Insights Into Regulation of Human RAD51 Nucleoprotein Filament Activity During

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Insights into Regulation of Human RAD51 Nucleoprotein Filament Activity During

Homologous Recombination

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Ravindra Bandara Amunugama, B.S.

Biophysics Graduate Program

The Ohio State University

2011

Dissertation Committee:

Richard Fishel PhD, Advisor

Jeffrey Parvin MD PhD

Charles Bell PhD

Michael Poirier PhD

Ravindra Bandara Amunugama

2011

ABSTRACT

Homologous recombination (HR) is a mechanistically conserved pathway that occurs during meiosis and following the formation of DNA double strand breaks (DSBs) induced by exogenous stresses such as ionization radiation. HR is also involved in restoring replication when replication forks have stalled or collapsed. Defective recombination machinery leads to chromosomal instability and predisposition to tumorigenesis. However, unregulated HR repair system also leads to similar outcomes.

Fortunately, eukaryotes have evolved elegant HR repair machinery with multiple mediators and regulatory inputs that largely ensures an appropriate outcome.

A fundamental step in HR is the homology search and strand exchange catalyzed by the RAD51 recombinase. This process requires the formation of a nucleoprotein filament (NPF) on single-strand DNA (ssDNA). In Chapter 2 of this dissertation I describe work on identification of two residues of human RAD51 (HsRAD51) subunit interface, F129 in the Walker A box and H294 of the L2 ssDNA binding region that are essential residues for salt-induced recombinase activity. Mutation of F129 or H294 leads to loss or reduced DNA induced ATPase activity and formation of a non-functional NPF that eliminates recombinase activity. DNA binding studies indicate that these residues may be essential for sensing the ATP nucleotide for a functional NPF formation.

An intriguing structural motif in the nucleotide-binding subunit interface of

RAD51 is known as the ATP cap. The RAD51 family of recombinases contains a conserved aspartate that forms a salt bridge with the terminal phosphate of ATP, which is the likely source of a nonphysiological cation requirement for the formation of an active

RAD51 NPF. In Chapter 3, I describe the biochemical analyses of a lysine substitution mutation of this conserved aspartate. The prototypical bacterial recombinase RecA as well as most RAD51 paralogs contains a conserved lysine at the analogous position. The

HsRAD51(D316K) substitution mutant possess a reduced protein turnover from DNA that results in improved recombinase functions. Structural analyses indicates that

HsRAD51(D316K) and its archaebacterial homolog form extended active NPF without the requirement of salt. These studies suggest that HsRAD51(D316) salt bridge may function as a conformational sensor that enhances RAD51 turnover at the expense of recombinase activity.

In Chapter 4, I examination the role of the RAD51 paralog complex RAD51B-

RAD51C as a potential mediator of RAD51 catalyzed D-loop formation. Modeled structures indicate that RAD51, RAD51B and RAD51C appear to have similar domain orientations within a NPF. Furthermore, RAD51B-RAD51C form stable complexes on ssDNA and partially stabilizes RAD51 NPF from the anti-recombinogenic activity of

BLM. At sub-stoichiometric levels, RAD51B-RAD51C may modestly stimulate RAD51 mediated D-loop formation in presence of RPA.

Collectively, these studies provide additional mechanistic and structural insight into the regulation of the RAD51 NPF during the homology search and strand exchange processes that is critical for efficient HR in human cells.

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Dedicated to Parents, my wife and my son

ACKNOWLEDGMENTS

It had always been my passion to study biochemical mechanisms of DNA damage repair and it was no surprise that from the first day of my rotation in Fishel Lab I decided to join and pursue the work that I have done so far. I would like to extend my heartfelt gratitude to my PhD advisor Richard Fishel for his continuous support throughout my graduate education in his lab. I greatly appreciate the freedom he allowed me to have to independently address research problems. Many a times I learned the ropes the hard way. But that gave me confidence to pursue a future career as an independent scientist.

The fact that Rick would often challenge my scientific opinions compelled me to be well versed with the literature. I would also like to thank him for carefully reading and editing the drafts of my manuscripts. Finally, I look forward to having him as a mentor for many years ahead for guidance and support.

I would like to thank the members of my committee Chuck Bell, Jeffrey Parvin and Michael Poirier for their guidance and advise throughout my graduate career. I thank

Steve Kowalczykowski, Lorraine Symington, Wolf-Dietrich Heyer and Steve West for stimulating discussions, advice and opinions on the confusing world of Recombination.

I am extremely grateful to Kang-Sup Shim (KS) for advice and teaching me the techniques initially and for many long hours of thoughtful discussions. Also I would like to thank Naduparambil Jacob who has been a wonderful collaborator and a colleague

over the years. I am thankful to Sarah Javaid, Kristine Yoder, Michael Mcilhatton and

Samir Acharya for their friendship, advice and helpful discussions. I also like to acknowledge Tom Clanton former director of the Biophysics graduate program, Ralf

Bundschuh current co-director and Susan Hauser for their assistance.

I am grateful to my parents and my brother for believing in me and all their support during my early education. It was the inspiration that I received from my father, being an academic himself, which made me choose the path of academia. Also, I am thankful to my friends Indi, Sharon, Chamika, Mekhala, Suresh, Harshi, Shekar and

Poorani for their friendship and numerous fun times we had that never made us miss home. I am also grateful to my mother-in-law for taking care of our son for a whole year that allowed my wife and me to continue with our education peacefully.

Finally, no words can express the how grateful I am to my dear wife Nirodhini for all her love, support and most importantly her patience throughout our busy years. I am also thankful to my dear son Rehan, who brought us an enormous amount of joy, happiness and meaning to our lives. I want you two to know that even though I have had many shortfalls in trying to be the ideal husband and father, you two are the most important people in my life and the motivation that drives me forward.

VITA

1977...... Born- Paris, France

2003...... B.S. Honors, Biochemistry and Molecular

Biology, University of Colombo, Sri Lanka

2003-2004………………………………….. Teaching Assistant in Biochemistry,

Molecular Biology and Chemistry,

University of Colombo, Sri Lanka

2004-2005 ...... Graduate Teaching Assistant, Department of

Chemistry, University of Iowa, USA

2005-Present ...... Graduate Research Associate, Department

of Molecular Virology, Immunology and

Medical genetics, The Ohio State

University, USA

vii

PUBLICATIONS

1. Amunugama R. and Fishel R. (2011) Subunit interface residues F129 and H294 of human RAD51 are essential for recombinase activity. Plos One 6(8), e23071.

2. Charbonneau, N., Amunugama, R., Schmutte, C., Yoder, K., and Fishel, R. (2009) Evidence that hMLH3 functions primarily in meiosis and in hMSH2-hMSH3 mismatch repair. Cancer Biol Ther 8(14), 1421-20.

3. Su, X., Jacob, N. K., Amunugama, R., Hsu, P.-H., Fishel, R., and Freitas, M. A. (2009) Enrichment and characterization of histones by two-dimensional hydroxyapatite/reversed-phase liquid chromatography-mass spectrometry. Analytical Biochemistry 388, 47-55.

4. Su, X., Jacob, N. K., Amunugama, R., Lucas, D. M., Knapp, A. R., Ren, C., Davis, M. E., Marcucci, G., Parthun, M. R., Byrd, J. C., Fishel, R., and Freitas, M. A. (2007) Liquid chromatography mass spectrometry profiling of histones. J Chromatogr B Analyt Technol Biomed Life Sci 850, 440-54.

5. Ikura, T., Tashiro, S., Kakino, A., Shima, H., Jacob, N., Amunugama, R., Yoder, K., Izumi, S., Kuraoka, I., Tanaka, K., Kimura, H., Ikura, M., Nishikubo, S., Ito, T., Muto, A., Miyagawa, K., Takeda, S., Fishel, R., Igarashi, K., and Kamiya, K. (2007) DNA damage-dependent acetylation and ubiquitination of H2AX enhances chromatin dynamics. Mol Cell Biol 27, 7028-40.

FIELD OF STUDY

Major Field: Biophysics

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TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGMENTS ...... v

VITA...... vii

LIST OF TABLES ...... xii

LIST OF FIGURES ...... xiii

CHAPTER 1: Homologous Recombination in Eukaryotes ...... 1

1.1 ABSTRACT ...... 2

1.2 INTRODUCTION TO HOMOLOGOUS RECOMBINATIONAL REPAIR ...... 3 1.3 MEIOSIS...... 4 1.4 DSB REPAIR IN SOMATIC CELLS ...... 6 1.5 RAD52 EPISTASIS GROUP ...... 12 1.6 RECOMBINATION MEDIATORS ...... 17 1.7 DSB REPAIR IN CHROMATIN ...... 33 1.8 POSTSYNAPTIC REMOVAL OF RAD51 ...... 43 1.9 SECOND-END CAPTURE ...... 44 1.10 DOUBLE HOLLIDAY JUNCTION DISSOLUTION ...... 44 1.11 HOLLIDAY JUNCTION RESOLUTION ...... 45

1.12 HOMEOLOGOUS RECOMBINATION: The interplay between mismatch repair and HR ...... 49 1.13 CONCLUDING REMARKS ...... 50 1.14 FOOTNOTES ...... 51 1.15 REFERENCES ...... 59

CHAPTER 2: Subunit Interface Residues F129 and H294 of Human RAD51 Are Essential For Recombinase Function ...... 90

2.1 ABSTRACT ...... 91

2.2 INTRODUCTION ...... 92

2.3 MATERIALS AND METHODS ...... 94

2.4 RESULTS ...... 98

2.5 DISCUSSION ...... 103

2.6 ACKOWLEDGEMENTS AND FOOTNOTES ...... 105

2.5 REFERENCES ...... 113

CHAPTER 3: The RAD51 ATP Cap Regulates Nucleoprotein Filament Stability 116

3.1 ABSTRACT ...... 117

2.2 INTRODUCTION ...... 118

3.3 MATERIALS AND METHODS ...... 121

3.4 RESULTS ...... 129

3.5 DISCUSSION ...... 138

3.6 ACKOWLEDGEMENTS AND FOOTNOTES ...... 142

3.7 REFERENCES ...... 162

CHAPTER 4: Human RAD51B-RAD51C Paralog Heterodimer Complex Enhances and Stabilizes RAD51 Nucleoprotein Filament for D-loop Formation ...... 165

4.1 ABSTRACT ...... 166

4.2 INTRODUCTION ...... 167

4.3 MATERIALS AND METHODS ...... 170

4.4 RESULTS ...... 174

4.5 DISCUSSION ...... 181

4.6 ACKOWLEDGEMENTS AND FOOTNOTES ...... 187

4.7 REFERENCES ...... 202

CHAPTER 5: CONCLUSION...... 208

REFERENCES ...... 212

COMPLETE BIBIOGRAPHY ...... 213

LIST OF TABLES

1.1 Comparison of DSB Repair Factors in Budding Yeast and Human...... 58 2.1 Summary of ATP hydrolysis and nucleotide binding data of HsRAD51 wild type (WT) and HsRAD51(F129V) and HsRAD51(H294V) mutant proteins...... 112 3.1 Summary of ATP hydrolysis and nucleotide binding data of HsRAD51 wild type (WT) and HsRAD51(D316K) mutant protein...... 159 3.2 Dissociation rate constants of HsRAD51 wild type (WT) and HsRAD51(D316K) ssDNA and dsDNA interactions ...... 160 3.3 X-ray crystallographic data and structure refinement statistics...... 161

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LIST OF FIGURES

1.1. DNA double strand break repair (DSBR) by homologous recombination (HR)...... 52 1.2. Alternative DSB repair mechanisms ...... 53 1.3. Break induced replication (BIR) and telomere restoration ...... 54 1.4. HR restores collapsed or stalled replication forks ...... 55 1.5. BRCA2 and its proposed role in HR...... 56 2.1. Cation-induced conformational rearrangement of conserved amino acid residues of RadA ...... 106 2.2. Mutation of HsRAD51(F129) and HsRAD51(H294) residues affect ATP turnover ...... 107 2.3. HsRAD51(F129V) and HsRAD51(H294V) are deficient in D-loop formation and strand exchange ...... 109 2.4. F129 and H294 of HsRAD51 are critical for DNA binding in the presence of ATP ...... 111 3.1 ATP Binding and Hydrolysis by HsRAD51 ATP Cap Substitution Mutation ...... 143 3.2 Purification of HsRAD51 and ADP-ATP exchange analysis ...... 145 3.3 HsRAD51(D316K) Exhibits Salt and RPA Independent Strand Exchange Activity ...... 146 3.4 HsRAD51(D316K) Preferentially Binds to Single Stranded DNA ...... 148 3.5 Competition DNA binding by HsRAD51 and HsRAD51(D316K) ...... 150 3.6 Strand Exchange Activity of HsRAD51 and HsRAD51(D316K) with Different Adenosine Nucleotides ...... 152 3.7 HsRAD51(D316K) Displays a Slow Turnover from ssDNA and dsDNA ...... 153 3.8 HsRAD51(D316K) Catalyzes D-loop Formation in the Presence of ATP and Magnesium ...... 155

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3.9 Structural comparison of MvRAD51 and RAD51 ATP Cap Aspartate→Lysine Substitution Mutation ...... 157 4.1 Homology based modeled structures of RAD51, RAD51B and RAD51C indicate similar domain orientations ...... 188 4.2 Sequence alignment of RAD51 homologs and RAD51 paralogs; RAD51B and RAD51C ...... 189 4.3 RAD51B-RAD51C heterodimer forms stable complexes on DNA ...... 190 4.4 RAD51B-RAD51C enhances RAD51 catalyzed D-loop formation in the presence of RPA ...... 191 4.5 Purified proteins used in the D-loop assays ...... 193 4.6 Order of addition of RAD51 and RAD51B-RAD51C for D-loop formation ...... 194 4.7 RAD51B-RAD51C protects RAD51 nucleoprotein filament against anti-recombinase function of BLM ...... 196 4.8 RAD51B-RAD51C does not suppress the overall ATPase activity of the RAD51/RAD51B-RAD51C nucleoprotein filament ...... 198 4.9 RAD51B-RAD51C RAD51B-RAD51C does not alleviate inhibitory effects of RPA ...... 199 4.10 Proposed model for RAD51B-RAD51C mediated stabilization of the RAD51 nucleoprotein filament ...... 201

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CHAPTER 1

Homologous Recombination in Eukaryotes

1.1 ABSTRACT

Homologous recombination (HR) is a mechanistically conserved pathway that occurs during meiosis and mitosis that ensures maintenance of genomic integrity. During meiosis, HR results in DNA crossover events between homologous chromosomes that produce the genetic diversity inherent in germ cells. The physical connection established between homologs during the crossover event is essential to facilitate correct chromosome segregation. HR is also involved in maintenance of somatic cell genomic stability by restoring replication after a stalled replication fork has encountered a DNA lesion or strand break as well as following exogenous stresses such as ionization radiation that induce DNA double strand breaks (DSBs). The importance of HR can be gauged by the conservation of HR genes and functions from bacteria to man. Here we review the players and mechanics of eukaryotic HR.

1.2 INTRODUCTION TO HOMOLOGOUS RECOMBINATIONAL REPAIR

DNA double-stranded breaks (DSBs) are generated spontaneously by radiation and chemical damage as well as intentionally as part of the chromosome pairing process during meiosis. Genome instability resulting from DSB recombination repair (RR) defects have been linked to a variety of human cancers including hereditary breast cancer

(BRCA1/2) as well as hematopoietic and other solid tumors (Ataxia telangiectasia mutated, ATM; Nijmegen breakage syndrome, NBS; Fanconi anemia, FANC; Bloom‟s syndrome, BLM) among others (Miki et al. 1994; Ellis et al. 1995; Savitsky et al. 1995;

Tavtigian et al. 1996a; D'Andrea and Grompe 2003). Unlike many repair pathways, RR engenders a complex cascade of responses that include cellular signaling integrated with the physical processes of DSB repair (Hoeijmakers 2001; Lobrich and Jeggo 2007). The

DSB repair reaction itself involves a complex cascade of enzymatic reactions that must manage the chromatin composite on the broken donor DNA in order to search and pair with the assembled chromatin of a homologous acceptor DNA. Deficiencies in any one of the multitudes of steps will affect the outcome of the RR process and ultimately affect genome stability. Understanding of the biophysical events associated with the DSB repair reaction is important since combinatorial chemical strategies are under development, which rely on targeting redundant or overlapping repair pathways that ultimately results in a synergistic therapeutic response in cancer patients (Farmer et al.

2005).

1.3 MEIOSIS

All sexually reproducing organisms undergo meiosis: a process that reduces the cellular diploid content to produce haploid gametes. RR has been co-opted and is essential for the completion of meiosis. Meiosis begins with replication that forms sister chromosomes (chromatids) and is followed by a pairing process that spatially associates chromosome homologs (Zickler and Kleckner 1999). The segregation of chromosome homologs is completed in the first meiotic division (meiosis I) and the segregation of chromatids is completed in the second meiotic division (meiosis II); ultimately producing haploid gametes. The mechanism, regulation, and checkpoint functions of meiosis II chromatid segregation appear similar to the well-defined processes associated with mitosis (Marston and Amon 2004). In contrast, meiosis I requires suppression of the tendency to segregate sister chromatids and instead the homologous chromosomes are separated. More than 50% of all spontaneous miscarriages are due to errors in chromosome segregation (non-disjunction) at the first meiotic division (Hassold et al.

2007). Moreover, 90% of Down syndrome cases can be attributed to errors in maternal meiosis (Hassold et al. 1995). With few exceptions, the critical meiosis genes appear identical in all eukaryotes (Heyting 1996; Keeney 2001).

The pairing of homologous chromosomes in meiosis I is a complex process fraught with many pitfalls that may ultimately result in infertility. Homologous chromosome pairing is initiated in Prophase I by the SPO11 gene product (Klapholz et al.

1985; Keeney 2001) which actively introduces hundreds of DSBs into the sister chromatids (Kleckner 1996). The homologous recombination (HR) repair of these DSBs

4 by the nearest sister is suppressed by the formation of meiosis-specific lateral elements between the chromatids (Cromie and Smith 2007). This leaves the homologous chromosome as the only DNA sequences available for HR repair to restore the exact integrity of the genome (Figure 1.1). The DSBs are first resected by a 5‟3‟ exonuclease (Alani et al. 1990; Liu et al. 1995). The resulting 3‟ single-stranded DNA

(ssDNA) end is then used in a classic homologous pairing and strand invasion reaction with the chromosome homolog to form a D-loop. Strand invasion requires RAD51 and/or the meiosis-specific DMC1, which are homologs to the prototypical bacterial recombination-initiation protein RecA (Masson et al. 1999; Masson and West 2001). The ssDNA binding (SSB) protein RPA is an essential cofactor in this process (Kantake et al.

2003; Wang and Haber 2004). Mutation of SPO11 or RAD51 result in a dramatic reduction of homologous chromosome pairing, a high frequency of meiosis I non- disjunction, and gamete inviability. In mice, upward of 400 DSB sites are formed that contain RAD51 and RPA beginning in leptotene (Moens et al. 2002). That the DSBs are almost always faithfully repaired is a testament to the accuracy and dependability of the process in the preservation of the many sexual species on earth.

Approximately 90% of the DSB sites are resolved following repair in a process that conversion of one parental homolog DNA sequence to the other parental homolog sequence with concurrent loss of that parental homolog DNA sequence (gene conversion;

(Housworth and Stahl 2003). These events leave the remaining chromosome of both parents intact (Housworth and Stahl 2003). The remaining 10% (40-50 in human) introduce visible chromosomal crossovers known as chiasmata that exchange entire arms

5 of genetic information reciprocally from one parental chromosome to the other (Kneitz et al. 2000). Ultimately, there are two significant events associated with meiotic DSB repair: 1.) genetic information is exchanged between chromosomes that is the basis of modern genetics (Stahl 1979), and 2.) homologous chromosomes become linked via chiasmata that are essential for proper chromosome segregation.

1.4 DSB REPAIR IN SOMATIC CELLS

Eukaryotes have four main pathways that repair DSBs generated spontaneously in somatic cells: HR, Non-homologous end joining (NHEJ), Alternative NHEJ (Alt-NHEJ, also known as microhomology mediated end joining, MMEJ) and single-strand annealing

(SSA; Figure 1.2; (Ciccia and Elledge 2010). The pathway of choice depends on the nature of DSB, the species, cell type and the stage of the cell-cycle stage where the DSB occurs (Heyer et al. 2010).

As discussed above, HR is a template dependent repair process and was long ago recognized to require the formation of a DNA cross-over structure at the site of homology between chromosomes (termed: Holliday Junction; (Holliday 1964). In the 1980‟s a

DSB repair model that involves the formation of a double Holliday Junction (dHJ) was developed based on transformation studies in budding yeast (Saccharomyces cerevisiae) where a linear plasmid was faithfully integrated into a homologous region of the host genome (Figure 1.1; (Orr-Weaver and Szostak 1983b; Orr-Weaver and Szostak 1983a).

Even though the DSB repair model also explains meiotic recombination products, during mitotic recombination very few crossover events are observed since the sister chromatid

6 appears to be largely used as the template. The use of a sister chromatid strongly suggests that mitotic recombination mainly occurs during late S and G2 phases of the cell cycle (Heyer et al. 2010). Importantly, crossover events during mitotic recombination could lead to loss of heterozygosity (LOH), which is a common process during tumorigenesis (Wu and Hickson 2006; Heyer et al. 2010; Moynahan and Jasin 2010).

Synthesis dependent strand annealing (SDSA) model was proposed to account for the low numbers of crossover recombination events (Figure 1.2; (Strathern et al. 1982;

Nassif et al. 1994; Ferguson and Holloman 1996). In SDSA once DNA synthesis occurs on the invading strand of a D-loop, it is unwound and displaced such that it may anneal with the second end to prime DNA synthesis on the latter (Figure 1.2). This pathway leads exclusively to noncrossover products. SDSA aside, dHJ based recombination products have been observed between homologous chromosomes in yeast during mitotic

DSB repair, but as a minor pathway (Bzymek et al. 2010).

During NHEJ the broken DSB ends are prevented from resection by the

Ku70/Ku80 heterodimer (Mahaney et al. 2009). The strong affinity of Ku70/Ku80 for

DSB ends appears to recruit DNA Ligase IV that is capable of sealing the DSB (Figure

1.2). The alternative NHEJ (Alt-NHEJ) pathway appears to resect a 5-25 nt region where microhomology may be used prior to ligation of the ends (Figure 2; (Hartlerode and

Scully 2009; Ciccia and Elledge 2010).

SSA occurs in regions flanked by direct repeat DNA sequences (Figure 1.2;

(Krogh and Symington 2004). Hence, this pathway is seen in higher eukaryotes where direct repeated sequences are prevalent (Lin et al. 1984; Maryon and Carroll 1991;

Fishman-Lobell et al. 1992; Krogh and Symington 2004). In SSA, both 5 flanking the

DSB are resected by nucleases and the resulting 3 overhangs annealed by RPA and

RAD52 (Heyer et al. 2010). Since, strand invasion and exchange are not involved, this process is independent of RAD51. 3 single-stranded overhangs are subsequently resected and ligated by nucleases and ligases, respectively (Krogh and Symington 2004).

NHEJ, Alt-NHEJ and SSA are mutation-prone pathways due to the lack of fidelity and loss of genetic information during the repair process.

Collapsed or Stalled Replication Forks - When a replication fork collapses or if a telomere becomes uncapped, a single ended DSB is formed (Lundblad and Blackburn

1993; Garvik et al. 1995; Hackett and Greider 2003). This end may be processed to produce a 3-overhang that can invade a homologous region of the sister chromatid, the homologous chromosome or a homologous region of another chromosome to initiate

DNA synthesis. This process is called Break-Induced Replication (BIR; Figure 1.3).

Use of any other template other than the sister chromatid leads to LOH during BIR.

During mitotic recombination however BIR is disfavored over SDSA apparently because of its slower kinetics (Malkova et al. 2005).

Stalled replication machinery or lesions on leading or lagging strand may lead to formation of DNA gaps or DSBs that often results in replication fork collapse (Heller and

Marians 2006; Branzei and Foiani 2008). In vertebrate cells replication fork collapse occurs in every cell cycle (Tsuzuki et al. 1996; Sonoda et al. 1998). Translesion synthesis (TLS), template switching, or HR can restore replication. TLS is error prone

8 due to the low fidelity of the polymerase employed (Prakash et al. 2005). However, template switching and HR are error-free. Many replication mutants with defective checkpoint activation are dependent on HR gene products for viability (Andreassen et al.

2006; Branzei and Foiani 2007). Uncontrolled HR however during replication fork collapse can lead to gross genomic instability. These observations suggest that cell cycle checkpoints tightly regulate HR pathway to ensure genomic integrity (Andreassen et al.

2006; Branzei and Foiani 2007).

If the nascent strand encounters a nick during replication, the fork may stall and the incomplete replicated strand may undergo resection which can invade the sister chromatid, once the latter is ligated (Figure 1.4, left). After DNA synthesis a resulting partial Holliday Junction may be resolved to reinitiate DNA synthesis at the fork. If the leading strand stalls due to a lesion on its template, the newly synthesized strands may pair via reverse branch migrate to form a chicken foot structure (a pseudo Holliday

Junction; Figure 1.4, middle; (Higgins et al. 1976). Following a short DNA synthesis to fill-in the chicken-foot ssDNA tail, the replication fork may be reinitiated by forward branch migration. A lesion on the lagging strand template would lead to a template switch mechanism where the blocked strand may invade the nascent complementary strand to bypass the lesion by DNA synthesis (Figure 1.4, right). Resolution of the resulting Holliday Junction may then restore replication.

DSB recognition and end resection- The substrate for HR is a ssDNA region with a 3- end generated by resection of the 5 strand of the duplex (Mimitou and Symington

2009a). In the event of a DSB by ionization radiation (IR) or chemical agents the terminal nucleotides are often modified structurally or exist as protein bound entities

(McKee and Kleckner 1997; Lobachev et al. 2004; Mimitou and Symington 2009b).

Such modifications posses an obstacle for downstream repair pathways and are effectively removed by nucleases and proteases or both.

The Mre11-Rad50-Xrs2 (MRX) complex in yeast and the homologous MRE11-

RAD50-NBS1 (MRN) complex in higher eukaryotes recognize DSB‟s (Martin et al.

1999; Lisby et al. 2004; Dupaigne et al. 2008; Wu et al. 2008a; Lisby and Rothstein

2009). Even though MRE11 possesses an inherent 35 nuclease activity, the initial

53 resection is initiated by the Sae2 (CtIP, in mammals) endonuclease in complex with MRX/MRN. Sae2/CtIP appears to process possible adducts on DSB ends for other nucleases to act upon (Krejci et al. 2003; Clerici et al. 2005; Lengsfeld et al. 2007). The cell cycle mediated phosphorylation of Sae2/CtIP by cyclin-dependent kinases (CDKs) appears to determine the choice between HR and NHEJ (Huertas et al. 2008; Huertas and

Jackson 2009). In mammalian cells, CtIP is ubiquitinated by BRCA1 during S and G2 phases of the cell cycle, which appears to facilitate its association with DSB sites (Huen and Chen 2010; Huen et al. 2010).

For extensive resection of the 5-strand the Exo1 and Dna2 nucleases as well as the Sgs1-Top3-Rmi1 (STR) complex (BLM-TOP3-RMI1-RMI2 in mammals; (Khavari et al.) is recruited (Zhu et al. 2008; Shim et al. 2010). The MRX complex is implicated in direct recruitment of Exo1 and Dna2 (Zhu et al. 2008). Deletion of Exo1, Dna2 or Sgs1 leads to reduction of resection and the generation of poor HR substrates (Mimitou and

Symington 2008; Zhu et al. 2008). Furthermore, Deletion of EXO1 in mammalian cells caused impaired recruitment of RPA and ATR at the DSB sites (Schaetzlein et al. 2007).

The initial resection complex that includes the MRX along with STR and Dna2 has been reconstituted in vitro (Cejka et al. 2010; Niu et al. 2010). Even though, Top3 and Rmi1 stimulate resection by recruiting Sgs1, they are not required for the 5-strand resection processes (Cejka et al. 2010; Niu et al. 2010). Stimulation of Sgs1 helicase activity by

RPA occurs in a species-specific manner, as the bacterial single strand binding protein

(SSB) is unable to stimulate Sgs1 (Cejka et al. 2010; Niu et al. 2010; Nimonkar et al.

2011). RPA also suppresses the inherent 3-endonuclease function of Dna2 while stimulating the 53 exonuclease required for DSB resection (Cejka et al. 2010; Niu et al. 2010). Two functional human resection complexes have been reconstituted in vitro, one comprising MRN-EXO1-BLM-RPA and the other MRN-DNA2-BLM-RPA

(Nimonkar et al. 2011). BLM exhibits direct protein-protein interactions with both

EXO1 and DNA2 (Nimonkar et al. 2008; Nimonkar et al. 2011). Furthermore, the nuclease activity of EXO1 is stimulated by BLM, RPA and MRN.

In the absence of a recombinase or a homologous sequence, resection could continue over several thousand nucleotides at rate of approximately 4 kb/hr in yeast (Zhu et al. 2008; Symington and Gautier 2011). During meiotic recombination resection tracts average ~850 nt (Chung et al. 2010; Zakharyevich et al. 2010). However, the resection length required for recombination between sister chromatids during mitotic recombination has not been determined (Symington and Gautier 2011).

