DNA DOUBLE-STRAND BREAK REPAIR BY THE

MRE11/RAD50 COMPLEX IN T4 PHAGE

PRIYA JAYARAMAN

NATIONAL UNIVERSITY OF SINGAPORE

2011

DNA DOUBLE-STRAND BREAK REPAIR BY THE

MRE11-RAD50 COMPLEX IN T4 PHAGE

PRIYA JAYARAMAN

(B.Tech, Vellore Institute of Technology, India)

A THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOLOGICAL SCIENCES

NATIONAL UNIVERSITY OF SINGAPORE

2011

I

ACKNOWLEDGEMENTS

This research work would not have been feasible without the training, support and

guidance provided by my supervisor, Dr. Kim Chu-Young. I am extremely grateful to

him for giving me the opportunity to be a part of his research group. His constant

encouragement during the challenging parts of the project instilled in me a sense of

confidence and determination.

I am thankful to National University of Singapore for granting me a research scholarship

and providing me with the facilities to carry out this research project.

To Dr. Kinya Hotta, I am thankful for his guidance on cloning and protein expression

techniques. I am grateful to Dr. Ganesh S. Anand and his labmembers-Moorthy, Suguna,

Srinath and Jeremy- for their invaluable input during deuterium exchange experiments

and data analysis. I am grateful to Michelle Mok and Lim Teck Kwang from the Protein

and Proteomics Center for their assistance in carrying out the mass spectrometry

experiments. I would also like to thank Dr. Yan Jie from single molecule biophysics lab,

department of physics, NUS, for carrying out the AFM experiments.

I am extremely fortunate to have a family, which has constantly supported my decisions

and encouraged me to pursue research, especially my parents and brother. Special thanks

to my uncle and aunt (Babu mama and Lalitha mammi) here in Singapore, who ensured

that I did not miss home and were always ready to have me over, even if it meant odd

timings after experiments.

Grad school has been one of the steepest learning phases of my life. This learning

experience could not have been as exciting and enjoyable without my labmates and

friends. I thank my labmates Chen Xi, Raymond, Satya, Roopsha, Soumya and Minyi for

the scientific discussions, moral support and fun times in lab. I would also like to thank

my friends Anusha, Ayshwarya, Ashish, Karthik, Madhuvika, Rika and Shaveta who

made my grad school years memorable.

II

TABLE OF CONTENTS

Title Page No

ACKNOWLEDGEMENT I

TABLE OF CONTENTS II

ABSTRACT V

LIST OF FIGURES VI

LIST OF TABLES XIII

ABBREVIATIONS XIV

Chapter1: INTRODUCTION

1.1 DNA damage 2

1.2 Single Strand DNA Repair 4

1.3 Double Strand DNA repair 7

1.4 Cell Cycle Checkpoint Response control by ATM and ATR during DSB 8

1.5 DNA damage and diseases 10

1.6 Mre11/Rad50/Nbs1 complex (MRN) 14

1.7 Scope of the project and AIM 23

1.8 T4 Bacteriophage 25

1.9 Hydrogen/Deuterium Exchange Followed by Mass spectrometry 29

III

Chapter 2: MATERIALS AND METHODS 34

2.1 Cloning, protein expression and purification 35

2.2 Circular Dichroism 35

2.3 Nuclease Assay 35

2.4 Isothermal Titration Calorimetry (ITC) 36

2.5 Atomic Force Microscopy 39

2.6 Hydrogen/Deuterium Exchange followed by Mass Spectrometry 40

Chapter 3: RESULTS AND DISCUSSION

3.1 Protein Expression and Purification 42

3.2 C-terminal domain of T4Mre11 is essential for its nuclease activity 46

3.3 Solution behavior of T4MR complex using HDXMS 48

3.3.1 Formation of the T4MR complex — A conformationally 52

stable state

3.3.2 AMP-PNP captures the complex in an open transition state 52

3.3.3 Conformational changes across lobe II of T4Rad50 54

and T4Mre11 capping domain

3.3.4 Conformational changes across the His loop 57

of T4Rad50 and nuclease motif of T4Mre11

3.3.5 Flexibility of Rad50 coiled-coil segment 59

undergoes changes upon AMP-PNP binding

IV

3.3.6 Effect of AMP-PNP on the distal coiled-coil segments 62

3.4 Hydrolysis of ATP is essential for substrate processing 63

by T4Mre11 dsDNA

Chapter 4: CONCLUSION 65

REFERENCES 71

APPENDIX A: Sequence of T4Rad50 and T4Mre11 80

APPENDIX B: Analytical Ultracentrifugation of the proteins 81

APPENDIX C: Peptide list for Rad50 82

APPENDIX D: Peptide list for Rad50 85

APPENDIX E: Peptide list for Mre11 86

V

ABSTRACT

The T4 phage proteins Mre11 and Rad50, also known as gp46 and gp47, are responsible

for the detection of DNA double-strand break and initiation of its repair. Mre11 carries

out the nuclease and strand annealing activities at the site of damage while Rad50, an

ATPase, regulates the nuclease activity of Mre11. Mutation in either protein can lead to

the loss of DNA repair function and, in humans, this leads to various forms of cancer,

neurological disorders, and immunological deficiencies. Although crystal structures of

Mre11-Rad50 complex have been reported, the precise mechanism of communication

between the two partner proteins in executing the DNA repair remains poorly understood.

We carried out in vitro biophysical characterization of the solution behavior of the T4

Mre11/Rad50 proteins using hydrogen-deuterium exchange followed by mass

spectrometry. Based on our study, we propose a new model for domain arrangement of

T4Mre11/Rad50 proteins in the complex. Our model is found to differ from the models

proposed so far.

VI

LIST OF FIGURES

Figure Page No

1.1 DNA damage response pathway in mammalian cells. Based on the

nature of damage, either of these pathways could function

independently or in combination with one another

3

1.2 DNA damage can be broadly classified into three: single strand breaks

(SSB), bulky lesions and double strand breaks (DSB). A. Single-strand

damage involves breaks in one of the DNA strands or chemical

modification of a single base (red hexagon). Such damages follow the

single-strand repair or base excision repair pathway (section 1.2) B.

Lesions (cyan sphere) that involve chemical modification of the

backbone leads to disruption of the helix backbone and these damages

are repaired by nucleotide-excision repair pathway (section 1.2). C.

DSBs: Bifunctional reagents that lead to interstrand cross-links (grey

dumbbell) or external agents like radical oxygen species lead to break

of both the strands and the damage is subsequently repaired by non-

homologous or homologous recombination pathway based on the

development stage of the cell (section 1.3).(Source: McKinnon PJ, 2009

(3)).

3

1.3 Base excision repair. SSBs arising due to DNA glycosylase action or

direct damage lead to recruitment of poly ADP-ribose polymerase 1

(PARP1) at the damage site, which subsequently activates scaffolding

protein XRCC1, DNA polymerase (Polβ), DNA ligase (LIG3) and

apurinic/apyrimidinic endonuclease 1 (APE1). Based on the length of

nucleotides that is to be excised the protein machinery involved differs.

Repair of longer stretches is carried out by LIG1 and PCNA. Also, in

5

VII

some cases based on the nature of damage at the SSB, tyrosyl DNA

phosphodiesterase 1(TDP1) or aprataxin (APTX) is recruited. TDP1

acts on 3′ modified ends or topoisomerase I DNA adducts while APTX

acts on 5′ ends resulting from abortive ligation events. (Source:

McKinnon PJ, 2009 (3))

1.4 Nucleotide Excision Repair pathway. The primary steps involved in

NER are recognition of the damage, excision and repair synthesis. Two

different pathways within NER differ in recognition step. When the

damage is recognized during transcription and involves transcription

factors (TFIIH) it is termed as transcription coupled repair (TCR). Else,

it is referred to as global genomic repair. Post recognition, the repair

steps followed in both the pathways is similar. The colored blocks on

the right highlight the disorders resulting from dysfunctional proteins of

the NER pathway. (Source: McKinnon PJ, 2009 (3) )

6

1.5 Double strand break repair pathway. Homologous recombination occurs

in proliferating cells. The replication protein A (RPA) and recombinase

Rad51 coat the broken double strand ends and initiates repair by

formation of the holliday junction with the help of the sister chromatid,

which serves a template ensuring a error-free repair. NHEJ occurs in

differentiated cells that lacks a reference template. It involves key

proteins like KU70 and KU80, which modify the damaged end and

prepare it for ligation. Ligation is further carried out by ligase IV

(LIG4), XRCC4, and other proteins. (Source: McKinnon PJ, 2009 (3).

7

1.6 Cell cycle checkpoint activation by ATM and ATR. ATM and ATR

activate check point kinases (CHK) and cell cycle proteins, which arrest

the cell in different stages of cell cycle. Once the repair is completed

successfully, the cell cycle proceeds by activation of cyclin dependent

kinases (CDK). Or else, the cell is directed towards proteasomal

9

VIII

degradation (Source: Branzei D et al. 2008 (9) )

1.7 Role of MRN in DNA repair and cell cycle control. Mre11 leads to

activation of several downstream pathways depending on the nature of

damage. It has been found to play an important role in homologous

recombination (HR), non-homologous end joining (NHEJ) and

activation of apoptotic pathway. This figure emphasizes key role of

MRN complex in maintaining cellular stability. (Source: Stracker. TH.

et al. 2011 (31))

14

1.8 Domain arrangement of Mre11 proteins: (a) The three domains are

highlighted in box form and the flexible linker as the dotted line. The

green segments within the nuclease domain box indicate the

phophodiesterase motifs. (b) Structural presentation of the domains in

Mre11 (Pink). The nuclease motifs harboring the active site are

highlighted in green which occur at the loop segments.

16

1.9 Domain arrangement of Rad50 proteins: (a) Domain arrangement of

Rad50. (b) Structural presentation of the domains in Mre11 (Pink).

Motifs and domains are highlighted in the respective colors shown in

the block diagram (a). (c) MR with truncated Mre11 bound to AMP-

PNP from Pyrococcus furiosus(PDB id: 3QKU). (d)Overlap of free

PfRad50 and AMP-PNP bound PfRad50, highlighting the rotation of

the subdomains (PDB id: 3QKT and 3QKU).

17

1.10 A. Molecular arrangement of MRN complex, a. Assembly of

Mre11/Rad50/Nbs on the DNA substrate. Rad50 functions as a dimer

and hence harbors two Mre11 interaction sites. The N and C domains of

Rad50 are arranged in an antiparallel fashion b. AFM image of MR

complex in DNA free and bound state. The images highlight the

different orientations of the coiled-coil segment (Source: Stracker. TH.

et al. 2011 (31)) B. Comparison of length of coiled-coil segment in

19

IX

different species. (courtesy: Scott Nelson)

1.11 Crystal structures of MR reported: (a) MR complex from Thermotoga

maritima(PDB id: 3QG5).(b) ATPγS bound Methanococcus jannaschii

(PDB id: 3AV0).

21

1.12 Proposed models of MR complex from different species. The models

highlight the orientation of the different domains in the free complex

(left) and AMP-PNP bound complex (right).

22

1.13 T4 Phage structural organization (53). 25

1.14 Exchangeable hydrogens in a protein. Red: Amide hydrogens with slow

exchange rate, Green: covalently attached hydrogens, which do not

exchange, Blue: side chain hydrogens, which have high exchange rate

and hence, are undetectable. (Source: www.hxms.com)

29

1.15 Workflow for HDMS (Adapted from Busenlehner LS et al. (58)). 31

1.16 Deuterium incorporation quantification. i. Steps involved in the HDX-

MS experiments. ii. Detailed picture of centroid calculation. As shown

in the table, centroid of the mass peak is noted at different time points

(A). The centroid values are used to calculate the relative increase in

deuteration levels (B). The deuterium levels are then plotted vs time (C)

(Adapted from Zheng X et al., 2008 (60) )

31

1.17 A. Linderstrøm-Lang model for deuterium exchange for proteins at

neutral pH subjected to fluctuations. kex –experimental rate constant for

exchange, ki- intrinsic amide exchange rate in the fully unfolded state,

ko -rate of opening and kc -rate of closing. B. The mass spectra profile

for EX1 and EX2 kinetics(Adapted from Zheng X et al., 2008 (60)).

33

X

2.1 Mre11 sequence. Deletion of residues in red corresponds to Mre11321

and deletion of both blue and red residues corresponds to Mre11297

37

3.1 T4Mre11WT

Intein tag purification 1: lysate; 2, 3: flow through; 4, 5:

cleavage buffer through the column; 6, 7: the first two chitin column

beads; 8: the second chitin column beads (to remove fusion protein and

intein); 9, 10: elutions; 11, 12: the second elution sample. Molecular

weight of T4Mre11:39kDa.

44

3.2 T4Mre11WT

Purification profiles. A. Anion exchange profile for

T4Mre11WT

B. Gel filtration profile T4Mre11WT

C. SDS-PAGE for

fractions of the gel filtration peak of T4Mre11WT

45

3.3 T4Rad50 purification: A: Gel filtration profile and B: the corresponding

peak fraction purity check using SDS-PAGE. T4Rad50 molecular

weight: 63.3kDa.

45

3.4 T4MR complex purification profile. A: Gel filtration peak of the

complex and B: SDS-gel. The complex gel filtration revealed three

peaks: peak1 and 2 were found to contain the complex; peak3

corresponds to the excess Mre11 protein.

46

3.5 Nuclease assay for testing the activity of T4 Mre11(WT), Mre11(321),

Mre11(297) truncation constructs . dsDNA (30mer) incubated with

Mre11 and activity was arrested at different time points. The samples

were loaded onto TBE gel and stained using gel green for visualization.

DNA substrate incubated with T4 Mre11(WT) for 0min , 10min ,

30min, 8h, 23h (Lanes 1-5), T4 Mre11(321) 0min, 10min, 30min, 8h,

23h (Lanes 6-10)

T4Mre11(297) 0min, 10min, 30min, 8h, 23h (Lanes 11-15).

47

XI

3.6 A. Circular Dichroism spectra of the Mre11 truncated proteins. 48

3.7 Binding isotherms for Mre11 proteins and Rad50. (a) T4Mre11WT (b)

T4Mre11(297) (c) T4Mre11(321). For all the constructs, 65 μM of

Mre11 was titrated against 6 μM of Rad50. The truncation proteins

failed to interact with Rad50. The Mre11 WT was found to show strong

binding to Rad50with a KD=10nM±0.17.

48

3.8a Heat Map of T4Mre11. The three bar represent the exchange

profile for the peptide in Free Mre11, MR complex and MR

Complex with AMP-PNP. Individual bars contain 5 time points

0, 0.5, 1, 2, 5 and 10 mins.

