Muller Lecture 3 2008

8/8/2019 Muller Lecture 3 2008

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Repair of DS BREAKS

Homology Directed Repair

Non-homology End Joining


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DS Break Repair Pathway

Retrieve Sequenceinformation fromundamaged DNA

Uses recombination Mutants defective in

recombination are UVsensitive (inefficientrepair)

DS DNA BREAKS (or

DSBs)

VERY toxic

Must be dealt with

High Mutagenic

potential

Exploit DSBs for

immunoglobulin

rearrangements


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Origin of DSBOrigin of DSB

Direct DNA Fracture

Replication fork encounters a ssbreak

Daughter strand gap (leading/laggingstrand progression halted by lesion)


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Direct DNA fraction:

Topoisomerase II

cleavages, Ionizing

radiation (minor)

One Ended DSB: Rep

fork encounters a ss DNA

break

Lesion on Template of

daughter strand

DNA replication: High risk of ss break conversion into DSB (very toxic)

Recombinational repair (DSB Repair) CRITICAL to minimizing this risk!!


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DSB REPAIR

Works ONLY when sister chromatid is availableto contribute homology AFTER DNA replication (S-phase)

What about BEFORE DNA Replication (no sisterchromatid available)? Cells use Non-Homologous End Joining or NHEJ

4N2N

S Phase


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- Involves simple end joining. Originally in Eukarya

more recently in Prokaryotes

- No homology but may involve microhomology of a

as few as 1-2 bp.

- Broken ends directly joined

- Misaligned ends may hook up by microhomology

- SS DNA tails snipped off

- Ku protein align ends (highly conserved in bacteria

yeast and man)

- Ku mediated NHEJ pretty messy not efficient


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Core machinery: NHEJ

DSB directly rejoin Ideal for blunt end breaks

Less ideal with non-compatible ends

Microhomology (


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DNA ends 1st bound by Kuheterodimer

Then attracts DNA-PKcs

Forms DNA-PK complex

Then attracts Ligase IVcomplex

SEALED

Eukaryotic NHEJ http://web.mit.edu/engelward-lab/animations/NHEJ.html


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Model for NHEJ

Showing Base related events.

Free DNA ends attract Ku

[protects from degradation]

Ku recruits DNA PK (red)

2 DNA PK subunits

autophoshorylate and phos. Ku

subunits

Phos. DNA PK released

Phos. Ku activates unwindase

Regions of microhomology (short)

hybridize to align

Trim up the whiskers at end


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NHEJ of difficult ends Not all ends readily religatable

Bizarre ends, 5 OH, Phosphates, damaged sugar moities or bases 3Phos. Or 5 OH ends can be processed by polynucleotide kinase

(interacts also with XRCC4)

Artemis nuclease: structure specific can cut hairpins and 3overhangs

Used in V(D)J joining of Immunoglobulin genes

Defects in NHEJ due to Artemis mutation = immunodeficiencysyndrome

WRN Protein has exonuclease activity (mutated in Werners

syndrome)

Eukaryotic NHEJ

DNA repair of DS breaks is foundation of

V(D)J Joining to create antibody diversity

in vertebrates based on NHEJ!


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B cells produce ABs that specifically bind

and recognize a HUGE diversity of

antigens the AB diversity is based on

Recombination and somatic mutation and

clonal selection.

Called

AB composed of 2

copies each L & H

chains.Variable region

defines AG binding

(composed on VLand VH domains

VLVH


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V(D)J Recombination OverviewGenomic Region Light Chain (kappa locus)

Ca. 300 copies of V

gene versions

Any pair of V genes

can fuse with any pair

of J segments

(allows >1200

possible outcomes)

These new segmentsfused to Constant

region by RNA

splicing

Genomic Region Light Chain (kappa locus): Similar to Heavy chain (but H has an additional region called D (for

diversity) which increases diversity (ex: 100 V genes, 12 D genes, 4 J regions = 4800 possible permutations.. The

completed AB can be any pairing of H and L variables. Yields a big number!


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V(D)J Recombination Mechanism

Recombination signal sequences flank the

V(D)J targets.