1.5 RAD52 EPISTASIS GROUP

Many of the proteins involved in the RR pathway are genes of the RAD52 epistasis group

(Table 1.1). The name was derived from radiation sensitivity genetic screening analysis of budding yeast (Game and Mortimer 1974; McKee and Lawrence 1980; Symington

2002; San Filippo et al. 2008). Among eukaryotes this group of genes are structurally and functionally conserved.

RAD51- RAD51 is unequivocally the central component in HR pathways (Table 1.1). It preserves a high sequence homology to the prototypical bacterial recombinase RecA

(West 2003; Krogh and Symington 2004; San Filippo et al. 2008). In eukaryotes, homologous pairing and strand exchange is primarily mediated by RAD51 (West 2003;

San Filippo et al. 2008). RAD51 exists as a heptamer in solution (Shin et al. 2003).

Yeast Rad51 and human RAD51 are 43kDa and 37kDa in size, respectively (Shinohara et al. 1992; Benson et al. 1994). The main catalytic ATPase core region that includes the

Walker A/B regions and ssDNA binding domains are conserved among the

RecA/RAD51 recombinases (Aboussekhra et al. 1992; Basile et al. 1992; Shinohara et al.

1992; Wu et al. 2004). However, RAD51 possesses an N-terminal extension that is absent in RecA, whilst in RecA a C-terminal extension is found that is not found in

RAD51 (Baumann and West 1998). These extensions have been implicated as possible double-stranded DNA (dsDNA) binding sites (Aihara et al. 1997; Aihara et al. 1999).

Formation and maintenance of a stable nucleoprotein filament (NPF) is required for the DNA homology search and strand exchange by RecA/RAD51 recombinases

(Kowalczykowski 1991; Bugreev and Mazin 2004; San Filippo et al. 2008). RAD51 nucleates on both ssDNA and dsDNA in vitro, forming an extended NPF (Ogawa et al.

1993; Sung and Robberson 1995). Biochemical studies have shown that incorporation of monovalent salts in the reaction buffer biases RAD51 nucleation on ssDNA (Liu et al.

2004b; Shim et al. 2006). This observation appears to presage the possibility that other recombination mediators might be responsible for efficient nucleation of RAD51 on ssDNA. A single RAD51 molecule binds to 3-4 nt or bp, extending the helical pitch by

~50% compared to canonical B-form DNA (Ogawa et al. 1993; Sung and Robberson

1995). Interestingly, when calcium was substituted for magnesium human RAD51 displayed enhance the strand exchange activity in vitro (Bugreev and Mazin 2004). This effect has been attributed to the suppression of ATP hydrolysis by the calcium that appears to enhance the lifetime of the ATP-bound active NPF (Bugreev and Mazin 2004).

This calcium-mediated stimulation is unique to human RAD51 (Bugreev and Mazin

2004).

Budding yeast containing a rad51 deletion are viable (Symington 2002).

However, knockout of RAD51 in vertebrates leads to chromosomal instability and embryonic lethality (Tsuzuki et al. 1996; Sonoda et al. 1998). Although no mutations have been reported in the open reading frame, in many cancers and cancer cell lines the expression of RAD51 is increased, presumably providing a replicative advantage to the rapidly dividing cells via its role in the HR repair of collapsed forks (Richardson et al.

2004; Klein 2008; Schild and Wiese 2010).

A catalytically conserved lysine residue in the Walker A box [yeast Rad51(K191) and human RAD51(K133)] is essential for ATP binding and hydrolysis. Mutation of this conserved lysine to alanine [Rad51(K191A)] leads to a null phenotype and is a dominant negative phenotypes in diploid cells (Morgan et al. 2002; Krogh and Symington 2004).

Expression of Rad51(K191R) in rad51 null yeast strains leads to resistance to DSB causing agents, suggesting nucleotide binding is sufficient for HR repair in vivo (Sung and Stratton 1996). Human RAD51(K133R) binds ATP but is unable to hydrolyze ATP, similar to the yeast mutation (Morrison et al. 1999; Chi et al. 2006). Overexpression of human RAD51(K133R) in chicken DT40 RAD51 knock out cells confers partial resistance to IR radiation (Morrison et al. 1999). RAD51(K133R) forms a stable NPF on

DNA and has enhanced recombinase activity in vitro (Chi et al. 2006). However, mouse embryonic stem cells generated that expressed RAD51(K133R) display increased sensitivity to DSBs and reduced efficiency of spontaneous sister-chromatid exchanges

(Stark et al. 2002).

ATP binds at the interface region of two adjacent RAD51 monomers within the

NPF (Wu et al. 2004; Wu et al. 2005). The bottom subunit provides the catalytic Walker

A (P-loop) domain while the top subunit shields the nucleotide with an ATP cap containing a conserved proline residue (Wu et al. 2005). For the homology search and strand exchange, ATP binding but not necessarily ATP hydrolysis is required

(Kowalczykowski 1991). When an ATP or a non-hydrolysable ATP analog binds at the subunit interface the NPF adopts an active extended conformation. Several biochemical studies have shown that incorporation of monovalent cations such as ammonium or

14 potassium as well as divalent cations such as calcium extends the NPF ~30% further (Wu et al. 2005; Qian et al. 2006). The NPF exists in the collapsed configuration with ADP

(Qian et al. 2005). The extended conformation is believed to facilitate homology sampling on duplex DNA. The ATP hydrolysis rate of RAD51 is several folds slower compared to the bacterial RecA (Tombline and Fishel 2002). However, this hydrolysis is still important for the dissociation of recombinase from both newly formed heteroduplex

DNA, as well as fortuitously bound RAD51 on duplex DNA (San Filippo et al. 2008).

The RAD51 NPF is not a static structure. Breast cancer susceptibility gene product 2 (BRCA2) is known to nucleate RAD51 NPF formation on ssDNA at the dsDNA-ssDNA junction (Galkin et al. 2005; Yang et al. 2005; Carreira et al. 2009;

Jensen et al. 2010). Many helicases in yeast and human have been identified both in vivo and in vitro that act as anti-recombinases capable of dissociating RAD51 from ssDNA.

These include Srs2 in yeast, BLM and RECQ5 in human (Krejci et al. 2003; Veaute et al.

2003; Bugreev et al. 2007; Hu et al. 2007; Antony et al. 2009). Biophysical studies also indicated that Srs2 augments the Rad51 ATPase activity within the NPF to facilitate rapid protein turnover (Antony et al. 2009). Moreover, both ensemble and single molecule experiments show a direct correlation of recombinase turnover from DNA and its ATPase activity (Kowalczykowski 1991; Bianco et al. 1998; Bugreev and Mazin 2004; Bugreev et al. 2005; Chi et al. 2006; Hilario et al. 2009).

Homology search during the synapsis phase by RAD51/RecA family is by random collision that involves transient non-specific interactions with dsDNA, presumably bound at the secondary DNA binding site (Kowalczykowski 1991; Bianco et

15 al. 1998). The transient triplex DNA formed during the homology search are paramenic and are not topologically interwound (Kowalczykowski 1991). Biophysical studies using fluorescence resonance energy transfer (FRET) and selective substitution of guanine to inosine on both ssDNA and its identical strand on the duplex DNA, showed that human

RAD51 facilitated homology search by a rapid A:T base flipping mechanism (Gupta et al. 1999). Once a homologous sequence is found, strand exchange occurs to produce a topologically interwould plectonemic heteroduplex DNA product (Kowalczykowski

1991; Bianco et al. 1998).

DMC1- DMC1 (Disrupted Meiotic cDNAs) was first isolated from a budding yeast meiotic cDNA library screen (Bishop et al. 1992). DMC1 is only expressed during meiosis and displays considerable homology to RecA/RAD51 family of recombinases

(Table 1.1; (Bishop et al. 1992). During meiosis both Rad51 and Dmc1 co-localize at

DSBs in yeast (Bishop 1994). Disruption of yeast Dmc1 leads to a number of abnormal meiotic phenotypes, including accumulation of DSBs, reduced reciprocal recombination, abnormal synaptonemal complex formation and defective meiotic prophase arrest

(Bishop et al. 1992). Dmc1 exhibits both overlapping yet non-redundant functions to

Rad51 (Tsubouchi and Roeder 2003). Yet, overexpression of Rad51 enables yeast cells to circumvent the defective meiotic phenotype of Dmc1 mutants (Tsubouchi and Roeder

2003). Human DMC1 exists as an octamer in solution (Passy et al. 1999) and functions similar to RAD51 in assays for recombination and ATPase activity in vitro (Sehorn et al.

2004; Bugreev et al. 2005; Sauvageau et al. 2005).

1.6 RECOMBINATION MEDIATORS

RPA- Replication Protein A (RPA) is a heterotrimeric (70 kD, 32 kD, 14 kD) SSB that binds to ssDNA with high affinity (Table 1.1; (Wold and Kelly 1988; Erdile et al. 1990).

It was first shown to stimulate strand exchange in vitro with strand exchange protein 1

(SEP1) (Heyer et al. 1990; Alani et al. 1992). A similar stimulatory effect was shown later with yeast Rad51 (Sung 1994). RPA has a dual stimulatory role during RAD51 mediated strand exchange. During the presynaptic phase it binds to ssDNA to prevent secondary structure formation that could potentially lead to inhibitory effects during

RAD51 NPF formation (Sugiyama et al. 1997). During the isoenergetic strand exchange phase RPA ensures unidirectional heteroduplex extension by binding to the displaced ssDNA (Eggler et al. 2002; Van Komen et al. 2002). However, during recombination assays in vitro where only RPA and RAD51 are present, if RPA is added to ssDNA prior to the addition of RAD51, strand exchange is inhibited due to the nanomolar affinity of RPA for ssDNA. This inhibition can be overcome by the addition of recombination mediators such as Rad52 or Rad55/Rad57 in yeast Rad51 recombinase reactions, and by the addition of BRCA2 or the RAD51 paralog heterodimer RAD51B-

RAD51C in human RAD51 recombinase reactions (Sung 1997b; Kanaar and

Hoeijmakers 1998; New et al. 1998; Shinohara and Ogawa 1998; Sigurdsson et al. 2001;

Yang et al. 2005; Jensen et al. 2010).

RAD52- Rad52 plays an essential role in HR and SSA and its deletion leads to severe sensitivity to DSB causing agents and defects during meiosis in budding yeast (Table 1.1;

(Symington 2002; San Filippo et al. 2008). Electron microscopic (EM) evidence indicates that both yeast and human RAD52 form oligomeric ring structures (Shinohara et al. 1998;

Stasiak et al. 2000; Ranatunga et al. 2001). The EM structure of human RAD52 indicated that the N-terminus is responsible for the formation of a heptameric ring structure and the C-terminus then self-assembles the heptameric rings into a higher ordered structure (Stasiak et al. 2000; Ranatunga et al. 2001). However, two independent

X-ray crystallographic analyses revealed that the yeast Rad52 N-terminal residues 1-201

(1-209 of human RAD52) formed an undecameric (11-subunit) ring (Kagawa et al. 2002;

Singleton et al. 2002). The overall structure resembles a mushroom top with positively charged residues lining a groove on the outside of the ring (Singleton et al. 2002). Even, though no DNA containing structures of RAD52 have been solved, the dimensions of the groove indicate that it is large enough to bind ssDNA in a sequence independent manner that would position the bases away from the protein surface for possible annealing with complementary bases (Singleton et al. 2002; West 2003). The ssDNA binding property of purified yeast Rad52 was first demonstrated by Rothstein and colleagues in 1996 and found to reside in the N-terminus (Mortensen et al. 1996). A similar ssDNA binding pattern was observed when ssDNA-RAD52 complexes were probed for hydroxyl radical hypersensitivity (Parsons et al. 2000).

During meiotic and mitotic DSB repair RAD51 recruitment is dependent on

RAD52 (Gasior et al. 1998; Sugawara et al. 2003; Wolner et al. 2003; Lisby et al. 2004).

Yeast Rad52 has been shown to stimulate Rad51 mediated strand exchange activity by mediating Rad51 NPF formation on RPA coated ssDNA filaments (Sung 1997a; Kanaar

18 and Hoeijmakers 1998; New et al. 1998; Shinohara and Ogawa 1998). This mediator activity is critical to overcome the inhibitory action of RPA, when RPA is added prior to

Rad51. Several methods including yeast-two hybrids, co-immunoprecipitation of whole cell extracts and direct protein-protein affinity pull-down techniques have shown a direct interaction between Rad52 and Rad51 (Shinohara et al. 1992; Milne and Weaver 1993;

Sung 1997a; Krejci et al. 2002). The interaction with Rad51 is mediated by the C- terminal 409-412 residues of Rad52 (Boundy-Mills and Livingston 1993; Krejci et al.

2002). Overexpression of Rad51 alleviates the defective RR phenotype of a rad52(D409-412) C-terminal deletion mutation (Krejci et al. 2002). In vitro and in vivo evidence suggest that human RAD52 interacts with the cognate RAD51 via residues 291-

330, a region that does not share homology with yeast Rad51 (Shen et al. 1996). These results appear to imply a species-specific interaction between RAD51 and RAD52.

Rad52 has also been implicated in yeast Rad51 independent events such as BIR and SSA

(Symington 2002; Krogh and Symington 2004; McEachern and Haber 2006).

Both yeast and human RAD52 have been shown to interact with RPA (Hays et al.

1998; Shinohara et al. 1998; Jackson et al. 2002). Yeast two-hybrid assays indicated

Rad52 interaction with all three subunits of yeast RPA (Hays et al. 1998). However, human RAD52 was shown to bind directly to large (70 kD) and middle (32 kD) subunits of RPA (Jackson et al. 2002). Interestingly, RAD52 interaction with RPA inhibits higher order self-association of RAD52 (Jackson et al. 2002). The strand annealing activity of both yeast and human RAD52 facilitates the second-end capture during HR repair

(McIlwraith and West 2008; Wu et al. 2008b).

Even though Rad52 plays an essential role in HR in budding yeast, in vertebrate cells or in cells with BRCA2 homologs, loss of RAD52 gene leads to few phenotypic defects in RR. RAD52 knockdown in mouse embryonic cells and in chicken B-cell line

DT40 cells lines does not cause an apparent sensitivity to IR or DSB causing chemical agents (Rijkers et al. 1998; Yamaguchi-Iwai et al. 1998). In the corn smut Ustilago maydis (which contains the Brh2 BRCA2 homolog) no defects in HR were found in

Rad52 mutants (Kojic et al. 2008). A recent study of human breast cancer cell line suggests that knock-down of RAD52 acts as a synthetically lethal in the presence of

BRCA2 deficiency (Feng et al. 2011). This finding elevates RAD52 as a target for anti- tumorigenic therapy for breast cancer (Feng et al. 2011; Liu and Heyer 2011). A model has been proposed where RAD52 functions in an alternative pathway to BRCA2 (Liu and

Heyer 2011). Human RAD52 does not appear to possess any RR mediator activity in vitro (San Filippo et al. 2008; Jensen et al. 2010). However, chicken DT40 cells were non-viable and exhibited severe HR defects in a double knockdown of RAD52 and the

RAD51 paralog XRCC3 (Fujimori et al. 2001). In addition, U. maydis Rad52 mutants demonstrated an enhanced UV and IR sensitivity when its sole RAD51 paralog rec2 was mutated (Kojic et al. 2008). Collectively, this implies that in human cells RAD52 might function as a recombination mediator in conjunction with any one or a combination of the

RAD51 paralogs, RAD51B, RAD51C, RAD51D, XRCC2 or XRCC3 (Liu and Heyer

2011).

BRCA2 - Germline mutations of BRCA2 gene predispose individuals to highly penetrant, autosomal dominant to breast and ovarian cancers as well as predisposition to other types of cancers (Wooster et al. 1994; Wooster et al. 1995; Tavtigian et al. 1996b; Jasin 2002).

Mutations of BRCA2 in metazoans lead to gross chromosomal rearrangements, accumulation of chromosomal breaks, developmental arrest, meiotic defects and increased hypersensitivity of DSB and inter-strand cross-linking (ICL) causing agents

(Sharan et al. 1997; Yu et al. 2000; Moynahan et al. 2001; Xia et al. 2001; Siaud et al.

2004; Martin et al. 2005). Biallelic BRCA2 mutations that lead to expression of truncated forms of BRCA2 predispose individuals to Fanconi Anemia (FA) and the designation as

FANCD1 (Howlett et al. 2002). The similar radiation sensitivity and developmental defective phenotypes observed in RAD51 and BRCA2 deficient cell types suggest that

BRCA2 is intricately involved in RAD51 mediated RR repair (Table 1.1). These assertions have been solidified by an observed interaction between BRCA2 and RAD51 using the yeast two-hybrid system (Mizuta et al. 1997; Sharan et al. 1997).

BRCA2 possesses two spatially distinct RAD51 binding regions. First region involves repeated sequences of BRC motifs and the second RAD51 interaction motif is located at the C-terminus of the protein (C-terminal RAD51 binding domain, CTRB;

Figure 1.5A; (Mizuta et al. 1997; Sharan et al. 1997; Wong et al. 1997; Chen et al. 1998;

Davies and Pellegrini 2007; Esashi et al. 2007; San Filippo et al. 2008). Each BRC repeat consists of about 35 amino acids and several of the residues within each motif is conserved. This conservation is seen among metazoan BRCA2 orthologues (Bork et al.

1996; Bignell et al. 1997). However, the number of repeats in each organism varies. For

21 instance, humans, mice and chicken have eight BRC repeats, Drosophila has four repeats,

Caenorhabditis elegans and U. maydis have a single BRC repeat and the plant species rice and Arabidopsis thaliana have eight and four repeats, respectively (Lo et al. 2003).

The BRC repeats are not functionally equivalent (Holloman 2011). Mutations within

BRC repeats lead to abrogation of RAD51 binding and thus manifests defective DNA repair (Chen et al. 1999). Structural analysis of the human BRCA2 BRC-4 repeat with the core region of RAD51 revealed an interface on BRC-4 that mimics a binding motif of

RAD51 (Pellegrini et al. 2002). This surface was suggested to function as an oligomerization site for RAD51 to facilitate RAD51 NPF formation (Pellegrini et al.

2002). Studies with U. maydis Brh2 also suggested a similar recruitment mechanism of

RAD51 (Yang et al. 2005). All BRC repeats of human BRCA2 bind to RAD51 with variable affinity but with a binding stoichiometry of 1:1 (Carreira and Kowalczykowski

2011). For example, BRC-1, -2, -3 and -4 bind to free RAD51 with a higher affinity compared to BRC-5, -6, -7 and -8 (Carreira and Kowalczykowski 2011). BRC repeats can also modulate the loading of RAD51 onto DNA (Carreira et al. 2009; Shivji et al.

2009; Carreira and Kowalczykowski 2011). RAD51 filament formation on dsDNA leads to a dead-end complex that are recombination deficient both in vivo and in vitro (Solinger et al. 2002; Shah et al. 2010). BRC-1, -2, -3 and -4 are able to suppress the ATP turnover rate of RAD51 and facilitate nucleation on RPA coated ssDNA, while suppressing

RAD51 binding to dsDNA (Jensen et al. 2010; Carreira and Kowalczykowski 2011)

(Figure 1.5B.). This in turn leads to enhanced recombinase activity (Carreira and

Kowalczykowski 2011). The later group, BRC-5, -6, -7 and -8 however, do not enhance

22 preferential filament formation of RAD51 (Carreira and Kowalczykowski 2011). The collective action of these two BRC groups can facilitate RAD51 nucleation and nascent filament formation on ssDNA prior to dissociation of BRCA2 (Carreira and

Kowalczykowski 2011). Similar attenuation of Rad51 ATPase activity is seen with C. elegans BRCA2 homologue BRC-2 (Petalcorin et al. 2006).

The CTRB region that interacts with RAD51 is seen only in vertebrates and this region binds RAD51 filaments, but not free RAD51, likely to stabilize the NPF (Davies and Pellegrini 2007; Esashi et al. 2007). Cyclin dependent kinase (CDK) phosphorylation of BRCA2(S3291) appears to inhibit its interaction with the RAD51 filament leading to its disassembly (Esashi et al. 2005; Davies and Pellegrini 2007). As might be predicted, during S phase when RR is highly active, there is very low

BRCA2(S3291) phosphorylation that gradually increases with the approach of M phase

(Esashi et al. 2005). These results suggest that the CTRB region functions as a cell cycle regulator of RR. In addition, the C-terminus of BRCA2 is essential for the nuclear transport of RAD51 from the cytoplasm since RAD51 lacks a nuclear localization signal

(NLS; (Davies et al. 2001; Gildemeister et al. 2009). Thus, in human pancreatic cancer cell line CAPAN-1 that expresses a truncated version of BRCA2 [BRCA26174delT)]

(Goggins et al. 1996), BRCA2 transportation into the nucleus is compromised and the levels of nuclear RAD51 is greatly diminished (Davies et al. 2001). Finally, it has been recently shown that CTRB region of BRCA2 is essential for protection of stalled replication forks against the MRE11 nuclease by stabilizing the RAD51 nucleoprotein filament (Hashimoto et al. 2010; Schlacher et al. 2011).

BRCA2 has also been shown to interact with DMC1 through a mammalian specific 26- amino acid interaction motif containing BRCA2 residues 2386-2411 (Thorslund et al.

2007). This motif contains three critical amino acids BRCA2 F2406, P2408 and P2409

(PhePP motif) that is essential for DMC1 interaction (Thorslund et al. 2007). The N- terminus of BRCA2 has also been shown to interact with RPA in a DNA independent manner by co-immunoprecipitation (Wong et al. 2003) and the cancer predisposing mutation BRCA2(Y42C) compromises this interaction (Wong et al. 2003).

PALB2 (Partner and localizer of BRCA2), also known as FANCN due to its involvement in Fanconi Anemia (Reid et al. 2007), interacts with the N-terminus of

BRCA2 and was shown to be essential for stable nuclear localization, recombination and checkpoint functions of the latter (Xia et al. 2006). The BRCA2(Y42C) also disrupts the interaction with PALB2 (Xia et al. 2006). In addition, PALB2 has been shown to stimulate RAD51 catalyzed D-loop formation by physically interacting with RAD51 and displays a cooperative effect in the presence of RAD51AP1, another stimulator of

RAD51 recombinase activity (Buisson et al. 2010; Dray et al. 2010).

DSS1 (the deleted in split hand/split foot gene) is a small acidic protein first shown to interact with BRCA2 by yeast and mammalian two-hybrid assays (Marston et al. 1999). Disruption of DSS1 leads to compromised RAD51 foci formation and DSB repair in both fungal and mammalian species (Kojic et al. 2003; Gudmundsdottir et al.

2004). DSS1 binds to DNA binding region of BRCA2 (Yang et al. 2002), and in U. maydis Dss1 prevents dimerization of Brh2 allowing the formation of a monomeric

24 functional protein (Zhou et al. 2007). These seemingly contradictory phenotypes will require some resolution in the coming years.

Other than the role of BRCA2 in DSB repair it is also suggested to be involved in post-replication repair of ssDNA gaps by a template switch mechanism due to damage in the original template (Jensen et al. 2010). In fact, studies with Brh2 have confirmed the template switch mechanism in vitro (Mazloum and Holloman 2009).

Recently three independent groups were able to successfully express and purify the full length BRCA2 that had initially been a challenge as a result of its sheer size

(3418 amino acids; (Jensen et al. 2010; Liu et al. 2010; Thorslund et al. 2010). The full length BRCA2 was shown to bind six RAD51 molecules and enabled their nucleation on

RPA coated ssDNA by binding to 3 tailed structures (Jensen et al. 2010; Liu et al. 2010;

Thorslund et al. 2010). Furthermore, as with BRC fragment analysis the full length

BRCA2 suppressed RAD51 filament formation on dsDNA and stabilized RAD51-ssDNA

NPF by suppressing the ATPase activity of the recombinase (Jensen et al. 2010;

Thorslund et al. 2010). EM evidence of BRCA2 bound forked DNA structures illustrates its involvement in DNA replication coupled repair (Thorslund et al. 2010).

RAD51 PARALOGS

Yeast RAD51 paralogs - RAD51 paralogs are products of gene duplication events of

RecA/RAD51 genes that function as accessory proteins in HR repair (Table 1.1; (Lin et al.

2006). Budding yeasts encodes two Rad51 paralogs, Rad55 and Rad57 (Kans and

Mortimer 1991; Lovett 1994). Mutants of Rad55 or Rad57 are cold sensitive to DNA damage (Hays et al. 1995). Overexpression of either Rad51 or Rad52 suppresses the

DNA repair defect of these mutants, consistent with the notion that Rad55 and Rad57 function as accessory proteins in recombination (Hays et al. 1995; Johnson and

Symington 1995). In addition, Rad55 has been shown to interact with Rad57 and Rad51 both in vivo and in vitro (Hays et al. 1995; Johnson and Symington 1995). Furthermore, inclusion of the stable heterodimer Rad55-Rad57 in sub-stoichiometric amounts suppresses the inhibitory effects with of RPA in vitro, suggesting an involvement during presynapsis (Sung 1997b). Even though, Rad55-Rad57 paralog heterodimer complex does not exhibit recombinase activity (Sung 1997b), mutation of a Walker A box conserved lysine to alanine in Rad55, but not in Rad57 results in defective meiotic recombination phenotypes (Johnson and Symington 1995). In response to DSB causing genotoxic stress cell cycle checkpoint kinase mediated phosphorylation of the Rad55 S2,

S8, S14 and S378 residues has been shown essential for activating homologous recombination (Bashkirov et al. 2000; Herzberg et al. 2006; Janke et al. 2010). In the fission yeast Schizosaccharomyces pombe, mutants of Rhp55 and Rhp57 exhibited similar mutator phenotypes as budding yeast Rad55 or Rad57 mutants, that indicated structural and functional homology between Rad55 and Rad57 with Rhp55 and Rhp57, respectively (Khasanov et al. 1999; Tsutsui et al. 2000).

Vertebrate RAD51 paralogs - Vertebrates encode five RAD51 paralogs RAD51B

(RAD51L1/ hRec2/ R51H2), RAD51C (RAD51L2), RAD51D (RAD51L3/ R51H3),

XRCC2 and XRCC3 that share 20 to 30% homology to RAD51 as well as to one other

(Table 1.1; (Thompson and Schild 2001; Symington 2002). Similar to yeast Rad51 paralogs, these gene products appear to be the result of gene duplication events of an ancestral RecA/RAD51 gene (Lin et al. 2006). However, these paralogs show a high degree of evolutionary divergence from RAD51 as well as from each other (Thompson and Schild 2001). XRCC2 and XRCC3 (X-ray repair cross complementing) were first identified in their ability to complement the extreme sensitivity of irs1 and irs1SF hamster cell lines (Jones et al. 1995; Tebbs et al. 1995; Thacker et al. 1995). Homology searches further identified RAD51B (Albala et al. 1997; Rice et al. 1997; Cartwright et al. 1998), RAD51C (Dosanjh et al. 1998) and RAD51D (Cartwright et al. 1998; Pittman et al. 1998). Yeast two- and three-hybrid analyses, co-immunoprecipitation techniques and biochemical studies have indicated interaction among the five RAD51 paralogs

(Schild et al. 2000; Symington 2002). For example, RAD51B forms a stable heterodimer with RAD51C (Dosanjh et al. 1998; Miller et al. 2002; Lio et al. 2003), while RAD51 interacts with XRCC3 and weakly with RAD51C (Liu et al. 1998; Schild et al. 2000). The latter interaction is improved in the presence of XRCC3 (Schild et al.

2000). RAD51D forms a stable complex with XRCC2 (Braybrooke et al. 2000). In

HeLa cells two discrete complexes containing XRCC3 and RAD51C and the other containing RAD51B, RAD51C, RAD51D and XRCC2 were found (Masson et al. 2001a;

Masson et al. 2001b). Knockout mutants of the RAD51 paralogs in chicken DT40 are viable yet exhibit increased sensitivity to cross-linking agents and ionizing irradiation as well as reduced RAD51 foci formation upon damage induction (Takata et al. 2000;

Takata et al. 2001). These phenotypes can be partially corrected by overexpression of

RAD51 (Takata et al. 2001).

Compared to RAD51, the RAD51 paralogs display weaker DNA stimulated

ATPase activities (Braybrooke et al. 2000; Sigurdsson et al. 2001; Lio et al. 2003; Shim et al. 2004). RAD51B and RAD51C bind to ssDNA, dsDNA and 3-tailed dsDNA (Lio et al. 2003). Moreover, RAD51C has an apparent strand exchange activity perhaps by destabilization of dsDNA (Lio et al. 2003). RAD51D preferentially binds to ssDNA

(Braybrooke et al. 2000). RAD51B-RAD51C heterodimer possesses in vitro recombination mediator activity for RAD51 catalyzed strand exchange by suppressing the inhibitory effect of RPA (Sigurdsson et al. 2001). Furthermore, RAD51B-RAD51C is able to suppress the anti-recombinogenic activity of BLM during RAD51 mediated D- loop formation (R. Amunugama and R. Fishel, unpublished data). XRCC2 has been shown to enhance the ATP processing activity of RAD51 by facilitating ADP to ATP exchange by reducing the affinity for ADP (Shim et al. 2004). Both in vivo complexes

RAD51B, RAD51C, RAD51D and XRCC2 and RAD51C-XRCC3 bind to ssDNA, 3- and 5-tailed dsDNA, forked DNA structures and Holliday Junctions (Masson et al.

2001a; Masson et al. 2001b; Compton et al. 2010).

