCTG CACACCCTG

CAGGGGTGTG CACACCCTG

A

B

C

D

77

Experimental approach

The experimental system for analyzing rearrangements of the Line 78 palindrome is as

follows (Fig. 4.2):

Rearrangements of the Line78 palindrome that occur meiotically or post-meiotically in

germ cells or those that occur during very early embryonic development are revealed by

breeding analysis. Here, genomic DNA isolated from the tails of the original ∆4bp mouse and

its offspring and analyzed by Southern blotting and DC-PCR (Digestion-Circularization PCR) (Fig.

4.3 C and D). Rearrangements also occur in somatic cells; these are seen either by culturing

embryonic fibroblasts or Ab-MLV transformed pre-B cells(Cunningham, Cote et al. 2003). The

latter system allows for determination of the rate of rearrangement of the Line78 palindrome.

In order to determine the rate of rearrangement of the ∆4bp Line78 palindrome in

somatic cells, permanent cell lines were established by Abelson murine leukemia virus (Ab-MLV)

infection of ∆4bp Line78 fetal livers (Fig. 4.2, steps 1 & 2). Test cultures were generated by

seeding a cloning well with a single cell bearing the ∆4bp Line78 structure at day 0 (Fig. 4.2,

step 3). The test culture was carried continuously throughout the experiment. The test culture

was then sampled at various time points by generating single cell subclones via limiting dilution

(Fig. 4.2, step 4). Subclones were expanded, DNA was extracted and each was analyzed for

rearrangement via Southern blotting (for large rearrangements) and DC-PCR (to monitor

changes near the symmetry centre). A rate of rearrangement per population doubling is

determined according to the following equation, x/y=(1-p)n (Fig. 4.2, step 5) and refined using

the Maximum Likelihood Estimate (see Chapter 2). Analysis of conversion events in the ∆4bp

Line78 palindrome was performed by DC-PCR (Fig. 4.3 C and D) followed by sequencing.

78

1

2

3

1

4

x/y=(1-p)n

5

Figure 4.2: Schematic of the Line78 rate of rearrangement assay. Cells are harvested from Line78 fetal livers (1). Permanent cell lines are generated by Ab-MLV infection, resulting in transformed pre-B cells (2). A test culture is generated from a single cell (3). The test culture was sampled by limiting dilution (4). Genomic DNA was isolated from the subcloned samples. Samples were scored as either input or rearranged based on the banding pattern in a Southern blot. Rate of rearrangement was calculated according to the equation x/y=(1-p)n (5), where x/y is equal to the fraction of subclones with an input Line78 transgene out of the total subclones analyzed, n is the number of population doublings since the culture was seeded with a single cell, and p is the rate of rearrangement per population doubling.

79

A

D

SacI SacI

2.4 15.6 8.3

EcoRI EcoRI ecI

5.6 15 8

B

Les1 Erc1

(PstI)

(PstI) ecI

PstI PstI

PstI ecI

ACLF1

ACLR1

Les1 Erc1

C

PstI

Les1 Erc1 ACLF1

Left

Les1 Erc1

PstI

Right

ACLR1

(PstI) (PstI) PstI

PROBE

2.4 2.4 3.5 3.5

PstI BamHI BamHI BamHI BamHI

3.5 3.5 4.8

BamHI BamHI BamHI BamHI

80

Figure 4.3: Schematic of the Line78 transgene and outlying areas. A: Grey rectangles represent lacZ repeats; the two defective copies of lacZ are differentiated from each other by the black arrowhead. Wavy lines represent chromosomal sequences. Pst1 sites in brackets represent sites of transgene insertion. These sites were not conserved. The black box represents the probe fragment which contains the lacZ sequence. The hatched boxes represent probe binding sites. B: Map of the integrated Line 78 palindrome showing the diagnostic digests used in this study. Sizes of probe-positive fragments are given. Dashed lines indicate fragments where the cut site lies outside of the transgene. C: Location of primers used for DC-PCR. Transgene-specific primers are shown in gray. Chromosome-specific primers are shown in black; left and right side primers are not identical. D: DC-PCR strategy. Chromosome-specific primers ACLF1 or ACRF1 are used to amplify the left and the right side of the palindrome, respectively. A first PCR reaction is performed with Les1, followed by a second round with Erc1.

81

Results

The 4bp mutation originated in the germline. As with the „tcc‟ mismatch, it was

originally thought to be intact based on Southern blotting. Because it was highly unusual for a

Line78 mouse to produce even a single litter where all transgene-bearing offspring remained

unrearranged, the structure of the transgene in the parent was further investigated. Sequence

analysis was performed and the 4bp was detected. Three cell lines were generated from 4bp

containing mice and all three contain the mutation, as verified by sequence analysis.

