Human Rad51 Research Articles

Understanding Rad51 function is a prerequisite for progress in cancer research.

The human protein Rad51 is double-edged in cancer contexts: on one hand, preventing tumourigenesis by eliminating potentially carcinogenic DNA damage and, on the other, promoting tumours by introducing new mutations. Understanding mechanistic details of Rad51 in homologous recombination (HR) and repair could facilitate design of novel methods, including CRISPR, for Rad51-targeted cancer treatment. Despite extensive research, however, we do not yet understand the mechanism of HR in sufficient detail, partly due to complexity, a large number of Rad51 protein units being involved in the exchange of long DNA segments. Another reason for lack of understanding could be that current recognition models of DNA interactions focus only on hydrogen bond-directed base pair formation. A more complete model may need to include, for example, the kinetic effects of DNA base stacking and unstacking ('longitudinal breathing'). These might explain how Rad51 can recognize sequence identity of DNA over several bases long stretches with high accuracy, despite the fact that a single base mismatch could be tolerated if we consider only the hydrogen bond energy. We here propose that certain specific hydrophobic effects, recently discovered destabilizing stacking of nucleobases, may play a central role in this context for the function of Rad51.

Human RAD51 paralogue RAD51C fosters repair of alkylated DNA by interacting with the ALKBH3 demethylase.

The integrity of our DNA is challenged daily by a variety of chemicals that cause DNA base alkylation. DNA alkylation repair is an essential cellular defence mechanism to prevent the cytotoxicity or mutagenesis from DNA alkylating chemicals. Human oxidative demethylase ALKBH3 is a central component of alkylation repair, especially from single-stranded DNA. However, the molecular mechanism of ALKBH3-mediated damage recognition and repair is less understood. We report that ALKBH3 has a direct protein-protein interaction with human RAD51 paralogue RAD51C. We also provide evidence that RAD51C–ALKBH3 interaction stimulates ALKBH3-mediated repair of methyl-adduct located within 3′-tailed DNA, which serves as a substrate for the RAD51 recombinase. We further show that the lack of RAD51C–ALKBH3 interaction affects ALKBH3 function in vitro and in vivo. Our data provide a molecular mechanism underlying upstream events of alkyl adduct recognition and repair by ALKBH3.

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells.

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.

Non-enzymatic roles of human RAD51 at stalled replication forks

The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not D-loop activity, to reveal mechanistic aspects of RAD51’s roles in the response to replication stress. Here, we find that cells lacking RAD51’s enzymatic activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly, we find that RAD51’s strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication fork regression, supporting a model in which fork regression depends on a non-enzymatic function of RAD51. We also show RAD51 promotes replication restart by both strand exchange-dependent and strand exchange-independent mechanisms.

A Novel Cell-Penetrating Antibody Fragment Inhibits the DNA Repair Protein RAD51

DNA damaging chemotherapies are successful in cancer therapy, however, the damage can be reversed by DNA repair mechanisms that may be up-regulated in cancer cells. We hypothesized that inhibiting RAD51, a protein involved in homologous recombination DNA repair, would block DNA repair and restore the effectiveness of DNA damaging chemotherapy. We used phage-display to generate a novel synthetic antibody fragment that bound human RAD51 with high affinity (KD = 8.1 nM) and inhibited RAD51 ssDNA binding in vitro. As RAD51 is an intracellular target, we created a corresponding intrabody fragment that caused a strong growth inhibitory phenotype on human cells in culture. We then used a novel cell-penetrating peptide “iPTD” fusion to generate a therapeutically relevant antibody fragment that effectively entered living cells and enhanced the cell-killing effect of a DNA alkylating agent. The iPTD may be similarly useful as a cell-penetrating peptide for other antibody fragments and open the door to numerous intracellular targets previously off-limits in living cells.

Emerging Roles of RAD52 in Genome Maintenance.

The maintenance of genome integrity is critical for cell survival. Homologous recombination (HR) is considered the major error-free repair pathway in combatting endogenously generated double-stranded lesions in DNA. Nevertheless, a number of alternative repair pathways have been described as protectors of genome stability, especially in HR-deficient cells. One of the factors that appears to have a role in many of these pathways is human RAD52, a DNA repair protein that was previously considered to be dispensable due to a lack of an observable phenotype in knock-out mice. In later studies, RAD52 deficiency has been shown to be synthetically lethal with defects in BRCA genes, making RAD52 an attractive therapeutic target, particularly in the context of BRCA-deficient tumors.