50

3.8b Heat Map of T4Rad50. The three bar represent the exchange

profile for the peptide in Free Rad50, MR complex and MR complex

with AMP-PNP. Individual bars contain 5 time points 0, 0.5, 1, 2, 5 and

10 mins

51

3.9. Amide exchange pattern for the MR complex. The structure of the

T4MR heterotetramer was generated based on MjMR complex bound to

ATPγS (PDB ID: 3AV0) as template using MODELLER.(a) Overall

amide exchange pattern of the Mre11 and Rad50 segments after

complex formation in comparison to the apo proteins. BLUE: represents

peptide segments showing decrease in amide exchange RED: Increase

in amide exchange. (b) Amide exchange pattern of MR-AMP-PNP

complex when compared to the MR complex. (c) Binding of AMP-

PNP at Rad50 dimer interface. The nucleotide binding motifs of Rad50

(RED) and nuclease motifs Mre11 (BLUE) show increase and decrease

in amide exchange, respectively.

52

XII

3.10. Proposed models of MR complex from different species. The models

highlight the orientation of the different domains in the free complex

(left panel) and AMP-PNP bound form (right panel).

54

3.11 Allosteric relay at lobe II of T4Rad50 and capping domain of T4Mre11.

(a) Cross sectional view of the lobe II loop of T4Rad50 and T4Mre11

capping domain interaction segment. The lobe II loop lies adjacent to

the signature motif (green) bound to the ATP-γS.(b) Closer view of

residues speculated to be involved in electrostatic interaction. R460

from T4Rad50 lies adjacent to the T268 and K286 from

T4Mre11.(c)Time course of amide HDX (0.5-10mins) for peptide

fragments 262-272 of Mre11 (m/z = 547.3, z = +2), (d) 282-298(m/z =

954.45, z = +3) and e. Rad50 fragment 454-468 (m/z= 851.4).

57

3.12 Allostery across the T4Rad50 His loop and T4Mre11 nuclease motif.

(a) Cross sectional view of T4Rad50 His-loop and Mre11 nuclease

motif II (51-63) and III (79-97) interaction. The Walker A motif

(Green) which binds to ATP occurs adjacent to the His-loop (Red). (b)

Closer view of the interaction and the residues that could be involve in

the electrostatic interaction.(c) Time course of amide exchange for

Mre11peptide fragment 51-63 (m/z = 525.95, z = +3), (d) Rad50

fragment 532-548 (in complex-AMP_PNP) but 533-548 in others

(e)Mre11 fragment 79-97 (m/z = 550.02, z= +4).

59

3.13 Conformational changes at the T4Mre11-Rad50 interface II. (a) Time

series (0-10mins) spectra of T4Rad50 peptide fragment 168-188 in apo

T4Rad50 protein showing bimodal distribution indicating presence of

more than one conformation. (b) Comparison of amide exchange at

10min time point (168-188) in free protein, after complex formation

and post AMP-PNP binding. The spectra shows absence of biomodal

62

XIII

distribution after complex formation and an increase in amide exchange

rate post AMP-PNP binding when compared to complex. (c) Mapping

of the segments which show changes in amide exchange profile post

AMP-PNP binding in the C-terminal domain of Mre11 and coiled-coil

segment of Rad50 at the interaction interface. These segments undergo

conformational changes which are otherwise not visible in the crystal

structure. (d) Time series of amide exchange of T4Rad50 C-terminal

coiled-coil segment 385-399 (m/z = 591.35, z = +3), e. 400-409 (m/z =

623.34, z = +2) (f) Mre11 C-terminal fragment 310-326 (m/z = 969.7,

z= +2).

3.14 Atomic Force Microscope analysis of T4Mre11 and Rad50 and DNA

binding interaction. (a) The linear PhiX174 DNA that was used in

binding interactions. (b and c) Reaction mixtures of linear PhiX174

DNA incubated with purified MR complex at DNA/protein ratios of

1:100(b) and 1:1000(c), respectively. No obvious binding activity was

observed in both conditions. (d) Reaction mixtures of linear PhiX174

DNA incubated with purified MR complex at DNA/protein ratio of

1:100 in the presence of 1 mM ATPγS. Dots on backbone of DNA

fragments may indicate MR complex bound to DNA. (e and f)

Reactions with linear PhiX174 DNA, incubated with purified MR

complex at DNA/protein ratios either of 1:30(e), or 1:100(f) in the

presence of 1 mM ATP. Flower structure composed of multiple inner

loops within one DNA molecule was observed at DNA/protein ratio of

1:30. Bundle formation was observed at DNA/protein ratio of 1:100.

Arrows indicate the protein bound to DNA. The scale bars are 500 nm.

65

4.1 Model for T4MR complex (a) Complex in free form, is prevented from

binding to DNA substrate as the Rad50 dimers are closer. (b) Complex

bound to AMP-PNP which locks the complex in an open conformation

similar to what has been observed in PfMR complex.

69

XIV

LIST OF TABLES

1.1 Disease caused by defective DNA repair proteins. The table highlights

the disease state, the corresponding phenotype or physiological effect

and the defective proteins responsible for the disease. (Source: Jackson

SP et al., 2009 (2)).

12

1.2 Chemotherapeutics available so far for treatment of cancer which

potentially target the cell cycle proteins (Source: Bolderson E et

al.(30)).

13

1.3 T4 gene segments homologous to eukaryotes and bacteria. (Source:

Bernstein H et al. (55))

28

2.1 Primer sequences for cloning T4Rad50 into pET28 vector. 36

XV

LIST OF ABBREVIATIONS

ABC- ATP Binding Cassette

AFM- Atomic Force Microscopy

AMP-PNP- Adenylyl-imidodiphosphate

AT-Ataxia Telangiectasia

ATM- Ataxia Telangiectasia Mutated

ATP- Adenosine triphosphate

ATR- Ataxia Telangiectasia and Rad3-related protein

AUC- Analytical Ultracentrifugation

BER- Base excision repair

CDK-Cyclin dependent kinases

CHK2- Checkpoint kinases-2

CD- Capping domain of Mre11

DNA- Deoxyribonucleic acid

dsDNA- double strand DNA

DSB- Double strand break

XVI

DTT- Dithiothreitol

EDTA- Ethylenediaminetetraacetic acid

ITC- Isothermal Titration Calorimetry

MR- Mre11/Rad50 complex

MRN- Mre11/Rad50/Nbs1complex

MjMR- Methanococcus Jannaschii MR complex

NBS- Njimegen Breakage Syndrome

NER- Nucleotide Excision Repair

NHEJ- Non-homologous end joining

ND- Nuclease domain of Mre11

PARP- Poly (ADP-ribose) polymerase

PfMR- Pyrococcus furiosus MR complex

RBD- Rad50 binding domain

SDS-PAGE- Sodium dodecyl sulphate- polyacrylamide gel electrophoresis

ssDNA- single strand DNA

SSB- Single strand break

TmMR- Thermotoga maritima MR complex

1

CHAPTER 1

INTRODUCTION

2

1.1 DNA Damage and Repair

Genomic stability, which is essential for maintenance of homeostasis in cells, is

constantly put at risk by DNA damage. Such damage can result from endogenous agents

such as reactive oxygen species generated during cellular processes or from exogenous

agents such as UV radiation or chemotherapeutics (1-4). DNA damage can be broadly

categorized into single-strand breaks, double-strand breaks, base modifications, and

bulky lesions (5). Deletion, mismatch of bases, and translocation of fragments are some

of the most frequently occurring DNA damages. Repair of such damages is essential to

prevent production of aberrant gene products and transmission of miscoded genetic

information to progeny, which leads to developmental abnormalities or cancer,

neurological , and immunological disorders (2, 6-8). Extensive studies have been carried

out to decipher the molecular events occurring during the repair process and to identify

constituent proteins involved in the signaling events (5, 8, 9). The repair pathways differ,

based on the kind of damage and the developmental stage of the affected cell (10). If the

damage is extensive and irreversible, cells are directed to the apoptotic pathway for

elimination from the system (6). Figures 1.1 and 1.2 depict the four major responses to

DNA damage in mammalian cells and the repair pathways followed depending on the

severity of the damage.

3

Figure 1.1: DNA damage response pathway in mammalian cells. Based on the nature of

damage, either of these pathways could function independently or in combination with

one another.

Figure 1.2: DNA damage can be broadly classified into three: single strand breaks

(SSB), bulky lesions and double strand breaks (DSB). A. Single-strand damage involves

breaks in one of the DNA strands or chemical modification of a single base (red

hexagon). Such damages follow the single-strand repair or base excision repair pathway

(section 1.2) B. Lesions (cyan sphere) that involve chemical modification of the

backbone leads to disruption of the helix backbone and these damages are repaired by

nucleotide-excision repair pathway (section 1.2). C. DSBs: Bifunctional reagents that

lead to interstrand cross-links (grey dumbbell) or external agents like radical oxygen

species lead to break of both the strands and the damage is subsequently repaired by non-

homologous or homologous recombination pathway based on the development stage of

the cell (section 1.3).(Source: McKinnon PJ, 2009 (3))

A B C

4

1.2 Single-Strand DNA Repair

Single-strand DNA (ssDNA) repair involves excising either small number of nucleotides

(when the damage is restricted to a single base) through the base excision repair (BER)

pathway or a long stretch of DNA through the nucleotide excision repair (NER) pathway,

respectively.

BER: This involves a lesion-specific DNA glycosylase and an endonuclease that excises

the base followed by recruitment of other enzymes such as APE1 endonuclease,

scaffolding protein XRCC1 (X-ray repair complementing defective repair in Chinese

hamster cells 1) and poly ADP-ribose polymerase (PARP). These proteins along with

DNA polymerase, fill in the gap. The strands are then ligated by ligase III/I based on the

length of the patch (Figure 1.3) (11, 12).

NER: There are of two kinds. When the repair occurs during transcription and involves

Transcription factor II H (TFIIH), transcription and repair complex, it is referred to as

transcription-coupled repair (TCR). In other cases, the repair is termed as global genomic

repair (GGR) (13, 14). These two pathways mainly differ in the proteins involved in the

initial damage recognition process. XPC (xeroderma pigmentosum, complementation

group C)/XPE (also known as DNA binding protein 1) is known to initiate the genomic

repair pathway while CSB (Cockayne Syndrome B) is required to initiate transcription-

coupled repair. Subsequent steps of repair are similar in both the pathways. The damage

verification and DNA unwinding is carried out by TFIIH, XPD, XPG, and XPA proteins,

by formation of a pre-incision complex. The nuclease activity of XPG causes incision to

5

occur, which is followed by gap-filling by DNA polymerase and rejoining by ligase I or

III (Figure 1.4).

Figure 1.3: Base excision repair. SSBs arising due to DNA glycosylase action or direct

damage lead to recruitment of poly ADP-ribose polymerase 1 (PARP1) at the damage

site, which subsequently activates scaffolding protein XRCC1, DNA polymerase (Polβ),

DNA ligase (LIG3) and apurinic/apyrimidinic endonuclease 1 (APE1). Based on the

length of nucleotides that is to be excised the protein machinery involved differs. Repair

of longer stretches is carried out by LIG1 and PCNA. Also, in some cases based on the

nature of damage at the SSB, tyrosyl DNA phosphodiesterase 1(TDP1) or aprataxin

(APTX) is recruited. TDP1 acts on 3′ modified ends or topoisomerase I DNA adducts

while APTX acts on 5′ ends resulting from abortive ligation events. (Source: McKinnon

PJ, 2009 (3))

6

Figure 1.4: Nucleotide Excision Repair pathway. The primary steps involved in NER are

recognition of the damage, excision and repair synthesis. Two different pathways within

NER differ in recognition step. When the damage is recognized during transcription and

involves transcription factors (TFIIH) it is termed as transcription coupled repair (TCR).

Else, it is referred to as global genomic repair. Post recognition, the repair steps followed

in both the pathways is similar. The colored blocks on the right highlight the disorders

resulting from dysfunctional proteins of the NER pathway. (Source: McKinnon PJ, 2009

(3) )

1.3 Double-Strand DNA Repair

Double-strand break (DSB) repair occurs through one of two major pathways,

homologous recombination (HR) or non-homologous end joining (NHEJ). Multiple

7

proteins are involved in sensing the DSB, which help in arresting the cell cycle and

activating the necessary repair pathway.

Figure 1.5: Double strand break repair pathway. Homologous recombination occurs in

proliferating cells. The replication protein A (RPA) and recombinase Rad51 coat the

broken double strand ends and initiates repair by formation of the holliday junction with

the help of the sister chromatid, which serves a template ensuring a error-free repair.

NHEJ occurs in differentiated cells that lacks a reference template. It involves key

proteins like KU70 and KU80, which modify the damaged end and prepare it for

ligation. Ligation is further carried out by ligase IV (LIG4), XRCC4, and other proteins.

(Source: McKinnon PJ, 2009 (3).

HR functions mainly in proliferating cells during the S and G2 phases of the cell cycle.

This repair process involves a sister chromatid which is used as template for correcting

8

the damaged DNA and hence in most cases error-free. The repair is carried out by

Mre11-Rad50-Nbs1 (MRN) protein complex— the protein of our interest. MRN in

association with BRCA1 C-terminal interacting protein (CTIP), generates a 3’ single-

stranded DNA (ssDNA). Replication protein A (RPA) and the recombinase Rad51 coat

the ssDNA generated, initiates formation of a holliday junction intermediate using the

template DNA from the sister chromatid for the repair to proceed (Figure 1.5).

NHEJ is a DSB repair pathway observed in non-replicating or differentiated cells that

lack a reference template. This increases chance of error during the repair process. NHEJ

repair involves modification of the two broken DNA ends so that they are compatible for

direct ligation. The end modification is carried out by DNA protein kinase complex

(DNA-PK), KU70, KU80 (ATP-dependent DNA helicases), and associated end-

processing factors (Figure 1.5). The ligation is catalyzed by DNA ligase IV (LIG4) in

conjunction with XRCC4 and Cernunnos.

1.4 Cell Cycle Checkpoint Response Control by ATM and ATR during DSB

DSB repair is associated with signaling events that prevent cell proliferation until the

repair is completed. This involves activation of specific cell cycle checkpoint kinases

(CHKs) that control the activity of cyclin dependent kinases (CDKs) known to regulate

the cell cycle. Two key DNA damage signaling proteins, ataxia telangiectasia mutated

kinase (ATM) and ataxia telangiectasia and RAD3-related kinase (ATR) phosphorylate

various cell cycle checkpoint effectors (15). ATM and ATR function cooperatively in

response to DSB and studies show that they are non-redundant in their physiological

roles. DNA-PK is another essential kinase involved in NHEJ; however it is found to be

9

functionally redundant with ATM. Once dsDNA damage is sensed, it leads to activation

of these repair proteins through MRN complex. Upon activation, they reach the site of

damage and activate CHK. ATM phophorylates checkpoint kinase2 (CHK2) and Polo-

like kinase-1 (PLK1) and ATR phosphorylates claspin and again PLK1 (Figure 1.6).

CHK arrests the cells in G2 phase by inactivation of CDKs (16, 17).