RAD54 - RAD54 is a highly conserved eukaryotic gene of the RAD52 epistasis group

(Table 1.1; (Tan et al. 2003; Heyer et al. 2006; Mazin et al. 2010). RAD54 homologs have been identified in a number of eukaryotes including yeast, Drosophila, plants, zebrafish, chicken, mice and human (Kanaar et al. 1996; Muris et al. 1996; Bezzubova et

28 al. 1997; Kooistra et al. 1997; Petrini et al. 1997; Thoma et al. 2005; Shaked et al. 2006).

A RAD54 homolog has been identified in the archaebacterium Sulfolobus solfataricus, but not in eubacteria (Seitz et al. 2001; Heyer et al. 2006). RAD54 homologs of budding yeast and human share a 66% similarity and 48% homology (Kanaar et al. 1996; Petrini et al. 1997). Budding yeast Rad54 was first discovered in a genetic screen to isolate mutants sensitive for ionizing radiation (Snow 1967; Game and Mortimer 1974). Similar to Rad51 and Rad52 mutants, Rad54 mutants were hypersensitive to ionizing radiation as well as DNA crosslinking and alkylating agents that eventually cause DSBs (Krogh and

Symington 2004). Rad54 mutants display only minor defects in meiotic recombination in yeast due to the presence of the meiotic homolog Rdh54/Tid1 (Klein 1997). RAD54 knock-down in mice causes hypersensitivity to ionizing radiation at embryonic stages but not in adult stages due to rescue by NHEJ repair (Essers et al. 2000). However, all development stages of RAD54 deficient mice are hypersensitive to DNA crosslinking agents (Essers et al. 2000). Mutational analysis of Walker A box conserved lysine indicated that in both mice and yeast ATP hydrolysis of Rad54 is essential for its function in vivo (Clever et al. 1999; Petukhova et al. 1999; Agarwal et al. 2011).

Rad54 expression levels increases during late G1 phase of the cell cycle (Cole et al. 1989; Johnston and Johnson 1995), presumably for HR repair of DSBs during late S and G2 phases (Takata et al. 1998). Rad54 expression levels are up-regulated during

DSB formation and Rad54 foci formation is dependent on Rad51 (Cole et al. 1987; Lisby et al. 2004). However, Rad51 foci formation is not dependent on Rad54, indicating that

Rad54 acts downstream of Rad51 (Lisby et al. 2004). Similarly, RAD54 co-localizes

29 with RAD51 foci following ionizing radiation in mammalian cells (Tan et al. 1999;

Essers et al. 2002).

RAD54 belongs to the Swi2/Snf2 SF2 (Super Family 2) of proteins (Tan et al.

2003; Heyer et al. 2006; Mazin et al. 2010). The Snf2/Swi2 proteins are commonly known for dsDNA dependent ATPase, ATP dependent chromatin remodeling, DNA translocase and DNA supercoiling activities (Tan et al. 2003; Heyer et al. 2006; Mazin et al. 2010). Like other members of SF1 and SF2 members RAD54 possesses several signature helicase motifs: I, Ia, II, III, IV, V, and VI that constitute the two tandem RecA- like lobes that utilize the energy of ATP binding and hydrolysis for function (Gorbalenya and Koonin 1993; Singleton et al. 2007). However, unlike helicases the SF2 family of proteins does not unwind but translocates on dsDNA (Heyer et al. 2006; Mazin et al.

2010). Also unlike helicases, the ATPase activity of RAD54 is not stimulated by ssDNA nor does it translocate on ssDNA (Swagemakers et al. 1998; Singleton et al. 2007). Both yeast and human RAD54 are strict dsDNA dependent ATPases, with a catalytic turnover rate ranging from 3000-6000 per minute (Mazin et al. 2010). The binding affinity for branched DNA structures such as PX-junctions (partial Holliday Junctions) is approximately 200 times higher than for ssDNA or dsDNA (Bugreev et al. 2006).

RAD54 function has been implicated in all three stages of recombination: presynapsis, synapsis and post-synapsis (Tan et al. 2003; Heyer et al. 2006). These include interaction with RAD51 to stabilize the ssDNA NPF, stimulation of homology search and strand exchange by catalyzed by RAD51, chromatin remodeling during the homology

30 search, disruption of RAD51-dsDNA filaments, branch migration of Holliday Junctions and interaction with specific endonuclease to stimulate resolution of Holliday Junctions.

RAD54 interacts with RAD51 in a species-specific manner through its N-terminal domain (Jiang et al. 1996; Clever et al. 1997; Golub et al. 1997; Haseltine and

Kowalczykowski 2009). This interaction is seen both with free RAD51 as well as with the RAD51 ssDNA NPF (Mazin et al. 2003). The ATPase activity of RAD54 is not required for RAD51 NPF stability. This was shown in vivo and in vitro using a RAD54

ATPase deficient mutation where a Walker A box lysine to arginine substitution allows only ATP binding (Mazin et al. 2003; Wolner and Peterson 2005).

Role of RAD54 in stimulating RAD51 mediated three-strand exchange activity and D-loop formation was first demonstrated with recombinant yeast proteins (Petukhova et al. 1998). This stimulation is seen with many RAD54 orthologs and occurs in a species-specific manner (Alexiadis and Kadonaga 2002; Sigurdsson et al. 2002; Mazina and Mazin 2004; Haseltine and Kowalczykowski 2009). Sub-stoichiometric amounts of

RAD54 are sufficient to greatly stimulate the RAD51 recombinase activity in vitro

(Petukhova et al. 1998; Sigurdsson et al. 2002; Mazina and Mazin 2004), indicating that the protein functions in a catalytic manner. In fact, for RAD51-mediated strand exchange stimulation, the ATPase activity of RAD54 is required (Petukhova et al. 1999).

Conversely, RAD51 improves the ATP hydrolysis and translocation ability of RAD54 on dsDNA (Van Komen et al. 2000; Mazina and Mazin 2004).

RAD54 facilitates dissociation of RAD51 from heteroduplex DNA following strand exchange in an ATP dependent manner (Solinger et al. 2002; Li et al. 2007).

Overexpression of Rad51 in Rdh54 deficient background leads to arrest of cell growth in yeast due to accumulation of Rad51 on undamaged chromatin (Shah et al. 2010).

Furthermore it was revealed that Rad54 is specialized for removal of Rad51 from damaged induced foci, while Rdh54 is involved in disassembly of Rad51 from undamaged toxic dead-end dsDNA complexes (Shah et al. 2010).

During the post-synaptic phases Rad54 enhances the heteroduplex extension of

Rad51 (Solinger and Heyer 2001). Human RAD54 has a relatively higher affinity for

Holliday Junctions and PX-junctions compared to dsDNA and it exhibits branch migration activity in a multimeric functional complex (Bugreev et al. 2006; Mazina et al.

2007). Furthermore, budding yeast Rad54 and human RAD54 have been shown to physically interact with the Holliday Junction the resolvase Mus81-Mms4 and MUS81-

EME1, respectively, to stimulate Holliday Junction resolution (Mazina and Mazin 2008;

Matulova et al. 2009). Even though, the branch migration activity of RAD54 was not required for MUS81-EME1 stimulation, ATP was required (Mazina and Mazin 2008).

RAD51AP1 - RAD51AP1 (RAD51 Associated Protein 1) previously known as PIR5, enhances RAD51 mediated joint molecule formation by physically interacting with both

RAD51 as well as joint DNA structures and dsDNA molecules (Kovalenko et al. 1997;

Modesti et al. 2007; Wiese et al. 2007). Knockdown of RAD51AP1 in human cells increases genotoxic stress to DSB inducing agents (Modesti et al. 2007; Wiese et al.

2007). However, the detailed role of RAD51AP in RR remains a mystery.

1.7 DSB REPAIR IN CHROMATIN

DSB induced histone modifications - In higher eukaryotes DNA is compacted into chromatin and the basic unit is a nucleosome, which consists of 146 bp of DNA wrapped approximately 1.7 times in left-handed superhelical turns around a tetramer of histones

H3 and H4 with two H2A-H2B dimers (Luger et al. 1997). There are several levels of compaction of chromatin in vivo (Kornberg 1977; Luger et al. 1997). The basic nucleosome structure is arranged into an array of nucleosomes resembling „beads on a string‟ (Thoma and Koller 1977). This array is further compacted into a 30nm filament solenoid through inter-nucleosomal interactions and interactions with linker histones H1 or H5 (Horn and Peterson 2002).

In order to perform DNA replication, repair and transcription the chromatin must be dynamic. The dynamic character is achieved by post-translational modifications

(PMTs) of amino acid residues on both the solvent exposed histone tails as well as the core regions (Kouzarides 2007). The PMTs include phosphorylation, methylation, acetylation, ubiquitination, SUMOylation, ADP-ribosylation and proline-isomerization

(Strahl and Allis 2000; Kouzarides 2007). Collectively these modifications have been termed the „Histone Code‟ (Strahl and Allis 2000; Jenuwein and Allis 2001; Rice and

Allis 2001). Once the histones are modified their affinity for DNA may change. For example, biochemical studies have shown that nucleosomes containing acetylated histone are more mobile than unmodified nucleosomes (Manohar et al. 2009; Simon et al. 2011).

These nucleosomes can then be evicted or pushed from the region of DNA that needs to be replicated, repaired or transcribed by chromatin remodelers using free energy of ATP

33 binding and/or hydrolysis (Gavin et al. 2001; Saha et al. 2002; Flaus and Owen-Hughes

2003; Kassabov et al. 2003; Eberharter and Becker 2004; Ulyanova and Schnitzler 2005;

Lorch et al. 2006).

The initial chromatin mediated response to a DSB is phosphorylation of the C- terminus of either major H2A variant in budding yeast on S129 (often referred to as

H2AX for simplicity (Morrison et al. 2004) or the H2AX variant in mammals on S139

(Rogakou et al. 1998; Downs et al. 2000; Rogakou et al. 2000). H2AX constitutes approximately 10% of the nucleosomal H2A complement and the Phosphorylated form is referred to as H2AX (Rogakou et al. 1998). The phosphorylation is ATM and MDC1 mediated and in yeast it spans several kilobases from the DSB, whist in mammals

H2AX spreads to megabase distances flanking the DSB (Rogakou et al. 1999; Downs et al. 2000; Harper and Elledge 2007). MDC1 directly interacts with H2AX via its C- terminal BRCT motifs (Stucki et al. 2005). Upon DSB formation MDC1 is phosphorylated by Casein Kinase 2 (CK2) on its N-terminal S-D-T tri-amino acid repeats and these phospho-domains interact with FHA and BRCT repeats of NBS1 in the MRN complex, that facilitates its recruitment to the DSB site (Chapman and Jackson 2008;

Melander et al. 2008; Spycher et al. 2008). Phosphorylated MDC1 once recruited to the

DSB site functions as a positive feedback regulator by binding to H2AX via its BRCT domain and to ATM through its FHA domain, respectively, to facilitate ATM mediated additional phosphorylation of H2AX to amplify the DNA damage signal (Lou et al.

2006). Sustaining H2AX flanking a DSB is critical for the recruitment of downstream repair factors (Celeste et al. 2002).

H2AX functions as a molecular beacon to recruit the cohesion complex that is involved in linking sister-chromatids during the post-replicative phase of the cell cycle

(Strom et al. 2004; Unal et al. 2004; Strom et al. 2007). This process prevents loss of heterozygosity (LOH) during mitotic HR by allowing the broken strand to use the sister- chromatid as the donor template. Studies on budding yeast indicate that H2AX recruits

ATP-dependent chromatin remodeling factor INO80 through direct interaction (Morrison et al. 2004; van Attikum et al. 2004). Furthermore, ssDNA formation that functions as the substrate for RAD51 nucleoprotein filament assembly is compromised in arp8 INO80 subunit mutants in yeast (van Attikum et al. 2004). The histone acetyl transferase

(HAT) NuA4 has also shown to interact with H2AX and appears to acetylate histone H4 following DSB formation (Downs et al. 2004).

H2AX(Y142) is also constitutively phosphorylated by the WSTF (Williams-

Beuren syndrome transcription factor) kinase (Xiao et al. 2009). Following that advent of a DSB H2AX(Y142) is dephosphorylated by the EYA1/EYA3 phosphatases (Cook et al. 2009; Krishnan et al. 2009). Interestingly, MDC1 interaction with H2AX depends on a dephosphorylation of H2AX(Y142) and the relative amount of dephosphorylation determines the recruitment of either pro-apoptotic or DNA repair factors to the damaged site (Cook et al. 2009). This has suggested that the H2AX(Y142) phosphorylation- dephosphorylation is a “molecular switch” in response to DSB damage (Stucki 2009).

An essential role for histone ubiquitination during the DSB repair response emerged after the discovery of the E3 ubiquitin ligase RNF8 at DSB foci (Huen et al.

2007; Kolas et al. 2007; Mailand et al. 2007). RNF8 is recruitment to DSB is mediated

35 by the interaction between its FHA domain with the phosphorylated motifs of MDC1

(Huen et al. 2007; Kolas et al. 2007; Mailand et al. 2007). RNF8 was shown to catalyze the ubiquitinylation of histone H2A and H2AX upon DSB formation, and knockdown of

RNF8 or disruption of FHA domains leads to failure to recruit the checkpoint activating proteins BRCA1 and 53BP1 to the DSB (Kolas et al. 2007; Mailand et al. 2007).

Furthermore, depletion of the E2 ubiquitin adapter UBC13 compromises RNF8 function (Huen et al. 2007; Kolas et al. 2007). UBC13 is an essential ubiquitin adapter for HR that is also recruited to DSBs (Ikura et al. 2007; Zhao et al. 2007). However,

RNF8 mediated ubiquitination is not sufficient to sustain the damage signal at DSB foci and another ubiquitin E3 ligase, RNF168, that acts with UBC13 to amplify the ubiquitination signal via K63 linked ubiquitination of H2A and H2AX (Doil et al. 2009;

Stewart et al. 2009). Another non-proteolytic E3 ligase, HERC2 is recruited to damage- induced foci and also forms a complex with RNF8 and RNF168 to extend their retention at the repair site (Bekker-Jensen et al. 2010). The K63 linked polyubiquitinated histones function as substrates for BRCA1 A-complex binding that includes BRCA1/BARD1,

ABRAXAS, RAP80, BRCC36 through the ubiquitin-interaction motif (UIM) of RAP80

(Wang and Elledge 2007). ABRAXAS is thought to recruit RAP80 to the DSB site

(Wang et al. 2007). Just as ubiquitination of H2AX is critical for DSB repair factor recruitment, deubiquitination is also tightly regulated during the damage response. In fact, the deubiquitinating enzymes BRCC36 and OTUB1 are simultaneously recruited damage-induced foci (Sobhian et al. 2007; Nakada et al. 2010). BRCC36 is part of the

BRCA1 A-complex and OTUB1 physically binds to UBC13 and inhibits RNF8 and

RNF168 mediated polyubiquitination (Sobhian et al. 2007; Nakada et al. 2010).

SUMOylation also appears to be essential for effective DSB damage response.

Recently it was shown that SUMO1, SUMO2 and SUMO3 are recruited to damage- induced foci along with the E3 ligases PIAS1 and PIAS4 (Galanty et al. 2009; Morris et al. 2009). SUMOylation is critical for productive assembly of BRCA1, 53BP1 and

RNF168 at damage-induced foci and PIAS mediated SUMOylation of BRCA1 leads to increased ubiquitin ligase activity of BRCA1/BARD1 heterodimer in vitro (Galanty et al.

2009; Morris et al. 2009).

Histone H2B(K120) has recently been shown to be ubiquitinylated by the E3 ligase RNF20-RNF40 heterodimer following DSB damage. The H2B(K120ub) modification leads to the recruitment of chromatin remodeling factor SNF2h to the DSB

(Moyal et al. 2011; Nakamura et al. 2011).

Other histone modifications include constitutively methylated histone H3(K79)

[H3(K79me)] that has been shown to provide an interacting domain for 53BP1 upon relaxation of higher order chromatin structure during a DSB (Huyen et al. 2004).

Furthermore, 53BP1 also interacts with the dimethylated form of histone H4(K20)

[H4(K20me2)] via its tandem tudor domains (Botuyan et al. 2006). HAT acetylation of histones is common during DNA replication and repair (Kouzarides 2007; Henikoff

2008). Acetylation is found not only on the histone tails but also on the core regions. For example, histones H3(K9) as well as H3(K56) are acetylated by GCN5/ KAT2A and p300 in response to DSB damage in human cells (Das et al. 2009; Tjeertes et al. 2009). It

37 is believed that H3(K56ac) influences the mobility of nucleosomes by neutralizing the positive charge on the lysine and mobilizing the entry-exit region of the nucleosome (Xu et al. 2005; Henikoff 2008). Finally, TIP60/Esa1 has been found to acetylate H4 and

H2AX during DSB repair (Ikura et al. 2000; Bird et al. 2002; Kusch et al. 2004; Murr et al. 2006).

ATP dependent chromatin remodeling - A yeast system that employed a galactose inducible HO endonuclease-provoked single DSB at a defined position in the “mating- type” (MAT) locus followed by chromatin immunoprecipitation allowed monitoring of chromatin remodeler and HR repair machinery recruitment at the recipient and donor sites in real-time (Azar 2011; Hicks et al. 2011). Once HO endonuclease cleaves the specific site within the MAT locus, that is otherwise protected by highly positioned nucleosomes, the break can be repaired by either NHEJ or HR (Osley et al. 2007). The later pathway is employed if the donor sequence HMRa or HML is present (Lee et al.

1998; Hicks et al. 2011). These studies have identified several chromatin remodelers recruited to DSB site including INO80, SWI/SNF, RSC, SWR1, RAD54 and TIP60

(Cairns et al. 1996; Ikura et al. 2000; Koyama et al. 2002; Cai et al. 2003; Martens and

Winston 2003; Wolner et al. 2003; Doyon et al. 2004; Kusch et al. 2004; Mizuguchi et al.

2004; Morrison et al. 2004; van Attikum et al. 2004; Chai et al. 2005; Shim et al. 2005;

Tsukuda et al. 2005; Wolner and Peterson 2005; Kruhlak et al. 2006; Papamichos-

Chronakis et al. 2006; Kent et al. 2007; Shim et al. 2007; Sinha et al. 2009)].

INO80 (Inositol auxotroph 80) is a multi-subunit chromatin remodeling complex that was first characterized in a budding yeast mutant strain that exhibited defective transcription activation following inositol depletion (Table 1.1; (Ebbert et al. 1999).

Relatively widely studied compared to other ATP-dependent chromatin remodelers, it is composed of several subunits that are shared by yeast and other eukaryotes (Osley et al.

2007). These core subunits include INO80 ATPase, two AAA+ ATPases (Rvb1 and

Rvb2 in yeast, RuvB-like 1 and RuvB-like 2 in human), actin and actin-related proteins

Arp4, Arp5 and Arp8, and INO80 subunits (Ies2 and Ies6) (Conaway and Conaway

2009). In addition, the yeast Ino80 complex contains unique polypeptides Taf14, HMG,

Nhp10 and Ies1, Ies3, Ies4, and Ies5 whereas the human homolog contains YY1, Uch37 and NFRKB (Conaway and Conaway 2009). The Ino80ATPase subunit of INO80 appears to essential for its cellular function and for ATP dependent chromatin remodeling in vitro (Shen et al. 2000; Shen et al. 2003; Jonsson et al. 2004). Ino80 is recruited to the

HO endonuclease DSB within an hour (Morrison et al. 2004; van Attikum et al. 2004;

Tsukuda et al. 2005). In yeast, deletion of Ino80ATPase, Arp5 or Arp8 subunits leads to increased sensitivity to DSB inducing agents (Morrison et al. 2004; van Attikum et al.

2004; Tsukuda et al. 2005). Recruitment of Ino80 to the DSB is dependent on a specific interaction between the Nhp10 (an HMG-like subunit of the Ino80 complex) and -H2AX and the loss of either component leads to compromised DSB repair (Morrison et al.

2004). However, two conflicting observations were reported with respect to recruitment of repair mediators at the DSB. One group observed that resection of 5 strands at the break was compromised in Arp8 and H2A mutants (van Attikum et al. 2004), while the

39 other reported that even though the resection occurred regularly in the Ino80 mutant,

Rad51 recruitment was delayed (Tsukuda et al. 2005).

SWI/SNF chromatin remodeling complex was first identified in two genetic screens in budding yeast (Table 1.1). The first gene regulates the mating type switch

(SWI) and the other regulates sucrose non-fermenting (SNF) phenotypes (Neigeborn and

Carlson 1984; Stern et al. 1984; Breeden and Nasmyth 1987). The multi-subunit complex consists of 9-12 subunits and has shown to possess ATP-dependent chromatin remodeling activity in vitro (Cairns et al. 1994; Cote et al. 1994). Several homologs of

SWI/SWF have been identified in metazoans; for example in Drosophila BRM (Brahma) and BAPs (BRM-Associated Proteins) were characterized as SWI/SNF homologs and in human BRM, BRG1 (Brahma-Related Gene1) and BAFs (BRM- or BRG1- Associated

Factors) are found as SWI/SNF complexes (Khavari et al. 1993; Dingwall et al. 1995;

Tamkun 1995; Reisman et al. 2009). SWI/SNF remodels nucleosomes both by nucleosome sliding and nucleosome ejection (Saha et al. 2006). Its activity is implicated both in transcription and DSB repair (Martens and Winston 2003; Chai et al. 2005; Osley et al. 2007). SWI/SNF involvement in DSB repair is extensively studied in yeast.

Although, Rad51 NPF formation does not require chromatin remodelers for homology searches and capture even on positioned nucleosomal surfaces in vivo or in vitro

(Sugawara et al. 2003; Wolner and Peterson 2005; Papamichos-Chronakis et al. 2006;

Sinha and Peterson 2008), Swi/Snf remodelers are essential for recombinational repair within heterochromatin (Sinha et al. 2009). When nucleosomal donor sequences are constrained by Sir2, Sir3 and Sir4 structural proteins that are found at telomeres and

40 silent mating-type loci, remodeling activity of Swi/Snf was required for efficient joint molecule formation (Sinha et al. 2009). Interestingly, Rad54, Ino80, RSC and Swr1 were incapable of promoting joint molecule formation within heterochromatin (Sinha et al.

2009). Several mammalian SWI/SNF complexes have also been shown to interact with

DSB repair response proteins such as BRCA1, p53 and with Fanconi Anemia pathway proteins (Bochar et al. 2000; Otsuki et al. 2001; Lee et al. 2002).

RSC (Remodel Structure of Chromatin) is another multi-subunit ATP dependent chromatin remodeler that is rapidly recruited to DSB site upon damage (Chai et al. 2005).

It is homologous to the SWI/SNF complex (Cairns et al. 1996). Its rapid recruitment to

DSB that coincides with MRX recruitment suggests that initial nucleosome remodeling at the DSB might facilitate DNA end processing by the MRX complex (Kent et al. 2007;

Osley et al. 2007; Shim et al. 2007). RSC is also required for loading of cohesins during

DSB repair to ensure repair occurs between sister chromatids during mitotic HR repair

(Shim et al. 2005; Shim et al. 2007). However, there is also evidence that suggest RSC might be involved in during the latter stages of HR repair pathway, particularly during

DNA ligation step after DNA synthesis (Chai et al. 2005).

SWR1 is closely related to INO80 that is recruited to DSB site in an H2AX dependent manner (Downs et al. 2004; van Attikum et al. 2007). However, its involvement in DSB repair is different than the role of INO80. SWR1 is known for exchanging histone H2A with the H2AZ variant (Krogan et al. 2003; Mizuguchi et al.

2004). SWR1 is also implicated in regulating H2AX levels. Inactivation of SWR1 ceases H2AZ incorporation at nucleosomes surrounding the DSB, however, this restores

H2AX levels and checkpoint adaptation; that functions antagonistically to INO80

(Papamichos-Chronakis et al. 2006).

Other than the acetylation of histones H4 and H2AX by TIP60 following DNA damage (Ikura et al. 2000; Bird et al. 2002; Kusch et al. 2004; Murr et al. 2006), the

Drosophila TIP60 has been shown to exchange acetylated histone H2Av (H2AX homolog) with unmodified H2Av using the domino/p400 ATPase (Kusch et al. 2004; Xu et al. 2010). The conjunctional action of TIP60/p400 that leads to nucleosomal destabilization at the DSB is required for efficient RNF8 mediated ubiquitination of histones and recruitment of BRCA1 and 53BP1 for DSB response (Xu et al. 2010).

Furthermore, the exchange of acetylated H2Av with unmodified H2Av might lay a critical for attenuation of DSB signal propagation (Osley et al. 2007).

RAD54 appears to have the ability to remodel chromatin (Haushalter and

Kadonaga 2003). RAD54 from budding yeast, Drosphila and human has been shown to posses ATP-driven chromatin remodeling activity in vitro using assembled mononucleosomes and nucleosomal arrays (Alexiadis and Kadonaga 2002; Alexeev et al.

2003; Jaskelioff et al. 2003; Zhang et al. 2007). The RAD51 nucleoprotein filament stimulates chromatin remodeling activity as well as the dsDNA-dependent ATPase activity of RAD54 (Tan et al. 2003; Heyer et al. 2006; Mazin et al. 2010). Strand exchange by RAD51 is greatly enhanced by RAD54 in chromatinized substrates

(Alexiadis and Kadonaga 2002; Zhang et al. 2007), even though for the initial homology capture by RAD51 nucleoprotein filament on a chromatin substrate RAD54 is not required (Sinha and Peterson 2008). Interestingly, a specific protein-protein interaction

42 between the amino terminus of histone H3 and RAD54 has also been reported, implying a specific role in RAD54 mediated chromatin remodeling (Kwon et al. 2007).

1.8 POSTSYNAPTIC REMOVAL OF RAD51

Postsynaptic RAD51 turnover plays a critical role in regulating the HR repair pathway

(Table 1.1). The deproteinization step in conventional strand exchange studies in vitro obliterates the possible analysis of RAD51 dissociation from dsDNA (Symington and

Heyer 2006). Furthermore, unlike RecA that dissociates from dsDNA upon ATP hydrolysis (Kowalczykowski 1991), RAD51 remains bound to heteroduplex DNA; probably due to its intrinsic slow ATP hydrolysis. These results suggested that eukaryotes accessory proteins have evolved to facilitate RAD51 removal from the nacent heteroduplex to allow the 3-end to prime DNA synthesis (Symington and Heyer 2006;

San Filippo et al. 2008). RAD54 has been shown to dissociate RAD51 from dsDNA

(Solinger et al. 2002; Heyer et al. 2006). Recent studies on the nematode C. elegans identified two more gene products, a helicase HELQ-1 (homologous to human HEL308) and the single RAD51 paralog RFS-1, that are essential for postsynaptic RAD51 turnover during meiotic homologous recombination (Ward et al. 2010). Both these gene products have been shown to have synthetic lethal interactions (Ward et al. 2010). It is suggested that once RAD54 removes the RAD51 from the 3 end of the invading strand HELQ-1 and RFS-1 might be involved in removing the remaining RAD51 from the heteroduplex

DNA (Ward et al. 2010; Williams and Michael 2010).

1.9 SECOND-END CAPTURE

When RAD51 nucleoprotein filament forms a D-loop the displace strand could potentially pair with the second 3 overhang that is produced. This process is called second-end capture. In yeast and mammalian cells this process is mediated by RAD52 through its inherent ability of pair with RPA-coated ssDNA (McIlwraith and West 2008;

Nimonkar et al. 2009; Shi et al. 2009; Sugiyama and Kantake 2009). In U. maydis Brh2 is also capable to catalyzing second-end capture in conditions where annealing by

RAD52 is inhibited (Mazloum and Holloman 2009).

1.10 DOUBLE HOLLIDAY JUNCTION DISSOLUTION

In the classical DSB repair model, after the second end capture, DNA synthesis and ligation results in a dHJ which can be either dissolved or resolved. During mitotic recombinational repair the former is the preferred pathway (Wu and Hickson 2006;

Bernstein et al. 2010; Heyer et al. 2010). dHJ dissolution is mainly mediated by RecQ helicases (Table 1.1; (Bernstein et al. 2010). The budding yeast RecQ homolog Sgs1

(fission yeast Rqh1) interacts strongly with the Top3 topoisomerase (Gangloff et al.

1994; Bennett et al. 2000; Fricke et al. 2001; Ahmad and Stewart 2005). In human cells the homologous RecQ helicase BLM strongly interacts with TOP3 (Johnson et al. 2000;

Wu et al. 2000). This helicase-topoisomerase complex has been shown to interact with yeast Rmi1/ Nce4 (RMI1/ BLAP75 in human cells) DNA binding protein. In budding yeast dHJ dissolution is mediated by Sgs1-Top3-Rmi1 (Chang et al. 2005; Mullen et al.

2005). In human cells the dHJ dissolution complex is comprised of BLM-TOP3-RMI1-

RMI2 (Yin et al. 2005; Bernstein et al. 2010).