The 4bp palindrome was observed through one generation in the germline. Of the 11

offspring who inherited the transgene, all 11 appeared unrearranged by Southern blot (Fig.

4.4). DC-PCR and sequence analysis revealed that they all contained the 4bp (data not

shown).

To determine the rate at which the 4bp palindrome rearranges due to palindrome

revision in somatic cells, 8 samplings were done with three test cultures. The first, 78D1307-1-

2-1, was sampled at 44, 66.7, 96 and 148 population doublings. The second, 78D1307-6-1-1,

was sampled at 136 population doublings. The final, 78D1310-1-2-1, was sampled at 29.3, 52

and 133.2 population doublings. There were a total of 96 samples analyzed and 95 appeared

unrearranged based on Southern blot. The calculated rate of rearrangement was 0.00065

rearrangement events per population doubling (95%CI: 0.00028-0.0013)(Table 4.1). A

Southern blot of genomic DNA isolated from a subset of the 96 samples is shown in Figure

4.5A. Each sample is represented twice, with a SacI digest in one lane and a BamHI and PstI

co-digest in the other. The sample represented in Lanes 7 and 8 is the single rearranged

sample; the others all exhibit an input Line78 band pattern. The same sample is represented

again in Lanes 3 and 4 of Figure 4.5B, digested with BamHI alone and EcoRI respectively. This

brief mapping exercise tells us that this transgene has an extensive deletion that spans into the

left and the right flanking regions, while leaving the centre of symmetry intact, as well as

82

Figure 4.4: Southern blot analysis of Line78 4bp rearrangements in the germline. A: A male mouse known to contain a 4 base pair deletion in the Line78 transgene adjacent to the symmetry centre was bred with a Balb/c female. Mouse tail DNA from the offspring was co-digested with BamHI and PstI and was probed as indicated above. An intact control in Lane 1 indicates the 3.5 kb and 2.4 kb bands. Offspring represented in lanes 4,7,8,11,12,14,15,17,19,20 and 21 exhibited input Line 78 transgene structure; those in lanes 2,3,5,6,9,10,13,16 and 18 did not contain the transgene. B: Digestion map showing BamHI (B) and Pst1 (P). Hatched boxes represent probe binding sites.

A

3.5kb

2.4kb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

B

2.4 2.4 3.5 3.5

B B B B P

83

Figure 4.5: Southern blot analysis of Line78 4bp rearrangements in somatic cells.

A: Cellular DNA was co-digested with BamHI and PstI (odd numbered lanes) or SacI (even numbered

lanes). An intact cell line in Lanes 1 and 2 indicates the 2.4kb band (BamHI/PstI and SacI) and 3.5kb band (BamHI/PstI only). Lanes 3-20 are samples. Lanes 7 and 8 represent a sample with a rearranged

transgene; the remaining lanes represent input Line78 structure. High molecular weight bands in the SacI digest lanes are fragments generated from a cut site that lies outside of the transgene.

B: Cellular DNA was digested with BamHI (Lanes 1 and 3) or EcoRI (Lanes 2 and 4). Lanes 1 and 2

represent an intact Line78 transgene. Lanes 3 and 4, the same sample as lanes 7 and 8 from 1B, representing a grossly rearranged transgene.

C: Digestion map showing BamHI (B) and Pst1 (P) co-digestion and SacI (S). Hatched boxes represent probe binding sites. Corresponds to 4.5A.

D: Digestion map showing EcoRI (E) and BamHI (B). Hatched boxes represent probe binding sites.

Corresponds to 4.5B.

1 2 3 4

15

8

5.6 4.8

3.5

1

11

3.5kb

2.4kb

2

11

3

11

4

11

5

11

6

11

7

11

8

11

9

11

10

011

11

0

11

12

0

11

13 14 15 16 17 18 19 20 A

B

3.5 3.5 2.4 2.4

2.4

3.5 3.5 4.8

B B B B P

C

S S

B B B B

E E

D

5.6 15 8

84

Table 4.1: Rate analysis of the 4bp Line 78 palindrome.