Once repair has been completed, the checkpoint proteins are eliminated to allow the cell

cycle to proceed. PLK1 promotes degradation of claspin and WEE1 protein kinases, that

allows the G2-to-M transition by increasing CDK activity (18, 19). Figure 1.6 highlights

the checkpoint kinases and cyclin-dependent kinases involved in the cell cycle

checkpoint activation during repair.

Figure 1.6: Cell cycle checkpoint activation by ATM and ATR. ATM and ATR activate

check point kinases (CHK) and cell cycle proteins, which arrest the cell in different

stages of cell cycle. Once the repair is completed successfully, the cell cycle proceeds by

activation of cyclin dependent kinases (CDK). Or else, the cell is directed towards

proteasomal degradation (Source: Branzei D et al. 2008 (9) )

10

1.5 DNA Damage and Diseases

DNA damage and defective repair proteins are found to be one of the key underlying

causes for various diseases (20-23).

Cancer: Chromosomal translocations arising due to errors in DNA repair pathway result

in the formation of defective gene products, for example, fusion of proto-oncogene loci

to antigen receptors arising from aberrant antigen receptor recombination (24).

Mismatched DNA and mutated bases are found to cause colorectal and endometrial

cancer (25, 26). Carcinogens are found to act by causing mutations, which lead to

uncontrolled cell growth. High cell proliferation rate leads to replication stress and

increased DNA damage, which directs the cells towards the apoptotic pathway.

Mutational or epigenetic inactivation of the proteins involved in the apoptotic pathway

leads to malignancies. Table 1.1 summarizes some of the malignancies caused by

inactivation of repair proteins.

Neurodegenerative diseases: DNA lesions in neuronal cells lead to diseases like ataxia,

Alzheimer’s, Parkinson’s and Huntington’s. High concentration of reactive-oxygen-

species (ROS) in the brain arising due to high mitochondrial respiration, defective repair

genes, and ageing lead to DNA damage in neurons. The damages are usually repaired

through error-prone SSB or NER or NHEJ pathway and improper repair of defective

genes increases the chances of neuronal cell death (23). Terminally differentiated

neurons in adults are more severely affected by such damages due to the lack of

replaceable neuronal cells. Ataxia is one such example where neurodegeneration occurs

due to the defective ATM gene (27),(28)). (Table 1.1 and reference 5 give a detailed note

on DNA damage based neurological disorders.)

11

Immune deficiencies and infertility: DNA repair proteins play an important role in

genome rearrangements during immune system development. In particular, defects that

affect the V (D) J recombination have been found to result in lymphoma or leukemia of

B cells and T cells. Defects in NHEJ repair proteins are found to cause immune

deficiencies. Ataxia Telangiectasia (AT) and Njimegen Breakage Syndrome (NBS)

patients are found to suffer from severely impaired immunity due to defects in class

switching (Table 1.1) (29). Damage to DNA repair elements is also found to affect

human spermatogenesis leading to infertility.

Table 1.2 lists a set the chemotherapeutics being developed to target components of the

repair pathway for treatment of cancer. These agents have been successful to different

extent. Their toxic side effects on healthy cells have prevented their usage for

elimination of tumors. Prevalence of alternative repair pathways in tumor cells have

been suggested to be one of major causes for limited effects of these chemotherapeutics.

In short, DNA damage repair pathway components are extensively studied to understand

the molecular basis of these diseases that arise due to their malfunctioning.

Mre11/Rad50/Nbs1 complex is one such component in eukaryotes known to cause

cancer and neurological diseases.

12

Table 1.1:Disease caused by defective DNA repair proteins. The table highlights the

disease state, the corresponding phenotype or physiological effect and the defective

proteins responsible for the disease. (Source: Jackson SP et al., 2009 (2)).

13

Table 1.2: Chemotherapeutics available so far for treatment of cancer which potentially

target the cell cycle proteins (Source: Bolderson E et al.(30) ).

1.6 Mre11/Rad50/Nbs1 (MRN) Complex

MRN complex is the first responder of the dsDNA damage response pathway. It

activates ATM and ATR kinases which subsequently recruit the cell cycle check point

kinases. Figure 1.7 depicts the role of MRN in multiple DNA repair pathways. MRN

complex is ubiquitous and essential for survival of a cell.

14

Figure 1.7: Role of MRN in DNA repair and cell cycle control. Mre11 leads to

activation of several downstream pathways depending on the nature of damage. It has

been found to play an important role in homologous recombination (HR), non-

homologous end joining (NHEJ) and activation of apoptotic pathway. This figure

emphasizes key role of MRN complex in maintaining cellular stability. (Source:

Stracker. TH. et al. 2011 (31))

Clinical significance of MRN complex: Mutations in Mre11 is found to cause

neurological diseases that phenotypically match ataxia telangiectasia (AT) and is hence

referred to as ataxia telangiectasia like disorder (ATLD) while mutation in Nbs1 causes

Nijmegen breakage syndrome (NBS) (28). AT is characterized by muscle hypotonia,

truncal swaying, abnormality in eye movement, cerebellar dysfunction, inability to sense

vibration, reduction in sensory conduction velocity, and axonal degeneration of

peripheral nerves (28). Mre11 mutation is found to be hypomorphic, which leads to

15

partial loss of function of the MR complex. Patients with this mutation do not have

telangiectasia. However, they develop ataxia-like phenotype with a slower onset of

neuronal degeneration. Mre11 inactivation mutations are also found to cause colorectal

cancer (26, 32). Mutation in NBS gene leads to formation of N-terminal truncated protein.

The N-terminal of Nbs is known to be essential for interaction with Mre11 and Rad50

for subsequent activation of the ATM kinase. This truncation is found to cause

microcephaly with occasional medulloblastoma brain tumors. Mutations in Rad50 gene

have been reported to cause NBS-like disorder with increased radiation sensitivity and

immunodeficiency (33, 34).

Structure and function: MR complex consists of two components: Mre11, a nuclease,

and Rad50, an ATPase which drives the nuclease. Mre11 provides ATP-independent

ssDNA (linear and circular) endonuclease activity and, in the presence of Rad50 and

Mn2+

, it acts as an ATP-dependent 3’ to 5’ dsDNA (linear) exonuclease (35-40). Mre11 is

composed of three domains: nuclease domain (ND), capping domain (CD) and C-

terminal Rad50 binding domain (RBD or CTD) (41-43) (Figure 1.8) . The highly

conserved ND harbors six phophodiesterase motifs while the variable CD determines the

nature of DNA processed by the complex (42, 43). . Dimerization of Mre11 enables DSB

repair to take place by tethering the dimer to two DNA ends and bridging them in a linear

manner (44). (Figure 1.8)

16

A. Mre11 domain arrangement

B. Structural representation

Figure1.8: Domain arrangement of Mre11 proteins: (a) The three domains are

highlighted in box form and the flexible linker as the dotted line. The green segments

within the nuclease domain box indicate the phophodiesterase motifs. (b) Structural

presentation of the domains in Mre11 (Pink). The nuclease motifs harboring the active

site are highlighted in green which occur at the loop segments.

Rad50 belongs to the ABC-ATPase superfamily with DNA binding activity (45). The N-

and C-terminal domains of Rad50 contain the ATP binding Walker motifs; Walker A and

B, respectively. These two nucleotide-binding domains (NBD) are separated by a long,

flexible coiled-coil segment which controls the tethering of DNA strands during repair

(Figure 1.9A) (38, 46). The length of the coiled-coil segment varies from 200-to-700

residues in different species (Figure 1.10B).

Nuclease

domain (ND)

Capping

domain (CD)

Rad50 binding

domain (RBD)

17

A. Rad50 domain arrangement

Figure 1.9: Domain arrangement of Rad50 proteins: (a) Domain arrangement of Rad50.

(b) Structural presentation of the domains in Mre11 (Pink). Motifs and domains are

highlighted in the respective colors shown in the block diagram (a). (c) MR with

truncated Mre11 bound to AMP-PNP from Pyrococcus furiosus(PDB id: 3QKU).

(d)Overlap of free PfRad50 and AMP-PNP bound PfRad50, highlighting the rotation of

the subdomains (PDB id: 3QKT and 3QKU).

C lobe

(NBD)

Coiled-coil

segment

N lobe

(NBD)

B. Structural representation of Rad50 domains C. AMP-PNP-bound PfRad50

D. Overlap of AMP-PNP-bound

and unbound PfRad50

N-Lobe C-Lobe

18

A conserved zinc-finger motif located at the center of this segment mediates dimerization

of two MR complexes during sister chromatid bridging (Figure 1.9A and 1.10A) (46, 47).

The N- and C-domain, also referred to as lobe I and II, house the conserved P, Q, D and

H loops and a characteristic signature motif of the Rad50 family (48) (Figure 1A and

1.9B). Each of these loops aid in conformational changes occurring in the Rad50 dimer

post ATP binding. The coiled-coil domain interacts with Mre11 and allows tethering of

homologous template.

ATP molecules are held by the signature motif of one and the Walker A motif of another

monomer (Figure 1.9C). ATP binding leads to conformational changes across the

ATPase domain and the coiled-coil domain (Figure 1.9D). This conformational change is

linked to unwinding of DNA substrate, which enables Mre11 to process the substrate.

Crystal structures of PfRad50 reveal that ATP binding at the dimer interface leads to ~30º

rotation of coiled coil segment (Figure 1.9D) (41-43).

A

19

Figure 1.10: A. Molecular arrangement of MRN complex, a. Assembly of

Mre11/Rad50/Nbs on the DNA substrate. Rad50 functions as a dimer and hence harbors

two Mre11 interaction sites. The N and C domains of Rad50 are arranged in an

antiparallel fashion b. AFM image of MR complex in DNA free and bound state. The

images highlight the different orientations of the coiled-coil segment (Source: Stracker.

TH. et al. 2011 (31)) B. Comparison of length of coiled-coil segment in different species.

(courtesy: Scott Nelson)

MR complex structure: Several Mre11 and Rad50 structures have been reported recently,

but only two of these are of the MR complex. These were the free MR complex structure

from Thermotoga maritima (TmMR) and ATPγS bound structure from Methanococcus

jannaschii (MjMR) (Figure 1.11A,B) (42, 43). These complex structures revealed that

MR is functionally a heterotetramer and the proteins share two interfaces: one across the

RBD of Mre11 and coiled-coil segment of Rad50, and another across the CD of Mre11

and lobe II of Rad50 (42, 43) (Figure 1.11). The CD of Mre11 is connected through a

long unstructured segment to the RBD. Both CD and ND domains were found to be

essential for Mre11 nuclease activity (42). The free TmMR crystal structure revealed that

in the absence of ATP, the complex exists in an open conformation with the Rad50

monomers seperated from each other (Figure 1.11A and 1.12A) (42). SAXS studies

revealed that, upon ATPγS binding, the TmMR complex adopts a compact conformation

arising due to dimerization of the Rad50 monomers (43). Crystal structure of ATPγS-

B

20

bound MjMR and AMP-PNP-bound Pyrococcus furiosus MR complex (PfMR)

supported this observation (Figure 1.11B, 1.9C) (43, 49).

Based on the structural and biochemical data on MR from different species, multiple

models of DNA repair by the complex have been proposed (Figure 1.12). These models

corroborate the effect of ATP on MR complex. In the absence of ATP, the complex

remains in the open loading form or the substrate accessible form where the separation of

Rad50 monomers opens up the Mre11 nuclease site accessible. This is referred to as the

open clamp mode (Figure 1.12). Binding of ATP leads to dimerization of the Rad50

NBDs which decreases the accessibility to the Mre11 nuclease site. This state is referred

to as the closed clamp mode.

However, the models proposed to date differ in the proposed orientation of Rad50 NBDs

with respect to the Mre11 domains. In the PfMR model constructed based on SAXS data,

Mre11 ND and CD were proposed to be away from the nucleotide-binding region of

Rad50 (50). On the other hand, the TmMR and MjMR structures show the presence of an

interface between CD and NBD of Rad50 and hence in these models the NBDs of Rad50

are oriented in an opposite fashion when compared to PfMR model (Figure 1.12B) (42,

43, 49).

Further, MjMR crystal structure and mutational studies indicated two possible

orientations of Rad50 NBD with respect to Mre11 ND in the non-ATP-bound form which

is highlighted in Figure 1.12C (43).

21

a. TmMR (Free form)

b. MjMR (ATPγS bound form)

Figure 1.11 Crystal structures of MR reported: (a) MR complex from Thermotoga

maritima(PDB id: 3QG5).(b) ATPγS bound Methanococcus jannaschii (PDB id: 3AV0).

Interface I

Interface II

Interface II

Interface I

22

a. TmMR model

b. PfMR model

c. MjMR model

I

II

Figure 1.12: Proposed models of MR complex from different species. Pink: Mre11

domains. Blue: Rad50 domains. Mre11 nuclease domain ( ), capping domain ( ),

linker region ( ), RBD ( ), n-terminal domain ( ), c-terminal domain ( ), coiled-

coil segment ( , ). ATP/AMP-PNP/ATPγS( ). (a) TmMR model (ref) (b) PfMR

model (ref) (c) Two models of MjMR model (ref). The models highlight the orientation

of the different domains in the free complex (left) and AMP-PNP bound complex (right).

Mre11-Rad50 /DNA binding mode Mre11-Rad50-AMP-PNP

23

1.7 SCOPE OF THIS STUDY

Several structural models have been put forth to describe how the MR proteins function,

yet ambiguity in the orchestration of DNA repair by the MR complex remains. Presence

of multiple models for functioning of MR proteins underscores the dynamic nature of

these proteins in solution. This emphasized the need to study the time-course behavior of

MR proteins. Therefore, we chose to conduct a detailed in vitro biophysical

characterization of the solution behavior of the MR proteins in free and complex form

under different conditions over time. For our study, we used the MR complex from

bacteriophage T4 also known as gp46/47 which was recently characterized (51). The

reasons for choosing T4 proteins have been highlighted in section 1.8. We explored the

behavior of T4MR proteins using a heavy isotope labeling technique — amide deuterium

exchange followed by mass spectrometry (HDXMS) (52). A detailed description about

this technique is highlighted in section 1.9. HDXMS serves as an ideal technique to

capture the short time-scale conformational changes occurring in the order of minutes

within the system which are critical for the allosteric regulation of Mre11 nuclease

activity. Using the available database of crystal structures of the MR proteins, we studied

the deuteration pattern of proteins in the unassociated form, in complex form, and AMP-

PNP-bound form. This study enabled us to identify the most probable orientation of the

T4Rad50 and T4Mre11 domains and the allosteric regulation sites in T4Mre11 and

T4Rad50. Some of these sites have been shown to affect the nuclease activity of Mre11

in other species.

24

AIM

In vitro biophysical characterization of the solution behavior of the T4

Mre11-Rad50 complex and propose a model for DNA repair by the

same

25

1.8 T4 Bacteriophage MR complex

T4 is an obligate parasite of Escherichia Coli and one of the key model organisms

studied for over 60 years to understand fundamental processes in molecular biology. It is

composed of an icosahedral-shaped head harboring the genetic material required for its

replication, followed by a neck segment with collar and a hollow tail segment surrounded

by a contractile sheath, which allows it to attach to host cell for transfer of the genetic

material.