1.11 HOLLIDAY JUNCTION RESOLUTION

Weisberg and colleagues reported the first biochemical evidence for an enzyme that has

Holliday Junction resolution activity in 1982. They identified the T4-endonuclease activity of bacteriophage T4 that was capable of cutting the branched structures of the phage genome before it was packaged into new phage particles (Mizuuchi et al. 1982;

Liu and West 2004; West 2009; Svendsen and Harper 2010). Soon afterwards, Holliday

Junction resolvases were identified in several organisms including budding yeast mitochondrial Cce1 (from cell-free extracts), fission yeast Ydc2 and endonuclease I of the bacteriophage T7 (de Massy et al. 1984; Symington and Kolodner 1985; West and

Korner 1985; Kleff et al. 1992; Whitby and Dixon 1997; Oram et al. 1998). The first prokaryotic resolvase to be identified was E.coli RuvC (Connolly et al. 1991; Sharples and Lloyd 1991). Biochemical characterization of RuvC revealed that the resolvase bound the Holliday Junction as a dimer and unfolds the antiparallel stacked-X structure

(observed in the presence of divalent cation Mg++) into an open planar structure

(observed in the absence of divalent ions) and nicks strands of the same polarity

(Connolly and West 1990; Connolly et al. 1991; Dunderdale et al. 1991; Iwasaki et al.

1991; Sharples and Lloyd 1991; Takahagi et al. 1991; West 1997). RuvC activity in bacterial cells are closely associated with the RuvA, the tetrameric protein that binds to

Holliday Junctions and unfolds it into an open-planar structure and RuvB, an hexameric

ATP hydrolysis driven translocase, which promotes branch migration (Parsons et al.

1995a; Parsons et al. 1995b; Hargreaves et al. 1998; Ariyoshi et al. 2000; Yamada et al.

2002; West 2003).

Holliday Junction resolvase activity in mammalian systems was first observed in extracts prepared from homogenized calf thymus tissues (Elborough and West 1990).

Similar resolvase activity was later observed in extracts prepared from cultured cells

(Hyde et al. 1994; Constantinou et al. 2001). Given the complexity of eukaryotic genomes and the stringency of maintaining its integrity, eukaryotic cells have evolved multiple pathways and resolvases to process Holliday Junctions (Klein and Symington

2009). This in turn made identification of single mutants defective in Holliday Junction resolution challenging in eukaryotic model organisms (Symington and Holloman 2008).

In 2001, Mus81-Eme1 (Mus81-Mms4 in budding yeast) heterodimer was identified in fission yeast as an endonuclease capable of cleaving Holliday Junctions as well as branched DNA structures (Table 1.1; (Boddy et al. 2001; Chen et al. 2001; Hollingsworth and Brill 2004). Mus81 is homologous to the XPF subunit of the ERCC1-XPF nucleotide excision repair endonuclease (Boddy et al. 2001; Chen et al. 2001). Eme1 is a non-catalytic subunit (Boddy et al. 2001; Chen et al. 2001; Svendsen and Harper 2010).

Mus81-Eme1 depletion caused some meiotic defects and stalled replication forks (Boddy et al. 2001; Chen et al. 2001). Meiotic defective cells could be rescued by ectopic expression of bacterial Holliday Junction resolvases (Boddy et al. 2001). In 2003, human

MUS81-EME1 was characterized as a replication fork/flap endonuclease that is essential to maintain the integrity of replication, even though it possessed inefficient Holliday

Junctions resolvase in vitro (Ciccia et al. 2003). In the case of budding yeast, Mus81 deletion only exhibited modest decrease in crossover formation during meiosis, implying an that Mus81-Eme1 is not the sole resolvase complex of Holliday Junctions (de los

Santos et al. 2001; de los Santos et al. 2003). Similar minor meiotic defects were observed in MUS81 knockout studies in mice (McPherson et al. 2004; Dendouga et al.

2005). Search for a RuvC type Holliday Junction resolvase in eukaryotic cells had been quite challenging by conventional sequence homology based queries due to the absence of conservation of primary amino acid sequences (West 2009). However, tertiary structure level conservation was seen among most of the identified Holliday Junction resolvases categorizing them into two main superfamilies of integrase and nuclease (West

2009). Identification of a Holliday Junction resolvase in eukaryotes that resembled bacterial RuvC was inadvertently assigned to RAD51C-XRCC3 heterodimer that was isolated from mammalian cells (Liu et al. 2004a; Symington and Holloman 2008).

However, the absence of an apparent nuclease domain and the inability of recombinant

RAD51C-XRCC3 to recapitulate the Holliday Junction resolvase activity led to questions regarding the assignment of the Holliday Junction resolvase to the RAD51C-XRCC3 heterodimer (Sharan and Kuznetsov 2007; Svendsen and Harper 2010). In 2008, after a tedious effort by the West laboratory (the group that suggested RAD51C-XRCC3 was a

Holliday Junction resolvase), the eukaryotic ResA Holliday Junction resolvase was identified (Ip et al. 2008). It appears that ResA co-purifies with RAD51C-XRCC3 isolated from human cells (Ip et al. 2008; Symington and Holloman 2008). Two complementary approaches were followed in identification of the ResA complex. One

47 was from fractionation of HeLa cell extracts from column chromatography followed by separating the final fraction on an SDS-PAGE gel (Ip et al. 2008). Following renaturation, one of the isolated bands still possessed Holliday Junction resolution activity in vitro, after mass spectroscopic (MS) analysis this novel protein was named

GEN1 (Ip et al. 2008). In the other approach, fission yeast library was screened where each gene was TAP (tandem affinity purification) tagged at the C-terminus (Ip et al.

2008). Using MS, Western blotting and in vitro Holliday Junction cleavage assays, along with Mus81-Eme1, the GEN1 ortholog in yeast Yen1 was identified (Ip et al. 2008).

Both GEN1 and Yen1 are members of Rad2/ XPG family of nucleases (West 2009) and they cleave Holliday Junctions symmetrically, resembling bacterial RuvC activity (Ip et al. 2008).

Recently another group of Holliday Junction resolvases, namely SLX1-SLX4 complex were identified (Table 1.1; (Andersen et al. 2009; Fekairi et al. 2009; Munoz et al. 2009; Svendsen et al. 2009). Even though Slx1 was conserved, conventional homology searches did not indicate a conservation of Slx4 outside of the yeast genome

(Fricke and Brill 2003; Klein and Symington 2009; Svendsen and Harper 2010). By more refined in silico analyses, human SLX4 was identified as BTBD12 which was the ortholog Drosophila MUS312 and fungal Slx4 (Fekairi et al. 2009; Munoz et al. 2009;

Reymer et al. 2009; Svendsen et al. 2009). In a separate study BTBD12 was identified as a phosphorylation substrate of ATM/ATR kinases (Matsuoka et al. 2007; Svendsen et al.

2009). SLX1 possesses the endonuclease domain for Holliday Junction cleavage, while

SLX4 acts as a protein interacting scaffold that interacts with multiple nucleases that

48 cleave Holliday Junctions both symmetrically and asymmetrically (Klein and Symington

2009; Svendsen and Harper 2010). In fact, SLX4 has been implicated in multiple genome maintenance pathways including replication and repair (Fekairi et al. 2009; Klein and Symington 2009; Munoz et al. 2009; Reymer et al. 2009; Svendsen et al. 2009).

Because SLX4 interacts with other Holliday Junction resolvases such as MUS81-EME1, when SLX1-SLX4 complex was isolated from human cells symmetrical cleavage of

Holliday Junctions was not observed (Matsuoka et al. 2007; Fekairi et al. 2009; Svendsen et al. 2009). However, bacterially expressed the SLX1-SLX4 heterodimer with a truncated SLX4 region that does not interact with MUS81-EME1, did possess symmetrical cleavage ability of Holliday Junctions (Matsuoka et al. 2007; Fekairi et al.

2009; Klein and Symington 2009; Svendsen et al. 2009). Among the many interacting partners of mammalian SLX4 are proteins of diverse functions. These include the endonucleases SLX1, ERCC4-ERCC1 and MUS81-EME1; mismatch repair heterodimer

MSH2-MSH3; telomere proteins TRF2/RAP1 and polo-like kinase PLK1 (Matsuoka et al. 2007; Fekairi et al. 2009; Klein and Symington 2009; Munoz et al. 2009; Svendsen et al. 2009).

1.12 HOMEOLOGOUS RECOMBINATION: the interplay between mismatch repair and HR

Mismatch repair (MMR) is a conserved process that plays an important role in maintaining genome integrity by correcting DNA mismatches formed during replication and recombination (Kolodner 1996; Kolodner and Marsischky 1999; Harfe and Jinks-

Robertson 2000). MMR repair ensures that HR occurs between perfectly homologous sequences and suppresses recombination between sequences that contain partial homology (homeologous recombination) (Harfe and Jinks-Robertson 2000). Genetic studies in budding yeast indicates that even a single mismatch reduces the recombination rates by at least four-folds compared to recombination between substrates of perfect homology (Datta et al. 1996; Datta et al. 1997). In yeast Msh2-Msh6, Msh2-Msh3,

Mlh1-Pms1 MMR heterodimers as well as Rad1-Rad10 and Exo1 nucleases and the helicases Sgs1 and Srs2 have been implicated in suppressing homeologous recombination

(Nicholson et al. 2000; Myung et al. 2001; Spell and Jinks-Robertson 2004; Welz-

Voegele and Jinks-Robertson 2008; Heyer et al. 2010). In mice and human cells a BLM

(Sgs1 homolog) deficiency still suppresses homeologous recombination. However, in combination with an Msh2 deficiency the amount of homeologous recombination events increased (Larocque and Jasin 2010). RecQ helicase WRN helicase has also been implicated in suppressing homeologous recombination by exhibiting strong interaction with the MSH2-MSH6, MSH2-MSH3 and MLH1-PMS2 heterodimers (Saydam et al.

2007). To date a reaction that suppresses homeologous recombination in vitro has not been developed.

1.13 CONCLUDING REMARKS

HR is mechanistically conserved in prokaryotes and eukaryotes. However, because of the genome complexity in eukaryotes, additional mediators are required for the successful repair of DSBs. Defective HR leads to genomic instability and tumorigenesis.

Paradoxically, unregulated HR also leads to the same outcome. Therefore, eukaryotic cells have evolved elegant mechanisms to regulate each step of HR to ultimately produce an accurate repair outcome.

1.14 FOOTNOTES

This chapter was submitted for publication as: Homologous Recombination in

Eukaryotes. Amunugama R. and Fishel R.

Figure 1.1 DNA double strand break repair (DSBR) by homologous recombination (HR). DSBR is initiated by D-loop formation (strand invasion) by the 3 ssDNA overhang that results from strand resection. The invading DNA strand primes DNA synthesis. During double Holliday junction (dHJ) formation the second end is captured and the strands are ligated after DNA synthesis. Branch migration can either dissolve dHJs that result in non-crossover products or stabilize dHJs to undergo resolution. dHJ resolution can result in either crossover or non-crossover products.

Figure 1.2 Alternative DSB repair mechanisms. Non-homologous end joining (NHEJ) or Alternative NHEJ (Alt-NHEJ) occurs by either direct ligation of the broken DNA strand or ligation after minimal processing. Both NHEJ and Alt-NHEJ are error- prone repair mechanisms. DSB repair within direct repeat sequences could occur by single-strand annealing (SSA). SSA causes loss of a repeat sequence due to direct resection, annealing and ligation. During DSB repair through synthesis dependent strand annealing (SDSA) synthesized nascent strand is displaced by D-loop dissociation, anneals with the other 3-ssDNA overhang to complete DNA synthesis. SDSA results in non-crossover products. After the initial D-loop formation and DNA synthesis the D- loop can also be cleaved to produce crossover products.

Figure 1.3 Break induced replication (BIR) and telomere restoration. BIR initiates from a single-ended strand invasion. If one arm of the chromatid is lost after the DSB or if a telomere is uncapped, a 3-overhang is formed. DNA synthesis can continue to the end of the chromatid either by migration of the D-loop or after D-loop cleavage.

Figure 1.4 HR restores collapsed or stalled replication forks. Nicks of template strands lead to replication fork collapse that can be repaired by D-loop formation and Holliday Junction cleavage to restore replication. A lesion on leading strand may result in replication fork regression and DNA synthesis on the leading strand. Subsequent branch migration restores the replication fork.

Figure 1.5 BRCA2 and its proposed role in HR. (A) Schematic representation of the functional and structural domains of human BRCA2. (B) Upon formation of a DSB, the 5-strand is resected to leave a 3-ssDNA. RPA binds and prevents secondary structure formation. BRCA2 binds at the dsDNA-ssDNA junction and initiates RAD51 nucleation on RPA coated ssDNA while limiting nucleation on dsDNA. Continued RAD51 nucleoprotein filament growth results in a functional nucleoprotein filament that performs a homology search and strand exchange.

continued

Figure 1.5: continued

Table 1.1 Comparison of DSB Repair Factors in Budding Yeast and Human.

HR repair process Budding yeast Human

5 resection Mre11-Rad50-Xrs2 (MRX) MRE11-RAD50-NBS1(MRN) Exo1 EXO1 Dna2 DNA2 Sae2 CtIP Sgs1-Top3-RmiI BLM-TOP3-RMII-RMI2 RPA RPA

Pre-synapsis & Rad522 BRCA2 Synapsis Rad51 RAD51 Rad55-Rad57 RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 Rad54 RAD54, RAD54B RAD51AP1 Dmc1 DMC1

DNA synthesis DNA polymerase δ DNA polymerase δ DNA polymerase η

Strand displacement Srs2 BLM Mph1 RTEL1 FANCM

HJ1 dissolution Sgs1-Top3-Rmi1 BLM-TOP3-RMI1-RMI2

HJ1 resolution Yen1 ResA (GEN1) Mus81-Mms4 MUS81-EME1 Slx1-Slx4 SLX1-SLX4

Chromatin remodeling Ino80 INO80 Swi/Snf SWI/SNF Swr1 SWR1 RSC TIP60

1Holliday Junction 2 Rad52 Epistasis Group in Red

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CHAPTER 2

Subunit Interface Residues F129 and H294 of Human RAD51 Are Essential For

Recombinase Function

2.1 ABSTRACT

RAD51 mediated homologous recombinational repair (HRR) of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. RAD51 forms a nucleoprotein filament (NPF) that catalyzes the fundamental homologous pairing and strand exchange reaction (recombinase) required for HRR. Based on structural and functional homology with archaeal and yeast RAD51, we have identified the human RAD51 (HsRAD51) subunit interface residues HsRad51(F129) in the Walker A box and HsRad51(H294) in the L2 ssDNA binding region as potentially important participants in salt-induced conformational transitions essential for recombinase activity. We demonstrate that the

HsRad51(F129V) and HsRad51(H294V) substitution mutations reduce DNA dependent

ATPase activity and are largely defective in the formation of a functional NPF, which ultimately eliminates recombinase catalytic functions. Our data are consistent with the conclusion that the HsRAD51(F129) and HsRAD51(H294) residues are important participants in the cation-induced allosteric activation of HsRAD51.

2.2 INTRODUCTION

Failure to repair DNA double strand breaks (DSBs) leads to tumorigenesis and genomic instability. Homologous recombination (HR) is an evolutionary conserved repair pathway utilized to restore DSBs. HR mediated DSB repair is initiated by resection of the 5-end of the break to leave a 3-single-stranded DNA (ssDNA) overhang (Mimitou and Symington 2008; Zhu et al. 2008). In eukaryotes, RAD51 forms a nucleoprotein filament (NPF) on the newly formed ssDNA region aided by other recombination mediators such as RAD52 in yeast and BRCA2 in vertebrates (Yang et al. 2002;

Sugawara et al. 2003; Lisby et al. 2004; Yang et al. 2005; Carreira et al. 2009; Jensen et al. 2010). The key function of the RAD51 NPF is to catalyze the homology search and initiate strand exchange. Deletion of Rad51 in mice results in embryonic lethality, while

RAD51 knock down in chicken DT40 cell lines results in increased chromosomal instability (Tsuzuki et al. 1996; Sonoda et al. 1998). Even though, no mutations of

RAD51 have been found in cancers, its expression is elevated in many cancer cell lines; perhaps to provide an advantage to rapidly dividing cells by repairing DSBs that would lead to replication fork collapse (Richardson et al. 2004; Klein 2008; Schild and Wiese

2010).

Despite the functional conservation with bacterial RecA, human RAD51

(HsRAD51) possesses an essential cation salt requirement for efficient strand exchange

+ in vitro. The most effective cation is ammonium (NH4 ), which is unlikely to be physiologically significant. However, potassium (K+) also enhances HsRAD51 recombinase functions to a lesser extent (Liu et al. 2004; Shim et al. 2006) and has been

shown to induce single stranded DNA-HsRAD51 complexes that structurally mimic active RecA NPFs (Liu et al. 2004). The effect of salt has been attributed to an induced preferential single stranded DNA (ssDNA) binding by RAD51 over double stranded

DNA (dsDNA) (Shim et al. 2006). However, crystallographic analysis of the methanococcus voltae RadA (MvRAD51) has revealed important K+-induced amino acid and structural rearrangements within the intersubunit region of the NPF (Figure 2.1A and

1B). For example, the MvRAD51(F107) residue in the highly conserved Walker A box rotates away from the ATP binding interface to accommodate K+ cations (Figure 2.1b;

(Wu et al. 2004). This rotation of MvRAD51(F107) induces ordering of the L2 ssDNA binding domain and a conformational transition of MvRAD51(H280) that results in the formation of a direct hydrogen bond with the -phosphate group of the ATP analogue; which is suggested to be involved in polarizing the water molecule involved in ATP hydrolysis. These two residues are conserved in RAD51 homologs from archaebacteria to human (Figure 2.1C).

Here, we have examined substitution mutations of the analogous HsRAD51 residues to MvRAD51(F107) and MvRAD51(H280), HsRAD51(F129) and

HsRAD51(H294). We find that HsRAD51(F129V) and HsRAD51(H294V) affect ATP hydrolysis (ATPase) activity but not adenosine nucleotide binding or ADPATP exchange. Moreover, they alter DNA binding properties in the presence of ATP and salt

+ + cations (K or NH4 ) that ultimately results in defective recombinase functions. These data further delineate the importance of salt-induced allosteric changes at the subunit interface of HsRAD51 that promotes the formation of a functional NPF.

2.3 MATERIALS AND METHODS

HsRAD51 Protein Expression and Purification- The mutant RAD51 genes F129V and

H294V were constructed using wild type HsRAD51 gene in the pET24d vector system

(Novagen) using conventional PCR. All mutations were confirmed by DNA sequencing.

Wild type and mutant hRAD51 proteins were expressed and purified following previously published protocols (Baumann et al. 1997; Tombline and Fishel 2002).

Briefly, hRAD51 was expressed in E. coli BLR strain and precipitated using spermidine-

HCl. Resuspended pellet was purified using Reactive-Blue-4-agarose (Sigma-Aldrich),

Heparin sepharose (GE Healthcare), hydroxyapatite (Bio-Rad) and Mono Q (GE

Healthcare) column chromatography. Purity of the fractions was verified by SDS-PAGE analysis. HsRPA was expressed in BL21(AI) cells using pET11d-tRPA purified as described (Henricksen et al. 1994), except for the resuspension of cells where HI buffer was supplemented with 100mM KCl.

DNA Substrates- X174 single-stranded (ss) virion DNA and replicative form I (RFI) were purchased from NEB. X174 RFIII was obtained by linearizing RFI with ApaLI restriction enzyme and gel purifying with Qiaquick Gel Extraction kit (Qiagen). For surface plasmon resonance (SPR) analysis, a 5′ biotinylated oligo dT50 was used as ssDNA and for dsDNA 5′ biotinylated 50-mer 5-TCG AGA GGG TAA ACC ACA-

ATT ATT GAT ATA AAA TAG TTT TGG GTA GGC GA was annealed with its complement. D-loop assay substrates were prepared as described (Van Komen et al.

2002). 94

ATPase assay- ATP hydrolysis was measured as previously described (Tombline and

Fishel 2002). Reactions were performed in 10 L volumes in Buffer A containing 20mM

HEPES (pH 7.5), 10% glycerol, 100g/mL BSA, 1mM DTT and 1mM MgCl2 and when indicated, 150mM KCl. Each reaction mixture contained RAD51 (1M) and 6M (nt or bp) of X174 ssDNA or dsDNA. Reactions were initiated by the addition of protein and incubation at 37°C. After 1 hr 400L of 10% activated charcoal supplemented with

10mM EDTA was added to terminate the reaction and incubated on ice for another 2 hrs.

After centrifuging for 10 min 50L duplicate aliquots were taken for counting [32P] free phosphate by Cerenkov method. Kinetic parameters were obtained by fitting data into

Michaelis-Menten equation using the software Kaleidagraph (Synergy software).

ATPS/ADP binding assay- Experimental procedure was as previously described

(Tombline et al. 2002). Reactions were performed in Buffer A and 150mM KCl, when indicated. RAD51 (1M) and 6M (nt) X174 ssDNA were used with the indicated amount of [-35S]ATPS or [3H]ADP. After a 30 min incubation at 37°C, reactions were kept on ice until filtered. Reaction was added into 4mL of ice-cold reaction buffer and filtered through HAWP nitrocellulose filters (Millipore) presoaked in the same buffer.

Another 4mL of buffer was used to wash the membrane and the filter was dried for 2 hrs.

Radioactivity was counted after an overnight incubation in liquid scintillation fluid.

ADP-ATP exchange assay- Reactions were performed in Buffer A with 150mM KCl.

RAD51(1M) and 6M (nt) X174 ssDNA was incubated with 3 M of [3H]ADP in a

60L reaction volume at 37°C for 10 mins. ADP to ATP exchange was initiated by addition of cold ATP to a 5mM final concentration. 10L aliquots were withdrawn at indicated time points, filtered and analyzed as in ADP binding.

SPR analysis- Biotinylated DNA was immobilized on a streptavidin-coated chip (GE

Healthcare) for the analysis. Protein binding and dissociation were analyzed at 25°C with a 5L/min flow rate on a Biacore 3000 (GE Healthcare). Reactions were performed in Buffer containing 20mM HEPES (pH 7.5), 10% glycerol, 1mM DTT and 1mM MgCl2,

0.005% surfactant P-20 (GE Healthcare), 2.5mM of the indicated nucleotide and 150mM

2+ KCl when indicated. For experiments with Ca , 1mM CaCl2 was used instead of MgCl2.

DNA binding was determined by injecting 100nM, 200nM, 400nM, 800nM and 1.6M of protein simultaneously into ssDNA and dsDNA containing flow channels to ensure saturated binding.

D-loop assay- 2.4 M (nt) labeled 90-mer was incubated with HsRAD51 (0.8M) in buffer A with 1mM ATP supplemented with the indicated amounts of MgCl2 or CaCl2 at

37°C for 10 min. Reaction was initiated by adding 35M (bp) supercoiled pBS-SK(-) plasmid and incubated further for 15 min. Samples were deproteinized by addition of 1%

SDS and 1 mg/mL proteinase K to a final concentration and incubated for 15 min at

37°C, mixed with 1/5 volume of gel loading dye and analyzed on 0.9% agarose gel in

TAE buffer, run at 4V/cm at 25°C. D-loops were quantified after drying and exposure to

PhosphoImager (Molecular Dynamics) screens.

DNA Strand Exchange Assay- HsRAD51 (5M) and 30M (nt) X174 circular ssDNA were pre-incubated in buffer containing 20mM HEPES (pH 7.5), 10% Glycerol, 1mM

DTT, 1mM MgCl2 supplemented with 2.5mM of ATP at 37°C for 5 min before addition of 150mM of the indicated salt and 15M (bp) linear X174 dsDNA. After another 5 min incubation 2M of HsRPA was added and the incubation was continued. After 3 hrs samples were deproteinized by addition of 3L of stop buffer containing 2% SDS and 10 mg/mL proteinase K, and analyzed on 0.9% agarose gel in TAE buffer. Electrophoresis was carried out at 4V/cm at 25°C with 0.1g/mL ethidium bromide.

2.4 RESULTS

Mutation of HsRAD51 inter-subunit residues F129 and H294 affect ATP turnover

HsRAD51(F129) and HsRAD51(H294) were mutated to Val (V) to minimize initial structural perturbations. The respective proteins were then purified to near homogeneity

(Fig. 2.2A). The RecA/RAD51 family of proteins exhibits DNA dependent ATPase activity (Weinstock et al. 1981a; Weinstock et al. 1981b; Pugh and Cox 1987; Baumann et al. 1996; Tombline and Fishel 2002). HsRAD51 possesses a modestly higher ATP turnover (kcat) in the presence of ssDNA compared to dsDNA, although it is about 150 times slower than the bacterial RecA (Weinstock et al. 1981a; Weinstock et al. 1981b;

Pugh and Cox 1987; Tombline and Fishel 2002). The HsRAD51(F129V) substitution mutation displays an ~2-fold reduction in kcat compared to the wild type HsRAD51, while the HsRAD51(H294V) substitution mutation nearly abolishes the ATPase activity (Fig.

2.2B, Table 1).

Reduced kcat could be due to several factors: an ATP binding deficiency, an ADP release deficiency or a deficiency in the catalytic step. To rule out the first two possibilities, we examined ATP binding, ADP binding and ADP-ATP exchange of the mutant proteins. We found no significant difference in ATP binding (Fig. 2.2C, Table 1),

ADP binding (Fig. 2.2D, Table 1) or ADP-ATP exchange (data not shown). Previous studies have confirmed a small but reproducible salt induced ATPase catalytic rate enhancement in the presence of ssDNA compared to dsDNA (Shim et al. 2006). Only

RAD51(F129V) displayed a similar salt (150 mM KCl) induced catalytic rate enhancement in the presence of ssDNA (Fig. 2.2E). Together these observations are

consistent with the conclusion that HsRAD51(H294) residue and to a significantly lesser extent the HsRAD51(F129) residue are required for appropriate ATP catalysis.

HsRAD51(F129V) and HsRAD51(H294V) are deficient in D-loop formation and strand exchange

We examined the recombinase activity of the HsRAD51(F129V) and HsRAD51(H294V) substitution mutations. HsRAD51 catalyzes D-loop formation in vitro between a 32P- labeled 90-mer and a homologous supercoiled plasmid (Fig. 2.3A). Efficient D-loop formation occurs when ATP is present in a slow-hydrolysable state (e.g. in the presence of calcium) or when non-hydrolysable ATP analogues such as AMP-PNP are substituted for ATP (Bugreev and Mazin 2004; Chi et al. 2006). Importantly, the Walker A box mutant HsRAD51(K133R) that binds ATP normally but is defective in ATP hydrolysis, catalyzes efficient D-loop formation with ATP and Mg2+ (Chi et al. 2006). These results have suggested that D-loop catalysis requires HsRAD51 to form and maintain an active

ATP-bound NPF (Bugreev and Mazin 2004; Ristic et al. 2005; Chi et al. 2006; San

Filippo et al. 2008).

In the presence of ATP, HsRAD51 catalyzed D-loop formation with Ca2+ but not with Mg2+ (Fig. 2.3B). Previous work has demonstrated that Ca2+ induces the formation of a stable ATP-bound NPF while Mg2+ allows the hydrolysis of ATP that results in a mixed (ADP/ATP) NPF that is largely inactive (Bugreev and Mazin 2004).

HsRAD51(F129V) and HsRAD51(H294V) did not catalyze D-loop formation in either

Mg2+ or Ca2+ (Fig. 3B). Thus, even though HsRAD51(H294V) is ATPase deficient, and both HsRAD51(F129V) and HsRAD51(H294V) display ATP binding that is comparable

to the wild type, these mutant proteins are incapable of catalyzing D-loop formation.

Collectively these results indicate that suppression of ATP hydrolysis alone is not sufficient to confer enhanced D-loop recombinase activity.

Catalysis of strand exchange between a duplex DNA substrate and a homologous single stranded circular DNA substrate is a hallmark of RecA/RAD51 proteins (Cox and

Lehman 1981; Menetski et al. 1990; Kowalczykowski 1991; Kowalczykowski and

Eggleston 1994; Baumann et al. 1996; San Filippo et al. 2008). Unlike D-loop formation

+ + that occurs in low salt, strand exchange requires specific cations (either NH4 or K ) to activate HsRAD51 activity (Sigurdsson et al. 2001; Liu et al. 2004; Shim et al. 2006).

Using X174 virion DNA and ApaL1 linearized X174 dsDNA we compared the strand exchange activity of the HsRAD51(F129V) and HsRAD51(H294V) mutant proteins to wild type HsRAD51. We also examined the stimulatory effect of the human single- stranded binding complex RPA (HsRPA). Neither HsRAD51(F129V) nor

HsRAD51(H294V) were able to form joint molecules in the presence of salt and/or RPA

(Fig. 2.3C). We can conclude that the HsRAD51(F129) and HsRAD51(H294) residues are critical for cation-induced HsRAD51 recombinase functions.

HsRAD51(F129) and HsRAD51(H294) are essential for ATP-dependent DNA binding

We examined real-time HsRAD51 ssDNA and dsDNA binding by surface plasmon resonance (SPR, Biacore). Biotinylated dT50 ssDNA and 50 bp dsDNA were immobilized on a streptavidin-coated flow-cell surface. SPR measures the change in the refractive index that reflects protein binding and/or dissociation from DNA. We analyzed ssDNA and dsDNA binding in salt conditions similar to those used in for strand

100

exchange. We titrated the protein from 100 nM to 1.6 M in the presence of ADP, ATP and in the absence of adenosine nucleotide. For clarity the binding and dissociation curves shown correspond to 800 nM of protein (Fig. 2.4). After protein injection nucleoprotein filament formation was analyzed for 150 sec. For steady-state protein disassembly, protein free buffer was injected into the flow cells for 300 sec. Because the ssDNA and dsDNA were introduced into separate channels we could simultaneously examine protein binding and dissociation to the two DNA substrates. In the absence of a adenosine nucleotide only the wild type HsRAD51 and HsRAD51(H294V) bound to ssDNA (Fig. 2.4A, left panel). In the presence of ADP, the wild type and mutant variants bound to ssDNA, but dissociated rapidly (Fig. 2.4A, middle panel). These results are consistent with previous studies that have suggested protein turnover associated with

ADP-bound RAD51 (Bugreev and Mazin 2004; Hilario et al. 2009). Interestingly, in the presence of ATP, wild type HsRAD51 bound ssDNA while both HsRAD51(F129V) and

HsRAD51(H294V) were largely defective in ssDNA binding (Fig. 2.4A, right panel).