Abstract 2570: Colonic adenocarcinoma-associated DNA-binding site mutations in Rad52 perturbs its ssDNA binding polarity and function in homologous recombination

Abstract Double strand DNA breaks (DSB) are repaired by the Homologous recombination (HR) pathway. Defective HR leads to accumulation of mutations that result in genomic instability and a plethora of associated cancers including, breast, ovarian, and skin cancers. In HR, the DSB is recognized and resected to yield single-stranded DNA (ssDNA) that are immediately coated by replication protein A (RPA). The Rad51 recombinase, which is the central engine for HR, needs to bind to ssDNA buried under RPA and form a nucleoprotein filament. Cells possess mediator proteins that either promote or inhibit the exchange of RPA for Rad51 on ssDNA. RAD52 is one such pro-recombinogenic mediator and functions analogous to the Breast Cancer type 2 Susceptibility Protein (BRCA2) to promote the formation of the Rad51 filament. However, the mechanistic details underlying how Rad52 mediates RPA-Rad51 exchange on ssDNA remains a mystery. Mutations in mediator proteins such as BRCA2 and RAD52 are prevalent in many cancers. For example, the COSMIC database shows there have been 139 sampled cases of mutations of Rad52 in solid tumors. To understand how Rad52 fulfills its mediator functions, it is imperative to first understand how Rad52 associates with DNA. The recent crystal structure of human RAD52 (PDB ID) proposed two ssDNA binding site for RAD52: an outer and an inner binding site, where Arg55 is the interface amino acid between the two sites. To systematically characterize how Rad52 associates with ssDNA, we conducted stopped flow experiments with fluorescently labeled ssDNA. Our results show that Rad52 binds cooperatively to the 5′ end of DNA. Additionally, we created mutations in Rad52-ssDNA binding sites to investigate their role in polarity and cooperativity in depth. These residues and adjacent amino acids are hotspots for mutagenesis in cancers. Abolishing the outer binding site results in a loss of cooperative binding and higher order Rad52-ssDNA complex formation. Mutating the inner binding site abolishes ssDNA binding entirely. Additionally, abolishing the interface arginine residue results in non-selective, rapid binding. This exact amino acid has been mutated in two cases of colonic adenocarcinoma. Our result suggests that both sites are required for maintaining cooperativity and polarity of Rad52 binding to ssDNA. A detailed mechanistic insight into Rad52-ssDNA binding will aid in the development of small-molecule inhibitor drugs that can be used as effective chemotherapeutic agents against cancers in the future. Note: This abstract was not presented at the meeting. Citation Format: Emma A. Tillison, Nilisha Pokhrel, Edwin Antony. Colonic adenocarcinoma-associated DNA-binding site mutations in Rad52 perturbs its ssDNA binding polarity and function in homologous recombination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2570.

Optimization of Drug Candidates That Inhibit the D-Loop Activity of RAD51.

RAD51 is the central protein in homologous recombination (HR) repair, where it first binds ssDNA and then catalyzes strand invasion via a D-loop intermediate. Additionally, RAD51 plays a role in faithful DNA replication by protecting stalled replication forks; this requires RAD51 to bind DNA but may not require the strand invasion activity of RAD51. We previously described a small-molecule inhibitor of RAD51 named RI(dl)-2 (RAD51 inhibitor of D-loop formation #2, hereafter called 2 h), which inhibits D-loop activity while sparing ssDNA binding. However, 2 h is limited in its ability to inhibit HR in vivo, preventing only about 50 % of total HR events in cells. We sought to improve upon this by performing a structure-activity relationship (SAR) campaign for more potent analogues of 2 h. Most compounds were prepared from 1-(2-aminophenyl)pyrroles by forming the quinoxaline moiety either by condensation with aldehydes, then dehydrogenation of the resulting 4,5-dihydro intermediates, or by condensation with N,N'-carbonyldiimidazole, chlorination, and installation of the 4-substituent through Suzuki-Miyaura coupling. Many analogues exhibited enhanced activity against human RAD51, but in several of these compounds the increased inhibition was due to the introduction of dsDNA intercalation activity. We developed a sensitive assay to measure dsDNA intercalation, and identified two analogues of 2 h that promote complete HR inhibition in cells while exerting minimal intercalation activity.