Figure 1.13: T4 Phage structural organization (53).

The T4 genome is composed of 168.9 kbp long linear double-stranded DNA, which

codes for over 300 gene products. The DNA sequence was found to be redundant at the

termini, leading to increase in length to about 172 kb, which is known to be a replication

strategy to protect DNA during replication (54). It is also the first virus found to contain

26

introns in its genome, which was one of the key indicators of its ancestral relationship to

eukaryotes.

T4 and T2 phage were among the first model organisms used to demonstrate existence of

DNA as a genetic material, triplet genetic code, discovery of mRNA, importance of

recombination in DNA replication, DNA repair mechanisms, restriction and modification

of DNA, splicing of introns, and many more fundamental biological concepts. Analysis

of the T4 nucleotide-synthesizing complex, the replisome, and recombination complexes

has given us insights into macromolecular interactions and how biological “molecular

machines” function (53).

Ease of production and isolation of T4 proteins in the 1970s allowed study of duplex

DNA replication mechanisms and the proteins involved. Later studies on eukaryotic and

bacterial replication systems were found to correspond highly with what was known from

the T4 phage system. Berstein et al. carried out an extensive genetic homology study of

the T4 genes with the bacterial and eukaryotic counterparts and showed that they share

equal ancestry with eukaryotes and bacteria. Table 1.3 lists some of the T4 genes

homologous to bacterial and eukaryotic genes (55). Our study focuses on the gene

products 46 and 47 (highlighted in Table 1.3), which are homologues of human Rad50

and Mre11 proteins, respectively. T4Rad50 and Mre11 share 38% and 28% sequence

identity with corresponding human homologues. gp46 and 47 are among 130

uncharacterized genes from T4 phage genome. All the recombination-dependent DNA

replication enzymes and proteins in T4 phage have been characterized, except gp46/gp47

complex. In this thesis, gp47 and gp46 are referred to as T4Mre11 and T4Rad50,

respectively.

27

As highlighted in earlier sections, extensive biochemical and structural studies have been

carried out on archae and bacterial counterparts of Mre11 and Rad50 proteins. Crystal

structures of MR complexes from Pyrococcus furiosus (thermophilic archae),

Thermotoga maritima (thermophilic bacteria) and Methanococcus jannaschii

(hyperthermophilic archae) have been published (41-43). Biochemical characterization of

the T4MR proteins revealed that it behaves in a similar fashion as other MR proteins (53).

However, there have been no structural or biophysical studies carried out yet on the

T4MR proteins

Ease of production: T4Rad50 coiled-coil segment is composed of only 200 residues as

compared to the humanRad50, which is composed of 900 residues. Thus, functional

heterotetrameric T4MR complex, with a molecular weight of 200kDa, is one of the

smallest across the three kingdoms (Figure 1.10B). T4Mre11 and Rad50 protein yields

are much higher (10-25mgs from 1l of E.Coli culture) when compared to yeast and

human MR complex where the yield typically ranges in microgram quantities (51, 56, 57).

Human Rad50, which is co-expressed with Mre11 in insect cell lines, is found to be

difficult to express due to the length and flexibility of the coiled-coil segment. However,

T4Rad50 owing to a smaller size is found to be comparatively more stable and easier to

handle in laboratory conditions.

28

Table 1.3: T4 gene segments homologous to eukaryotes and bacteria. (Source: Bernstein

H et al. (55))

29

1.9 Hydrogen/Deuterium Exchange followed by Mass Spectrometry (H/DX-MS)

Figure 1.14: Exchangeable hydrogens in a protein. Red: Amide hydrogens with slow

exchange rate, Green: covalently attached hydrogens, which do not exchange, Blue: side

chain hydrogens, which have high exchange rate and hence, are undetectable. (Source:

www.hxms.com)

Amide hydrogen/deuterium exchange mass spectrometry is a sensitive technique used to

study protein-protein, protein-ligand interactions, protein folding and denaturation in

solution (52, 58, 59). Proteins constantly undergo exchange of hydrogens with the

solvent. There are two different kinds of hydrogen atoms available for exchange in a

polypeptide chain – main chain amide hydrogens and side chain hydrogens. The main

chain hydrogens, which are covalently linked to carbon atoms do not exchange while

side chain hydrogens have a high back exchange rate and hence are not monitored

during experiments (Figure1.14).

30

The exchange kinetics of a protein is a function of its structure, pH of the solution, and

the adjacent side chain groups of an exchangeable amide. The exchange kinetics is

monitored by diluting concentrated protein solution in D2O, which leads to replacement

of amide hydrogen during solvent exchange by deuterium, a heavier isotope. The proton

abstraction can be catalyzed by D3O+

(acid), OD−

(base), D2O or buffer ions. At

physiological pH, at which the proteins function, it is catalyzed by OD (59).

A-H + OD− A

− + H-OD

A−

+ D3O+

A-D + D2O

This exchange reaction is quenched by lowering the pH to 2.5 and temperature to 4ºC.

The deuterated sample is further subjected to digestion by pepsin (which functions at low

pH). The pepsin-generated peptide fragments are resolved using reversed-phase HPLC

and further analyzed using ESI-MS (electrospray ionization mass spectrometry).

The number of deuterium incorporated is calculated by the given equation (58):

Where D is the deuterons incorporated after correcting for those lost during the digestion

and separation, N is the number of exchangeable amides (total no. of amides minus N

terminal and prolines if any), m0%, m100% and m refer to average of the molecular masses

(centroid of the MS peak) of the non-deuterated, completely-deuterated control samples

and sample at time point t, respectively (58). The corrected deuterons number is then

plotted versus time using first-order reaction parameters (Figure 1.16).

31

Figure 1.15: Workflow for HDMS (Adapted from Busenlehner LS et al. (58) ).

Figure 1.16: Deuterium incorporation quantification. i. Steps involved in the HDX-MS

experiments. ii. Detailed picture of centroid calculation. As shown in the table, centroid

of the mass peak is noted at different time points (A). The centroid values are used to

i ii

32

calculate the relative increase in deuteration levels (B). The deuterium levels are then

plotted vs time (C) (Adapted from Zheng X et al., 2008 (60) ).

The solvent accessibility of different segments of a protein differs according to its tertiary

structure. Residues occurring in the compactly folded hydrophobic core would be less

solvent accessible compared to those present on the surface, which gives rise to different

exchange patterns in peptide spanning the two regions. This difference in exchange rate

serves as a tool to monitor conformational changes in a protein when its environment

changes (58, 59).

Regional unfolding and refolding events in solution contribute to the intrinsic exchange

rate of an amide. Figure 1.17 shows the Linderstrøm-Lang model for deuterium exchange

kinetics in solution. There are two kinds of kinetics described by the model: EX1 and

EX2. In EX1 kinetics, the rate of local folding (kc) is faster than rate of intrinsic amide

exchange rate (ki), hence rate of exchange (kex) depends on intrinsic exchange rate and

equilibrium constant between open and closed state (ko/kc). In EX2 kinetics, rate of

intrinsic exchange is faster than rate of folding and hence rate of exchange (kex) is

determined by rate of opening (ko) (refer to equation in Figure 1.17). EX2 is characterized

by random distribution of amides while peptides showing EX1 are characterized by

regions, which either get completely or not at all deuterated, thus generating a bimodal

isotopic pattern in the mass spectra (58, 60).

Hydrogen bonding, solvent accessibility, and backbone flexibility affect the kinetics of

hydrogen exchange. Comparison of the exchange profiles of protein fragments in native

state and the ligand-bound state provides insight into local and global conformational

changes occurring within the protein.

33

Figure 1.17: A. Linderstrøm-Lang model for deuterium exchange for proteins at neutral

pH subjected to fluctuations (58). kex –experimental rate constant for exchange, ki-

intrinsic amide exchange rate in the fully unfolded state, ko -rate of opening and kc -rate of

closing. B. The mass spectra profile for EX1 and EX2 kinetics (Adapted from Zheng X et

al., 2008 (60)).

Our study focuses on identifying the conformational changes occurring in Mre11 and

Rad50 once the MR complex formation occurs and after AMP-PNP binds to the complex.

The hydrogen exchange profiles of the free proteins, the complex, and AMP-PNP-bound

complex were compared to identify the regions undergoing changes deuteration pattern.

A B

34

CHAPTER 2

MATERIALS AND METHODS

35

2.1 Cloning, protein expression and purification

T4Mre11 cloned into pTYB1 plasmid for intein-assisted tag-free protein production

(New England Biolabs, Ipswich, MA, U.S.A.) was obtained from Scott Nelson’s

laboratory (51). C-terminal truncated T4Mre11: Mre11297

(1-297), Mre11321

(1-321), were

prepared by amplifying and inserting respective sequences into pTYB1 (Figure 2.1).

These truncations are designed based on the available PfMre11 crystal structure (PDB id:

1ii7) and secondary structure prediction tools- SABLE and FASS03 (61, 62). Full length-

T4Mre11WT

, T4Mre11297

, and T4Mre11321

were expressed in BL21 (DE3) (Invitrogen,

Carlsbad, CA, U.S.A.). Cells were grown to an OD600 of 1.0 and expression was induced

using 0.15 mM IPTG for 16 h at 18°C. Cells were harvested by centrifugation at 6,000

rpm for 30 min, frozen at –80°C, and lysed in the Mre11 lysis buffer (20 mM Tris-HCl

pH 7.8 and 1 mM EDTA) with 500 mM NaCl. After centrifugation at 12,000 rpm for 45

min, the cell-free extract was loaded onto a chitin column and washed overnight with the

Mre11 lysis buffer with 1 M NaCl. Following the wash, on-column intein cleavage and

T4 Mre11 release was performed by quickly flushing the column with cleavage buffer

containing 75 mM β-mercaptoethanol and incubating the column at 4°C for 24 h. After

cleavage, T4Mre11 was eluted from the column using the Mre11 lysis buffer. The eluted

proteins were dialyzed into buffer A (20 mM Tris-HCl pH 7.5, 1 mM DTT, 1 mM

EDTA, 10% glycerol) for anion exchange chromatography using Hitrap Q XL (GE

Healthcare, Piscataway, NJ, U.S.A.). Full-length and truncated Mre11 were eluted at 27%

buffer B (buffer A with 1 M NaCl). The eluted Mre11 fractions were further purified by

gel-filtration using Superdex 200 10/300 GL (GE Healthcare) with a running buffer

containing 20 mM sodium phosphate pH 7.5, 0.1 mM EDTA, and 5% glycerol.

36

Figure 2.1: Mre11 sequence. Deletion of residues in red corresponds to Mre11321

and

deletion of both blue and red residues corresponds to Mre11297

T4Rad50 cloned in pTYB1 vector was found produce unstable protein. Hence, it was re-

cloned into pET28 vector with N-terminal his-tag using EcorI and NdeI restriction sites.

For this purpose, the Nde I site within the T4Rad50 gene was mutated from CATATG to

CATAAG. This was done in two set of reactions using Rad50_pET_F1 and

Rad50_pET_R2 primers for one reaction and Rad50_pET_F2 + Rad50_pET_R1 as

primers for the other (sequence listed in table 2.1). Two fragments hence obtained were

digested, purified, and mixture was used as template for amplification using

Rad50_pET_F1 and Rad50_pET_R1 to generate the entire Rad50 coding fragment with

mutated Nde1 site. This fragment was further cloned into Nde I/ EcoR I of pET28 vector

using the standard protocol.

Table 2.1: Primer sequences for cloning T4Rad50 into pET28 vector.

BL21 (DE3) cells carrying Rad50 gene were grown to an OD600 of 0.5 and induced using

0.15 mM IPTG for 16 h at 18°C. Cells were harvested by centrifugation at 6,000 rpm for

297 321 339

37

30min, frozen at –80°C, and lysed in the Rad50 lysis buffer (50 mM sodium phosphate

pH 7.4, 300 mM NaCl) with 10 mM imidazole and 10% glycerol. After centrifugation at

12,000 rpm for 45 min, the cell-free extract was loaded onto a cobalt column (Thermo

Scientific) and washed with the Rad50 lysis buffer. T4Rad50 was eluted using lysis

buffer containing 150 mM imidazole, and further purified by gel-filtration (Superdex 200

10/300 GL) in buffer containing 20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1 mM EDTA.

For complex preparation, purified Mre11 and Rad50 were mixed at a 2:1 molar ratio, and

the mixture was purified using gel filtration in similar fashion as Rad50. For the complex

with AMP-PNP sample, the protein was gel filtration purified into buffer containing 20

mM Tris-HCl pH 7.2, 5 mM MgCl2, 1 mM MnCl2, 5% glycerol. The protein after

purification was concentrated to desired level and then incubated with 1 mM AMP-PNP

for 30 min before performing the deuterium exchange experiments.

2.2 Circular Dichroism

Full length Mre11 and its truncated versions were concentrated to 100 μM in 20 mM

sodium phosphate pH 7.5, 0.1 mM EDTA. The CD spectrum was measured using a Jasco

J810 spectropolarimeter equipped with a Peltier temperature controller and a quartz cell

of 0.1 cm path length. The spectrum was recorded at a bandwidth of 0.1nm and

acquisition time of 50 nm/sec. Three scans were performed at wavelength ranging from

190 nm to 260 nm at 25° C. The sample spectra were corrected by subtracting the buffer

spectrum. The data is presented in millidegrees.

38

2.3 Nuclease Assay

Mre11 nuclease assay was performed in 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM

DTT, 5 mM MgCl2, 1 mM MnCl2, 2 mM ATP at 37°C. Mre11, the truncated protein

were incubated with Rad50 and dsDNA substrate (5’CTT ACT GTG TCA TCT CTT

ACT GTG TCA TCT 3’) at a concentration of 0.3 μM in separate tubes. Samples were

collected at 0 min, 10 min, 30 min, 8 h, and 23 h, and loaded onto 20% TBE-PAGE gel

and stained using gel green for analysis.

2.4 Isothermal Titration Calorimetry (ITC)

Microcal VP-ITC (GE Healthcare) was used to estimate the KD and stoichiometry for the

interaction of Rad50 and T4Mre11 variants mentioned above using standard procedure as

described earlier (50, 62-66). Titrations were performed with the Mre11 proteins and

Rad50 at 25°C in 20 mM Tris-HCl pH7.2, 150 mM NaCl, 1mM EDTA at 25ºC. The

proteins were dialyzed into the buffer at 4ºC before titrations to ensure that the heat

change due to buffers is minimized. The proteins were then concentrated using Vivaspin

concentrators (GE Healthcare) and degassed under vacuum by gentle stirring for five

minutes just before use. T4Mre11 proteins (65 µM in the syringe) were titrated in 10 µl

increments against Rad50 (6 µM in the well) with a time interval of 270s. The baseline of

the isotherm was smoothened and baring the first peak, other peaks were integrated to

obtain the heat change. The stoichiometry (N), the dissociation constant (KD) and the

enthalpy ΔH were calculated using ORIGINTM

software package provided by Microcal

(GE Healthcare).