7mer and 9mer sites Spacer between the 7/9 is either 12 or 23 bp

These bind recombinase

Recombination always occurs between

inverted repeats of 12 bp spacer (one end)and the 23 bp spacer on the other end.


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Recombination Signal

Sequences or RSS

structures

Recombination result with H and Light: Note that they are

flanked with inverted repeats.


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V(D)J Recombination MechanismRecombinase = RAG1, RAG2

RAGs make ss DNA cleavages as

shown & free 3OH attacks opposing

strand to produce a hairpin structureshown

Other Cellular repair proteins (NHEJ

Factors) complete the job,..

Note: its sloppy and involves a few

insertions or additions mutations to

create more diversity.

VERY Similar to transposition processes


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Prokaryotic NHEJ

Recently shown in some bacteria

In Eukarya: Homologous Recombination Recovers DSB

(especially yeast): Limited to S/G2 NHEJ more predominant in higher Eukarya (acts

throughout cell cycle)

Homologues to KU identified in Bacteria (but not

all e.g. enterobacteria like E. coli lack)


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Stars = homologues Ku detected

E. coli


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DSB Repair by NHEJ in Prokaryotes

DS Break forms (Replicative break, Ionizing

Radiation, adduct formation, etc.

Ku Locates site: Serves as end bridging or

alignment factor

Processing enzymes recruited by Ku ringsaround DNA

Ku recedes to all enzyme action (gap filling, exo,

end processing, etc., makes termini suitable for

ligation

Ligation by NHEJ specific ligase

Complex dissociates

NOTE: Prokaryotic Ku is a homodimer whileEukaryotic Ku is heterodimer 70/80


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Ku = homodimer

Ligase: modular with Polymerase, Ligase, nuclease domains.

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Pseudomonas Ligase Domain structure

Ref: PLOS Genetics 2006 review by Bowater and Doherty

tt ://g ti . l j ur l . rg/ r iv /1553-7404/2/2/ f/10.1371_j ur l. g .0020008-L.

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DS BREAK REPAIR: Homology DrivenDS BREAK REPAIR: Homology Driven

Recombination RepairRecombination Repair

Recover Sequence information by

homology to another region of genome

Accomplished by Finding homologous sequences (best if sister

chromatid)

Tends to be error free/high fidelity (some

exceptions)


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DS DNA Break Repair:

Related to Homologous Rcbn

General comments about Homol. Rcbn. (orHR)

Prokaryotes: HR is RecBCD pathway

Well studied model: See Ch. 10 Watson

DS Breaks (DNA damage) initiate HR

RecA ptn= Binds ss DNA drives pairing & strandinvasion (helps in homology search with SS Binding

protein) RecBCD: Helicase/nuclease process DNA breaks to

generate ss ends for HR invasion

RuvABC: binds Holiday Junctions to resolve


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Resolution

(RuvC)

RecBCD pathway in E. coliDSB somewhere in genome.

Helicase unwinds toward Chi site (every 5KB or so)

Chi: 5GCTGGGTGG

RecA promotes D-loop invasions; helps find homology

Once D-loop hybrids form, RecA/SSB desorb and release

Nick= RecBCD: allows tail to bp

with SS region in other DNA

Gaps/nicks sealed ligase

Branch migration

Resolution gives different

products.

RecA protein: ss DNA

binding ptn that promotes

strand exchange

-Coats ss DNA but not DS

DNA

NOTE: RecA cooperateswith a SSB ptn


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Holiday Junction Resolution Products

Outcomes from Holiday Junction

Cleavages: 2 possible.


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General comments about HR (cont)

Eukaryotes: HR is essential for life

1. Meiosis: Links up homol. Chromosomes for

segregation

2. Recombines parental alleles for offspring

diversity3. Deals with DNA damage: NEXT.


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Homology Directed Repair

Pathways

Synthesis-Dependent Strand Annealing

Classic Double Holiday Junctions: Less evidence

for this in higher eukaryotes

Single Strand Annealing Pathway

Will discuss these two pathways


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Synthesis Dependent Annealing

at DSBs Predominant mechanism Low error rates

Gene Conversion model Mechanistically complex with many

factors will cover from standpoint of

DNA templating process.