Human RAD51 paralogue SWSAP1 fosters RAD51 filament by regulating the anti-recombinase FIGNL1 AAA+ ATPase

RAD51 assembly on single-stranded (ss)DNAs is a crucial step in the homology-dependent repair of DNA damage for genomic stability. The formation of the RAD51 filament is promoted by various RAD51-interacting proteins including RAD51 paralogues. However, the mechanisms underlying the differential control of RAD51-filament dynamics by these factors remain largely unknown. Here, we report a role for the human RAD51 paralogue, SWSAP1, as a novel regulator of RAD51 assembly. Swsap1-deficient cells show defects in DNA damage-induced RAD51 assembly during both mitosis and meiosis. Defective RAD51 assembly in SWSAP1-depleted cells is suppressed by the depletion of FIGNL1, which binds to RAD51 as well as SWSAP1. Purified FIGNL1 promotes the dissociation of RAD51 from ssDNAs. The dismantling activity of FIGNL1 does not require its ATPase but depends on RAD51-binding. Purified SWSAP1 inhibits the RAD51-dismantling activity of FIGNL1. Taken together, our data suggest that SWSAP1 protects RAD51 filaments by antagonizing the anti-recombinase, FIGNL1.

S-DNA and RecA/RAD51-Mediated Strand Exchange in Vitro.

S-DNA (stretched DNA) is an elongated base-paired DNA conformation under high tension. Because the RecA/Rad51 family DNA recombinases form helical filaments on DNA and mediate the formation of the DNA triplex (D-loop), in which the DNA is stretched, and because the extension of these nucleoprotein filaments is similar to the extension of S-DNA, S-DNA has long been hypothesized as a possible state of DNA that participants in RecA/Rad51-mediated DNA strand exchange in homologous recombination. Such a hypothesis, however, is still lacking direct experimental studies. In this work, we have studied the polymerization and strand exchange on S-DNA mediated by Escherichia coli RecA, human Rad51, and Saccharomyces cerevisiae Rad51 by single-molecule magnetic tweezers. We report that RecA/Rad51 polymerizes faster on S-DNA than on B-DNA with the same buffer conditions. Furthermore, the RecA/Rad51-mediated DNA triplex forms faster from S-DNA than from B-DNA together with the homologous single-stranded DNA. These results provide evidence that S-DNA can interact with RecA and Rad51 and shed light on the possible functions of S-DNA.

Human RAD52 protein regulates homologous recombination and checkpoint function in BRCA2 deficient cells.

Cancer cells exhibit HR defects, increased proliferation and checkpoint aberrations. Tumour suppressor proteins, BRCA2 and p53 counteract such aberrant proliferation by checkpoint regulation. Intriguingly, chemo-resistant cancer cells, exhibiting mutated BRCA2 and p53 protein survive even with increased DNA damage accumulation. Such cancer cells show upregulation of RAD52 tumour suppressor protein implying that RAD52 might be providing survival advantage to cancer cells. To understand this paradoxical condition of a tumour suppressor protein facilitating cancer cell survival, in the current study, we investigate the role of RAD52 overexpression in BRCA2 deficient cells. We provide evidence that RAD52 protein alleviates HR inhibition imposed by p53 in BRCA2 deficient cells. In addition, we study the role of RAD52 protein during short replication stress in BRCA2 deficient cells. BRCA2 deficient cells exhibit excessive origin firing and checkpoint evasion in the presence of prevailing DNA damage. Interestingly, overexpression of RAD52 rescues the excessive origin firing and checkpoint defects observed in BRCA2 deficient cells, indicating RAD52 protein compensates for the loss of BRCA2 function. We show that RAD52 protein, just as BRCA2, interacts with pCHK1 checkpoint protein and helps maintain the checkpoint control in BRCA2 deficient cells during DNA damage response.

RAD-ical New Insights into RAD51 Regulation.

The accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions; however, they can be repaired using homologous recombination. Homologous recombination is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, which is a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, which are collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here, we discuss the current perspective on the in vivo and in vitro function of the RAD51 paralogs, and their relationship with cancer in vertebrate models.