39

2.5 Atomic Force Microscopy

The linear PhiX174 DNA was used for AFM analysis, which was obtained by digesting

PhiX174 plasmid (New England Biolabs Inc., Ipswich, MA, U.S.A) with restriction

enzyme StuI (New England Biolabs Inc., Ipswich, MA, U.S.A). The fragment was

further purified using QIAquick PCR purification kit (QIAGEN, CA, U.S.A). The DNA

sample was prepared in imaging buffer (20 mM Tris-HCl, 10 mM MgCl2, 25 mM NaCl,

pH 8.0) at concentration of 0.1ng/µl. Mre11–Rad50–DNA mixtures of certain

DNA/protein ratios was prepared by incubating 2 ng linear PhiX174 DNA with

corresponding amount of purified T4Mre11 and Rad50 complex in a final volume of 20

µl at 37°C for 20 min. ATP (1 mM) or ATPγS (1 mM ) was added together with DNA

and protein prior to incubation for Mre11–Rad50–DNA mixtures in the presence of ATP

or ATPγS. The mixtures were then diluted 10-fold in imaging buffer, and 50 µl portion

was deposited on freshly cleaved mica. After about 12 min, mica was washed with

deionised water, and dried with a stream of compressed nitrogen gas. Samples were

imaged with a Molecular Imaging atomic force microscope (Agilent Technologies,

Santa Clara, CA, U.S.A) operated in AAC mode in air at room temperature. Silicon

AFM probes, BudgetSensors Tap 300 (Innovative Solutions Bulgaria Ltd., Sofia,

Bulgaria) were used. AFM images were processed only by flattening to remove the

background slop using the Gwyddion software.

2.6 Hydrogen/Deuterium Exchange followed by Mass Spectrometry (H/DX–MS)

Purified T4Mre11, T4Rad50 and complex were concentrated to 35, 25 and 50 µM,

respectively, using VivaspinTM

concentrator (Sartorius, Goettingen, Germany) and

40

concentrations were estimated using UV spectrophotometer (280nm). Four samples were

prepared in respective sample buffers:

a. 35 µM of T4Mre11 in 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA

b. 25 µM of T4Rad50 in 20 mM Tris-HCl pH 7.2, 150 mM NaCl, 1mM EDTA

c. 5 µM T4MR complex without AMP-PNP in 20 mM Tris-HCl pH 7.2, 150 mM NaCl,

1mM EDTA.

d. 50 µM complex with AMP-PNP in 20 mM Tris-HCl pH 7.2, 150 mM NaCl, 1 mM

MnCl2, 5 mM MgCl2. The complex was incubated with 1mM of AMP-PNP for 30mins

before performing the experiment.

Preliminary runs were carried out to determine the optimum concentration required for

each sample. 2 µl of the samples were incubated in 18 µl respective sample buffers

prepared in D2O (final concentration-90%) for various time intervals (0.5, 1, 2, 5 and

10min) at 20ºC. The exchange reaction was quenched using 30 µl of prechilled 0.1%

TFA to lower the pHread to 2.5. 50 µl of the quenched sample was injected into nano-

UPLC sample manager (Waters, Milford, MA, USA) maintained at 4°C, wherein, the

sample was allowed to pass through a pepsin column (Porozyme, ABI, Foster City, CA)

using 0.05% formic acid as solvent at a flow rate of 100 µl/min. The digested peptides

were then trapped in a 2.1 x 5 mm C18 column (ACQUITY BEH C18 VanGuard Pre-

column, 1.7 μm, Waters, Milford, MA), eluted using a gradient of 8-40% acetonitrile

containing 0.1% formic acid at a flow rate of 40 µl/min using nano ACQUITY Binary

Solvent Manager (Waters, Milford, MA). The eluted peptides were further resolved using

a reversed–phase, 1.0 x 100 mm, C18 column (ACQUITY UPLC BEH, Waters, Milford,

41

MA, USA). This was followed by mass spectrometric analysis using SYNAPT HDMS

(Waters, Manchester, UK) in MSE mode (67). Samples were tested for each time point in

duplicates. The instrument was continuously calibrated with Glu-fibrinogen peptide

(GFP) at 100fmol/µl.

Using the MSE

data, a peptide list was generated based on the undeuterated sample using

ProteinLynx Global Server (PLGS 2.4 beta test version, Waters, Milford, MA, USA) (68).

The peptides, which appeared in duplicate sets were selected and further analyzed using

HDX browser (prototype by Waters, Milford). Time point data for each peptide was

collated on HDX browser, extracted into a format compatible for analysis using HX

express program to quantify the deuterons incorporated (69, 70). Each peptide was

manually analyzed to eliminate peaks with poor signal to noise ratio. Control experiments

were carried out to estimate the back exchange rate wherein the sample was incubated in

D2O for 24 hours. Peptides from highly solvent exchangeable region, which underwent

100% exchange at 10 min time point, were chosen. Average of the back exchange rate

seen in these peptides was estimated to be 32.7% (71, 72). This exchange rate was found

to be consistent in multiple rounds of experiments. Hence, all the centroid values for the

peptides were corrected by a multiplication factor of 1.49. The deuteron numbers were

then plotted against time based on one-phase association model using PRISMTM

.(GraphPad Software, Inc., La Jolla, CA, U.S.A. (43, 73) ).

42

CHAPTER 3

RESULTS AND DISCUSSION

43

3.1 Protein Expression and Purification

Figure 3.1 highlights the purity of the Mre11WT

fractions obtained from intein-tag

purification. After incubation with the cleavage buffer, beads indicated the presence of

intein, fusion protein and Mre11 (lane 6 and 7). The elution was allowed pass through

another clean chitin column, which allowed us to get rid of the fusion protein and intein

(lane 8). The yield for recombinant wild type and truncated Mre11 proteins were

~10mg/L of culture.

Figure 3.1: T4Mre11WT

Intein tag purification 1: lysate; 2, 3: flow through; 4, 5:

cleavage buffer through the column; 6, 7: the first two chitin column beads; 8: the

second chitin column beads (to remove fusion protein and intein); 9, 10: elutions; 11, 12:

the second elution sample. Molecular weight of T4Mre11:39kDa.

Intein+Mre11

Intein

Mre11

Lane #

44

d

Figure 3.2: T4Mre11WT

Purification profiles. A. Anion exchange profile for T4Mre11WT

B. Gel filtration profile T4Mre11WT

C. SDS-PAGE for fractions of the gel filtration peak

of T4Mre11WT

.

Figure 3.3: T4Rad50 purification: A: Gel filtration profile and B: the corresponding peak

fraction purity check using SDS-PAGE. T4Rad50 molecular weight: 63.3kDa

A B

C

A B

45

Intein-tag T4Rad50, which initially gave us poor yields and unstable protein was found to

give significantly pure protein and higher yields after cloning into pET28 vector with his-

tag. The yield for his-tag T4Rad50 was found to be ~30mg/L of culture.

Figure 3.4: T4MR complex purification profile. A: Gel filtration peak of the complex

and B: SDS-gel. The complex gel filtration revealed three peaks: peak1 and 2 were found

to contain the complex; peak3 corresponds to the excess Mre11 protein.

The gel filtration purification of T4MR complex revealed presence of three peaks. Based

on analysis using SDS-PAGE, peak 1 and 2 were both found to contain the complex and

peak 3 was found to contain excess Mre11 (Figure 3.4). Peak1 and 2 were further

analyzed using analytical ultra centrifugation experiments (AUC) (refer to appendix B).

Peak 2 was found to correspond to a molecular weight of 189kDa indicating a

stoichiometric ratio of 2:2 of the proteins when in complex (Mre11-39kDa, rad50-

62kDa). Peak1 was found to give an anomalous molecular weight and hence eliminated

from further analysis. Addition of ATP to the complex obtained from peak 2 did not

influence the molecular weight (appendix B). Peak2 was hence used for all subsequent

analyses.

1

2

3

37kDa

75kDa

Peak1 Peak2

A B

46

3.2 C-terminal domain of T4Mre11 is essential for its nuclease activity

We sought to first identify the minimal fragment of T4Mre11 required for MR

functioning. Hence the two Mre11 constructs truncated at C-terminal were designed:

T4Mre11321

and T4Mre11297

. The wild type Mre11 (Mre11WT

) is 339 residues long.

These proteins were tested for exonuclease activity on a 30mer dsDNA in the presence of

Rad50, ATP, Mg2+

and Mn2+

. Mre11WT

digested the substrate in ten minutes but the

truncated Mre11s failed to do so even after 8 hours (Figure 3.5). Circular dichroism

results indicated that the lack of activity was not due to loss of structure (Figure 3.7).

Subsequently, binding studies were carried out to test if the truncated proteins were able

to bind T4Rad50 using isothermal titration calorimetry (ITC). The binding isotherms

confirmed that the activity of truncated proteins was lost due to their inability to bind

Rad50 (Figure 3.7). These observations highlighted that the C-terminal of T4Mre11 was

necessary for binding Rad50 and the nuclease activity. This requirement for the Mre11 C-

terminal segment for the MR complex formation was also observed in other MR protein

complexes (42, 50).

Figure 3.5: Nuclease assay for testing the activity of T4 Mre11(WT)

, Mre11(321)

, Mre11(297)

truncation constructs . dsDNA (30mer) incubated with Mre11 and activity was arrested at

different time points. The samples were loaded onto TBE gel and stained using gel green for

visualization. DNA substrate incubated with T4 Mre11(WT)

for 0min , 10min , 30min, 8h, 23h

(Lanes 1-5), T4 Mre11(321)

0min, 10min, 30min, 8h, 23h (Lanes 6-10) T4Mre11(297)

0min,

10min, 30min, 8h, 23h (Lanes 11-15).

A

B

C D

T4Mre11WT T4Mre11

321 T4Mre11297

47

A.

Figure 3.6: Circular Dichroism spectra of the Mre11 truncated proteins. Mre11WT

(♦),Mre11 321

(●) and Mre11

297(▲)

B.

Figure 3.7: Binding isotherms for Mre11 proteins and Rad50. (a) T4Mre11WT

(b)

4Mre11(297)

(c) T4Mre11(321)

. For all the constructs, 65 μM of Mre11 was titrated against

6 μM of Rad50. The truncation proteins failed to interact with Rad50. The Mre11 WT

was

found to show strong binding to Rad50with a KD=10nM±0.17.

a. T4Mre11WT c.T4Mre11321 d.T4Mre11297

48

3.3 Solution behavior of T4MR complex using HDXMS

Overview

Deuterium exchange experiments were carried out on four samples: T4Mre11, T4Rad50,

T4MR complex, AMP-PNP-bound T4MR complex. AMP-PNP was used to study the

ATP induced conformational changes occurring in the MR complex. For T4Mre11, 24

common peptide fragments covering more than 90% of the sequence was analyzed

(Appendix D). 33 common peptide fragments covering 91% of the Rad50 sequences

were analyzed (Appendix E). The exchange percentage was calculated by dividing the

average number of deuterons exchanged at each time point for each peptide with the

number of exchangeable amides for the given peptide (% deuteration = 100* D/N). The

percentage exchange for all the peptides was then tabulated to plot the heat map using the

HXMS online tool (74). The exchange rate for both the proteins are shown using a color

scheme in Figure 3.8. Violet and brown represent the lowest (10%) and the highest

exchange rates (>90%), respectively. Sequence alignment of T4Mre11/Rad50 with

Mre11/Rad50 from other species is shown in Appendix C.The deuterium exchange

profiles of the peptide fragments were mapped onto a model structure which was built

using MODELLER with MjMR (PDB id: 3AV0) as a template (75) (Figure 3.9).The

pattern of exchange of the T4MR complex in comparison to free proteins and the AMP-

PNP-bound T4MR complex as compared to the free complex are shown in Figure 3.9A

and 3.9B, respectively. This section discusses the deuterium exchange profile of the

peptide fragments, which show difference in free, complex, and AMP-PNP bound forms.

49

Figure 3.8 A : Heat Map of T4Mre11. The three bar represent the exchange profile for

the peptide in Free Mre11, MR complex and MR complex with AMP-PNP. Individual

bars contain 5 time points 0, 0.5, 1, 2, 5 and 10 mins.

Mre11

MR

MR-AMP-PNP

MR

30secs

to

10mins

Capping domain

C-terminal domain

Nuclease domain

% Deuteration

50

Figure 3.8 B: Heat Map of T4Rad50. The three bar represent the exchange profile for the

peptide in Free Rad50, MR complex and MR complex with AMP-PNP. Individual bars

contain 5 time points 0, 0.5, 1, 2, 5 and 10 mins.

Rad50

MR

MR- AMP-PNP

N-Terminal

domain

Coiled-coil

domain

C-Terminal

domain

Mre11 interaction site

Mre11 interaction site

% deuteration

%Deuteration

51

Figure 3.9: Amide exchange pattern for the MR complex. The structure of the T4MR

heterotetramer was generated based on MjMR complex bound to ATPγS (PDB ID:

3AV0) as template using MODELLER.(a) Overall amide exchange pattern of the Mre11

and Rad50 segments after complex formation in comparison to the apo proteins. BLUE:

represents peptide segments showing decrease in amide exchange RED: Increase in

amide exchange. (b) Amide exchange pattern of MR-AMP-PNP complex when

compared to the MR complex. (c) Binding of AMP-PNP at Rad50 dimer interface. The

nucleotide binding motifs of Rad50 (RED) and nuclease motifs Mre11 (BLUE) show

increase and decrease in amide exchange, respectively.

c

Rad50 dimer

interface

Mre11 nuclease

motifs

Mre11-Rad50-AMP-PNP

a b

52

3.3.1 Formation of the T4MR complex — A conformationally stable state

The amide exchange levels of the segments of T4Rad50 and T4Mre11 segments forming

the complex interface were found to be lower in comparison to free protein state,

especially, the NBD of T4Rad50 and ND of T4Mre11 (Figure 3.9A). The peptide

fragments encompassing the characteristic Rad50 motifs, including — Walker A (three

deuterons), Q loop (one deuteron), D loop (one deuton) and the H loop (one deuteron),

showed corresponding decreases in deuteron uptake (Figure 3.9A). The decrease of

deuterons in these T4Rad50 segments after MR complex formation suggested it is driven

to a less solvent accessible closed dimer state. In T4Mre11, peptide fragments spanning

the recognition loops harboring the active site residues for nuclease activity showed a

decrease in deuteron uptake after complex formation and a further decrease post AMP-

PNP binding (Figure 3.9C, Appendix D, highlighted in yellow). This decrease suggested

that the T4Rad50 NBDs infact lie close to T4Mre11 ND. These observations were found

to support the open state of MjMR model II wherein the Rad50 dimers occur close to

each other and adjacent to the Mre11 nuclease domains (Figure 3.10, C.II), and not in the

open Rad50 conformation or in the conformation where Mre11s sit away from the Rad50

NBDs as seen other MR models (Figure 3.10) (42, 65).