Only wild type HsRAD51 displayed dsDNA binding in the presence of ATP as well as in the absence of a nucleotide (Fig. 2.4B). These results suggest that the HsRAD51(F129) and HsRAD51(H294) residues are important for the ATP induced DNA binding that ultimately results in an active NPF required for recombinase function.

The abnormal DNA binding did not appear to fully account for the altered steady- state ATPase activity of HsRAD51(F129V) and HsRAD51(H294V) (compare Fig. 2.2E,

Table 2.1 with Fig. 2.4). This is particularly true for HsRAD51(H294V) which showed reduced but not absent binding to ssDNA, yet near background ATPase activity. The dissociation of ssDNA binding activity from ATPase activity suggests that some other

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function(s) of these proteins are compromised. Based on the location of these residues in the crystal structures we postulate that cation induced conformational transitions are defective in HsRAD51(F129V) and HsRAD51(H294V) (see Figs. 2.1A and 2.1B).

These conformational transitions do not influence ADP/ATP binding or ADPATP exchange, but do affect the ability of the mutant proteins to interact appropriately with

DNA and to properly catalyze ATP hydrolysis.

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2.5 DISCUSSION

Structural studies of the MvRAD51 and S.cerevisiae RAD51 (ScRAD51) have detailed significant conformational transitions associated with the NPF. The ScRAD51 residues analogous to HsRAD51(F129) and HsRAD51(H294), ScRAD51(F187) and

ScRAD51(H352), were found in two alternate conformations within the NPF (Conway et al. 2004; Qian et al. 2005). In one conformation ScRAD51(H352) was positioned above the ATP binding site while in the other ScRAD51(F187) excluded ScRAD51(H352) by moving it out of the active site. Importantly, K+ cations induce similar conformational transitions of MvRAD51 of the analogous residues to HsRAD51(F129) and

HsRAD51(H294), MvRAD51(F107) and MvRAD51(H280). In the presence of K+ the

MvRAD51(F107) rotates away from the ATP catalytic interface and MvRAD51(H280) rotates in to form a hydrogen bond interaction with the -phosphate of ATP, while at the same time ordering the L2 ssDNA binding domain (Wu et al. 2004).

Our studies support the importance of these conformational transitions. While

HsRAD51(F129V) can bind and hydrolyze ATP, it displays significantly reduced ssDNA binding in the presence of ATP. These results suggest an impaired ability to incite the conformational transitions necessary to the form an active NPF on ssDNA. We speculate that this impairment involves an inability of the smaller Val residue to incite ordering of the L2 ssDNA-binding domain that is normally provoked by cation-induced rotation of the Phe residue away from the ATP catalytic interface. In contrast, HsRAD51(H294V) binds but does not hydrolyze ATP, binds ssDNA in the absence of adenosine nucleotide, yet displays significantly reduced ssDNA binding in the presence of ATP. We speculate

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that these impairments are the result of an inability of the Val residue to form an appropriate hydrogen bond interaction with the -phosphate of ATP in spite of the fact that L2 ssDNA-binding domain ordering has been provoked by the MvRAD51(F107) residue (Wu et al. 2005). Modeling of the HsRAD51 structure strongly support such an allosteric communication between ATPase site and L2 ssDNA binding region (Renodon-

Corniere et al. 2008; Reymer et al. 2009). A recent mutational analysis also revealed that the subunit interface residue ScRAD51(H352) is critical for functional NPF formation and strand exchange activity (Grigorescu et al. 2009). Ultimately, the defects associated with both HsRAD51(F129V) and HsRAD51(H294V) result in defective D-loop and strand exchange functions. Because both proteins bind ADP and ATP similar to the wild type HsRAD51, the functional defects are unlikely to be a result of improper folding and/or aggregation.

For RecA/RAD51 proteins ATP binding but not necessarily hydrolysis, is sufficient to catalyze strand exchange (Menetski et al. 1990; Kowalczykowski 1991;

Rehrauer and Kowalczykowski 1993; Kowalczykowski and Eggleston 1994; Shan et al.

1996; Chi et al. 2006). It has been suggested that RAD51 might utilize binding of ATP and the resulting release of the hydrolysis product as a conformational switch for regulating recombinase function similar to other members of the AAA+ superfamily

(Erzberger and Berger 2006). Our data is consistent with the hypothesis that the

HsRAD51(F129) and HsRAD51(H294) residues along with salt cations may play a significant role in this conformational switch during HRR.

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2.6 ACKNOWLEDGEMENTS AND FOOTNOTES

This chapter was published as: Subunit Interface Residues F129 and H294 of Human

RAD51 Are Essential For Recombinase Function. Amunugama R. and Fishel R.

Plos One (2011) 6(8):e23071.

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Figure 2.1 Cation-induced conformational rearrangement of conserved amino acid residues of RadA. (A) Subunit interface of MvRadA (MvRAD51) structure in the absence of potassium cation (PDB code 1T4G). (B) Subunit interface region of

MvRAD51 structure in the presence of potassium cation (PDB code 1XU4). Structural figures were generated using Pymol. (C) Sequence alignment of WalkerA/P-loop and L2 ssDNA binding region of H. sapiens (Hs), S. cerevisiae (Sc) and M. voltae (Mv) recombinases. HsRAD51 residues F129 and H294 are indicated with asterisks.

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Figure 2.2 Mutation of HsRAD51(F129) and HsRAD51(H294) residues affect ATP turnover. (A) Purification of wild type and HsRAD51 substitution mutant proteins. Protein (1 g) analyzed by 12% SDS-PAGE. (B) Steady-state ATPase activity with ssDNA in the presence of 150 mM KCl. (C) ATPS binding by wild type and HsRAD51 substitution mutant proteins in the presence of ssDNA and 150 mM KCl. (D) ADP binding by wild type and HsRAD51 substitution mutant proteins in the presence of ssDNA and 150 mM KCl. (E) ATP turnover (kcat) with ssDNA and dsDNA in the + presence and absence of KCl (K ). kcat values were calculated by Michaelis-Menten analysis. Error bars indicate standard deviation from at least three independent experiments.

Continued

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Figure 2.2: continued

108

Figure 2.3. HsRAD51(F129V) and HsRAD51(H294V) are deficient in D-loop formation and strand exchange. (A) In vitro D-loop assay reaction schematic. (B) 0.8M of HsRAD51, HsRAD51(F129V), or HsRAD51(H294V) and [P32]-labeled ssDNA (90mer; 2.4 M nt) were preincubated for 10 min at 37°C in the reaction buffer containing 1 mM ATP and 1mM MgCl2 or CaCl2. Reactions were initiated by the addition of supercoiled pBS SK(-) plasmid DNA (35M bp). After 15 min, reactions were terminated by the addition of proteinase-K and SDS. Joint molecules (JMs) were analyzed on a 0.9% agarose gel. (C) Analysis of salt and RPA requirement for strand exchange. Reaction schematic shown above: HsRAD51 (5M) and X174 circular ssDNA (30M nt) were pre-incubated with 2.5mM ATP and 1mM MgCl2 at 37°C for 5 min prior to the addition of 150mM NaNH4HPO4 (if indicated) and linear X174 dsDNA (15M bp). After 5 min, HsRPA (2M) was added (if indicated) and the incubation was continued for 3 hrs. Samples were deproteinized and analyzed on 0.9% agarose gel with 0.1g/mL ethidium bromide.

Continued

109

Figure 2.3: continued

110

Figure 2.4 F129 and H294 of HsRAD51 are critical for DNA binding in the presence of ATP. (A) ssDNA binding analysis of wild type and HsRAD51 substitution mutant proteins by surface Plasmon resonance (SPR, Biacore) in the absence of an adenine nucleotide, in the presence of ADP and in the presence of ATP. Association and dissociation curve corresponding to 800nM of each protein is shown. (B) dsDNA binding analysis of wild type and HsRAD51 substitution mutant proteins.

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Table 2.1 Summary of ATP hydrolysis and nucleotide binding data of HsRAD51 wild type (WT) and HsRAD51(F129V) and HsRAD51(H294V) mutant proteins.

Kinetic Parameter WT F129V H294V -1 kcat (min ) ssDNA 0.180 ± 0.004 0.094 ± 0.004 0.010 ± 0.001 ssDNA, KCl 0.190 ± 0.004 0.119 ± 0.009 0.008 ± 0.001

dsDNA 0.167 ± 0.002 0.095 ± 0.009 0.021 ± 0.002 dsDNA, KCl 0.135 ± 0.007 0.101 ± 0.008 0.013 ± 0.002

Km (M) ssDNA 17.48 ± 3.25 6.68 ± 1.39 2.59 ± 0.10 ssDNA, KCl 9.34 ± 1.42 6.40 ± 0.032 6.40 ± 3.31

dsDNA 6.54 ± 0.67 4.23 ± 0.38 5.38 ± 0.50 dsDNA, KCl 5.52 ± 0.26 4.90 ± 0.24 8.82 ± 0.26

KD(M) ATPS ssDNA 2.49 ± 0.46 1.53 ± 0.69 0.817 ± 0.14 ssDNA, KCl 1.18 ± 0.22 1.15 ± 0.55 1.40 ± 0.31

Bmax(M) ATPS ssDNA 1.02 ± 0.02 0.85 ± 0.03 0.63 ± 0.01 ssDNA, KCl 0.83 ± 0.04 0.87 ± 0.04 0.77 ± 0.03

KD (M) ADP ssDNA, KCl 1.24 ± 0.32 1.27 ± 0.11 2.66 ± 0.10 Bmax (M) ADP ssDNA, KCl 0.82 ± 0.05 1.03 ± 0.02 0.95 ± 0.01

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2.7 REFERENCES

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Lisby M, Barlow JH, Burgess RC, Rothstein R. 2004. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118: 699-713. Liu Y, Stasiak AZ, Masson JY, McIlwraith MJ, Stasiak A, West SC. 2004. Conformational changes modulate the activity of human RAD51 protein. J. Mol. Biol. 337: 817-827. Menetski JP, Bear DG, Kowalczykowski SC. 1990. Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis. Proc. Natl. Acad. Sci. U. S. A. 87: 21-25. Mimitou EP, Symington LS. 2008. Sae2, Exo1 and Sgs1 collaborate in DNA double- strand break processing. Nature 455: 770-774. Pugh BF, Cox MM. 1987. Stable binding of recA protein to duplex DNA. Unraveling a paradox. J. Biol. Chem. 262: 1326-1336. Qian X, Wu Y, He Y, Luo Y. 2005. Crystal structure of Methanococcus voltae RadA in complex with ADP: hydrolysis-induced conformational change. Biochemistry (Mosc). 44: 13753-13761. Rehrauer WM, Kowalczykowski SC. 1993. Alteration of the nucleoside triphosphate (NTP) catalytic domain within Escherichia coli recA protein attenuates NTP hydrolysis but not joint molecule formation. J. Biol. Chem. 268: 1292-1297. Renodon-Corniere A, Takizawa Y, Conilleau S, Tran V, Iwai S, Kurumizaka H, Takahashi M. 2008. Structural analysis of the human Rad51 protein-DNA complex filament by tryptophan fluorescence scanning analysis: transmission of allosteric effects between ATP binding and DNA binding. J. Mol. Biol. 383: 575- 587. Reymer A, Frykholm K, Morimatsu K, Takahashi M, Norden B. 2009. Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 106: 13248-13253. Richardson C, Stark JM, Ommundsen M, Jasin M. 2004. Rad51 overexpression promotes alternative double-strand break repair pathways and genome instability. Oncogene 23: 546-553. Ristic D, Modesti M, van der Heijden T, van Noort J, Dekker C, Kanaar R, Wyman C. 2005. Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function. Nucleic Acids Res 33: 3292-3302. San Filippo J, Sung P, Klein H. 2008. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77: 229-257. Schild D, Wiese C. 2010. Overexpression of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability. Nucleic Acids Res 38: 1061- 1070. Shan Q, Cox MM, Inman RB. 1996. DNA strand exchange promoted by RecA K72R. Two reaction phases with different Mg2+ requirements. J. Biol. Chem. 271: 5712- 5724. Shim KS, Schmutte C, Yoder K, Fishel R. 2006. Defining the salt effect on human RAD51 activities. DNA Repair (Amst) 5: 718-730.

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Sigurdsson S, Trujillo K, Song B, Stratton S, Sung P. 2001. Basis for avid homologous DNA strand exchange by human Rad51 and RPA. J. Biol. Chem. 276: 8798-8806. Sonoda E, Sasaki MS, Buerstedde JM, Bezzubova O, Shinohara A, Ogawa H, Takata M, Yamaguchi-Iwai Y, Takeda S. 1998. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J. 17: 598-608. Sugawara N, Wang X, Haber JE. 2003. In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol. Cell 12: 209-219. Tombline G, Fishel R. 2002. Biochemical characterization of the human RAD51 protein. I. ATP hydrolysis. J. Biol. Chem. 277: 14417-14425. Tombline G, Shim KS, Fishel R. 2002. Biochemical characterization of the human RAD51 protein. II. Adenosine nucleotide binding and competition. J. Biol. Chem. 277: 14426-14433. Tsuzuki T, Fujii Y, Sakumi K, Tominaga Y, Nakao K, Sekiguchi M, Matsushiro A, Yoshimura Y, MoritaT. 1996. Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. U. S. A. 93: 6236-6240. Van Komen S, Petukhova G, Sigurdsson S, Sung P. 2002. Functional cross-talk among Rad51, Rad54, and replication protein A in heteroduplex DNA joint formation. J. Biol. Chem. 277: 43578-43587. Weinstock GM, McEntee K, Lehman IR. 1981a. Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Characterization of ATP hydrolysis. J. Biol. Chem. 256: 8829-8834. -. 1981b. Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Steady state kinetic analysis of ATP hydrolysis. J. Biol. Chem. 256: 8845-8849. Wu Y, He Y, Moya IA, Qian X, Luo Y. 2004. Crystal structure of archaeal recombinase RADA: a snapshot of its extended conformation. Mol. Cell 15: 423-435. Wu Y, Qian X, He Y, Moya IA, Luo Y. 2005. Crystal structure of an ATPase-active form of Rad51 homolog from Methanococcus voltae. Insights into potassium dependence. J. Biol. Chem. 280: 722-728. Yang H, Jeffrey PD, Miller J, Kinnucan E, Sun Y, Thoma NH, Zheng N, Chen PL, Lee WH, Pavletich NP. 2002. BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure. Science 297: 1837-1848. Yang H, Li Q, Fan J, Holloman WK, Pavletich NP. 2005. The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA-ssDNA junction. Nature 433: 653-657. Zhu Z, Chung W-H, Shim EY, Lee SE, Ira G. 2008. Sgs1 Helicase and Two Nucleases Dna2 and Exo1 Resect DNA Double-Strand Break Ends. Cell 134: 981-994.

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CHAPTER 3

The RAD51 ATP Cap Regulates Nucleoprotein Filament Stability

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3.1 ABSTRACT

RAD51 mediates homologous recombination (HR) by forming an active DNA nucleoprotein filament (NPF). A conserved aspartate that forms a salt bridge with the

ATP -phosphate is found at the nucleotide-binding interface between RAD51 subunits of the NPF known as the ATP cap. The salt bridge accounts for the nonphysiological cation(s) required to fully activate the RAD51 NPF. In contrast, RecA homologs and most RAD51 paralogs contain a conserved lysine at the analogous structural position.

We demonstrate that substitution of human RAD51(D316) with lysine

[HsRAD51(D316K)] decreases NPF turnover and facilitates considerably improved recombinase functions. Structural analysis shows that archaebacterial Methanococcus voltae RadA(D302K) [MvRAD51(D302K)] and HsRAD51(D316K) form extended active NPFs without salt. These studies suggest that the HsRAD51(D316) salt bridge may function as a conformational sensor that enhances turnover at the expense of recombinase activity. It predicts that at least some of the lysine-containing RAD51 paralogs may function to stabilize the RAD51 NPF.

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3.2 INTRODUCTION

Failure to repair DNA double-stranded breaks (DSBs) may result in genomic instability and tumorigenesis (Bernstein and Rothstein 2009; Jackson and Bartek 2009; Ciccia and

Elledge). HR is an error free pathway that utilizes a template strand to restore DSB ends

(Krogh and Symington 2004). A key component in HR mediated repair is RAD51 (West

2003). Deletion of RAD51 in mice results in embryonic lethality, while RAD51 knockdown in chicken DT40 cell lines results in increased chromosomal instability (Tsuzuki et al. 1996; Sonoda et al. 1998). RAD51 forms a presynaptic NPF that catalyzes homologous pairing and strand-exchange. In addition, RAD51 associates with RAD52,

RAD54 and BRCA2 during recombinational repair (San Filippo et al. 2008). Eukaryotic

HR is further tuned by the presence of several RAD51 paralogs, where some have been shown to enhance human RAD51 (HsRAD51) functionality in vitro (Schild et al. 2000;

Masson et al. 2001; Shim et al. 2004).

Despite functional conservation with the prototypical bacterial homolog RecA,

RAD51 requires unusual salt conditions for an efficient strand exchange in vitro (Liu et

+ al. 2004; Shim et al. 2006). For example, the ammonium (NH4 ) cation appears to be most efficient at enhancing RAD51 recombinase activity, yet is unlikely to occur at physiologically relevant conditions (Liu et al. 2004; Shim et al. 2006). Other cations such as potassium (K+) confer significantly reduced activity (Liu et al. 2004; Shim et al.

2006). Cations that enhance recombinase activity appear to induce an NPF that mimics the active extended RecA NPF (Liu et al. 2004). Recombinase-enhancing cations may also promote preferential binding of single-stranded DNA (ssDNA) over double-stranded

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DNA (dsDNA), which influences RAD51 ATPase activity (Tombline and Fishel 2002;

Liu et al. 2004; Shim et al. 2006). Structural analysis of MvRAD51 has revealed cation- induced protein conformational rearrangements at the inter-subunit region that results in an active NPF (Wu et al. 2004; Wu et al. 2005).

An evolutionarily intriguing feature of the RAD51/RecA NPF is the ATP cap located at the adenosine nucleotide-binding interface within the inter-subunit region (Wu et al. 2004; Wu et al. 2005). A proline residue that is conserved in all RecA/RAD51 homologs appears to sandwich the adenine nucleotide at the ATP binding interface (Fig.

3.1A). Five amino acids N-terminal to the conserved proline the RAD51 homologs possess a conserved aspartate residue that forms a salt bridge with the -phosphate of

ATP. In contrast, RecA homologs possess a conserved lysine at the analogous position that forms direct hydrogen bonds with the -phosphate (Fig. 3.1A; star). Importantly, four of the five HsRAD51 paralogs contain lysine at this corresponding position (Fig.

3.1A; star). The biophysical role of the HsRAD51 paralogs during HR is largely unknown.

HsRAD51 paralogs are difficult to purify in significant quantities for structural studies and tend to severely aggregate. Here we examined the structure and function of the conserved HsRAD51-paralog ATP cap lysine residue by constructing a substitution mutation where the conserved HsRAD51(D316) was replaced by lysine

[HsRAD51(D316K)]. We found that the HsRAD51(D316K) substitution enabled the uncomplicated formation and maintenance of an active NPF. As a functional consequence, HsRAD51(D316K) displayed reduced ATP hydrolysis (ATPase), enhanced discrimination of ssDNA versus dsDNA, and significantly enhanced recombinase

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functions. Crystallographic and EM structural analysis indicate that MvRAD51(D302K) and HsRAD51(D316K) form a stable extended NPF in the absence of salt, that mimics salt-induced conformations of the wild type protein. Our results are consistent with the conclusion that the conserved aspartate in the ATP cap functions as a regulatory switch that enhances HsRAD51 NPF turnover, and predict that analogous lysine-containing

HsRAD51 paralogs may function to increase NPF stability. The enhanced stability and recombinase activity of hRAD51(D316K) in physiologically relevant conditions should provide a useful reagent for biochemical studies of HR.

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3.3 MATERIALS AND METHODS

HsRAD51 Protein Expression and Purification- The HsRAD51(D316K) was constructed using PCR mutagenesis using primers 5- ATC TGC AAA ATC TAC AAA TCT CCC

TGT CT and its complement for mutagenesis (lysine encoding codon in bold), and for cloning into pET24d expression vector (Novagen) primers 5- TAT ACC ATG GCA

ATG CAG ATG CAG CTT GAA and 5- TTC GGA TCC TTA TCA GTC TTT GGC

ATC TCC CA were used that contain NcoI and BamHI restriction sites, respectively. For untagged native protein expression stop codons were introduced upstream of BamHI restriction site. Mutation was confirmed by DNA sequencing. HsRAD51wild type and

HsRAD51(D316K) proteins were expressed and purified following previously published protocols (Baumann et al. 1997; Tombline and Fishel 2002). Briefly, HsRAD51 was expressed in E. coli BLR strain and precipitated using spermidine-HCl. Resuspended pellet was purified using Reactive-Blue-4-agarose (Sigma), Heparin sepharose (GE

Healthcare), Hydroxyapatite (Bio-Rad) and Mono Q (GE Healthcare) column chromatography. Purity of the fractions was verified by SDS-PAGE analysis. HsRPA was expressed in BL21(AI) cells using pET11d-tRPA purified as described (Henricksen et al. 1994), except for resuspension of cells, HI buffer containing 30mM HEPES (pH

7.5), 1mM DTT, 0.25mM EDTA, 0.25% (w/v) inositol and 0.01% (v/v) NP-40 was supplemented with 100mM KCl.

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DNA Substrates- X174 single-stranded (ss) virion DNA, replicative form I (RFI) was purchased from NEB. X174 RFIII was obtained by linearizing RFI with ApaLI restriction enzyme and gel purifying with Qiaquick Gel Extraction kit (Qiagen). For surface plasmon resonance (SPR) analysis, a 5′ biotinylated oligo dT50 was used as ssDNA and for dsDNA 5′ biotinylated 50-mer 5′-TCG AGA GGG TAA ACC ACA-

ATT ATT GAT ATA AAA TAG TTT TGG GTA GGC GA was annealed with its complement and purified by HPLC on a Gen-Pak FAX column (Waters). For competition DNA binding experiments an oligo dT50 ssDNA and a 50bp dsDNA were used made by annealing the 50-mer 5'- AGA TCT ATA AAC GCA CCT TTG GAA

GCT TGG AAG TGG GCC GAA TCT CCC CA with its complement followed by

HPLC purification as above. D-loop assay substrates were prepared as described (Van

Komen et al. 2002).

ATPase assay- ATP hydrolysis was measured as previously described (Tombline and

Fishel 2002). 10 L reactions were performed in Buffer A containing 20mM HEPES (pH

7.5), 10% glycerol, 100g/mL BSA, 1mM DTT and 1mM MgCl2 and when indicated,

150mM KCl. Each reaction mixture contained HsRAD51(1M) and 6M (nt or bp) of

X174 ssDNA or dsDNA. Reactions were initiated by the addition of protein and were incubated at 37°C for 1 hr. The reaction was stopped by the addition of 400L 10% activated charcoal supplemented with 10mM EDTA and incubated on ice for 2 hrs.

Following centrifuging for 10 min, 50L duplicate aliquots were taken for counting [32P]

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free phosphate by Cerenkov method. Kinetic parameters were obtained by fitting data into Michaelis-Menten equation using Kaleidagraph software (Synergy).

ATPS/ADP binding assay- Experimental procedure was as previously described

(Tombline and Fishel 2002). Reactions were performed in Buffer A and 150mM KCl, when indicated. HsRAD51(1M) and X174 ssDNA (6M nt) were used with the indicated amount of [-35S]ATPS or [3H]ADP. Following a 30 min incubation at 37°C, reactions were kept on ice until filtered. The reaction mix was added into 4mL of ice cold reaction buffer and filtered through HAWP nitrocellulose filters (Millipore) presoaked in the same buffer. Another 4mL of buffer was used to wash the membrane and the filter was dried for 2 hrs. Radioactivity was counted after an overnight incubation in liquid scintillation fluid.

ADP-ATP exchange assay- Reactions were performed in Buffer A with 150mM KCl.

HsRAD51(1M) and X174 ssDNA (6M nt) was incubated with 3 M of [3H]ADP in a

60L reaction volume at 37°C for 10 min. ADPATP exchange was initiated by addition of cold ATP to a 5mM final concentration. 10L aliquots were withdrawn at indicated time points, filtered and analyzed as in ADP binding.

SPR analysis- Biotinylated DNA was immobilized on a streptavidin-coated chip (GE

Healthcare) for the analysis. Protein binding and dissociation were analyzed at 25°C with

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a 5L/min flow rate on a Biacore 3000 (GE Healthcare). Reactions were performed in

Buffer containing 20mM HEPES (pH 7.5), 10% glycerol, 1mM DTT and 1mM MgCl2 supplemented, 0.005% surfactant P-20 (GE Healthcare), 2.5mM of the indicated

2+ nucleotide and 150mM KCl when indicated. For experiments with Ca , 1mM CaCl2 was used instead of MgCl2. DNA binding was determined by injecting 100nM, 200nM,

400nM, 800nM and 1.6M of protein simultaneously into ssDNA and dsDNA containing flow channels to ensure saturated binding.

SPR data was performed at five different concentrations for each mutant under each set of conditions, and fit to a single exponential decay in order to obtain rate constants for dissociation (koff). The decay curve, ƒoff(t), is given by,

-koff t ƒoff(t) = A(C) e

Where A(C) is the amplitude, which depends on the concentration of HsRAD51; C, and t is the time. In some cases, binding is insufficient and/or dissociation occurs very rapidly, and cannot be accurately determined by SPR. For these cases we simply report not determinable (ND) for the value.

Gel mobility shift assay - HsRAD51 wild type or HsRAD51(D316K) at indicated

32 concentrations, was incubated with P- labeled oligo dT50 (81 nM nt) in buffer containing 20mM HEPES (pH 7.5), 10% Glycerol, 1mM DTT, 1mM MgCl2 supplemented with 2.5mM of ATP and 150mM KCl at 37°C for 15min. Samples were then kept on ice until resolved by 5% nondenaturing PAGE at 10V/cm in 0.3x TBE

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buffer at 4°C. Gels were dried and exposed to PhosphoImager (Molecular Dynamics) screens for imaging.

Competition DNA binding analysis- HsRAD51 wild type or HsRAD51(D316K) (730 nM) was incubated with 32P- labeled oligo dT50 (81 nM nt) and the indicated amounts (in bp) of 50bp cold dsDNA in buffer containing 20mM HEPES (pH 7.5), 10% Glycerol,

1mM DTT, 1mM MgCl2 supplemented with 2.5mM of ATP and when indicated, 150mM

KCl, at 37°C for 15min. Samples were kept on ice until resolved by 5% nondenaturing

PAGE at 10V/cm in TBE buffer at 4°C. Dried gels were exposed to PhosphoImager

(Molecular Dynamics) screens for quantification using ImageQuant software (GE

Healthcare).

D-loop assay- Labeled 90-mer (2.4 M nt) was incubated with HsRAD51(0.8M) in buffer A with 1mM ATP supplemented with the indicated amounts of MgCl2 or CaCl2 at

37°C for 10 min. Reaction was initiated by adding supercoiled pBS-SK(-) DNA (35M bp) and incubated further for 15 min. Samples were deproteinized by addition of 1%

SDS and 1 mg/mL proteinase K to a final concentration and incubated for 15 min at

37°C, mixed with 1/5 volume of gel loading dye and analyzed on 0.9% agarose gel in

TAE buffer, run at 4V/cm at 25°C. D-loops were quantified using ImageQuant software

(Molecular Dynamics) after drying and exposure to PhosphoImager screens.

DNA Strand Exchange Assay- HsRAD51 (5M) and X174 circular ssDNA (30M nt) were pre-incubated in buffer containing 20mM HEPES (pH 7.5), 10% Glycerol, 1mM 125

DTT, 1mM MgCl2 supplemented with 2.5mM of ATP at 37°C for 5 minutes before addition of 150mM of the indicated salt and linear X174 dsDNA (15M bp). Following a additional 5 min incubation HsRPA (2M) was added and the incubation was continued. After 3 hrs samples were deproteinized by addition of 3L of stop buffer containing 2% SDS and 10 mg/mL proteinase K, and analyzed on 0.9% agarose gel in

TAE buffer. Electrophoresis was carried out at 4V/cm at 25°C with 0.1mg/mL ethidium bromide. Gels were analyzed on a gel documentation station (Bio-Rad). For quantification of joint molecules ImageJ software (http://rsbweb.nih.gov/ij) was utilized.

For strand exchange reaction in RecA format, X174 circular ssDNA (5M nt) and linear X174 dsDNA (5M bp) was preincubated with HsRAD51(5M) in buffer containing 20mM HEPES (pH 7.5), 10% Glycerol, 1mM DTT, 1mM MgCl2 supplemented with 2.5mM of ATP and 150mM of the indicated salt (when added) at

37°C for 5 min. After 5 min, HsRPA (2M) was added and analyzed as described above.