4bp

Mutation Cell

Line

Days in

Culture

Population

Doublingsa (n)

Input/Totalb

(x/y)

Fraction

Input

Ratec

95% CId

78D1307-1-2-1 33 44 6/6 1.0 0.0 [0 – 0.004]

78D1307-1-2-1 50 66.7 7/7 1.0 0.0 [0 – 0.002]

78D1307-1-2-1 72 96 3/4 0.75 0.003 [0.000175 – 0.0129]

78D1307-1-2-1 111 148 24/24 1.0 0.0 [0 – 0.0003]

78D1307-6-1-1 102 136 11/11 1.0 0.0 [0 – 0.0007]

78D1310-1-2-1 22 29.3 6/6 1.0 0.0 [0 – 0.006]

78D1310-1-2-1 39 52 9/9 1.0 0.0 [0 – 0.002]

78D1310-1-2-1 100 133.3 29/29 1.0 0.0 [0 – 0.00025]

Pooled Data 95/96 0.99 0.00065 [0.00028 –

0.0013]

The rate of rearrangement was determined using the Maximum Likelihood Estimate (MLE) as described in detail in the legend to Table 1. The samplings did not differ significantly from each other (Likelihood ratio test, p-value 0.2884). For further explanation, please see sample MLE calculations. aCalculated from a doubling time of 18h. bSubclones with an input Line 78 transgene/total number of subclones analyzed. cRate of rearrangement per population doubling, calculated by MLE. dThe 95% Confidence Interval, calculated by MLE.

85

potential insertions. Extensive mapping of rearranged transgenes has been done previously

(Cunningham, Cote et al. 2003) and several categories of rearrangement have been identified.

The rearranged transgene from the 4bp does not fit into any of those categories, providing

further evidence for its complexity.

DC-PCRs was performed on all the subclones generated from the 44, 66.7, and 96

generation sampling of test culture 78D1307-1-2-1 as well as the 29.3 and 52 generation

sampling of test culture 78D1310-1-2-1. All samples generated an input sized 200 bp band

from both the left and the right side (data not shown). Sequence analysis was performed on 19

of these samples. All 19 contained the ∆4bp on the correct side, including the one rearranged

transgene. These results indicate that the ∆4bp effectively prevents further rearrangement of

the palindrome.

Discussion

This study demonstrates the effects of a small mutation on the stability of a perfect

transgenic palindrome in the mouse genome. A single mutation, a ∆4bp near the symmetry

centre, was studied. It will be compared to results from the „g‟ insertion palindrome and the

„tcc‟ mismatch palindrome (Appleby 2005).

We know that the rate of rearrangement of a perfect Line78 palindrome in a wild type

background is 0.0055 (95%CI: 0.0045-0.0067) rearrangement events per population doubling

(Cunningham, Cote et al. 2003). The „g‟ insertion palindrome rearranges at a rate 3-fold less

than that of a perfect palindrome, suggesting that a single base disruption in the symmetry of a

palindrome can measurably affect, but not prevent, its rearrangement (Appleby 2005). The

„tcc‟ mismatch has a much more dramatic effect on the stability of Line78; it rearranges at a

rate at least 35-fold less than the perfect palindrome, comparable to the effect of the ∆270bp

palindrome.

86

The rate of rearrangement of the ∆4bp palindrome is more complicated to analyze. Out

of the 96 samples analyzed, all but one were intact. Rate of rearrangement calculations show

that the ∆4bp palindrome rearranges at a rate 8-fold less than that of the intact transgene. On

the surface, it seems that the ∆4bp has only a moderate affect on centre break palindrome

revision, however this may not be the case. The one rearranged sample was mapped more

extensively than samples which appeared intact (Fig. 4.5B). It exhibited extensive deletions

from both arms of the palindrome while leaving the centre of symmetry intact. This type of

rearrangement cannot be accounted for by the centre break palindrome revision model.

Therefore, including this rearrangement event in the rate calculations results in a measure of

palindrome rearrangement not related to centre break palindrome revision. If that entire

sampling (78D1307-1-2-1 at 72 days; Table 4.1) is eliminated from the rate calculation, the rate

of rearrangement of the ∆4bp palindrome is 0.0 (95%CI: 0.0-0.0006) events per population

doubling. This indicates that the ∆4bp does indeed have an effect on cruciform formation and

therefore on centre break palindrome revision.

While stabilizing rearrangements of the Line78 palindrome have been seen in the

germline (Akgun, Zahn et al. 1997) and in somatic cells (Cunningham, Cote et al. 2003)

previously, the resultant spacers are quite large when compared to the ∆4bp. In these cases,

the interruptions in the palindrome were approximately 200 to 300 base pairs, while the ∆4bp

has a mere 10bp spacer. This is the smallest stabilizing rearrangement of the Line78

palindrome identified, even smaller than the 14bp spacer created by the „tcc‟ mismatch. It also

provides a good basis for rate comparisons with an intact Line78 transgene (Cunningham, Cote

et al. 2003) because they are in the same genetic background; the „g‟ insertion palindrome and

„tcc‟ mismatch palindrome studied are both in a WRN/∆hel background (Lebel and Leder 1998).