3.3.2 AMP-PNP captures the complex in an open transition state

Subsequently, we compared the T4Rad50 amide exchange pattern as a free protein

without AMP-PNP to Rad50 in complex and Rad50 in complex bound to AMP-PNP. It

was interesting to observe that AMP-PNP binding increased the deuteron uptake of

T4Rad50 when compared to free protein and complex state.

53

a. TmMR model

b. PfMR model

c. MjMR model

I

II

Figure 3.10: Proposed models of MR complex from different species. Pink: Mre11

domains. Blue: Rad50 domains. Mre11 nuclease domain ( ), capping domain ( ),

linker region ( ), RBD ( ), n-terminal domain ( ), c-terminal domain ( ), coiled-

coil segment ( , ). ATP/AMP-PNP/ATPγS ( ). (a) TmMR model (42) (b) PfMR

model (41) (c) Two models of MjMR model (43). The models highlight the orientation of

the different domains in the free complex (left panel) and AMP-PNP bound form (right

panel).

Mre11-Rad50 /DNA binding mode Mre11-Rad50-AMP-PNP

54

Again, the peptide fragments covering the key motifs at the Rad50 dimer interface –

signature motif (468–482), Walker A (27–46), Walker B (502–511), Q loop (133–148)

and D loop (512–525) showed an average increase of two deuterons in each segment

(Figure 3.9C). The increase, however, was not just restricted to the T4Rad50 dimer

interface but in segments away from the interface, showing that Rad50 becomes

conformationally more flexible (Figure 3.9B). Contrary to the T4Rad50 behavior,

T4Mre11 showed further decrease in deuteration rate post AMP-PNP binding as

compared to in the complex. Especially, peptide fragments covering the nuclease motifs,

motif II (51–63), III (79–97), IV (161–174), V, (178–191), VI(195–217) (Figure 3.9C,

Appendix D). Each of these segments showed at least one deuteron decrease in deuterium

uptake. These deuteration profiles suggested that AMP-PNP held the Rad50 monomers

away from each other in an open transient state but the Mre11 nuclease domains become

less flexible and more solvent inaccessible. These observations were in agreement with

AMP-PNP-bound models of MjMR and PfMR wherein the binding of AMP-PNP leads

to opening of the Rad50 dimer and rotation of the coiled-coil segment. However, the

orientation of Mre11 NDs in PfMR complex (Figure 3.10B) does not support the T4MR

amide exchange data. These deuteration patterns suggested that Mre11 in fact adopts a

more stable conformation for nuclease activity to proceed.

3.3.3 Conformational changes across lobe II of T4Rad50 and T4Mre11 capping

domain

Signature motif of T4Rad50 has been previously implicated in allosteric activation of the

complex (76).The amide exchange profile of lobe II loop (454–467) adjacent to signature

motif showed difference upon AMP-PNP binding. It was interesting to observe that the

55

T4Mre11 capping domain segments (260– 272 and 282– 298) which lies close the Rad50

loop also showed deuteration differences upon complex formation and ATP binding

(Figure 3.11A). T4Rad50 loop (454–464) showed a single deuteron decrease after the

MR complex formation and a deuteron increase after AMP-PNP binding. T4Mre11

capping domain segment 260–272 showed a decrease of two deuterons after complex

formation and an increase of two after AMP-PNP binding, giving a similar pattern to

what was observed with the T4Rad50 loop described earlier (Figure 3.11C). Fragment

282–298, on the other hand, showed an increase of more than two deuterons during the

complex formation and a decrease of three after AMP-PNP binding (Figure 3.11D).

Closer inspection of the amino acid sequence of in these segments (Mre11—260–272,

282–298 and Rad50—454–464) on either proteins showed that the residues were

oppositely charged suggesting a presence of ion-pair interaction similar to what has been

observed in the MjMR complex. We speculate the involvement of the side chain of R460

of T4Rad50 which lies adjacent to the signature motif, and the side chain of T268 and

D288 and the main chain of K286 and I287 in the T4Mre11 capping domain in the

Rad50-Mre11 interaction (Figure 3.11B). Mutation studies in this Mre11-Rad50

interaction interface in the MjMR complex have been shown to affect the nuclease

activity (65). These conformational changes in the capping domain segments of T4Mre11

post AMP-PNP binding suggest that T4Mre11 capping segment could also share an

interaction interface with T4Rad50 similar to how Mre11 and Rad50 are arranged in the

MjMR complex(65).

56

Figure 3.11: Allosteric relay at lobe II of T4Rad50 and capping domain of T4Mre11. (a)

Cross sectional view of the lobe II loop of T4Rad50 and T4Mre11 capping domain

interaction segment. The lobe II loop lies adjacent to the signature motif (green) bound to

the ATP-γS.(b) Closer view of residues speculated to be involved in electrostatic

interaction. R460 from T4Rad50 lies adjacent to the T268 and K286 from

T4Mre11.(c)Time course of amide HDX (0.5-10mins) for peptide fragments 262-272 of

Mre11 (m/z = 547.3, z = +2), (d) 282-298(m/z = 954.45, z = +3) and (e) Rad50 fragment

454-468 (m/z= 851.4). The solid lines on the graphs denote the best fit of the data to a

one-phase association non linear exponential curve fit (GraphPad Prism 5.0(San

Diego,CA).

Mre11 (262-274)

0 2 4 6 8 100

2

4

6

8Mre11

Mre11-Rad50

Mre11-Rad50-AMP-PNP

Time (Min)

Deu

tero

ns

Inco

rpo

rate

d

Mre11 (282-298)

0 2 4 6 8 100

5

10

15Mre11

Mre11-Rad50

Mre11-Rad50-AMP-PNP

Time (Min)

Deu

tero

ns

Inco

rp

orate

d

a b

c d

454-468

282-298

262-270 ATP-γS

Signature

motif

Mre11– 282-298 Mre11– 262-270

e Rad50− 454-468

262-270

Mre11

Rad50

T268

K286

I287

D288

R460

Mre11 /Rad50

Mre11-Rad50 complex

Mre11-Rad50-AMP-PNP

57

3.3.4 Conformational changes across the His loop of T4Rad50 and nuclease motif of

T4Mre11

MjMR complex structure had previously revealed an interaction between the nuclease

domain of Mre11 and the His switch of Rad50 (65). T4MR deuterium exchange study

identified a similar interaction. Binding of AMP-PNP was found to affect the deuteration

pattern of a peptide fragment spanning the His loop (532–548, Figure 3.12A and D) of

T4Rad50. This segment lies close to the Walker A (a T4Rad50 motif highlighted in

green, Figure 3.12A) which along with the signature motif from the adjacent T4Rad50

unit binds the second AMP-PNP molecule (magenta in Figure 3.12A) of the

heterotetramer. Two peptide fragments spanning these two nuclease motifs: 51–63 (II)

and 79–97 (III) showed differential deuteration patterns during the complex formation

and after AMP-PNP binding. Motif II showed a consistent protection while motif III

showed an increase (one deuteron) followed by a decrease in deuteron uptake levels

(three deuterons) (Figure 3.12C and E).The His loop peptide fragment: 532–548, showed

a decrease of close to two deuterons upon complex formation and one deuteron increase

after AMP-PNP binding (Figure 3.12D). The difference in the deuteration pattern

indicates that AMP-PNP binding to T4Rad50 could allosterically affect the nuclease

activity of Mre11 through the His loop interaction. The possible residues involved in

hydrogen bond interaction between the His loop and nuclease motifs are highlighted in

Figure 3.12B.

58

Figure 3.12: Allostery across the T4Rad50 His loop and T4Mre11 nuclease motif. (a)

Cross sectional view of T4Rad50 His-loop and Mre11 nuclease motif II (51-63) and III

(79-97) interaction. The Walker A motif (Green) which binds to ATP occurs adjacent to

the His-loop (Red). (b) Closer view of the interaction and the residues that could be

involve in the electrostatic interaction.(c) Time course of amide exchange for

Mre11peptide fragment 51-63 (m/z = 525.95, z = +3), (d) Rad50 fragment 532-548 (in

complex-AMP_PNP) but 533-548 in others (e)Mre11 fragment 79-97 (m/z = 550.02, z=

+4). The solid lines denote the best fit of the data to a one-phase association non linear

exponential curve fit (GraphPad Prism 5.0(San Diego,CA).

79-97

51-63

532-548 His loop

Walker A D538

Q542

R53 Y89 N84

ATP

a b

c d

e

Mre11

Rad50

Mre11 /Rad50

Mre11-Rad50 complex

Mre11-Rad50-AMP-PNP

Signature

motif

Mre11 (51-63) Rad50 (532-548)

Mre11 (79-97)

59

3.3.5 Flexibility of Rad50 coiled-coil segment undergoes changes upon AMP-PNP

binding

The N-terminal (189–198) and C-terminal (385–399 and, 400–409) base of coiled-coil

segment of T4Rad50 and the CTD of T4Mre11 (311–326) showed a 40–50% decrease in

deuteration after the complex formation (Figure 3.8A). This decrease corroborated our

binding studies and showed that the CTD of T4MRe11 forms an interface with the

T4Rad50 coiled-coil domain. It was even more interesting to observe a biomodal

hydrogen-deuterium exchange profiles in the Rad50 N-terminal coiled-coil segment

(168–188) that indicated that this region of Rad50 assumes multiple conformations. The

MR Complex formation was found to select one of the conformations (Figure 3.13A).

Furthermore, the coiled-coil segment is driven to yet another conformation upon AMP-

PNP binding, resulting in a slight increase of deuteration than in the complex but

decrease from the free T4Rad50 deuteration levels (Figure 3.13A and B). A similar

increase in deuteration levels was also observed in the C-terminal coiled-coil fragment

(385–399) post AMP-PNP binding) (Figure 3.13D). AFM studies on the human MR

complex previously showed that binding of AMP-PNP does not affect the flexibility of

the coiled-coil segment (77). Contrary to this, our deuterium exchange data on T4MR

reveals that the coiled-coil segment indeed undergoes small scale conformational changes

that suggest flexibility in the coiled-coil segment. The mutations in this interface in the

TmMR complex have been previously shown to affect the nuclease activity (42).

Although structural studies so far highlight the global translational shift in the position of

the coiled-coil segment, little is known about the internal conformational changes in the

coiled-coil segment. Amide exchange data revealed that there are internal changes

60

occurring within the coiled-coil base segment which when disrupted could affect the

nuclease activity of T4Mre11.

Mre11 CTD in MR Mre11 CTD in MR-

AMP-PNP

Rad50 coiled-coil

segment in MR

Rad50 coiled-coil

segment in MR-AMP-

PNP

a b

c Mre11-Rad50 Interface- Amide exchange pattern

Rad50 (168-188) Rad50 (168-188)

61

Figure 3.13: Conformational changes at the T4Mre11-Rad50 interface II. (a) Time series

(0-10mins) spectra of T4Rad50 peptide fragment 168-188 in apo T4Rad50 protein

showing bimodal distribution indicating presence of more than one conformation. (b)

Comparison of amide exchange at 10min time point (168-188) in free protein, after

complex formation and post AMP-PNP binding. The spectra shows absence of biomodal

distribution after complex formation and an increase in amide exchange rate post AMP-

PNP binding when compared to complex. (c) Mapping of the segments which show

changes in amide exchange profile post AMP-PNP binding in the C-terminal domain of

Mre11 and coiled-coil segment of Rad50 at the interaction interface. These segments

undergo conformational changes which are otherwise not visible in the crystal structure.

(d) Time series of amide exchange of T4Rad50 C-terminal coiled-coil segment 385-399

(m/z = 591.35, z = +3), e. 400-409 (m/z = 623.34, z = +2) (f) Mre11 C-terminal fragment

310-326 (m/z = 969.7, z= +2). The solid lines denote the best fit of the data to a one-

phase association non linear exponential curve fit (GraphPad Prism 5.0(San Diego,CA).

d e

f

Mre11 /Rad50

Mre11-Rad50 complex

Mre11-Rad50-AMP-PNP

62

3.3.6 Effect of AMP-PNP on the distal coiled-coil segments

The coiled-coil segment of Rad50 is known to communicate with the globular NBDs of

Rad50 and play an important role in maintaining the integrity of MR complex (46). In the

structural studies conducted so far, the coiled-coil segment of Rad50 except for the

minimal fragment required for Mre11 binding of Rad50 has been consistently eliminated

to avoid aggregation. However, the low working protein concentrations require for the

deuterium exchange experiments allowed us to retain the full length T4Rad50 to study

the effect of AMP-PNP in the distal regions of the coiled-coil segment. The deuterium

exchange profile of this segment (200–375) (Figure 3.8A) highlights that most of the

peptide fragments, except the fragment 200–216, do not show any change in deuteration

levels upon complex formation ( Appendix E ). Fragment 200–216 which lies adjacent to

the N-terminal base of coiled-coil of the interface II, was found to show close to 40%

decrease in the deuteration level. No change in the deuteron uptake level in the rest of the

segment suggested that there are no significant changes occurring in the distal coiled-coil

segment of Rad50 due to binding of Mre11. However, all of the segments covering the

coiled-coil segment (200–375) showed an increase in the deuteration level post AMP-

PNP binding, suggesting the occurrence of a conformational change along the entire

coiled-coil segment (Appendix E). These changes highlight that the distal coiled-coil

segments could play a role in the functioning of the complex which is supported by a

recent truncation study on the coiled-coil segment (78).

63

3.4 Hydrolysis of ATP is essential for substrate processing by T4Mre11 dsDNA

Furthermore, AFM was employed to examine the DNA-binding activity and structural

organization of the T4MR–DNA complex. The AFM images identified that the T4MR

complex does not bind the DNA substrate in the absence of ATP (Figure 3.14),

suggesting that the lack of T4Mre11 nuclease activity in the absence of ATP is due to the

inability of the MR complex to bind the substrate. Although the quaternary structure of

MR complex was not clearly discernible, we were able to identify that the MR complexes

bound multiple strands of DNA together into a bundle (Figure 3.14E and F). In line with

the biological activity of a MR complex to join together DNA ends for DSB repair, these

MR–DNA complexes were frequently observed at the ends of the DNA substrates

(Figure 3.14E). Interestingly, when ATPγS was used in place of ATP, the MR complex–

DNA bundle formation was not observed despite that the MR complexes were found to

be associated with DNA (Figure 3.14D). These results indicate that T4MR complex

requires ATP to bind to the DNA substrate, but hydrolysis of ATP is required to bring

DNA strands together. Unlike the MjMR complex where ATP-bound Rad50 is found to

downregulate the MjMre11 endonuclease activity, T4MR appears to behave similar to

other MR complexes such as PfMR, and TmMR) which require ATP for the

endonuclease activity.