Human Rad52 Promotes XPG-Mediated R-loop Processing to Initiate Transcription-Associated Homologous Recombination Repair

Given that genomic DNA exerts its function by being transcribed, it is critical for the maintenance of homeostasis that DNA damage, such as double-strand breaks (DSBs), within transcriptionally active regions undergoes accurate repair. However, it remains unclear how this is achieved. Here, we describe a mechanism for transcription-associated homologous recombination repair (TA-HRR) in human cells. The process is initiated by R-loops formed upon DSB induction. We identify Rad52, which is recruited to the DSB site in a DNA-RNA-hybrid-dependent manner, as playing pivotal roles in promoting XPG-mediated R-loop processing and initiating subsequent repair by HRR. Importantly, dysfunction of TA-HRR promotes DSB repair via non-homologous end joining, leading to a striking increase in genomic aberrations. Thus, our data suggest that the presence of R-loops around DSBs within transcriptionally active regions promotes accurate repair of DSBs via processing by Rad52 and XPG to protect genomic information in these critical regions from gene alterations.

Structural Basis of Homology-Directed DNA Repair Mediated by RAD52

SummaryRAD52 mediates homologous recombination by annealing cDNA strands. However, the detailed mechanism of DNA annealing promoted by RAD52 has remained elusive. Here we report two crystal structures of human RAD52 single-stranded DNA (ssDNA) complexes that probably represent key reaction intermediates of RAD52-mediated DNA annealing. The first structure revealed a “wrapped” conformation of ssDNA around the homo-oligomeric RAD52 ring, in which the edges of the bases involved in base pairing are exposed to the solvent. The ssDNA conformation is close to B-form and appears capable of engaging in Watson-Crick base pairing with the cDNA strand. The second structure revealed a “trapped” conformation of ssDNA between two RAD52 rings. This conformation is stabilized by a different RAD52 DNA binding site, which promotes the accumulation of multiple RAD52 rings on ssDNA and the aggregation of ssDNA. These structures provide a structural framework for understanding the mechanism of RAD52-mediated DNA annealing.

Construction of plasmids to make a specialized S. cerevisiae strain to investigate Saw1 protein recruitment in single‐strand annealing repair

DNA damaging agents such as ionizing radiation and reactive oxygen species resulting from cellular metabolism can lead to double‐strand breaks (DSBs) which, if left unrepaired, may result in genetic diseases and cancer. Living systems are equipped with DNA repair pathways that alleviate such damage as it occurs. One mode of repair is single‐strand annealing (SSA), a non‐conservative pathway that utilizes DNA repeats flanking the DSB site. DSBs are resected 5′ to 3′ exposing complementary strands of the repeats which become annealed by the protein Rad52, generating overhanging 3′ flaps deriving from the DNA originally located between the repeats. Flap cleavage is achieved by the Rad1‐Rad10 endonuclease which is recruited by the protein Saw1. The point‐in‐time at which Saw1 recruits Rad1‐Rad10 is not known but likely occurs following annealing. To investigate this question, we are constructing a specialized yeast strain containing two inducible and fluorescently tagged DSB sites to monitor SSA by fluorescence microscopy. Simultaneous induction of both DSB sites will lead to repair by SSA that results in convergence of the two fluorescent signals during annealing by Rad52. Using this assay system, we will be able to peg Saw1 arrival to the annealing step, making it possible to detect whether Rad52 is required for the recruitment of Saw1 to DSB sites. An additional goal of this research project will be to investigate whether human Rad52 can be swapped in, in place of the endogenous yeast Rad52.The first step toward this goal has been to construct two DNA plasmids that will enable creation of a transgenic yeast strain with the features described above. The first plasmid will integrate one of the inducible DSB sites and one of the DNA repeats. Plasmid 1 was constructed by ligating into vector “pLAY500”: 1) a HYG marker from plasmid “pAAH1”, 2) a cassette containing the partial HIS3 marker, his3Δ3′, situated next to an HO endonuclease restriction site, and 3) flanking yeast DNA sequence homologous to the intended site of integration in the yeast genome. The desired recombinant plasmid product was initially produced via standard cloning methods and its sequence confirmed by PCR screens and DNA sequencing, but it contained several mutations. We corrected these by site‐directed mutagenesis and confirmed the corrected sequence by DNA sequencing. Additionally, during site‐directed mutagenesis one of the integrated flanking sequences in Plasmid 1 was lost, but was reintroduced via restriction digest and ligation. The second plasmid is designed to remove an unwanted HO endonuclease site present in the target yeast strain by replacing this portion of the yeast chromosome with DNA sequence lacking the HO endonuclease cassette. Making Plasmid 2 entailed PCR amplification of several DNA fragments which were then linked to each other by adaptamer‐mediated PCR. The resulting linked PCR product was ligated into the pGEMT‐Easy vector and E. coli transformants were screened. Plasmids 1 and 2 will enable creation of the specialized yeast strain for fluorescence microscopy to elucidate the timing of the end‐processing steps in SSA.Support or Funding InformationThe National Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Two distinct conformational states define the interaction of human RAD51‐ATP with single‐stranded DNA