Electron microscopy- The typical DNA binding reaction for electron microscopy was performed in 20 μl strand exchange buffer containing either no salt, 100 mM ammonium sulfate or 200 mM potassium chloride, a 5 ng/μl concentration of the substrate DNA

(either ss or ds M13 DNA) and HsRad51 proteins at a 3:1 (nt or bp: protein) molar ratio.

The reactions were carried out at 37C for 15 min. For negative staining, an aliquot of the sample was diluted 1:4 in buffer and immediately adsorbed to glow-charged thin carbon foils for 3 min followed by staining with 2% unbuffered uranyl acetate for 5 min and followed by air drying. For direct mounting, the remaining sample volume was passed

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over 2-ml columns of 6% agarose beads (ABT inc, Burgos Spain) equilibrated with TE buffer (10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA-NaOH) to eliminate unwanted salts and unbound proteins, and fractions enriched for DNA-protein complexes were collected.

Aliquots of the fractions of interest were mixed with a buffer containing spermidine and adsorbed onto copper grids coated with a thin carbon film glow-charged shortly before sample application. After adsorption of the samples for 2–3 min, the grids were dehydrated through a graded ethanol series and rotary shadowcast with tungsten at 10−7 torr. Samples were examined in an FEI T12 TEM and a Philips CM12 TEM equipped with Gatan 2kx2k SC200 CCD cameras at 40 kV (shadowcast samples) or at 80 kV

(stained samples). Dimensions of particles in the images saved from the CCD cameras were analyzed using Digital Micrograph software (Gatan, Inc.). Adobe Photoshop software was used to arrange images into panels for publication.

MvRAD51 protein preparation and crystallization- The recombinant protein was over- expressed in BL21 Rosetta2 (DE3) cells (Novagen). In addition to the D302K mutation, this recombinant protein also carried a N4G mutation and lacked the first three amino acid residues. The soluble protein was purified as reported for the wild-type MvRAD51 protein (Wu et al. 2004; Qian et al. 2006b). In brief, the purification procedure involved steps of polymin P (Sigma) precipitation, high salt extraction and three chromatography steps using heparin (Amersham Biosciences), hydroxyapatite (BioRad) and Sephacryl S-

300 gel filtration (GE Healthcare) columns. The purified MvRAD51(D302K) protein was concentrated to ~30 mg/ml by ultra-filtration.

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The hexagonal MvRAD51(D302K) crystals (P61 space group) were grown by the hanging drop vapor diffusion method. The maximum dimension was 0.2 mm x 0.2 mm x

0.3 mm. The sample contained ~1 mM of concentrated protein and 2 mM of AMP-PNP.

The reservoir solutions contained 4-5% polyethylene glycol (PEG) 3,350, 50 mM MgCl2,

50 mM Tris-HCl buffer at pH 7.9, 0.4 M NaCl and 14% (w/v) sucrose. One crystal was transferred into a stabilizing solution composed of the reservoir solution supplemented with 28% (w/v) sucrose, looped out of the solution, and frozen in a nitrogen cryo-stream at 100 K. The diffraction data set was acquired at the Canadian Light Source beamline

08ID-1 and was processed using the XDS program (Kabsch 1993). The statistics of the diffraction data is listed in Supplementary Table 2.

The previously solved wild-type MvRAD51 model (PDB code 1T4G) was used as the starting model. After initial refinement, the electron density of the side chain of

Lys-302 was clearly visible in the resulting 2Fo – Fc map. The model was then iteratively rebuilt using XtalView (McRee 1999) and refined using CNS. The molecular figures were generated using Pymol (DeLano 2002). The coordinates and structure factors have been deposited in the Protein Data Bank (PDB code 3NTU).

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3.4 RESULTS

HsRAD51(D316K) substitution affects ATPase but not adenosine nucleotide binding or exchange. Comparison of RecA and RAD51 homologs identified a number of conserved residues (Fig. 3.1A). The HsRAD51(D316) residue was examined to test the effect of lysine in the ATP cap that is conserved in RecA and most HsRAD51 paralogs (Fig. 3.1A, red star). We purified the HsRAD51(D316K) protein (Fig. 3.2A) and examined the steady state ATPase activity (Fig. 3.1D; Table 3.1). We found that the ATPase rate was approximately 3-fold lower than the wild type protein. Our results are also consistent with previous studies that have suggested stimulatory cations may modestly enhance the discrimination between ssDNA- and dsDNA-stimulated ATPase (Fig. 3.1E, compare ssDNA to dsDNA ATPase with and without K+; (Liu et al. 2004; Shim et al. 2006).

However, we observe a reduced cation-induced difference in ssDNA and dsDNA stimulated ATPase with HsRAD51(D316K) compared to the wild type protein (Fig.

3.1E).

The difference in HsRAD51(D316K) ATPase compared to the wild type

HsRAD51 was not the result of altered ATP binding activity (Fig. 3.1F; Table 3.1), and there appeared to be no defect associated with ADPATP exchange, although kinetic differences at time points shorter than 30 sec would not have been detected (Fig. 3.2B).

We observed an approximately 4-fold increase in KD for ADP in the presence of ssDNA compared to the wild type HsRAD51 (Fig. 3.1G, Table 3.1). Importantly, the protein preparations appeared fully active since the ATP and ADP maximum binding (Bmax) as well as 1:1 stoichiometry appeared equivalent under a variety of conditions (Fig. 3.1F

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and 3.1G; Table 3.1). These observations suggest that defects in adenosine nucleotide binding and exchange are unlikely to fully account for the reduced HsRAD51(D316K)

ATPase activity.

HsRAD51(D316K) exhibits RPA and salt independent strand exchange activity.

Replication protein A (RPA) facilitates RAD51 mediated strand exchange at both presynaptic and postsynaptic stages (Sugiyama et al. 1997; Eggler et al. 2002; Van

+ Komen et al. 2002). Efficient strand exchange is facilitated by the NH4 cation, which is a characteristic of RAD51 even though such conditions are unlikely to be physiologically relevant (Sigurdsson et al. 2001a; Liu et al. 2004; Shim et al. 2006). We compared the strand exchange activity of wild type HsRAD51 and HsRAD51(D316K) in the presence

+ of HsRPA and NH4 cation (Fig. 3.3). Remarkably, HsRAD51(D316K) appeared significantly more active than wild type HsRAD51 and was capable of catalyzing efficient strand exchange in the absence of HsRPA (Fig. 3.3A). Moreover, we still

+ observed strand exchange products in the absence of both HsRPA and NH4 cation (Fig.

3.3A). These studies suggest that HsRAD51(D316K) may circumvent both the cation and ssDNA binding (SSB) protein requirements of wild type HsRAD51.

We examined the cation requirements of HsRA51(D316K) and observed efficient strand exchange activity in the presence of sodium (Na+) and K+ cations (Fig. 3.3B).

Moreover, peak strand exchange activity with HsRAD51(D316K) appears to occur with

+ + approximately half the NH4 or K cation concentration compared to the wild type

HsRAD51 (Fig. 3.3C).

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The enhanced strand exchange activity and reduced SSB protein requirement of

HsRAD51(D316K) appeared similar to bacterial RecA. The order-of-addition requirements for efficient RecA strand exchange are different from RAD51, the latter of which requires a series of steps that include the pre-formation of an NPF on the ssDNA substrate prior to the addition of salt and then the dsDNA donor. In contrast, all of the reaction components including ssDNA and dsDNA substrates may be included with

RecA, and there is no unusual cation requirement. Remarkably, HsRAD51(D316K) appears to efficiently catalyze strand exchange under RecA order-of-addition in the

+ + presence of K and significantly less efficiently in the presence of NH4 cation (Fig.

3.4A). These studies suggest that HsRAD51(D316K) may catalyze fundamental recombination reactions under more physiologically relevant conditions.

A gel based competition analysis was used to examine the relative binding of

HsRAD51 to ssDNA and dsDNA (Fig. 3.4B and 3.4C). In this system HsRAD51 induces a ssDNA NPF gel shift that can be used in a competition analysis with cold ssDNA or dsDNA competitor (Fig. 3.5A-C). As previously suggested for wild type

HsRAD51, salt (KCl) dramatically decreases the ability of cold dsDNA to compete for labeled ssDNA binding while modestly affecting the ability of cold ssDNA to compete for labeled ssDNA binding (Fig. 3.4B, compare WT and WT-KCl; (Shim et al. 2006). In contrast, the HsRAD51(D316K) ssDNA NPF is largely stable to cold dsDNA competition regardless of salt (KCl) while the ssDNA binding appears further enhanced to cold ssDNA competition in the presence of salt (KCl). These results strongly suggest that the HsRAD51(D316K) naturally displays a substantial discrimination between ssDNA and dsDNA that the wild type HsRAD51 only displays in the presence of salt.

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We also note that the HsRAD51(D316K) mutant protein is unable to catalyze strand exchange in the presence of ATPS, yet performs modest strand exchange in the presence of ADP (Fig. 3.6). Since ATPS binding by wild type HsRAD51 and

HsRAD51(D316K) appear identical, it is likely that this poorly hydrolysable ATP analog induces a conformation that is incompatible with strand exchange activity. Together, these results are consistent with the conclusion that the HsRAD51(D316K) substitution significantly alters the salt requirement for triggering an appropriate adenosine nucleotide-dependent conformational transition necessary for strand exchange (see Fig.

3.1C).

HsRAD51(D316K) affects protein turnover.

RAD51 DNA interaction(s) during pre- and postsynaptic stages is believed to play an important role during HR (Solinger et al. 2002; Chi et al. 2006; Symington and Heyer

2006). We quantitatively examined the DNA binding and dissociation properties of the

HsRAD51 substitution mutant proteins using surface plasmon resonance (SPR) and biotin-streptavidin immobilized dT50 ssDNA or 50bp dsDNA. These studies were performed in physiologically relevant salt (150 mM KCl) with magnesium and ATP similar to strand exchange conditions (see Fig. 3.3). The protein was titrated between

100 nM to 1.6M to examine saturated binding on both ssDNA and dsDNA. After protein injection and NPF formation the sample was analyzed for steady-state protein disassembly (Fig. 3.7).

Previous studies have suggested a multimeric RAD51 binding that would significantly alter the simple concentration dependence of kon (Hilario et al. 2009). A 132

power-dependence of concentration on kon would make any calculations of KD problematic. In contrast the koff behaves as a simple single exponential decay and appears highly accurate for a variety of conditions.

The saturation and shape of the binding curves varied considerably for the wild type HsRAD51 as predicted by previous studies. (Shim et al. 2006). Two general observations emerged: 1.) HsRAD51 does not bind dsDNA in the presence of ADP, and

2.) the HsRAD51 koff increases 2-4 fold in the presence of adenosine nucleotide with the koff•ADP > koff•ATP (Fig. 3.7 and Table 3.2). These results are consistent with previous studies that suggested increased RecA and RAD51 turnover upon ATP hydrolysis

(Kowalczykowski 1991; Bianco et al. 1998; Bugreev and Mazin 2004; Bugreev et al.

2005; Chi et al. 2006).

In contrast, the HsRAD51(D316K) is completely deficient in both ssDNA and dsDNA binding in the absence of adenosine nucleotide (Fig. 3.7). This observation appears to largely mimic the properties of bacterial RecA and yeast Rad51 (Shibata et al.

1979; Pugh and Cox 1987; De Zutter and Knight 1999; Zaitseva et al. 1999). In contrast,

HsRAD51(D316K) binds strongly to ssDNA and dsDNA in the presence of both ADP or

ATP (Fig. 3.7 and Table 3.2). Moreover, the stability of the NPF is increased 7-10 fold

-1 compared to the wild type HsRAD51 in the presence of ATP (koff•ATP•ssDNA = 0.0005 s ;

-1 koff•ATP•dsDNA = 0.0006 s ; Table 3.2). Prolonged analysis confirmed the stability of the

NPF (data not shown). The decreased koff as well as the ability to bind dsDNA in the presence of ADP suggests that the HsRAD51(D316K) mutant protein displays a significantly reduced adenosine nucleotide-induced DNA turnover compared to wild type

HsRAD51.

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HsRAD51(D316K) catalyzes efficient D-loop formation independent of divalent cation.

RAD51 catalyzes the formation of D-loop joint molecules in the presence of calcium

(Ca++) or nonhydrolyzable ATP analogues (Bugreev et al. 2005; Chi et al. 2006).

Moreover, the P-loop substitution mutation of HsRAD51(K133R) that binds ATP but renders the protein ATPase deficient was found to catalyze efficient D-loop formation in the presence of magnesium (Mg++; (Chi et al. 2006). Together these results are consistent with the conclusion that the catalysis of D-loop joint molecules by RAD51 requires the formation and maintenance of an ATP-bound NPF, which can be enhanced by substitution of Ca++ for Mg++ or by using a hydrolysis-deficient HsRAD51 mutation. The intrinsic stability of the HsRA51(D316K) NPF suggested it might be more efficient at catalyzing D-loop formation.

As expected, we found that HsRAD51 catalyzes the formation of D-loops in the absence of salt, but requires Ca++ to induce an active ATP-bound NPF (Bugreev and

Mazin 2004). The yield of D-loops by wild type HsRAD51 in the presence of Mg++ divalent cation was 8.1 fmol (3%) , and 81 fmol (30%) in the presence of Ca++ (Fig. 3.8B, lanes 3 and 4; Fig. 3.8C). Importantly, HsRAD51(D316K) catalyzed the formation of D- loops in the absence of any divalent cation or in the presence of either Mg++ or Ca++ (107 fmol (38%) and 94 fmol (35%) respectively; Fig. 3.8B, lanes 5 and 6; Figs. 3.8C and

3.8D). Kinetic analysis revealed an increase in the initial rate of D-loop formation by

HsRAD51(D316K) (data not shown). D-loop catalysis by HsRAD51(D316K) in the presence of the nonhydrolyzable nucleotide analogue adenosine 5′-(β,γ- imido)triphosphate (AMP-PNP) appeared similar to the ATP-driven catalysis regardless

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of divalent cation cofactor (Fig. 3.8D). Moreover, in the presence of Ca++ the

HsRAD51(D316K) mutant protein appeared capable of efficiently catalyzing D-loop formation with ADP (Fig. 3.8D).

We examined the stability of the wild type HsRAD51 and HsRAD51(D316K) presynaptic NPF on ssDNA in the presence of ATP and either Mg++ or Ca++ (Fig. 3.8E and 3.8F). A significant decrease in koff was observed for wild type HsRAD51 in the presence of Ca++ as well as for HsRAD51(D316K) in the presence of both Ca++ and Mg++

(Fig. 3.8E and 3.8F). These results strongly suggest that HsRAD51(D316K) inherently forms and maintains an active ATP-bound NPF regardless of associated divalent cations.

Collectively these results underline previous conclusions that the ability to form and maintain an active adenosine nucleotide bound NPF conformation drives D-loop formation (Shibata et al. 1979; Cox and Lehman 1981; Bugreev and Mazin 2004).

MvRAD51(D302K) induces and active nucleoprotein filament.

We determined the structure of the MvRAD51(D302K) at 1.9Å resolution (Table 3.3).

The MvRAD51(D302K) protein forms a filament with a helical pitch of 104.3Å, similar to the helical pitch of active wild type MvRadA (104.8 Å; Fig. 3.9A). The L2 single strand binding region (D256 to R285) of MvRAD51(D302K) is largely ordered. The non-hydrolysable ATP analogue, AMP-PNP, is buried between RadA monomers as expected (Fig. 3.9B). The adenosine nucleotide moiety is sandwiched between the ATP cap (residues K302 to D308) and the side chain of R158. The amino group of the K302 side chain forms a total of four hydrogen bonds: one with the terminal phosphate of the

ATP analogue and three with the carbonyls of residues G279, H280 and A282. These

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four groups stabilized a Ca++ cation in a previously reported calcium-containing active form of MvRAD51 (Qian et al. 2006a). The K302 appears to provide further stabilization of the -phosphate in addition to the well-known P-loop residues and Mg++ cation (Wu et al. 2004). Importantly, the K302 side chain replaces both K+ cations including one that normally bridges the wild type D302 with the -phosphate of ATP and additionally coordinates an ordered helix (residues G275 to A282) analogous to helix G of EcRecA (Story and Steitz 1992) (Fig. 3.9C). The H280 side chain makes a direct hydrogen bond with the -phosphate of the ATP analogue and F107 is flipped away from the nucleotide binding pocket as is required to form and active MvRAD51 NPF.

Interestingly, the K248 of the E. coli RecA active nucleoprotein filament is at the analogous position of K302 (Fig. 3.9D)(Chen et al. 2008). The structure of

MvRAD51(D302K) closely resembles the recurrent active form of M. voltae and M. maripaludis RAD51-AMP-PNP complexes (Qian et al. 2005; Wu et al. 2005; Li et al.

2009), but differs from the recurrent inactive form of MvRAD51 (Wu et al. 2004) which has a largely disordered L2 region (compare Fig. 3.9C with Figs. 3.1B and 3.1C).

HsRAD51(D316K) maintains an active nucleoprotein filament in the absence of salt.

To confirm the structural analysis of MvRAD51(D302K) we examined the wild type

HsRAD51 and HsRAD51(D316K) NPFs by electron microscopy (EM) (Fig. 3.9E and

3.9F). These studies were performed in strand exchange conditions with ATP in the

+ + absence of salt or in the presence of equi-normal K or NH4 cations. The wild type

HsRAD51 protein exhibited a 30% increase in the helical pitch with visible striation of

+ + the filament in the presence of K or NH4 (Fig. 3.9E). In contrast, HsRAD51(D316K) 136

maintains an inherently extended conformation (helical pitch of 100-112Å) similar to the

MvRadA(D302K) filament and the salt-induced wild type HsRAD51 filament under all conditions (Fig. 3.9F). These studies are consistent with the biochemical and structural analyses that suggest HsRAD51(D316K) forms and maintains an active adenosine nucleotide bound NPF under a wide range of conditions.

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3.4 DISCUSSION

The ATP cap of RecA/RAD51 homologs is a structurally conserved region that overlays the ATP binding site of an adjacent protomer within an NPF. It contains a highly conserved proline residue that functions to sandwich the adenine moiety (Fig. 3.1A).

RAD51 homologs possess a conserved aspartate residue adjacent to the conserved proline in the ATP cap, while RecA homologs and a large majority of RAD51 paralogs have a lysine at the analogous position (Fig. 3.1A). Both the aspartate and lysine residues ultimately contact the -phosphate of ATP (Wu et al. 2005; Chen et al. 2008). The lysine residue does this directly, while the cation of the aspartate salt bridge mediates the - phosphate contact. Conformational transition(s) of the MvRAD51 F107 and H280 residues have been shown to be associated with the development of the aspartate salt bridge, which together appear necessary to form an active NPF (Wu et al. 2005). The available data suggest that the cation which occupies this salt bridge must have a sufficient ionic radius to provoke the formation and maintenance of an active ATP-bound

NPF (Shim et al. 2006). In its absence or under conditions in which ATP hydrolysis may readily occur, the RAD51 NPF appears largely inactive. Taken together, these results suggest that the aspartate salt bridge plays a significant role in the management and regulation of RAD51 recombinase functions.

In contrast, RecA homologs and RAD51 containing a lysine residue in the analogous position appear to more easily form and maintain an active NPF that translates to enhanced recombinase function(s) (Chen et al. 2008). The majority of RAD51 paralogs contain a lysine residue in this analogous position (Fig. 3.1A). The human

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RAD51 paralogs have been purified in a variety of heteromeric forms (Masson et al.

2001; Sigurdsson et al. 2001b; Shim et al. 2004). However, the quantities of these heteromeric forms and their tendency to aggregate have thus far inhibited a complete examination their biophysical and structural role in HR. The HsRAD51(D316K) substitution mutation has allowed us to examine the effect of a RAD51 protein containing an ATP cap lysine on recombinase functions.

A prediction of this analysis is that RAD51 paralogs containing an ATP cap lysine may function to stabilize the RAD51 filament. Such a Rad51 NPF stabilization has recently been demonstrated for the yeast paralogs Rad55-Rad57 (Liu et al.). It is possible that near stoichiometric quantities of these RAD51 paralogs might be required to induce such stability since they would have to occupy a large fraction of the NPF. Of the human RAD51 paralogs, only HsRAD51B and HsRAD51C appear to be expressed in quantities equivalent or in excess of HsRAD51 (Miller et al. 2002). Interestingly, the

HsRAD51B-HsRAD51C heterodimer appears prevalent in human cells and both contain an ATP cap lysine (Miller et al. 2002). Moreover, both HsRAD51B and HsRAD51C display modest binding to ssDNA and HsRAD51B contains the conserved proline in the

ATP cap region (Lio et al. 2003).

The mechanism of ATP cap regulation. The mechanism associated with ATP cap regulation of RAD51 might be inferred from the MvRAD51 structure (Wu et al. 2005).

In the MvRAD51 K+ cation-induced active NPF structure, the H280 residue formed a hydrogen bond with the -phosphate of the ATP analogue, while the F107 residue was moved out of the ATP binding interface to accommodate two K+ cations (one forming the

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D302 salt bridge). A similarly active MvRAD51 contains an offset calcium that occupies the space of both K+ cations but still forms a salt bridge with the -phosphate (Qian et al.

2006a). Together these observations suggest that activation of MvRAD51 requires salt occupation of the ATP cap and that cations, which disrupt the conformational changes associated with the ATP hydrolytic cycle further enhance activity. The

MvRAD51(K302) and HSRAD51(K316) residues appears to perform these functions naturally, but without the ability to perform post-synaptic turnover.

RPA independence. It has been suggested that RPA enhances strand exchange activity by disrupting ssDNA secondary structure formation and by stabilizing the newly formed joint molecule by binding to the newly released ssDNA (Sugiyama et al. 1997; Mazin and Kowalczykowski 1998). Since HsRAD51(D316K) demonstrates RPA independence in catalyzing strand exchange, it is possible that this protein possesses an enhanced ability to disrupt secondary structures on ssDNA and/or it binds the ssDNA product more efficiently from the triplex joint molecule. Indeed, HsRAD51(D316K) binds ssDNA

~15-fold better that the wild type HsRAD51 in the presence of ATP and ~3-fold better than wild type HsRAD51 in the presence of ADP.

Evolution of aspartate in the ATP cap. An intriguing evolutionary question is why

RAD51 recombinases have selected an aspartate residue in the ATP cap region instead of a lysine found in bacterial RecA? One possibility is that the preservation of the aspartate residue and associated salt bridge fundamentally provides the turnover required during

HR. Perhaps the ability to efficiently recycle HsRAD51 from an expanding presynaptic

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filament during the construction of an active NPF or the release of HsRAD51 from a completed recombination product is more important in eukaryotic genomes? In this scenario, the aspartate residue and associated salt bridge would function as a conformational sensor in delineating active and inactive protomers in the NPF before and/or after HR. Finally, the development of the HsRAD51(D316K) should significantly enhance biochemical studies of HR when physiologically relevant conditions are required.

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3.5 ACKNOWLEDGEMENTS AND FOOTNOTES

Crystallographic work was performed by Yujiong He and Yu Luo at the University of

Saskatchewan, Canada. EM studies were performed by Smaranda Willcox and Jack

Griffith at the University of North Carolina. Model fitting of DNA binding data was performed by Robert Forties and Ralf Bundschuh. This chapter was submitted for publication as: The RAD51 ATP Cap Regulates Nucleoprotein Filament Stability,

Amunugama R., He Y. , Willcox S., Forties R.A., Shim K-S., Bundschuh R., Yu Luo,

Griffith J., and Fishel R.

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Figure 3.1 ATP Binding and Hydrolysis by HsRAD51 ATP Cap Substitution Mutation. (A) Sequence alignment of the ATP cap region of H. sapiens (Hs), S. cerevisiae (Sc), M. voltae (Mv), and E. coli (Ec) recombinases. Sequence alignment of the ATP cap region of HsRAD51 paralogs is indicated below. The analogous position of HsRAD51(D302) is shown with an asterisk. (B) ATP cap region of MvRadA (MvRAD51) structure in the absence of potassium cation (PDB code 1T4G). (C) ATP cap region of MvRAD51 structure in the presence of potassium cation (PDB code 1XU4). Structural figures were generated using Pymol. (D) Steady-state ATPase activity of HsRAD51 wild type or HsRAD51(D316K) with ssDNA in the presence of 150 mM KCl. (E) ATP turnover values (kcat) with ssDNA and dsDNA in the presence and + absence of KCl (K ). kcat values were calculated by Michaelis-Menten analysis. (F) ATPS binding by HsRAD51 wild type or HsRAD51(D316K) in the presence of ssDNA and 150 mM KCl. (G) ADP binding by HsRAD51 wild type or HsRAD51(D316K) in the presence of ssDNA and 150 mM KCl. Error bars indicate standard deviation from at least three independent experiments.

Continued

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Figure. 3.1: continued

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Figure 3.2 Purification of HsRAD51 and ADP-ATP exchange analysis (A) Purification of HsRAD51 WT and HsRAD51(D316K) substitution mutant protein. 1 g of protein analyzed by 12% SDS-PAGE. (B) ADP-ATP exchange HsRAD51 WT and HsRAD51(D316K) mutant protein in the presence of ssDNA and 150 mM KCl.

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Figure 3.3 HsRAD51(D316K) Exhibits Salt and RPA Independent Strand Exchange Activity. (A) Analysis of salt and RPA requirement for strand exchange. Reaction schematic shown above: HsRAD51 wild type or HsRAD51(D316K) (5M) and X174 circular ssDNA (30M nt) were pre-incubated with 2.5mM ATP and 1mM MgCl2 at

37°C for 5 min prior to the addition of 150mM NaNH4HPO4 (if indicated) and linear X174 dsDNA (15M bp). After 5 min, HsRPA (2M) was added (if indicated) and the incubation was continued for 3 hrs. Samples were deproteinized and analyzed on 0.9% agarose gel with 0.1g/mL ethidium bromide. (B) Strand exchange activity of HsRAD51 and HsRAD51(D316K) as a function of cation normality. HsRAD51 (5M) and X174 circular ssDNA (30M nt) were pre-incubated with 2.5mM ATP and 1mM MgCl2 at 37°C for 5 min before addition of indicated amounts of salt and linear X174 dsDNA (15M bp). After another 5 min incubation HsRPA (2M) was added and the incubation was continued. After 3 hrs samples were deproteinized and analyzed on 0.9% agarose gel with 0.1g/mL ethidium bromide. (C) Quantification of the joint molecules in B.

Continued

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Figure 3.3: continued

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Figure 3.4 HsRAD51(D316K) Preferentially Binds to Single Stranded DNA. (A) Strand Exchange in RecA format. X174 circular ssDNA (5M nt) and linear X174 dsDNA (5M bp) was preincubated with HsRAD51 wild type or HsRAD51(D316K) (5μM) in buffer containing 20mM HEPES (pH 7.5), 10% Glycerol, 1mM DTT, 1mM

MgCl2 supplemented with 2.5mM of ATP and 150mM KCl at 37°C for 5 min. After 5 min, HsRPA (2M) was added and the incubation was continued. After 3 hrs samples were deproteinized and analyzed on 0.9% agarose gel with 0.1μg/mL ethidium bromide. (B) Quantification of competition DNA binding analysis (see Supp.Fig. 2) of HsRAD51 wild type and HsRAD51(D316K). HsRAD51 wild type or HsRAD51(D316K) (730 nM) 32 was incubated with P- labeled oligo dT50 (81 nM nt) and the indicated amounts of 50bp cold competitor dsDNA in buffer containing 20mM HEPES (pH 7.5), 10% Glycerol,

1mM DTT, 1mM MgCl2 supplemented with 2.5mM of ATP and when indicated, 150mM KCl, at 37°C for 15min. Samples were kept on ice until resolved by 5% nondenaturing PAGE at 10V/cm in TBE buffer at 4°C. Dried gels were exposed to PhosphoImager screens for quantification. (C) Competition DNA binding with oligo dT50 cold competitor DNA. Reaction conditions are similar to B.

Continued

148

Figure 3.4: continued

149

Figure 3.5 Competition DNA binding by HsRAD51 and HsRAD51(D316K). (A) HsRAD51 wild type or HsRAD51(D316K) at indicated concentrations, was incubated 32 with P- labeled oligo dT50 (81 nM) in buffer containing 20mM HEPES (pH 7.5), 10%

Glycerol, 1mM DTT, 1mM MgCl2 supplemented with 2.5mM of ATP and 150mM KCl at 37°C for 15min. Samples were then kept on ice until resolved by 5% nondenaturing PAGE at 10V/cm in TBE buffer at 4°C. A distinct ssDNA-HsRAD51(D316K) complex formed is indicated with an asterisk. (B) HsRAD51 wild type or HsRAD51(D316K) (730 32 nM) was incubated with P- labeled oligo dT50 (81 nM) and the indicated amounts of 50bp cold competitor dsDNA in buffer containing 20mM HEPES (pH 7.5), 10%

Glycerol, 1mM DTT, 1mM MgCl2 supplemented with 2.5mM of ATP and when indicated, 150mM KCl, at 37°C for 15min. Samples were kept on ice until resolved by 5% nondenaturing PAGE at 10V/cm in TBE buffer at 4°C. Dried gels were exposed to PhosphoImager screens for quantification. (C) Competition DNA binding with oligo dT50 cold competitor DNA. Reaction conditions are similar to B.