64

Figure 3.14: Atomic Force Microscope analysis of T4Mre11 and Rad50 and DNA

binding interaction. (a) The linear PhiX174 DNA that was used in binding interactions.

(b and c) Reaction mixtures of linear PhiX174 DNA incubated with purified MR

complex at DNA/protein ratios of 1:100(b) and 1:1000(c), respectively. No obvious

binding activity was observed in both conditions. (d) Reaction mixtures of linear

PhiX174 DNA incubated with purified MR complex at DNA/protein ratio of 1:100 in the

presence of 1 mM ATPγS. Dots on backbone of DNA fragments may indicate MR

complex bound to DNA. (e and f) Reactions with linear PhiX174 DNA, incubated with

purified MR complex at DNA/protein ratios either of 1:30(e), or 1:100(f) in the presence

of 1 mM ATP. Flower structure composed of multiple inner loops within one DNA

molecule was observed at DNA/protein ratio of 1:30. Bundle formation was observed at

DNA/protein ratio of 1:100. Arrows indicate the protein bound to DNA. The scale bars

are 500 nm.

a b

c d

e f

65

CHAPTER 4

CONCLUSION

66

Mre11–Rad50 complex has been extensively studied over the last two decades. The

assembly and coordinated functioning of this robust cellular DNA repair motor has been

unraveled to a great extent. Studies have highlighted both the conserved nature of

domains within the complex and diversity in its organization (79). Although archeal MR

crystal structures have been reported and several biophysical studies on truncated

versions of the complex have been carried out (38, 42, 44, 50, 65, 80), the precise pathway

of communication between the two proteins remains a mystery. More recently, multiple

evidences of a long range allosteric regulation of Mre11 nuclease activity by Rad50 have

been put forth (50, 65, 76). Our study on the T4MR complex highlights the segments of

Mre11 and Rad50 that undergo dynamic rearrangements during the complex formation

and after AMP-PNP binds to the Rad50 dimer interface. Some of these dynamic

segments have been previously shown to play a role in allosteric communication (43, 50).

However, those previous studies were limited by the use of Rad50 mutants lacking the

coiled-coil region to avoid coiled-coil-mediated aggregation at the protein concentration

required for the structural studies. The use of HDXMS technique allowed us to perform

the experiments a low protein concentration, allowing the use an intact protein in our

biochemical assays.

When analyzed at a whole-protein level using amide exchange patterns, both proteins are

conformationally stabilized during the MR complex formation. Furthermore, upon AMP-

PNP binding, the conformational flexibility of Rad50 increased, while Mre11 was

stabilized further. When analyzed at peptide level, the exchange patterns revealed that

small scale conformational changes were occurring at the MR complex interfaces and

distal domains of Rad50 and Mre11. Because structural studies were inherently

67

insensitive to smaller dynamic behaviors of proteins, previous studies failed to detect

those conformational shifts we were able to observe in the MR complex.

AFM studies of T4MR show that ATP is essential for DNA substrate binding, suggesting

that the binding site is not accessible for the substrate in the absence of ATP (Figure

3.14). This speculation was further supported by the lower amide exchange rates

observed at the Rad50 dimer interface which suggest that it exists in a closed

conformation, thereby preventing the substrate to access the nuclease site. ATP

hydrolysis is known to drive the open–close conformation transition of Rad50 (81). It is

interesting to observe that the AFM studies also showed that the T4MR complex still

binds the substrate in the presence of ATPγS in (Figure 3.14d). This could be explained

by the ability of ATPγS to hold the Rad50 dimers in an open conformation in a similar

fashion as AMP-PNP, which would allow the substrate to be seated in the binding pocket.

The closed conformational state (in the absence of ATP) is physiologically relevant as it

would block random substrates from binding the active site. This could prevent

anomalous activity of the MR complex, hence serving as a checkpoint for the T4MR

activity. Based on these observations, we propose a model for the functioning of the

T4MR complex (Figure 4.1). The model highlights the domain orientation of the

constituent proteins within the complex forming a closed conformation (Figure 4.1A) and

the AMP-PNP-bound form assuming an open conformation (Figure 4.1B). Another

interesting observation is the bundling of T4MR complex observed in the presence of

ATP in the AFM studies. This bundling behavior is similar to what has been observed in

human and S. cerevisiae MR complexes which form large multimers (79). This is,

68

however, not seen in prokaryotic MR complexes, suggesting that T4MR may function in

a similar fashion as eukaryotic MR complexes through the formation of oligomers.

Figure 4.1: Model for T4MR complex (a) Complex in free form, is prevented from

binding to DNA substrate as the Rad50 dimers are closer. (b) Complex bound to AMP-

PNP which locks the complex in an open conformation similar to what has been observed

in PfMR complex. This transition allows the substrate recognition to occur. Pink: Mre11

domains, Blue: Rad50 domains. Mre11 nuclease domain ( ), capping domain ( ),

linker region ( ), RBD ( ), N-terminal domain ( ), C-terminal domain ( ), coiled-

coil segment ( , ).

Until now, only a little attention has been paid to the coiled-coil domain in investigating

the function of the MR complex. The length of this domain is variable, and the sequence

is far less conserved compared to the globular domain. However, a high structural

similarity of this domain is observed amongst various eukaryotic and prokaryotic species.

Different orientations of the coiled-coil segment with respect to the globular domain have

been observed during AFM studies (79). These arrangements amongst various species

have been used to explain the diverse cellular activities carried out by MR complex (79).

Until a decade ago, the coiled-coil segment of Rad50 was thought to be a stiff structural

element. Hence, researchers were unable to explain some of the unfavorable orientations

a. T4Mre11-Rad50

AMP-PNP

b. T4Mre11-Rad50-AMP-PNP

DNA binding mode

69

observed in AFM studies (79). However, a study by John et al. revealed that the coiled-

coil segment of Rad50 harbors segments of higher flexibility (lower coiled-coil

probability) which could, for example, explain the occurrence of the ring formation of

coiled-coil segment observed in hRad50 structures (arising out attachment at the Zn motif

and globular domain)(82).Truncation studies of coiled-coil segment is also found to alter

the complex nuclease activities suggesting that this domain is essential. Our amide

exchange data supported John et al.’s observations. We observed an alternating fashion

of deuterium exchange pattern along the coiled-coil segment (Appendix E). In addition to

that, the data also revealed that this flexibility is further affected by the AMP-PNP-

binding event. A consistent increase in deuteron uptake levels all along the segment

suggests that the flexibility of the coiled-coil segment increases upon AMP-PNP-binding

(Figure 3.9B, appendix E). This confirmed that AMP-PNP binding leads to

conformational changes at the distal end of the coiled-coil segment. This increase in the

coiled-coil conformational flexibility explains the formation of some of the otherwise

unfavorable MR arrangements seen in AFM structures. It also explains the ability of MR

complexes to recruit and orient template DNA for repair during homologous

recombination or to orient the N and C lobes of Rad50 to facilitate the entry of the

damaged substrates of various sizes into the active site.

Sufficient evidences has been put forth to establish allosteric regulation of Mre11

nuclease activity by Rad50. And these were obtained using crystal structures and

mutational studies in the fragments which showed visible conformational changes.

However, our study revealed that, apart from the large time-scale conformational

changes, there are other small time-scale changes which play a crucial role in MR

70

allostery. These include the changes highlighted in our study across the interfaces and

coiled-coil segment. Thus, Mre11 activity is found to be controlled through multiple sites

involving the terminals and distal regions of the coiled-coiled domain, the signature motif

and His-switch of Rad50 as well as the Mre11 capping domain, and there could be others

which are not arising due to their non-visible nature (83). These findings suggest that

Rad50 could regulate the Mre11 activity allosterically through multiple pathways as

observed in other systems (84). MR complex is an example of protein machinery where

combination of both protein dynamics and conformational redistribution plays an

important role in allosteric regulation(85, 86).

Recent drug discovery efforts have shifted from targeting the active site to trapping the

protein targets in an inactive conformation (87). These drugs are designed primarily by

targeting allosteric regulation sites within the proteins. Occurrence of more than one

allosteric site increases the number of viable drug targets, hence increasing the chance of

successfully deactivation of the target proteins. Our findings here lay a platform for

designing allosteric drugs against MR complex. These drugs could be used to selectively

sensitize cancer cells by disrupting MR complex and increase the potency of

chemotherapeutic agents or radiotherapy, an approach which has been repeatedly

studied(88, 89).Combination therapy constituting ligands targeting the various allosteric

sites of the MR complex could be used for treatement.

71

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APPENDIX A:

T4Rad50 sequence:

MKNFKLNRVKYKNIMSVGQNGIDIQLDKVQKTLITGRNGGGKSTMLEAITFGLFGKPFRDVKKGQLINSTNKKELLVELWMEYDEKKYYIKRGQKPNVFEITVNGTRLNESASSKDFQAEFEQLIGMSYASFKQIVVLGTAGYIPFMGLSTPARRKLVEDLLEVGTLAEMDKLNKALIRELNSQNQVLDVKKDSIIQQIKIYNDNVERQKKLTGDNLTRLQNMYDDLAKEARTLKSEIEEANERLVNIVLDEDPTDAFNKIGQEAFLIKSKIDSYNKVINMYHEGGLCPTCLSQLSSGDKVVSKIKDKVSECTHSFEQLSTHRDNLKVLVDEYRDNIKTQQSLANDIRNKKQSLIAAVDKAKKVKAAIEKASSEFIDHADEIALLQEELDKIVKTKTNLVMEKYHRGILTDMLKDSGIKGAIIKKYIPLFNKQINHYLKIMEADYVFTLDEEFNETIKSRGREDFSYASFSEGEKARIDIALLFTWRDIASIVSGVSISTLILDEVFDGSFDAEGIKGVANIINSMKNTNVFIISHKDHDPQEYGQHLQMKKVGRFTVMV

T4Mre11 sequence:

MKILNLGDWHLGVKADDEWIRGIQIDGIKQAIEYSKKNGITTWIQYGDIFDVRKA

ITHKTMEFAREIVQTLDDAGITLHTIVGNHDLHYKNVMHPNASTELLAKYPNVK

VYDKPTTVDFDGCLIDLIPWMCEENTGEILEHIKTSSASFCVGHWELNGFYFYKG

MKSHGLEPDFLKTYKEVWSGHFHTISEAANVRYIGTPWTLTAGDENDPRGFWM

FDTETERTEFIPNNTTWHRRIHYPFKGKIDYKDFTNLSVRVIVTEVDKNLTKFESE

LEKVVHSLRVVSKIDNSVESDDSEEVEVQSLQTLMEEYINAIPDITDSDREALIQY

ANQLYVEATQ

81

APPENDIX B:

Analytical Ultracentrifugation data for Mre11 and complex

T4Mre11- 39kDa

T4Mre11- Rad50: 189kDa

T4Mre11- Rad50 – AMP-PNP: 220kDa

APPENDIX C:

Figure 1: Mre11 alignment

Motif I II

T4Mre11 ............MKILNLGDWHLGVKADDEWIRGIQI.DGIKQAIEYSKKNGITTWIQYGDIFDVRKAIT

pfMre11 ............MKFAHLADIHLGYEQFHKPQREEEFAEAFKNALEIAVQENVDFILIAGDLFHSSRPSP

hMre11 MSTADALDDENTFKILVATDIHLGFME.KDAVRGNDTFVTLDEILRLAQENEVDFILLGGDLFHENKPSR

scMRE11 MDYPDP....DTIRILITTDNHVGYNE.NDPITGDDSWKTFHEVMMLAKNNNVDMVVQSGDLFHVNKPSK

MjMre11 MMFVHIADNHLGYRQYNLDDREKDIYDSFKLCIKKILEIKPDVVLHSGDLFNDLRPP-

Motif III

T4Mre11 HKTMEFAREIVQ................................TLDDAGITLHTIVGNHDLHYKNVMHP

pfMre11 GTLKKAIALLQI.................................PKEHSIPVFAIEGNHDRTQRGP..S

hMre11 KTLHTCLELLRKYCMGDRPVQFEILSDQSVNFGFSKFPWVNYQDGNLNISIPVFSIHGNHDDPTGADALC

scMRE11 KSLYQVLKTLRLCCMGDKPCELELLSDPSQVFHYDEFTNVNYEDPNFNISIPVFGISGNHDDASGDSLLC

MjMre11 VKALRIAMQAFK................................KLHENNIKVYIVAGNHEMPRR..LGE

Motif

T4Mre11 NASTELLAKY..PNVKVY................DKPTTVDFDGCLIDLIPWMCEENTGEILEHIKTSSA

pfMre11 VLNLLEDFGLVYVIGMRKEKVENEYLTSERLGNGEYLVKGVYKDLEIHGMKYM....SSAWFEANKEI..

hMre11 ALDILSCAGFVNHFGRSMSVEKIDISPVLLQKGSTKIALYGLGSIPDERLY....RMFVNKKVTML....

scMRE11 PMDILHATGLINHFGKVIESDKIKVVPLLFQKGSTKLALYGLAAVRDERLF....RTFKDGGVTFE....

MjMre11 ESPLALLKDY..VKILDG................KDVINVNGEEIFICGTYYHKKSKREEMLDKLKNFES

Motif IV V

T4Mre11 .SFCVGHWELNGFYFYKGMKSHG...................LEPDFLKTYKEVWSGHFH..........

pfMre11 LKRLFRPTDNAILMLHQGVREVSEARGEDYFEIGLGD.....LPEGYL....YYALGHIH..........

hMre11 RPKEDENSWFNLFVIHQ......NRSKHGSTNFIPEQ.....FLDDFID..LVIW.GHEHECKIA.....

scMRE11 VPTMREGEWFNLMCVHQ......NHTGHTNTAFLPEQ.....FLPDFLD..MVIW.GHEHECIPN.....

MjMre11 ...EAKNYKKKILMLHQGINPYIPLDYE..............LEHFDLPKFSYYALGHIH..........

Motif VI

T4Mre11 ..T.ISEAAN...VRYIGTPWTLTAGDENDPR...............GFWMFDT...ETERTEFIPNNTT

pfMre11 ..KRYETSYSGSPVVYPGSLERWDFGDYEVRYEWDGIKFKERYGVNKGFYIVEDF.......KPRFVEIK

hMre11 PTKNEQ...QLFYISQPGSSVVTSLSPGEA..............VKKHVGLLRIKGRKMNMHKIPLHTV.

scMRE11 LVHNPI...KNFDVLQPGSSVATSLCEAEA..............QPKYVFILDIKGEAPKMTPIPLETI.