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Resection: chewback to 3 overhangs

Binding proteins

mediate (Rad51)

3 nucleofilament seeks

homology elsewhere in

genome

Strand

invasion

forms D loop

DNA Synthesis

across region ofhomology

Sister chromatid: intact wt DNA

Holliday junction

Branch migration releases the

extended strand

Trim

Fill

Ligate

http://web.mit.edu/engelward-lab/animations/SDSA.html


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END Lecture #3


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SS annealing model for repair of DSBsSS annealing model for repair of DSBs

QuickTime 2 and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Works with DNA repeats

(contiguous)

The overhangs (3) simply

anneal

Trim

Ligate

http://web.mit.edu/engelward-lab/animations/SSA.html


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SS annealing model for repair of DSBsSS annealing model for repair of DSBs

Important notes on SS Annealing Model:1. Need adjacent repeats: High

homology important

2. Some sequence loss between repeats

3. One of repeats deleted4. Human genome: lots repeats (Alu

elements x 106, 10% is repeat

sequence anyway)

5. Human genome repeats: highly

polymorphic

6. High sequence diversity in repeats =

reduced efficiency

7. In general: may be a minor pathway

for repair


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Homology Directed Repair or Non-

homologous end Joining?

Which to choose?

1. Cell cycle phase: Homologous recombination requires sister

chromatids (limited to S and G2).

Cell Cycle Dependent homology driven repair

Difficult to perform in bulk chromatin in interphase cells

2. IF Homologous Repair is NOT suppressed outside of S-G2.

Mutations will be more frequent as weak homology may be selected.

As diploids: can recover sequence off an allele but if

heterozygous, the parental allele may differ.3. Simple, DS breaks with flush ends are rapidly re-ligated since the

NHEJ pathway is rapid and recruited quickly to needed sites.

(homology directed repair is big and complex = slow)

Difficult breaks may be harder to fix and slower to re-ligate (?)


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How to analyze DSB Repair

In human cells: GFP Gene conversion

cassette

Combined with rare cutting restrictionenzymes to introduce specific cut sites

Analysis of gene silencing at repair patch

sites (methyl-C at CpG sites silenced

linked genes).

http://genetics.plosjournals.org/perlserv/?request=get-document&doi=10.1371%2Fjournal.pgen.0030110


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Qui i e and aTIFF (LZW) decompressor

are needed to see this picture.

SceI: rare cutting enzyme (no sites present in huDNA)

Thus-> transfect cells with SceI gene construct: uniquely cuts at this site to create a

sequence specific DS break

Primers allow us to distinguish Rec and Unrec products at genomic level

GFP signatures: Detects gene conversion event.


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DNA Replication Blockages:

Covalent Adduct

Hairpin

SS nicks

Stably bound

protein (topo)

Other Templating

[transcription shown]


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Enables Replication to Proceed Across DNA Damage or Potentially Blocking Lesions


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TLS

Failsafe backup for lesion misses

Has higher error rate than ideal

TLS still saves the fate of cell fromblockage in DNA replication

Requires specialized D. Pol.

Members of Y polymerases (1999)


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Y Pol properties

N- terminus well conserved cataltyic domain

C-term: less conserved (ptn:ptn interactions forlocalization)

Poorly processive

Synthesis is template dependent but NOT templateddirected Low fidelity (no 3-5 proofreading exonuclease)

Error prone process

All stimulated by PCNA (polymerase sliding clampaccessory ptn.


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TLS polymerases may

incorporate specific NT TLS Not Template Dependent but some ofthe Y pol are specific

Example: DNA Polymerase L Acts at T-T dimers

Tends to insert A residues opposite


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TLS in E. coli

Synthesis directly across lesion

Complex of UmuC and UmuD

TLS is so error prone that UmuCD normally not present

SOS Response pathway induces these genes (LexArepressor proteolyzed after UV)

Activates the SOS Pathway genes

Includes RecA (recombination protein)


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TLS DNA SYNTHESIS

Pol III + Sliding Clamp encounters

TT Dimer

Dissociation/fork stall

Translesion DNA Pol inserts bases

opposite dimer

Dissociation of TLS Pol

DNA pol III takes over

Release of TLS Pol due to

low processivity of

enzyme

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Muller Lecture 3 2008