Continued

150

Figure 3.5: continued

151

Figure 3.6 Strand Exchange Activity of HsRAD51 and HsRAD51(D316K) with Different Adenosine Nucleotides. HsRAD51 (5μM) and X174 circular ssDNA (30μM nt) were pre-incubated in buffer containing 20mM HEPES (pH 7.5), 10% Glycerol, 1mM

DTT, 1mM MgCl2 with 2.5mM of indicated adenosine nucleotide at 37°C for 5min before addition of 150mM NaNH4HPO4 and linear X174 dsDNA (15μM bp). After another 5 min incubation HsRPA (2μM) was added and the incubation was continued. After 3 hrs samples were deproteinized and analyzed on 0.9% agarose gel with 0.1mg/mL ethidium bromide.

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Figure 3.7 HsRAD51(D316K) Displays a Slow Turnover from ssDNA and dsDNA. (A) ssDNA binding analysis of wild type and HsRAD51(D316K) by surface Plasmon resonance (SPR, Biacore) in the presence of the indicated nucleotide. Association and dissociation curves correspond to 100nM, 200nM, 400nM, 800nM and 1.6M of protein. Fitted cures are overlaid in black lines. (B) dsDNA binding analysis of wild type and HsRAD51(D316K) in the presence of the indicated nucleotide. Protein concentrations are similar to A.

Continued

153

Figure 3.7: continued

154

Figure 3.8 HsRAD51(D316K) Catalyzes D-loop Formation in the Presence of ATP and Magnesium. (A) Reaction schematic. (B) 0.8M of HsRAD51 wild type or HsRAD51(D316K) and [P32]-labeled ssDNA (90mer; 2.4 M nt) were preincubated for

10 min at 37°C in the reaction buffer containing 1 mM ATP and 1mM MgCl2 or CaCl2. Reactions were initiated by the addition of supercoiled pBS SK(-) plasmid DNA (35M bp). After 15 min reactions were terminated by the addition of deproteinization solution and processes similar to Fig. 2. Joint molecules (JMs) were analyzed on a 0.9% agarose gel. (C) Quantification of JMs in B. Error bars indicate standard deviation from at least three independent experiments. (D) D-loop formation analysis with different nucleotides. 0.8M of HsRAD51 wild type or HsRAD51(D316K) and 2.4 M (nt) of P32- labeled ssDNA (90mer) were preincubated for 10 min at 37°C in the reaction buffer containing

1mM of the indicated nucleotide and 1mM MgCl2 , CaCl2 or without a cofactor . Reactions were initiated and analyzed as described in B. (E) ssDNA binding analysis of wild type HsRAD51 using SPR in the presence of magnesium (Mg2+-) ATP and calcium (Ca2+-) ATP. (F) ssDNA binding analysis of HsRAD51(D316K) in the presence of magnesium (Mg2+-) ATP and calcium (Ca2+-) ATP.

Continued

155

Figure 3.8:continued

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Figure 3.9 Structural comparison of MvRAD51 and RAD51 ATP Cap AspartateLysine Substitution Mutation. (A) Superimposed filament assembly of active wild type MvRAD51 structure in the presence of potassium (K+) cations (cyan) (PDB code 1XU4) and MvRAD51(D302K) (red) (PDB code 3NTU). Helical pitch is indicated with the respective color. (B) Enlarged view of the intersubunit ATP binding region of active wild type MvRAD51 and MvRAD51(D302K). Relative location of AMP-PNP-Mg2+-2K+ of the wild type MvRAD51 (blue) and the AMP-PNP-Mg2+ of the MvRAD51(D302K) (green) are shown. (C) ATP cap region of MvRadA(D302K). F129 and H280 are in active form conformation. - phosphate group of ATP analog forms a direct interaction with -amino group of K302. (D) Active filament form of E.coli RecA - K248 of the ATP cap forms a direct contact with the ALF4 group of the ATP analog (PDB code 3CMU). Electron microscopy (EM) analysis of nucleoproteins filaments of wild type HsRAD51 (E) and HsRAD51(D316K) (F) with ssDNA. Reaction conditions and product visualization are described in Experimental Procedures. Reaction conditions are indicated above each EM image and the corresponding helical pitch is indicated below. Insets indicate enlarged images of the helical configuration. Continued

157

Figure 3.9: continued

158

Table 3.1 Summary of ATP hydrolysis and nucleotide binding data of HsRAD51 wild type (WT) and HsRAD51(D316K) mutant protein.

Kinetic Parameter WT D316K -1 kcat (min ) ssDNA 0.180 ± 0.004 0.070 ± 0.005 ssDNA, KCl 0.190 ± 0.004 0.063 ± 0.005

dsDNA 0.167 ± 0.002 0.060 ± 0.007 dsDNA, KCl 0.135 ± 0.007 0.052 ± 0.005

Km (M) ssDNA 17.48 ± 3.25 3.08 ± 0.1 ssDNA, KCl 9.34 ± 1.42 2.14 ± 0.002

dsDNA 6.54 ± 0.67 2.17 ± 0.15 dsDNA, KCl 5.52 ± 0.26 1.70 ± 0.003

KD (M) ATPS ssDNA 2.49 ± 0.46 0.66 ± 0.21 ssDNA, KCl 1.18 ± 0.22 0.72 ± 0.21

Bmax (M) ATPS ssDNA 1.02 ± 0.02 0.90 ± 0.03 ssDNA, KCl 0.83 ± 0.04 0.87 ± 0.05

KD (M) ADP ssDNA, KCl 1.24 ± 0.32 0.26 ± 0.06 Bmax (M) ADP ssDNA, KCl 0.82 ± 0.05 0.86 ± 0.02

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Table 3.2 Dissociation rate constants of HsRAD51 wild type (WT) and HsRAD51(D316K) ssDNA and dsDNA interactions*

WT (s-1) D316K (s-1) ssDNA,no NTa 0.0014 NDb ssDNA, ADP 0.0070 0.0027 ssDNA, ATP 0.0053 0.0005 dsDNA,no NT 0.0017 ND dsDNA, ADP ND 0.0004 dsDNA, ATP 0.0041 0.0006

*The statistical error of fitting was less than 0.03 for each measure. However, binding drift suggested accuracy of ±10%. a NT- Nucleotide b ND- Not determinable as a result of low binding

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Table 3.3 X-ray crystallographic data and Structure refinement statistics

Data collection

Space group P61 Cell dimensions a, b, c (Å) 85.0, 85.0, 104.3  (°) 90, 90, 120 Resolution (Å) 50.0 - 1.9 (1.98 - 1.90)*

Rsym 0.049 (0.278) I / I 27.1 (10.0) Completeness (%) 99.1 (98.6) Unique Reflections 33350 (3828) Redundancy 9.5 (9.3) Refinement Resolution (Å) 50.0 – 1.90 No. reflections 33350

Rwork / Rfree 0.188 / 0.214 No. atoms 2501 Protein 2375 Ligand/ion 34 Water 92 B-factors 37.6 Protein 37.7 Ligand/ion 27.6 Water 37.7 R.m.s. deviations Bond lengths (Å) 0.015 Bond angles (°) 1.73

*Values in parentheses are for highest-resolution shell.

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3.5 REFERENCES

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Li Y, He Y, Luo Y. 2009. Conservation of a conformational switch in RadA recombinase from Methanococcus maripaludis. Acta Crystallogr. D. Biol. Crystallogr. 65: 602- 610. Lio YC, Mazin AV, Kowalczykowski SC, Chen DJ. 2003. Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro. J. Biol. Chem. 278: 2469-2478. Liu Y, Stasiak AZ, Masson JY, McIlwraith MJ, Stasiak A, West SC. 2004. Conformational changes modulate the activity of human RAD51 protein. J. Mol. Biol. 337: 817-827. Masson JY, Stasiak AZ, Stasiak A, Benson FE, West SC. 2001a. Complex formation by the human RAD51C and XRCC3 recombination repair proteins. Proc. Natl. Acad. Sci. U. S. A. 98: 8440-8446. Masson JY, Tarsounas MC, Stasiak AZ, Stasiak A, Shah R, McIlwraith MJ, Benson FE, West SC. 2001b. Identification and purification of two distinct complexes containing the five RAD51 paralogs. Genes Dev. 15: 3296-3307. Mazin AV, Kowalczykowski SC. 1998. The function of the secondary DNA-binding site of RecA protein during DNA strand exchange. EMBO J. 17: 1161-1168. McRee DE. 1999. XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125: 156-165. Miller KA, Yoshikawa DM, McConnell IR, Clark R, Schild D, Albala JS. 2002. RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51. J. Biol. Chem. 277: 8406-8411. Pugh BF, Cox MM. 1987. Stable binding of recA protein to duplex DNA. Unraveling a paradox. J. Biol. Chem. 262: 1326-1336. Qian X, He Y, Ma X, Fodje MN, Grochulski P, Luo Y. 2006a. Calcium stiffens archaeal Rad51 recombinase from Methanococcus voltae for homologous recombination. J. Biol. Chem. 281: 39380-39387. Qian X, He Y, Wu Y, Luo Y. 2006b. Asp302 determines potassium dependence of a RadA recombinase from Methanococcus voltae. J. Mol. Biol. 360: 537-547. Qian X, Wu Y, He Y, Luo Y. 2005. Crystal structure of Methanococcus voltae RadA in complex with ADP: hydrolysis-induced conformational change. Biochemistry (Mosc). 44: 13753-13761. San Filippo J, Sung P, Klein H. 2008. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77: 229-257. Schild D, Lio Y-c, Collins DW, Tsomondo T, Chen DJ. 2000. Evidence for Simultaneous Protein Interactions between Human Rad51 Paralogs. J. Biol. Chem. 275: 16443- 16449. Shibata T, Cunningham RP, DasGupta C, Radding CM. 1979. Homologous pairing in genetic recombination: complexes of recA protein and DNA. Proc. Natl. Acad. Sci. U. S. A. 76: 5100-5104. Shim KS, Schmutte C, Tombline G, Heinen CD, Fishel R. 2004. hXRCC2 enhances ADP/ATP processing and strand exchange by hRAD51. J. Biol. Chem. 279: 30385-30394. Shim KS, Schmutte C, Yoder K, Fishel R. 2006. Defining the salt effect on human RAD51 activities. DNA Repair (Amst) 5: 718-730.

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Sigurdsson S, Trujillo K, Song B, Stratton S, Sung P. 2001a. Basis for avid homologous DNA strand exchange by human Rad51 and RPA. J. Biol. Chem. 276: 8798-8806. Sigurdsson S, Van Komen S, Bussen W, Schild D, Albala JS, Sung P. 2001b. Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange. Genes Dev. 15: 3308-3318. Solinger JA, Kiianitsa K, Heyer WD. 2002. Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments. Mol. Cell 10: 1175-1188. Sonoda E, Sasaki MS, Buerstedde JM, Bezzubova O, Shinohara A, Ogawa H, Takata M, Yamaguchi-Iwai Y, Takeda S. 1998. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J. 17: 598-608. Story RM, Steitz TA. 1992. Structure of the recA protein-ADP complex. Nature 355: 374-376. Sugiyama T, Zaitseva EM, Kowalczykowski SC. 1997. A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. J. Biol. Chem. 272: 7940-7945. Symington LS, Heyer WD. 2006. Some disassembly required: role of DNA translocases in the disruption of recombination intermediates and dead-end complexes. Genes Dev. 20: 2479-2486. Tombline G, Fishel R. 2002. Biochemical characterization of the human RAD51 protein. I. ATP hydrolysis. J. Biol. Chem. 277: 14417-14425. Tombline G, Shim KS, Fishel R. 2002. Biochemical characterization of the human RAD51 protein. II. Adenosine nucleotide binding and competition. J. Biol. Chem. 277: 14426-14433. Tsuzuki T, Fujii Y, Sakumi K, Tominaga Y, Nakao K, Sekiguchi M, Matsushiro A, Yoshimura Y, MoritaT. 1996. Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. U. S. A. 93: 6236-6240. Van Komen S, Petukhova G, Sigurdsson S, Sung P. 2002. Functional cross-talk among Rad51, Rad54, and replication protein A in heteroduplex DNA joint formation. J. Biol. Chem. 277: 43578-43587. West SC. 2003. Molecular views of recombination proteins and their control. Nat Rev Mol Cell Biol 4: 435-445. Wu Y, He Y, Moya IA, Qian X, Luo Y. 2004. Crystal structure of archaeal recombinase RADA: a snapshot of its extended conformation. Mol. Cell 15: 423-435. Wu Y, Qian X, He Y, Moya IA, Luo Y. 2005. Crystal structure of an ATPase-active form of Rad51 homolog from Methanococcus voltae. Insights into potassium dependence. J. Biol. Chem. 280: 722-728. Zaitseva EM, Zaitsev EN, Kowalczykowski SC. 1999. The DNA binding properties of Saccharomyces cerevisiae Rad51 protein. J. Biol. Chem. 274: 2907-2915.

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CHAPTER 4

Human RAD51B-RAD51C Paralog Heterodimer Complex Enhances and Stabilizes

RAD51 Nucleoprotein Filament for D-loop Formation

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4.1 ABSTRACT

There are five RAD51 related proteins, RAD51B, RAD51C, RAD51D, XRCC2 and

XRCC3, termed RAD51 paralogs, that appear to enhance RAD51 mediated homologous recombinational (HR) repair of DNA double strand breaks (DSBs). Here we model the structures of human RAD51, RAD51B and RAD51C and show similar domain orientations within a hypothetical nucleoprotein filament (NPF). We then demonstrate that RAD51B-RAD51C heterodimer forms stable complex on ssDNA and partially stabilizes the RAD51 NPF against the anti-recombinogenic activity of BLM. RAD51B-

RAD51C also appears to stimulate RAD51 mediated D-loop formation in the presence of

RPA. However, RAD51B-RAD51C does not facilitate RAD51 nucleation on a RPA coated ssDNA. These findings provide further insight into RAD51 paralog involvement during presynaptic and synaptic phases of HR.

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4.2 INTRODUCTION

Homologous recombination (HR) ensures genomic stability during DNA double strand breaks (DSBs) caused by ionizing radiation (IR) and stalled or collapsed replication machinery (Symington 2002; Krogh and Symington 2004; Ciccia and Elledge 2010). In eukaryotes, RAD51 functions as the main recombinase in HR (Symington 2002; West

2003; Krogh and Symington 2004; San Filippo et al. 2008). However, unlike its robust bacterial counterpart RecA, RAD51 exhibits an inefficient homologous pairing and strand exchange activity in vitro (Liu et al. 2004; Shim et al. 2006; San Filippo et al.

2008). This observation would appear to necessitate the requirement of accessory proteins to facilitate efficient RAD51 mediated DSB repair by HR (San Filippo et al.

2008; Heyer et al. 2010).

In yeast, Rad55 and Rad57 display a limited homology to Rad51 (termed Rad51 paralogs; (Symington 2002; Krogh and Symington 2004) and appear to have evolved from gene duplication events (Lin et al. 2006). Mutations of either Rad55 or Rad57 lead to IR sensitivity that can be overcome by overexpression of Rad51 or expression of

Rad51 gain-of-function mutants that possess a higher affinity for DNA (Hays et al. 1995;

Johnson and Symington 1995; Fortin and Symington 2002). The Rad55 and RAD57 proteins were shown to form a stable heterodimer (Sung 1997). Moreover, the inclusion of Rad55-Rad57 during strand exchange reactions relieves the inhibitory effect RPA when Rad51 and RPA are introduced simultaneously with ssDNA (Sung 1997).

Five RAD51 paralogs, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have been identified in vertebrates that share 20-30% homology with RAD51 (Albala et al.

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1997; Rice et al. 1997; Cartwright et al. 1998; Dosanjh et al. 1998; Pittman et al. 1998;

Thompson and Schild 1999). All five RAD51 paralogs contain canonical Walker A/B domains and all exhibits DNA depended ATPase activities, although to a lesser extent compared to RAD51 (Braybrooke et al. 2000; Sigurdsson et al. 2001; Lio et al. 2003;

Shim et al. 2004). These paralogs exist in a variety of heterodimer complexes in vivo and in vitro that include RAD51B-RAD51C, RAD51D-XRCC2, RAD51B-RAD51C-

RAD51D-XRCC2 and RAD51C-XRCC3. Both XRCC3 and RAD51C have been shown to interact with RAD51 (Liu et al. 1998; Schild et al. 2000), although the RAD51C-

RAD51 interaction appears to be weak (Liu et al. 1998).

Mutations of most RAD51 paralog genes render cells susceptible to DSB causing agents. For example, Chinese hamster cell lines irs1 and irs1SF defective in XRCC2 and

XRCC3, respectively, are sensitive to IR and DNA crosslinking agents (Tebbs et al. 1995;

Liu et al. 1998). These cells also display chromosomal aberrations and missegregation

(Cui et al. 1999; Griffin et al. 2000). Knock out of XRCC2, RAD51D and RAD51B in mice lead to embryonic lethality (Shu et al. 1999; Deans et al. 2000; Pittman and

Schimenti 2000); a phenotype that is similar to RAD51 disruption (Tsuzuki et al. 1996;

Sonoda et al. 1998). In addition, a uterine leiomyoma contains a translocation in the intronic region of RAD51B that leads to premature termination of its transcript (Ingraham et al. 1999; Schoenmakers et al. 1999). Six monoallelic mutations within RAD51C have been shown to confer susceptibility to breast and ovarian cancer (Meindl et al. 2010), while one biallelic mutation caused abnormalities associated with Fanconi anemia (FA)

(Levy-Lahad 2010; Vaz et al. 2010). The intricate involvement of RAD51C in the FA

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pathway led to its provisional assignment as FANCO, the 14th FA complementation group (Kee and D'Andrea 2010).

RAD51B-RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-

XRCC3 complexes have been shown to bind ssDNA and, dsDNA with 3- or 5-tails, dsDNA with gapped regions and branched DNA structures (Masson et al. 2001a; Masson et al. 2001b; Sigurdsson et al. 2001; Compton et al. 2010). Previously it was demonstrated that RAD51B-RAD51C functions as a mediator during RAD51 catalyzed three-strand exchange assays by reducing the competition between RPA and RAD51 for ssDNA binding (Sigurdsson et al. 2001).

Here we demonstrate that modeled structures of human RAD51, RAD51B and

RAD51C appear capable of forming filaments with similar domain orientations.

Furthermore, RAD51B-RAD51C heterodimer partially stabilizes the RAD51 NPF against the anti-recombinogenic activity of BLM. At sub-stoichiometric concentrations,

RAD51B-RAD51C appears to enhance D-loop formation in the presence of RPA.

Interestingly, in contrast to its role in alleviation of inhibitory effect of RPA on three- strand exchange (Sigurdsson et al. 2001), RAD51B-RAD51C does not appear to facilitate RAD51 nucleation on an RPA coated ssDNA during D-loop formation in vitro.

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4.3 MATERIALS AND METHODS

Structure modeling and sequence alignments- Human RAD51, RAD51B and RAD51C structures were modeled using composite iterative threading assembly refinement software I-TASSER (Roy et al. 2010) without additional restraints and the optimized structures were superimposed on the Methanococcus voltae RadA (MvRAD51) crystal structure coordinates ( (Wu et al. 2005) PDB ID: 1XU4) using Pymol molecular imaging software (DeLano 2002). Protein sequence alignments were done using ClustalW2

(http://www.ebi.ac.uk/Tools/msa/clustalw2/)

Proteins expression and purification- Human RAD51B and RAD51C were cloned into pFast-Bac vector (Invitrogen) from cDNA. RAD51B carried a N-terminal six-histidine tag. 2 L of infected SF9 insect cells (Invitrogen) were harvested after 48 hours and resuspended in Buffer P containing 25 mM Tris-Cl (pH 8.0), 300 mM NaCl, 10mM imidazole, 10% Glycerol and protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride,

1 ug/mL leupeptin, 1 ug/mL pepstatin, 1 ug/mL apropitin). The resuspended pellet was flash frozen in liquid nitrogen and stored at -80 °C until purification.

Cell pellet was thawed on ice, sonicated a passed through a 21-guage needle. The insoluble material was separated by centrifugation at 41000 rpm at 4 °C for 1 hr in a

Beckman-Coulter Ti70 rotor). The soluble material was immediately loaded on NiSO4 charged 3 mL Ni-NTA (Nitrilotriacetic acid) Superflow (Qiagen) equilibrated with buffer

P. Column was washed with 10-column volumes of the same buffer followed by 10- column volumes of Buffer P containing 20 mM imidazole. RAD51B-RAD51C complex

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was eluted in a 50 mL linear gradient from 20 mM - 250 mM imidazole in Buffer P.

RAD51B-RAD51C containing fractions were dialyzed overnight into Buffer R containing 25 mM Tris-Cl (pH 8.0), 100 mM NaCl, 1 mM DTT, 0.5 mM EDTA and 10%

Glycerol. Dialyzed fraction was loaded onto a 10 mL SP-sepharose column (GE

Healthcare) equilibrated with buffer R and the flow through was collected loaded on a 10 mL Q-sepharose column equilibrated in the same buffer. After a 5-column volume wash

RAD51B-RAD51C was eluted in a linear gradient from 100 mM - 600 mM NaCl in

Buffer R. Pooled fractions were dialyzed back into Buffer R with 100 mM NaCl and loaded on a 10 mL Heparin sepharose (GE Healthcare) equilibrated with Buffer R washed with 5-column volumes of the same buffer. RAD51B-RAD51C was eluted in a linear gradient from 100 mM - 800 mM NaCl. Peak fractions containing RAD51B-

RAD51C were concentrated using an Amicon ultra-15 (Millipore) centrifugal filter unit at 4 °C. Concentrated fraction was dialyzed into storage buffer containing 25 mM Tris-

Cl (pH 7.5), 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 10% Glycerol and flash frozen in liquid nitrogen in small aliquots and stored in -80 °C.

Human RAD51 and RPA were expressed and purified as described in Chapter 2

(ref.(Amunugama and Fishel 2011) and Chapter 3). BLM was purified as described

(Bhattacharyya et al. 2009) with an additional chromatographic step on S-sepharose (GE

Healthcare).

DNA Substrates- For surface plasmon resonance (SPR) analysis, a 5′ biotinylated oligo dT50 was used as ssDNA and for dsDNA 5′ biotinylated 50-mer 5-TCG AGA GGG TAA

ACC ACA- ATT ATT GAT ATA AAA TAG TTT TGG GTA GGC GA was annealed

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with its complement and purified by HPLC on a Gen-Pak FAX column (Waters). D-loop assay substrates were prepared as described (Van Komen et al. 2002).

SPR analysis- Biotinylated DNA was immobilized on a streptavidin-coated chip (GE

Healthcare). Binding and dissociation were analyzed following the injection of the indicated amounts of protein at 25°C with a 5 L/min flow rate on a Biacore 3000 (GE

Healthcare). Reactions were performed in Buffer containing 25 mM Tris-OAc (pH 7.5),

1 mM ATP, 1 mM Mg(OAc)2, 2 mM DTT and 0.005% Tween-20.

D-loop assays- RAD51 (1M) was bound with RAD51B-RAD51C (at the indicated concentrations) on 90mer ssDNA (3 M nt) by incubating at 37 °C for 15 min in reaction buffer containing 25 mM Tris-OAc (pH 7.5), 1 mM ATP, 1 mM Mg(OAc)2, 1 mM

CaCl2, 2 mM DTT and BSA (100g/mL). After 15 min, RPA (200 nM) was added (as indicated) and D-loop formation was initiated by the addition of supercoiled dsDNA (50

M bp) and incubation at 37 °C for 15 min. D-loops were analyzed by electrophoresis on a 0.9% agarose gel after deproteinization. Gel was dried and exposed to a

PhosphoImager (Molecular Dynamics) screen for quantification.

Inhibition of D-loop formation- RAD51 (1 M) filaments were formed with RAD51B-

RAD51C (31.2 nM) if indicated, on 90-mer ssDNA (3 M nt) by incubating at 37 °C for

15 min in reaction buffer containing 25 mM Tris-OAc (pH 7.5), 1 mM ATP, 1 mM

Mg(OAc)2, 2 mM DTT, BSA (100 g/mL), 20 mM phosphocreatine and creatine

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phosphokinase (30 U/mL). After the 10 min incubation BLM at indicated concentrations and RPA (200 nM) were added and incubated for an additional 10 min at 37 °C. 1 mM

CaCl2 was then added and followed by another 10 min incubation at 37°C. D-loop formation was initiated by the addition of supercoiled dsDNA (50 M bp) and incubation at 37°C for 15 min. D-loops were analyzed by electrophoresis on a 0.9% agarose gel after deproteinization. Gel was dried and exposed to a PhosphoImager (GE Healthcare) screen for quantification.

ATPase assays- RAD51 (1 M) and RAD51B-RAD51C at the indicated concentrations with oligo dT50 (3 M nt), X174 ssDNA (3 M nt) or X174 dsDNA (3 M bp) in reaction buffer containing 25 mM Tris-OAc (pH 7.5), 1 mM Mg(OAc)2, 2 mM DTT,

BSA (100 g/mL) and 200 M ATP supplemented with 200 nCi of [-32P] ATP for 1 hr at 37°C. The reaction was stopped by the addition of 400 μL 10% activated charcoal supplemented with 10 mM EDTA and incubated on ice for 2 hrs. Following centrifuging for 10 min, 50 μL duplicate aliquots were taken for counting [32P] free phosphate by

Cerenkov method (Tombline and Fishel 2002). Hydrolyzed ATP percentage is indicated.

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4.4 RESULTS

Homology based modeled structures of RAD51, RAD51B and RAD51C indicate similar structural and functional domain orientations.

The crystal structure of human RAD51 or any of its paralogs has not been solved.

Utilizing a composite iterative threading assembly refinement technique (Roy et al.

2010), structures of RAD51, RAD51B and RAD51C were modeled. The predicted three-dimensional structures were based on the archaebacterial RadA structure solved in the presence of K+ (Wu et al. 2005). Structural alignments indicated similar orientations of the ATP cap and the ssDNA binding L2 region (Fig. 4.1). The RAD51B predicted structure contains an extra disordered region within L2 region (Fig. 4.1Band Fig. 4.2).

Interestingly, the RAD51(D316) residue aligns in an orientation that seems to coordinate with the K+ ion that functions as a salt bridge between the ATP cap of RadA and the ATP analog (Fig. 4.1A). In both RAD51B and RAD51C, the analogous residues to

RAD51(D316), RAD51B(K324) and RAD51C(K328), respectively, align at a position that would allow direct interaction with the -phosphate group of the ATP-analog (Fig.

4.1B and Fig. 4.1C). These structural predictions and alignments provide intriguing insights into the potential role of the ATP cap domain of RAD51, RAD51B and RAD51C proteins and how these proteins might collectively form a functional NPF.

RAD51B- RAD51C heterodimer forms stable complexes on ssDNA and dsDNA.

RAD51, RAD51B, RAD51C and the RAD51B-RAD51C heterodimer complex have been shown to bind ssDNA, DNA substrates with tails and dsDNA (Benson et al. 1994;

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Sigurdsson et al. 2001; Lio et al. 2003). The dsDNA binding of RAD51B and RAD51C appears less avid than RAD51 (Benson et al. 1994; Sigurdsson et al. 2001; Lio et al.

2003). We wanted to investigate the RAD51B-RAD51C heterodimer turnover from

DNA and compare it to the turnover properties of RAD51 to ascertain complex stability.

We employed real-time DNA binding/dissociation analysis using surface plasmon resonance (SPR). These studies were performed in 25mM KCl and 100mM KCl to determine the ionic conditions that allow RAD51B-RAD51C binding to DNA.

RAD51 displayed comparable levels of ssDNA and dsDNA binding in both conditions (Fig. 4.3, time scale 100s-300s). However, RAD51 turnover from ssDNA increased at the higher salt concentration (Fig. 4.3, time scale 300s-600s). The increased turnover can be attributed to the salt induced enhancement of the ATPase activity of the recombinase (Liu et al. 2004; Shim et al. 2006); a common manifestation of the RecA/

RAD51 family of proteins (Kowalczykowski 1991; San Filippo et al. 2008).

Interestingly, RAD51B-RAD51C binding to both ssDNA and dsDNA was significantly inhibited in 100 mM KCl (Fig. 4.3A and Fig.4.3B). The inhibition reduced dsDNA binding to nearly the control level (Fig. 4.3B). However, an intriguing feature emerged from RAD51B-RAD51C dissociation pattern. Even though the binding was comparably less than that of RAD51, the bound RAD51B-RAD51C appeared stably associated with the DNA, indicating little or no turnover. This feature was evident in both 25 mM and

100 mM salt on the ssDNA and 25 mM salt on the dsDNA (Fig. 4.3A and Fig.4.3B).

Collectively, these results indicate that RAD51B-RAD51C may form stable complexes on DNA.

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RAD51B-RAD51C enhances RAD51 catalyzed D-loop formation in the presence of RPA at sub-stoichiometric amounts.