The ∆4bp palindrome comes from a background that clearly is competent for rearrangement.

The original ∆4bp mouse had 9 siblings that had inherited the transgene. Three of the 9

87

appeared to have unrearranged transgenes according to DC-PCR and one of these was the

∆4bp; 7 out of the 9 siblings had a transgene that had been rearranged.

Line78 Rearrangements do not occur via replication slipped mispairing

It has been proposed that the centre break palindrome revision mechanism was

responsible for palindrome rearrangements in the Line78 transgene (Cunningham, Cote et al.

2003). However, this need not be an exclusive mechanism because in cases where the

symmetry centre was not intact, replication slipped mispairing provided a possible explanation.

There is good evidence for replication slipped mispairing as a mechanism for palindrome-

stimulated deletion in E. coli. Hairpin formation occurs preferentially from ssDNA on the

lagging strand (Trinh and Sinden 1991; Rosche, Trinh et al. 1995; Pinder, Blake et al. 1998;

Bzymek and Lovett 2001). As hairpins are known to stall replication forks (Lebofsky and

Bensimon 2005), this provides an opportunity for the nascent strand to misalign to short

homologous sequence on the opposite side of the hairpin, thereby deleting the majority of the

palindrome, including the axis of symmetry after the next round of replication (Weston-Hafer

and Berg 1989; Bzymek and Lovett 2001). The key difference between these two mechanisms

is that replication slipped mispairing only requires a hairpin to form from ssDNA, while centre

break palindrome revision requires a cruciform to form from dsDNA; thus only palindromes

capable of forming cruciforms undergo centre break palindrome revision. Neither the „g‟

insertion, „tcc‟ mismatch nor ∆4bp would be predicted to prevent rearrangement if the initiating

secondary structure occurs in ssDNA. Since only the ∆4bp and the „tcc‟ mismatch had a marked

stabilizing effect, we must conclude that rearrangement occurs by a centre break mechanism

and that a single „g‟ insertion is not enough of a sequence disruption to prevent cruciform

formation. Cruciform extrusion from dsDNA can be impeded by imperfections in a palindrome

or near-palindrome as the free energy cost of incorporating unpaired structures into a cruciform

must be met by a corresponding increase in negative superhelicity (Benham, Savitt et al. 2002).

88

The decrease in the rate of rearrangement for the „g‟ insertion mismatched palindrome suggests

that while it may have an increased free energy of cruciform formation cost, it is still below the

superhelicity threshold required to drive extrusion. In contrast, the increased free energy cost

for incorporating a 10bp or 14bp loop (∆4bp and „tcc‟ mismatch respectively) into a cruciform is

likely above threshold levels.

Another study examining inverted repeat transgenes in mice has yielded similar results.

The two transgenic lines, Line 109 and OX1-5, are prone to rearrangement at the site of the

inverted repeats, either insertions or deletions, and that these modifications stabilize the

transgene from further rearrangement in both the germline and in somatic cells (Collick, Drew

et al. 1996). In E. coli studies have shown that a plasmid containing a 101bp palindrome

undergoes deletions 10-fold more than a palindrome-free plasmid; when a 14bp spacer is

introduced into the centre of the palindrome, the stimulatory effect on deletion is eliminated

(Bzymek and Lovett 2001). Also, an 8 bp spacer alleviates the deleterious effects of a perfect

palindrome on propagation of palindrome-containing phage (Chalker, Okely et al. 1993). In a

similar study assaying E. coli propagation based on plaque size, it was found that the effects of

imperfections in the near-palindrome decreased as they moved away from the symmetry

centre. This implies that intrastrand base pairing after central melting is crucial to the formation

of cruciforms, which in turn is crucial to the centre break mechanism and Line78 palindrome

rearrangements.

Future Directions

The centre break mechanism is extremely efficient at producing stabilizing

rearrangements of the Line78 palindrome, as evidenced by the fact that a single four base pair

deletion, resulting in a 10 base pair loop, can confer such stability. In the future, breeding

experiments involving Line78 and various DNA repair strains, such as that which had been

attempted with the WRN/∆hel mice, will be undertaken. Alternatively, DNA repair factors can

89

be downregulated via siRNA in somatic cells. This will allow us to determine which proteins are

responsible for the centre break mechanism in mammalian cells.

An understanding of how cells prevent palindromic instability will help us understand

how such instability can lead to the various chromosomal aberrations such as translocations and

gene amplifications that can lead to genetic disease.

90

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