An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 kBT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.

Human RAD51 rapidly forms intrinsically dynamic nucleoprotein filaments modulated by nucleotide binding state.

Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which is required for homology search, strand exchange and protection of replication forks. Formation of nucleoprotein filaments (NF) is required for development and genomic stability, and its failure is associated with developmental abnormalities and tumorigenesis. Here we describe the structure of the human RAD51 NFs and of its Walker box mutants using electron microscopy. Wild-type RAD51 filaments adopt an ‘open’ conformation when compared to a ‘closed’ structure formed by mutants, reflecting alterations in helical pitch. The kinetics of formation/disassembly of RAD51 filaments show rapid and high ssDNA coverage via low cooperativity binding of RAD51 units along the DNA. Subsequently, a series of isomerization or dissociation events mediated by nucleotide binding state creates intrinsically dynamic RAD51 NFs. Our findings highlight important a mechanistic divergence among recombinases from different organisms, in line with the diversity of biological mechanisms of HR initiation and quality control. These data reveal unexpected intrinsic dynamic properties of the RAD51 filament during assembly/disassembly, which may be important for the proper control of homologous recombination.

RAD51C/XRCC3 Facilitates Mitochondrial DNA Replication and Maintains Integrity of the Mitochondrial Genome.

Mechanisms underlying mitochondrial genome maintenance have recently gained wide attention, as mutations in mitochondrial DNA (mtDNA) lead to inherited muscular and neurological diseases, which are linked to aging and cancer. It was previously reported that human RAD51, RAD51C, and XRCC3 localize to mitochondria upon oxidative stress and are required for the maintenance of mtDNA stability. Since RAD51 and RAD51 paralogs are spontaneously imported into mitochondria, their precise role in mtDNA maintenance under unperturbed conditions remains elusive. Here, we show that RAD51C/XRCC3 is an additional component of the mitochondrial nucleoid having nucleus-independent roles in mtDNA maintenance. RAD51C/XRCC3 localizes to the mtDNA regulatory regions in the D-loop along with the mitochondrial polymerase POLG, and this recruitment is dependent upon Twinkle helicase. Moreover, upon replication stress, RAD51C and XRCC3 are further enriched at the mtDNA mutation hot spot region D310. Notably, the absence of RAD51C/XRCC3 affects the stability of POLG on mtDNA. As a consequence, RAD51C/XRCC3-deficient cells exhibit reduced mtDNA synthesis and increased lesions in the mitochondrial genome, leading to overall unhealthy mitochondria. Together, these findings lead to the proposal of a mechanism for a direct role of RAD51C/XRCC3 in maintaining mtDNA integrity under replication stress conditions.

Expression, Purification, and Biochemical Evaluation of Human RAD51 Protein.

The RAD51 DNA strand exchange protein plays an important role in maintaining the integrity of the human genome. It promotes homology-directed DNA repair by exchanging strands between the damaged and the intact DNA molecules. It also plays an important role in stabilizing distressed DNA replication forks. When overexpressed or misregulated, however, RAD51 contributes to "rogue," genome destabilizing events that can lead to cancer, cell death, and to acquisition of chemotherapy resistance by cancerous cells. Human RAD51 is, therefore, an important and highly coveted anticancer drug target. Biochemical, biophysical, and structural studies of the human RAD51 and establishment of its structure-activity relationship require purification of large quantities of protein. In this chapter we describe a robust method for expression and purification of human RAD51 and the methods for assessing its activity based on the single-strand DNA-binding stoichiometry and its capacity to carry out the DNA strand exchange reaction.