We then investigated the D-loop stimulatory effect of RAD51B-RAD51C catalyzed by

RAD51 and RPA (Fig. 4.4A). Previous studies of RAD51 paralog stimulated strand exchange reactions catalyzed by RAD51 were observed when the paralogs were used at sub-stoichiometric amounts (Sung 1997; Sigurdsson et al. 2001). In the D-loop formation we incubated RAD51 and varying amounts of RAD51B-RAD51C with ssDNA in D-loop formation buffer containing Ca++ (Fig. 4.4), since this divalent cation is a requirement for human RAD51 catalyzed D-loop formation (See figure 4.6B; (Bugreev and Mazin 2004). RAD51B-RAD51C complex did not possess any D-loop forming ability alone (Fig. 4.4B, lanes 2 and 10). The co-incubation of RAD51B-RAD51C complex with RAD51 was necessary to see optimal D-loop formation compared to addition of the paralog complex prior to RAD51 or after formation of RAD51 nucleoprotein filament (Fig. 4.6A and 4.6B). For reactions in lanes 11-17 of figure 4.4B, purified human RPA (Fig. 4.5) was added Prior to initiation of D-loop formation by addition of supercoiled dsDNA. The stimulation by RAD51B-RAD51C occurred at sub- stoichiometric levels (Fig. 4.4B and 4.4C). In the presence of RPA, we observed an increase in D-loop formation that maximized at a molar ratio of ~ 1:30, RAD51B-

RAD51C : RAD51 (Fig. 4.4B, lanes 11-13 and Fig 4.4C). Increasing the concentration of

RAD51B-RAD51C further lead to decreased product formation. We expected addition of RPA to stimulate D-loop formation as it could potentially bind to the displaced ssDNA. These observations suggest that there may be some functional synergism between a RAD51/ RAD51B-RAD51C NPF and RPA.

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RAD51B-RAD51C protects RAD51 nucleoprotein filament against the anti-recombinase function of BLM.

Yeast Sgs1 and Human BLM of the RecQ family as well as yeast Srs2 have been shown to induce anti-recombinase activity by dissociating RAD51 from ssDNA thus preventing functional NPF formation for strand exchange (Krejci et al. 2003; Veaute et al. 2003;

Bugreev et al. 2007). We hypothesized that RAD51 NPF could be stabilized by the

RAD51B-RAD51C heterodimer as a result of its stable binding activity to ssDNA, as previously demonstrated by SPR.

The assay for RAD51 NPF stability was based on a previous approach to study the inhibitory effects of BLM on RAD51-mediated strand exchange activity (Bugreev et al. 2007). First the RAD51 NPFs were formed either in the absence or presence of the

RAD51B-RAD51C heterodimer in an ATP regeneration system that is required for recombinase NPF formation as well as for the subsequence BLM mediated step. The amount of RAD51B-RAD51C introduced into the reaction was based on the concentration required for optimal D-loop formation (Fig 4.4B). After the initial incubation, purified BLM (Fig. 4.5) and RPA were added, and the reactions were incubated further (Fig. 4.7A). RPA serves to prevent possible RAD51 rebinding events after it has been dislodged by BLM translocase activity on ssDNA (Bugreev et al. 2007).

D-loop formation was initiated by addition of Ca++ and supercoiled DNA. BLM translocation is greatly attenuated in the presence of Ca++ (Bugreev et al. 2007), therefore it was necessary to add the divalent cation prior to the addition of supercoiled dsDNA.

BLM translocation dissociates RAD51 from the NPF, that would lead to reduced D-loop

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formation. As expected, we observed a decreased RAD51 mediated D-loop formation with increasing concentration of BLM (Fig. 4.7B and 4.7C). As we hypothesized, a partial stabilization of the NPF was observed in the presence of RAD51B-RAD51C paralog complex, resulting modestly increased D-loop formation (compare lanes 2-8 and lanes 9-15 of Fig. 4.7B and 4.7C). This result suggests that RAD51B-RAD51C may confer a partial stability to the RAD51 NPF.

RAD51-RAD51C does not suppress the overall ATPase activity of the RAD51/ RAD51B-

RAD51C nucleoprotein filament.

Both RAD51 and RAD51B-RAD51C are DNA stimulated ATPases (Benson et al. 1994;

Sigurdsson et al. 2001; Tombline and Fishel 2002). The ATPase activity of RAD51B-

RAD51C, derives from both constituents RAD51B and RAD51C (Lio et al. 2003). Since we observed modestly increased D-loop formation with sub-stoichiometric amounts of

RAD51B-RAD51C in the RAD51 catalyzed reaction, we investigated if the paralog complex inhibited the ATP turnover rate of RAD51, as recombinase activity of RAD51 can be enhanced by suppressing the ATP hydrolysis of the protein (Bugreev and Mazin

2004)..

We examined the ATP hydrolysis activity of the RAD51/ RAD51B-RAD51C mixed filament in varying ratios with an oligo dT50, X174 ssDNA and X174 dsDNA substrates (Fig. 4.8). As expected, RAD51 indicated the highest ATP turnover rate with the oligonucleotide substrate (Fig. 4.8). Collectively, the ATP turnover rates of the mixed filament increased with the concentration of the paralog complex, even at ratios that induced enhanced D-loop formation (compare Fig. 4.8 with Fig. 4.4B and 4.4C).

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This suggests that the stimulatory effect of RAD51B-RAD51C on RAD51 mediated strand exchange activity is not a result of suppression of the ATP turnover rate of the latter.

RAD51B-RAD51C does not alleviate inhibitory effects of RPA to promote RAD51 mediated D-loop formation.

During DSB repair, RPA binds to ssDNA once the 5 ends are resected, to prevent formation of secondary structures (Sugiyama et al. 1997). RPA is eventually replaced by

RAD51 nucleation mediated by either Rad52 in yeast or BRCA2 in vertebrates (Kanaar and Hoeijmakers 1998; New et al. 1998; Shinohara and Ogawa 1998; Yang et al. 2005;

Jensen et al. 2010). During recombinase assays in vitro, pre-incubation of RPA and ssDNA leads to greatly reduced strand exchange activity , due to its strong affinity for the latter. Previously, it was shown that RAD51B-RAD51C heterodimer functions as a presynaptic mediator in a three-strand exchange assay (Sigurdsson et al. 2001). We examined if RAD51B-RAD51C could alleviate the inhibitory effect of RPA without the requirement of BRCA2 during RAD51 catalyzed D-loop formation.

In this approach we pre-incubated ssDNA with the indicated amounts of RPA and then RAD51 or RAD51 with RAD51B-RAD51C was added (Fig. 4.9). Pre-addition of

RPA lead to significantly reduced product formation compared to RPA addition after

RAD51/ RAD51B-RAD51C NPF had formed (compared lanes 2 and 6, and lanes 7 and

11 in Fig. 4.9A and Fig 4.9B). We did not observe any alleviation of RPA inhibition by

RAD51B-RAD51C (Fig. 4.9A and 4.9B).

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We also examined if RAD51B-RAD51C directly interacted with RPA. Pull- down experiments were performed with Ni-NTA beads exploiting the His-tag on

RAD51B (Fig. 4.9C). However, we were not able to see any direct physical interaction between RAD51B-RAD51C paralog complex and RPA (Fig. 4.9C). Even a pull down experiment performed at a low-salt concentration (50mM KCl) failed to indicate an interaction between RPA and RAD51B-RAD51C (data not shown).

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4.5 DISCUSSION

Evolution of RAD51 paralogs in eukaryotes.

Gene duplication allows one copy of the gene to retain the existing function while the other evolves to gain a new function by mutations (Lin et al. 2006; Conant and Wolfe

2008). The process also leads to separation of ancestral function (Conant and Wolfe

2008). Phylogenetic analyses indicate that the RecA/ RAD51 family of genes could be divided into three subfamilies; RAD, RAD and RecA that have all evolved from a common ancestral RecA (Lin et al. 2006). RAD contains genes of highly conserved function that include RAD51 and DMC1. RAD includes genes of divergent functions that include RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 (Lin et al. 2006). RecA is found in eubacteria (Lin et al. 2006). The budding yeast Rad51 paralogs, Rad55 and Rad57 genes appear to be orthologous to vertebrate XRCC2 and

XRCC3, respectively (Lin et al. 2006). Even though RAD51 paralogs share only 20-30% homology with RAD51, there are well conserved within vertebrate cells (Takata et al.

2000; Takata et al. 2001).

Homologous recombination mechanism is conserved in prokaryotes and eukaryotes. The structural and functional properties of the recombinases; bacterial RecA and eukaryotic RAD51 are similar (Ogawa et al. 1993; Story et al. 1993; Benson et al.

1994). However, compared to RecA, RAD51 possesses a greatly reduced recombinase function (Liu et al. 2004; Shim et al. 2006; San Filippo et al. 2008). This necessitates the requirement of other recombination mediator to perform efficient recombinational repair in more complex genomes. 181

Stabilization of the nucleoprotein filament

We previously reported (Chapter3) that the ATP cap of RAD51 is intricately involved in regulating the turnover of RAD51 thereby, affecting the recombinase function. The Mutation, D316K of the ATP cap of RAD51 attenuates the ATPase activity and leads to formation of a stable NPF that results from slow protein turnover from DNA. This in turn, causes an enhanced recombinase activity. In our modeled structures the ATP cap residues RAD51(D316), RAD51B(K324) and RAD51C(K328) are positioned in a similar orientation. Therefore, it is tempting to speculate that the slow protein turnover observed for RAD51B-RAD51C- DNA interaction (see Fig. 4.3), may be mediated by the ATP cap of the paralog complex. However, RAD51B-RAD51C complex does not possess any recombinase function (Fig. 4.4 and 4.6 and ref.(Sigurdsson et al. 2001)). Also, addition of RAD51B-RAD51C does not reduce the amount of

RAD51 required for strand exchange functions (data not shown; (Sigurdsson et al.

2001)). Increasing the amounts of RAD51B-RAD51C in the reactions in fact, leads to reduced strand exchange activity by RAD51 (Fig. 4.4). In our SPR studies we did not observe any apparent reduction in the protein turnover rate when mixed RAD51/

RAD51B-RAD51C NPFs were formed at ratios that were optimal for enhanced D-loop formation (data not shown). Therefore, the partial stabilization of the NPF we observe against the anti-recombination function of BLM may be as a cause of RAD51B-RAD51C functioning as a mediator of D-loop formation after binding to DNA interspersedly (Fig.

4.7).

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For human RAD51 catalyzed D-loop formation, ATP must be in an active nonhydrolyzable state, i.e. in the presence of Ca++ (Bugreev and Mazin 2004). In the cytosol

Ca++ ions are tightly regulated as they function as secondary messengers and they do not appear in concentrations appreciable for D-loop formation (Ritchie et al. 2011).

Therefore, we hypothesized that RAD51B-RAD51C may function as auxiliary proteins in stabilizing the NPF. However, our ATPase data do not support the fact that RAD51B-

RAD51C stabilizes the NPF by reducing the overall ATP hydrolysis of the RAD51/

RAD51B-RAD51C mixed NPF (Fig. 4.8). Structurally, this is unlikely as both RAD51B and RAD51C paralogs contain Walker A box domains that hydrolyze ATP (Fig. 4.2).

Therefore, it is likely that the partial stabilization we observe against BLM anti- recombination might be as a result of RAD51B-RAD51C being physically bound to

DNA, thereby slowing down the translocation of BLM. However, we cannot rule out the possibility of post-translational modifications or the involvement of other RAD51 paralogs in vivo that might improve the stabilization effect conferred by RAD51B-

RAD51C complex.

RAD51 foci formation during IR is the initial step of HR repair (Haaf et al. 1995;

Scully et al. 1997). BRCA2 is involved in transporting RAD51 to the nucleus, as the latter lacks a nuclear localization signal (NLS) and initiating recombinase nucleation on ssDNA (Gildemeister et al. 2009). The BRC repeats of BRCA2 stabilize RAD51 by suppressing its ATPase activity and facilitates nucleation on ssDNA while inhibiting

RAD51 filament formation on dsDNA (Carreira et al. 2009; Jensen et al. 2010; Carreira and Kowalczykowski 2011). However, in pancreatic epithelial tumor derived CAPAN-1 cells (Goggins et al. 1996), that encode a C- terminal truncated BRCA2 that lacks the

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NLS, RAD51 foci formation occurs to a lesser extent during S phase and even upon IR in a BRCA2 independent manner (Tarsounas et al. 2003; Gildemeister et al. 2009).

Surprisingly, BRCA2 independent RAD51 nuclear localization was attributed to

RAD51C, which contains a functional NLS (Gildemeister et al. 2009). Therefore,

RAD51B-RAD51C mediated stabilization of the NPF against anti-recombinases of the

RecQ family as we have shown here might be critical in event of BRCA2 absence.

Even though HR is an error-free repair pathway to repair DSBs, unregulated HR could lead to gross chromosomal rearrangements (Wu and Hickson 2006). Therefore, cells have evolved counter mechanism to suppress HR at untimely events. Both cellular and biochemical data indicate that the yeast Srs2 helicase disrupts Rad51-ssDNA filaments to suppress HR (Krejci et al. 2003; Veaute et al. 2003). Overexpression of

RecQ homolog Sgs1 in Srs2 deleted strains suppresses the hyperrecombinogenic phenotypes (Mankouri et al. 2002; Ira et al. 2003). Human protein BLM and RECQL5 has been reported to suppress RAD51 mediated strand exchange activity by dissociating

RAD51-ssDNA filaments (Bugreev et al. 2007; Hu et al. 2007). Recently, yeast Rad55-

Rad57 heterodimer complex was shown to stabilize Rad51 NPF to counteract the anti- recombinogenic activity of Srs2 helicase by a direct protein-protein mediated interaction

(Liu et al. 2011). Furthermore, a genetic study illustrated that the yeast Shu complex that contains Shu1, Shu2, Csm2 and Psy3 (Shu1 and Psy3 being Rad51 paralogs) were involved in suppressing the anti-recombinase activity of Srs2 by physically interacting with the helicase (Bernstein et al. 2011). A similar direct interaction between human

RAD51B-RAD51C paralog complex with the helicase BLM that might inhibit the anti- recombinase function is yet to be characterized. However, BLM has been shown to

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physically interact with RAD51D of the RAD51D-XRCC2 complex (Braybrooke et al.

2003).

Enhanced D-loop formation in the presence of RPA

RAD51C possesses an ability to destabilize dsDNA that might facilitate DNA strand exchange activity (Lio et al. 2003). In our D-loop assays the RPA induced stimulation appears more apparent in the presence of RAD51B-RAD51C (Fig. 4.4). We could rationalize this observation as RPA capturing the displaced strand that has been destabilized by RAD51C of the RAD51B-RAD51C complex to facilitate RAD51 catalyzed D-loop formation. In fact, previous studies have shown the RPA facilitating the strand exchange activity by binding to the leaving ssDNA (Eggler et al. 2002; Van

Komen et al. 2002).

We can conclude that the RAD51B-RAD51C mediated stimulation of D-loop formation occurs at the presynaptic phase (Fig. 4.6). From our results it is evident that

RAD51B-RAD51C requires co-incubation with RAD51 for optimal NPF formation to catalyze strand invasion (Fig. 4.6). Our D-loop assay does not indicate that RAD51B-

RAD51C has the ability to facilitate nucleation of RAD51, by binding to ssDNA prior to

RAD51. RAD51B-RAD51C also does not appear to bind later to the RAD51 NPF to facilitate D-loop formation (Fig. 4.6). Thus, our conclusion is consistent with a previous observation of RAD51B-RAD51C mediator activity using three-strand exchange assays

(Sigurdsson et al. 2001).

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In summary, we could conclude that RAD51 paralog complex RAD51B-RAD51C might enable regulation of the stability of the RAD51 NPF by acting as a pro- recombinase against anti-recombinogenic factors (Fig. 4.10). The dynamic property afforded to the RAD51 NPF by these recombination mediators is required for a highly regulated HR mechanism in the cell.

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4.6 ACKNOWLEDGMENTS AND FOOTNOTES

Purified BLM protein used in this study was kindly provided by Jeremy Keirsey and

Joanna Groden at The Ohio State University. Work in this chapter is in preparation for publication as: Human RAD51B-RAD51C paralog heterodimer complex enhances and stabilizes RAD51 nucleoprotein filament for D-loop formation, Amunugama R. and

Fishel R.

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Figure 4.1 Homology based modeled structures of RAD51, RAD51B and RAD51C indicate similar domain orientations. (A) Human RAD51 (HsRAD51) structure was modeled using homology based modeling software I-TASSER (Roy et al. 2010) and the optimized structures were superimposed on the Methanococcus voltae RadA (MvRAD51) crystal structure coordinates ((Wu et al. 2005) PDB ID: 1XU4) using Pymol molecular imaging software (DeLano 2002). The non-hydrolysable ATP analog AMP- PNP is bound at the interface between MvRAD51 top monomer (cyan) and the bottom monomer (wheat). The Walker A subunit of the bottom monomer is indicated in grey. L2 ssDNA binding regions of MvRAD51 and HsRAD51 are highlighted in salmon and brown colors, respectively. The ATP caps of MvRAD51 and HsRAD51 are indicated in red and magenta colors, respectively. D316 of HsRAD51 is indicated. Two potassium (K+) ions at the interface of MvRAD51 monomers are also illustrated. Modeled and superimposed structures of Human RAD51B (HsRAD51B) in Blue (B) and Human RAD51C (HsRAD51C) in Green (C). The domain color assignments are similar to A.

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Figure 4.2 Sequence alignment of RAD51 homologs and RAD51 paralogs; RAD51B and RAD51C. ClustalW sequence aligment of Methanococcus voltae RadA (MvRAD51), human RAD51 (HsRAD51), human RAD51B (HsRAD51B) and human RAD51C (HsRAD51C). Walker A, L2 ssDNA binding region and the ATP domains are highlighted in grey, wheat and pink colors, respectively.

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Figure 4.3 RAD51B-RAD51C heterodimer forms stable complexes on DNA. (A) ssDNA binding and dissociation curves obtained from surface plasmon resonance (SPR). The SPR curves correspond to 800nM concentration of either RAD51 or RAD51B- RAD51C heterodimer. DNA binding corresponds to the time scale 100s-300s and dissociation from 300s-600s. Binding response is given in Response Units. DNA binding and dissociation were analyzed in 25mM Tris-OAc (pH 7.5), 1mM ATP, 1mM

Mg(OAc)2, 2mM DTT and 0.005% Tween-20. Control (no protein) curve is red, RAD51B-RAD51C binding at 25mM K+ (KCl) curve is black, RAD51B-RAD51C binding at 100mM K+ curve is pink, RAD51 binding at 25mM K+ curve is blue and RAD51 binding at 100mM K+ curve is green. (B) dsDNA binding and dissociation of RAD51B-RAD51C heterodimer and RAD51 analyzed by SPR. The reaction conditions and the binding curve descriptions are same as in A.

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Figure 4.4 RAD51B-RAD51C enhances RAD51 catalyzed D-loop formation in the presence of RPA. (A) Schematic diagram of the experimental approach. Asterisk indicates the [32P] label on ssDNA. Utilized proteins and DNA substrates are illustrated with labels. B-C, RAD51B-RAD51C heterodimer. RAD51 (1M) filaments were formed with RAD51B-RAD51C at the indicated concentrations (if added), on 90mer ssDNA (3M nt) by incubating at 37°C for 15min in reaction buffer containing 25mM

Tris-OAc (pH 7.5), 1mM ATP, 1mM Mg(OAc)2, 1mM CaCl2, 2mM DTT and BSA (100g/mL). After 15min, RPA (200nM) was added (if indicated) and D-loop formation was initiated by the addition of supercoiled dsDNA (50M bp) and incubation at 37°C for 15min. D-loops were analyzed by electrophoresis on a 0.9% agarose gel after deproteinization. Gel was dried and exposed to a PhosphoImager screen for quantification. (C) Quantification of D-loops formed in B. Error bars indicate S.D.

continued

191

Figure 4.4:continued

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Figure 4.5 Purified proteins used in the D-loop assays. RAD51B-RAD51C (1g), RPA (RPA1-RPA2-RPA3) (1g), RAD51 (1g) and BLM (500ng) were analyzed by 15% SDS-PAGE.

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Figure 4.6 Order of addition of RAD51 and RAD51B-RAD51C for D-loop formation. (A) In scheme I, RAD51 (1M) filaments were formed with RAD51B- RAD51C at the indicated concentrations in B, on 90mer ssDNA (3M nt) by incubating at 37°C for 10min in reaction buffer containing 25mM Tris-OAc (pH 7.5), 1mM ATP,

1mM CaCl2, 2mM DTT and BSA (100g/mL). D-loop formation was initiated by the addition of supercoiled dsDNA (50M bp) and incubation at 37°C for 15min. In scheme II, RAD51 (1M) filaments were formed on 90mer ssDNA (3M nt) by incubating at 37°C for 10min in reaction buffer. RAD51B-RAD51C at the indicated concentrations in B were then added and incubated further for 10min at 37°C. In scheme III, RAD51B- RAD51C at the indicated concentrations in B were mixed with 90mer ssDNA (3M nt) and incubated at 37°C for 10min in reaction buffer. RAD51 (1M) was then added and incubated further for 10min at 37°C. In all schemes D-loop formation was initiated by the addition of supercoiled dsDNA (50M bp) and incubation at 37°C for 15min. D- loops were analyzed by electrophoresis on a 0.9% agarose gel after deproteinization. Gels were dried and exposed to PhosphoImager screens for quantification. (C) Quantification of relative amounts of D-loops formed in B. Quantification of D-loops formed in the presence of Mg++ is also shown. Error bars indicate S.D. Continued

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Figure 4.6: continued

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Figure 4.7 RAD51B-RAD51C protects RAD51 nucleoprotein filament against anti- recombinase function of BLM. (A) Experimental scheme. Asterisk denotes the [32P] label on ssDNA. Utilized proteins and DNA substrates are illustrated with labels. B-C, RAD51B-RAD51C heterodimer. (B) RAD51 (1M) filaments were formed with RAD51B-RAD51C (31.2 nM) if indicated, on 90mer ssDNA (3M nt) by incubating at 37°C for 15min in reaction buffer containing 25mM Tris-OAc (pH 7.5), 1mM ATP,

1mM Mg(OAc)2, 2mM DTT, BSA (100g/mL), 20mM phosphocreatine and creatine phosphokinase (30U/mL). After the 10min incubation BLM at indicated concentrations and RPA (200nM) were added and incubated further for 10min at 37°C. 1mM CaCl2 was then added and followed by another 10min incubation at 37°C. D-loop formation was initiated by the addition of supercoiled dsDNA (50M bp) and incubation at 37°C for 15min. D-loops were analyzed by electrophoresis on a 0.9% agarose gel after deproteinization. Gel was dried and exposed to a PhosphoImager screen for quantification. (C) Quantification of relative amounts of D-loops formed in B. Error bars indicate S.D.

continued

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Figure 4.7: continued

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Figure 4.8 RAD51B-RAD51C does not suppress the overall ATPase activity of the RAD51/RAD51B-RAD51C nucleoprotein filament. ATPase assays were performed by incubating RAD51 (1M) and RAD51B-RAD51C at the indicated concentrations with oligo dT50 (3M nt), X174 ssDNA (3M nt) or X174 dsDNA (3M bp) in reaction buffer containing 25mM Tris-OAc (pH 7.5), 1mM Mg(OAc)2, 2mM DTT, BSA (100g/mL) and 200M ATP supplemented with 200nCi of [-32P] ATP for 1hr at 37°C. The reaction was stopped by the addition of 400μL 10% activated charcoal supplemented with 10mM EDTA and incubated on ice for 2 hrs. Following centrifuging for 10 min, 50μL duplicate aliquots were taken for counting [32P] free phosphate by Cerenkov method. ATP hydrolyzed percentage is indicated.

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Figure 4.9 RAD51B-RAD51C does not alleviate inhibitory effects of RPA (A) RPA 200nM, 400nM, 800nM and 1600nM (lanes 2-5 and lanes 7-10) was incubated with 90mer ssDNA (3M nt) at 37°C for 10min in reaction buffer containing 25mM Tris-OAc

(pH 7.5), 1mM ATP, 1mM Mg(OAc)2, 1mM CaCl2, 2mM DTT and BSA (100g/mL) for 5min. After 5min, RAD51 (1M) and RAD51B-RAD51C (32.5 nM) if indicated, was added and incubation was continued for another 15min at 37°C. Reaction in lane 6, RAD51 was incubated with 90mer ssDNA (3M nt) at 37°C for 10min in reaction buffer. Reaction in lane 11, RAD51 (1M) and RAD51B-RAD51C (32.5nM) incubated with 90mer ssDNA (3M nt) at 37°C for 10min in reaction buffer, prior to addition of RPA and further incubation at 37°C for 5min. D-loop formation was initiated by the addition of supercoiled dsDNA (50M bp) and incubation at 37°C for 15min. D-loops were analyzed by electrophoresis on a 0.9% agarose gel after deproteinization. Gels were dried and exposed to PhosphoImager screens for quantification. (B) Quantification of D- loops formed in A. Error bars indicate S.D. (C) RAD51B-RAD51C does not interact with RPA directly. Pull down experiments were performed in three separate reactions; RPA (20nM) alone, RAD51B-RAD51C (20nM) alone or RPA (20nM) and RAD51B- RAD51C (20nM) were incubated in reaction buffer containing 25mM Tris-OAc (pH 7.5), 20mM Imidazole, 150mM KCl and 0.05% Tween-20 in 50L for 1hr at 4°C on a rotisserie shaker. Ni-NTA magnetic beads (Qiagen) were then added at a final concentration of 1% (w/v) and incubation continued for another 1hr. Beads were isolated using a spin magnet and the solution was discarded. Beads were then washed twice with 100L of wash buffer (reaction buffer containing 50mM Imidazole). Bound proteins were eluted in 50L of elution buffer (reaction buffer containing 250mM Imidazole). Eluted fractions was analyzed on 15% SDS-PAGE followed by silver staining (indicated as pull down). As markers purified RPA and RAD51B-RAD51C heterodimer were also run.

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continued Figure 4.9:continued

200

Figure 4.10 Proposed model for RAD51B-RAD51C mediated stabilization of the RAD51 nucleoprotein filament. Here we represent RAD51 mediated D-loop formation that can be enhanced by RAD51B-RAD51C paralogs. The stable nucleoprotein filament that forms could slow the action of anti-recombinases, a property that might be enhanced by additional cofactors. Proteins are illustrated with labels. B-C denotes RAD51B- RAD51C heterodimer.

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CHAPTER 5

Conclusion

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Formation and maintenance of a stable RAD51 nucleoprotein filament (NPF) is essential for efficient homologous recombination in eukaryotes (Bugreev and Mazin 2004; Chi et al. 2006; San Filippo et al. 2008). In this thesis I have addressed by biochemical and structural analyses how the structural elements of the ATP binding interface may have evolved to regulate the stability of the NPF. I have also tested the hypothesis that the

RAD51 paralog complex RAD51B-RAD51C may stabilize the NPF to enhance the recombinase function.

In Chapter 2, I examined two conserved residues in the ATP binding interface,

F129 of the Walker A box and H294 in the L2 ssDNA binding region, and found they are essential for the recombinase function of RAD51. Substitution mutations of these two residues lead to loss or reduced ATPase activity and complete abrogation of recombinase function. I also found that the F129 and H294 residues are essential RAD51 DNA binding in the presence of ATP, implying allosteric communication between the ATP binding and hydrolysis site and the L2 ssDNA binding region.

In chapter 3, I examined an intriguing structural motif in the ATP binding interface termed the ATP cap that appears to function in the regulation of RAD51 turnover from DNA. An aspartate residue resides at position 316 of the ATP cap in

RAD51. Substitution of lysine for this conserved aspartate confers reduced turnover and enhanced recombinase efficiency. We speculated that in complicated eukaryotic genomes the rapid turnover of RAD51 from postsynaptic structures might facilitate downstream homologous recombination repair events. Based on primary sequence analyses we also predicted that some of the RAD51 paralogs containing a lysine residue in the analogous ATP cap position might function to stabilize the NPF.

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In chapter 4, I describe several biochemical functions of the RAD51 paralog complex RAD51B-RAD51C that ultimately stimulate RAD51 mediated D-loop formation. Modeled structures indicate that RAD51, RAD51B and RAD51C could potentially bind to DNA with similar domain orientations. Furthermore, I showed that

RAD51B-RAD51C form stable complexes on DNA and can partially stabilize the

RAD51 NPF against anti-recombinase activity of BLM. At sub-stoichiometric amounts,

RAD51B-RAD51C could stimulate RAD51-mediated D-loop formation in the presence of ssDNA binding protein RPA.

Taken as a whole these studies suggest that RAD51 may have evolved faster turnover from postsynaptic DNA structures, at the expense of recombinases efficiency.

RAD51 paralogs, that are available in eukaryotes may compensate for the loss in efficiency by functioning as mediators to form stable NPF and enhance strand exchange activity. Even though, our results with RAD51B-RAD51C indicate partial stabilization, we cannot rule out the possibility that post-translational modifications of RAD51 or the

RAD51 paralogs, or the involvement of other RAD51 paralogs and/or mediators, might augment RAD51 function in vivo. In vertebrates, BRCA2 has been shown to nucleate

RAD51 on RPA coated ssDNA (Carreira et al. 2009; Jensen et al. 2010; Carreira and

Kowalczykowski 2011). During double strand break (DSB) repair by homologous recombination (HR) extensive ssDNA regions are form by exonucleolytic resection that ultimately function as substrates for the homology search and strand exchange reactions

(Symington and Gautier 2011). Our results suggest the possibility that RAD51 paralogs such as RAD51B-RAD51C may interspersedly bind to ssDNA further away from the

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nucleation site to continue growth and/or stabilization of the RAD51 NPF to promote an efficient homology search and strand exchange process.

211

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