MjMre11 ..KRILERFNDGILAYSGSTEIIYRNEYEDYK...........KEGKGFYLVDFSGNDLDISDIEKIDIE

T4Mre11 WHRRIHYPFK...................GKIDYKDFTNLSVR...........VIVTEVD....KNLT

pfMre11 VRPFIDVKIK..............GSEEEIRKAIKRLIPLI.........PKNAYVRLNIGWRKPFDLT

hMre11 .RQFFMEDIVLANHPDIFNPDNPKVTQAIQSFCLEKIEEMLENAER....PEKPLVRLRVD....YSGG

scMre11 .RTFKMKSISLQDVPHL....RPHDKDATSKYLIEQVEEMIRDANE....LPKPLIRLRVD....YSAP

MjMre11 CREFVEVNIK...................DKKSFNEAVNKIERC......KNKPVVFGKIK....REFK

T4Mre11 ...........KFESELEKVVHSLRVVSKIDN...SVESDDSEEVEVQSLQTLMEEYIN........AI

pfMre11 ...........EIKELLN..VEYLKIDTWR.....IKERTDEESGKIGLPSDFFTEFELKI.....IDI

hMre11 ........FEPFSVLRFSQKFVDRVANPKDII---GKLITKPSEGTTLRVEDLVKQYFQTAEKNVQLSL

scMre11 SNTQSPIDYQVENPRRFSNRFVGRVANGNNVV---DVEKLFSESGGELEVQTLVNDLLNKM....QLSL

MjMre11 ...........PWFDTLKDKILINKAIIVDDE...FIDMPDNVDIESLNIKELLVDYANRQ......GI

T4MRE11 P.DITDSDREALIQYAN.........QLYVEATQ 339

PfMre11 LGEKDFDD.FDYIIKLITEGKVEEGPLEEAVKKVSEEKGKT 402

hMre11 LTERGMGEAVQEFVDKEEKD.....AIEELVKYQLEKTQRF 484

ScMre11 LPEVGLNEAVKKFVDKDEKT.....ALKEFISHEISNEVGI 497

MjMre11 DGDLVLSLYKALLNNEN.........WKELLDEYYNTKFRG 366

Figure 2: Rad50 alignment

Motif Walker A (P-loop)

T4Rad50 .MKNFKLNRVKYKNIMSVGQNGIDIQLDKVQ.KTLITGRNGGGKSTMLEAITFGLFGKPFRDVKKGQLIN

pfRad50 ....MKLERVTVKNFRSHSDTVVEFKEG....INLIIGQNGSGKSSLLDAILVGLY...WPLRIKDIKKD

hsRad50 MSRIEKMSILGVRSFGIEDKDKQIITFFSP..LTILVGPNGAGKTTIIECLKYICTGDFPPGTKGNTFVH

scRad50 MSAIYKLSIQGIRSF..DSNDRETIEFGKP..LTLIVGMNGSGKTTIIECLKYATTGDLPP.SKGGVFIH

MjRad50 ..MSMILKEIRMNNFKSHVNSRIKFEKG....IVAIIGENGSGKSSIFEAVFFALFGAGSN..FNYDTII

Motif

T4gp46 S...TNKKELLVELWMEYDE.........KKYYIKRGQKPNVF........EITVNGTRLNESASSKDFQ

pfRad50 EFTKVGARDTYIDLIFEKDG..........TKYRITRRFLKGYSSGEIHAMKRLVGNEWKHVTEPSSKAI

hsRad50 DPKVAQETDVRAQIRLQFRDVNGELIAVQRSMVCTQKSKKTEFKTLEG.VITRTKHGEKVSLSSKCAEID

scRad50 DPKITGEKDIRAQVKLAFTSANGLNMIVTRNIQLLMKKTTTTFKTLEGQLVAINNSGDRSTLSTRSLELD

MjRad50 T...KGKKSVYVELDFEVNG.........NNYKIIREYDSGRG........GAKLYKNGKPYATTISAVN

Motif Q-loop

T4gp46 AEFEQLIGMSYASF......KQIVVLGTAGYTPFMGLSTPARRKLVEDLLEVGTLAEMDKLNKALIRELN

pfRAD50 SAFMEKL.IPYNIFLNAIYIRQGQIDAILE..........SDEAREKVVREVLNLDKFETAYKKLSELKK

hsRad50 REMISSLGVSKAVLNNVIFCHQEDSNWPLS..........EGKALKQKFDEIFSATRYIKALETLRQVRQ

scRad50 AQVPLYLGVPKAILEYVIFCHQEDSLWPLS..........EPSNLKKKFDEIFQAMKFTKALDNLKSIKK

MjRad50 KAVNEILGVDRNMFLNSIYIKQGEIAKFLSL.........KPSEKLETVAKLLGIDEFEKCYQKMGEIVK

Coiled-coil segment

T4Rad50 SQNQVLDVKKDSIIQQIKIYNDNVERQKKLTGDNLTRLQNMYDDLAKEARTLKSEIEEANERLVNIVLDE

pfRad50 TINNRIKEYRDILARTENIEELIKENEQELIKAERELSKAEKNSNVETLKMEVISLQNEKADLDRTLRKL

hsRad50 TQGQKVKEYQMELKYLKQYKEKACEIRDQITSKEAQLTSSKEIVKSYENELDPLKNRLKEIEHNLSKIMK

scRad50 DMSVDIKLLKQSVEHLKLDKDRSKAMKLNIHGYMKDIENYIQDGKDDYKKQKETELNKVIAQLSECEKHK

MjRad50 EYEKRLERIEGELNYKENYEKELKNKMSQLEEKNKKLMEINDKLNKIKKEFEDIEKLFNEWENKKLLYEK

COILED-COIL SEGMENT

T4gp46 LQEELDKIVKTKTNLVMEKYHRGILTDMLKDSGIKGAIIKKYIPLFNKQINHYLKIM.............

pfRAD50 EKENRERVKKEIKDLEKAKDFTEELIEKVKKYKALAREAALSKIGELASEIFAEFTEGKY..........

hsRad50 YREMMIVMRTTELVNKDLDIYYKTLDQAIMKFHSMKMEEINKIIRDLWRSTYRGQDIEYIEIRSDADEN.

scRad50 YHKEWVELQTRSFVTDDIDVYSKALDSAIMKYHGLKMQDINRIIDELWKRTYSGTDIDTIKIRSDE....

MjRad50 RLKEMSNLEKEKEKLTKFVEYLDKVRRIFGRNGFQAYLREKYVPLIQKYLNEAFSEFDLPY.........

Motif Signature motif Walker B

T4gp46 EADYVFTLDEEFNETIKSRGREDFSYASFSEGEKARIDIALLFTWRDIASIVSGVSISTLILDEVF....

pfRAD50 SEVVVRAEENKVRLFVVWEGKE.RPLTFLSGGERIALGLAFRLAMSLYLA....GEISLLILDEP.....

hsRad50 VSASDKRRNYNYRVVMLKGDTALDMRGRCSAGQKVLASLIIRLALAETFC....LNCGIIALDEP.....

scRad50 VSSTVKGKSYNYRVVMYKQDVELDMRGRCSAGQKVLASIIIRLALSETFG....ANCGVIALDEP.....

MjRad50 ....SFVELTKDFEVRVHAPNGVLTIDNLSGGEQIAVALSLRL...AIANALIGNRVECIILDEPT....

Motif D-loop H-loop

T4gp46 DGSFDAEGIKGVA.....NIINSMKNTN..VFIISHKDHDPQE

pfRAD50 TPYLDEERRRKLI.....TIMERYLKKIPQVILVSHDEELKDA

hsRad50 TTNLDRENIESLAHALVEIIKSRSQQRNFQLLVITHDEDFVEL

scRad50 TTNLDEENIESLAKSLHNIINMRRHQKNFQLIVITHDEKFLGH

MjRad50 VYLLDENRRAKLA.....EIFRKVKSIP.QMIIITHHRELED

APPENDIX D: Mre11 peptides

Peptide fragment

Sequence

no

m/z

Z

Exchang-

able Amides

MKILNLGDW 1-9 545.29 2 8 0.9±0.01 1.2±0.06 0.85±0.15

MKILNLGDWHL 1-11 670.36 2 10 1.5±0.02 1.4±0.07 1.1±0.1

MKILNLGDWHLGVKADDE 1-18 685.36 3 17 4.0±0.02 4.0±0.03 3.1±0.3

WIRGIQIDGIKQ 19-30 713.91 2 11 1.1±0.01 0.8±0.1 0.75±0.25

WIRGIQIDGIKQAIE 19-33 580.67 3 14 2.7±0.06 0.7±0.2 0.5±0.3

IQYGDIF 45-50 855.42 1 5 1.2±0.01 1.30±0.01 0.7±0.3

DVRKAITHKTME 51-62 476.92 3 11 5.7±0.06 5.1±0.19 5.5±0.3

DVRKAITHKTMEF (I) 51-63 525.95 3 12 5.9±0 5±0.06 4.6±0.3

HTIVGNHDLHYKNVMHPNA(II) 79-97 550.03 4 17 4.5±0.01 5.3±0.3 3.7±0.1

LAKYPNVKVYDKPTTVDF 102-119 700.05 3 15 4.6±0.01 4.1±0.02 5.22±0.1

CEENTGEILE 131-140 1136.48 1 9 3.5±0.15 4.06±0 3.1±0.2

ENTGEILE 133-140 904.42 1 7 2.6±0.02 3.1±0.05 2.3±0.1

FCVGHWELNGF 149-159 654.80 2 10 3.7±0.03 4.5±0.06 4.3±0.1

CVGHWELNGF 150-159 581.26 2 9 3.8±0.02 4.5±0.02 4.2±0.1

CVGHWELNGFY 150-160 662.79 2 10 4.6±0.1 5±0.02 4.7±0.2

FYKGMKSHGLEPDF (III) 161-174 828.40 2 12 4.2±0.03 5±0.2 4.2±0.1

YKEVWSGHFHTISE (IV) 178-191 860.414 2 13 2.5±0.1 3.8±0.1 1.3±0.1

VRYIGTPWTLTAGDENDPRGF(V) 195-215 789.06 3 18 6.9±0.05 8.2±0.01 6.2±0.1

IHYPFKGKIDYKDFTNL 238-254 700.38 3 15 3.8±0.1 3.7±0.1

PNNTTWHRRIHYPFKGKIDYKDFTNL 229-254 816.181 4 23

8.5±0.1

EVDKNLTKF 262-270 547.30 2 8 5.5±0.1 3.6±0.11 5.5±0.1

RVVSKIDNSVESDDSEE 282-292 954.447 2 16 10.2±0.1 12.61±0.1 10.4±0.2

EYINAIPDITDSDREAL 310-326 967.97 2 15 11.2±0.03 1.5±0.1 2.5±0.2

IQYANQL 327-333 849.44 1 6 5.7±0.02 0.29±0.1

Mre11-Rad50-

AMP-PNP

Mean±S.E

Mre11

Mean±S.E

Mre11-

Rad50

Mean±S.E

aAverage and standard deviations were calculated using measurements from three independent experiments.

Peptides highlighted are those harboring the nuclease motifs of Mre11.

APPENDIX E: Rad50 peptides

Peptide fragment

Sequence no

m/z

Z

Exchange

able

amides

R

R

M

r

e

SVGQNGID 16-23 789.37 1 7 4.0±0 3.5±0.2 3.6±0.3

DKVQKTLITGRNGGGKSTML 27-46 526.79 4 19 12.4±0.2 9.4±0.5 8.11±0.1

EAITFGL 47-53 750.40 1 6 0.3±0 0.3±0 0.34±0.03

ITFGLFGKPFRDVKKGQLINSTNKKELL 50-76 639.18 2 25 10.9±0.2 9.8±0.4 12.52±0

INSTNKKEL 67-75 523.79 3 8 2.4±0.1 1.8±0.1 4.0±0.3

WMEYDEKKYYIKRGQKPNVFE 80-100 551.08 5 20 3.4±0.1 3.1±0.1 4.7± 0.2

NESASSKDFQAEF 109-121 730.32 2 12 7.1±0.1 6.6±0.2 7.9±0.1

KQIVVLGTAGYTPFMG 133-148 841.45 2 15 8.5±0.2 7.6±0.3 9.7±0.1

LSTPARRKLVEDLL 149-161 886.51 2 11 2.5±0.1 1.5±0 2.9±0.1

DVKKDSIIQQ 189-198 587.33 2 9 5.7±0.1 0.6±0.1 1.1±0

KIYNDNVERQKKLTGDNL 200-217 537.79 4 17 11.1±0.3 4.8±0.1 8.69±0.1

LTRLQNM 217-223 438.24 2 6 1.8±0 1.9±0.1 2.7±0.1

YDDLAKEARTLKSEIEEANERLVN 224-247 702.37 4 23 1.3±0 1.3±0.1 2.7±0.1

DEDPTDAFNKIGQEAFL 251-267 955.45 2 15 3.2±0.1 2.7±0 6.3±0.3

FLIKSKIDSYNKVINM 266-281 638.36 3 15 1.0±0 1.0±0.1 3.07±0

YHEGGLCPTCL 282-292 596.76 2 9 1.4±0 1.4±0.1 1.7±0

SSGDKVVSKIKDKVSE 296-311 569.32 3 15 7.1±0 6.6±0.2 7.6±0

LSTHRDN 319-325 421.71 2 6 1.4±0 1.1±0.1 1.2±0.1

YRDNIKTQQSLAND 332-346 833.41 2 14 1.6±0.1 1.5±0.1 5±0.1

QSLANDIRNKK 341-350 643.86 2 9 1.3±0 1.1±0.1 2.2±0.1

Mre11-

Rad50

Mean±S.E

Rad50

Mean±S.E

MR-AMP-PNP

Mean±S.E

aAverage and standard deviations were calculated using measurements from three independent experiments.

Peptides highlighted are those spanning the coiled-coil segment of Rad50.

IAAVDKAKKVKAAIE 354-369 518.99 3 15 1.5±0.1 1.4±0.1 3.8±0.2

LQEELDKIVKTKTNL 385-399 886.51 2 14 6.5±0.1 1.3±0.1 4.2±0.3

VMEKYHRGIL 400-409 623.34 2 9 4.3±0.1 0.8±0.0 0.5±0.1

LKDSGIKGAIIKKYIPLFNKQINHY 413-437 581.15 5 24 6.5±0.2 1.9±0.1 6.7±0.3

TLDEEF 448-453 753.33 1 5 0.9±0.1 0.9±1 1.5±0.2

NETIKSRGREDFS 454-468 769.88 2 12 7.5±0.1 6.7±0.3 8±0.5

ASFSEGEKARIDIAL 468-482 803.93 2 14 2.7±0.08 2.8±0.0 5.3±0.1

FTWRDIAS 484-491 498.25 2 7 0.3±0 0.2±0

FTWRDIASIVSGVS 484-497 760.43 1 13

3.96±0.1

ILDEVFDGSF 502-511 571.26 2 9 1.1±0 1.6±0.1 5.4±0.1

DAEGIKGVANIINS 512-515 700.87 2 13 9.0±0.01 7.8±0.2 10±0.5

IISHKDHDPQEYGQHLQM 533-550 726.02 3 17 7.4±0 5.9±0.1 5.8±0.2