Rad52 protein associates with replication protein A (RPA)-single-stranded DNA to accelerate Rad51-mediated displacement of RPA and presynaptic complex formation

The Rad51 nucleoprotein filament mediates DNA strand exchange, a key step of homologous recombination. This activity is stimulated by replication protein A (RPA), but only when RPA is introduced after Rad51 nucleoprotein filament formation. In contrast, RPA inhibits Rad51 nucleoprotein complex formation by prior binding to single-stranded DNA (ssDNA), but Rad52 protein alleviates this inhibition. Here we show that Rad51 filament formation is simultaneous with displacement of RPA from ssDNA. This displacement is initiated by a rate-limiting nucleation of Rad51 protein onto ssDNA complex, followed by rapid elongation of the filament. Rad52 protein accelerates RPA displacement by Rad51 protein. This acceleration probably involves direct interactions with both Rad51 protein and RPA. Detection of a Rad52-RPA-ssDNA co-complex suggests that this co-complex is an intermediate in the displacement process.     

Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein

The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.     

Analysis of the human replication protein A:Rad52 complex: evidence for crosstalk between RPA32, RPA70, Rad52 and DNA

The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential for DNA replication, and plays important roles in DNA repair and DNA recombination. Rad52 and RPA, along with other members of the Rad52 epistasis group of genes, repair double-stranded DNA breaks (DSBs). Two repair pathways involve RPA and Rad52, homologous recombination and single-strand annealing. Two binding sites for Rad52 have been identified on RPA. They include the previously identified C-terminal domain (CTD) of RPA32 (residues 224-271) and the newly identified domain containing residues 169-326 of RPA70. A region on Rad52, which includes residues 218-303, binds RPA70 as well as RPA32. The N-terminal region of RPA32 does not appear to play a role in the formation of the RPA:Rad52 complex. It appears that the RPA32CTD can substitute for RPA70 in binding Rad52. Sequence homology between RPA32 and RPA70 was used to identify a putative Rad52-binding site on RPA70 that is located near DNA-binding domains A and B. Rad52 binding to RPA increases ssDNA affinity significantly. Mutations in DBD-D on RPA32 show that this domain is primarily responsible for the ssDNA binding enhancement. RPA binding to Rad52 inhibits the higher-order self-association of Rad52 rings. Implications for these results for the "hand-off" mechanism between protein-protein partners, including Rad51, in homologous recombination and single-strand annealing are discussed.     

Dynamic Regulatory Interactions of Rad51, Rad52, and Replication Protein-A in Recombination Intermediates

Rad51, Rad52, and replication protein-A (RPA) play crucial roles in the repair of DNA double-strand breaks in Saccharomyces cerevisiae. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. Rad52 recruits Rad51 into single-stranded DNAs (ssDNAs) that are saturated with RPA. Rad52 also promotes annealing of ssDNA strands that are complexed with RPA. Specific protein-protein interactions are involved in these reactions. Here we report new biochemical characteristics of these protein interactions. First, Rad52-RPA interaction requires multiple molecules of RPA to be associated with ssDNA, suggesting that multiple contacts between the Rad52 ring and RPA-ssDNA filament are needed for stable binding. Second, RPA-t11, which is a recombination-deficient mutant of RPA, displays a defect in interacting with Rad52 in the presence of salt above 50 mM, explaining the defect in Rad52-mediated ssDNA annealing in the presence of this mutation. Third, ssDNA annealing promoted by Rad52 is preceded by aggregation of multiple RPA-ssDNA complexes with Rad52, and Rad51 inhibits this aggregation. These results suggest a regulatory role for Rad51 that suppresses ssDNA annealing and facilitates DNA strand invasion. Finally, the Rad51-double-stranded DNA complex disrupts Rad52-RPA interaction in ssDNA and titrates Rad52 from RPA. This suggests an additional regulatory role for Rad51 following DNA strand invasion, where Rad51-double-stranded DNA may inhibit illegitimate second-end capture to ensure the error-free repair of a DNA double-strand break.     

Role of Saccharomyces single-stranded DNA-binding protein RPA in the strand invasion step of double-strand break repair

The single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB) and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5′ to 3′ exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E) mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA.     

Role of Saccharomyces Single-Stranded DNA-Binding Protein RPA in the Strand Invasion Step of Double-Strand Break Repair

The single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB) and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5′ to 3′ exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E) mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA.     

Yeast Rad52 and Rad51 recombination proteins define a second pathway of DNA damage assessment in response to a single double-strand break

Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest. We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta. rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants. Neither rad54Delta nor rad55Delta affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants. Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p.     

Rad52-mediated DNA annealing after Rad51-mediated DNA strand exchange promotes second ssDNA capture

Rad51, Rad52, and RPA play central roles in homologous DNA recombination. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. Rad52 has two distinct activities: to recruit Rad51 onto single-strand (ss)DNA that is complexed with the ssDNA-binding protein, RPA, and to anneal complementary ssDNA complexed with RPA. Here, we report that Rad52 promotes annealing of the ssDNA strand that is displaced by DNA strand exchange by Rad51 and RPA, to a second ssDNA strand. An RPA that is recombination-deficient (RPA(rfa1-t11)) failed to support annealing, explaining its in vivo phenotype. Escherichia coli RecO and SSB proteins, which are functional homologues of Rad52 and RPA, also facilitated the same reaction, demonstrating its conserved nature. We also demonstrate that the two activities of Rad52, recruiting Rad51 and annealing DNA, are coordinated in DNA strand exchange and second ssDNA capture.     

The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A

Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization. We further demonstrate that RPA has a critical postsynaptic role in DNA strand exchange, stabilizing the DNA pairing initiated by Rad51 protein. Stabilization of the Rad51-generated DNA pairing intermediates can be can occur either by binding the displaced strand with RPA or by degrading the same DNA strand using exonuclease VII. The optimal conditions for Rad51-mediated DNA strand exchange used here minimize the secondary structure in single-stranded DNA, minimizing the established presynaptic role of RPA in facilitating Rad51 filament formation. We verify that RPA has little effect on Rad51 filament formation under these conditions, assigning the dramatic stimulation of strand exchange nevertheless afforded by RPA to its postsynaptic function of removing the displaced DNA strand from Rad51 filaments.     

Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast

Repair of DNA double-strand breaks (DSBs) by homologous recombination requires members of the RAD52 epistasis group. Here we use chromatin immunoprecipitation (ChIP) to examine the temporal order of recruitment of Rad51p, Rad52p, Rad54p, Rad55p, and RPA to a single, induced DSB in yeast. Our results suggest a sequential, interdependent assembly of Rad proteins adjacent to the DSB initiated by binding of Rad51p. ChIP time courses from various mutant strains and additional biochemical studies suggest that Rad52p, Rad55p, and Rad54p each help promote the formation and/or stabilization of the Rad51p nucleoprotein filament. We also find that all four Rad proteins associate with homologous donor sequences during strand invasion. These studies provide a near comprehensive view of the molecular events required for the in vivo assembly of a functional Rad51p presynaptic filament.     

Interaction with Rad51 is indispensable for recombination mediator function of Rad52

In the yeast Saccharomyces cerevisiae, the RAD52 gene is indispensable for homologous recombination and DNA repair. Rad52 protein binds DNA, anneals complementary ssDNA strands, and self-associates to form multimeric complexes. Moreover, Rad52 physically interacts with the Rad51 recombinase and serves as a mediator in the Rad51-catalyzed DNA strand exchange reaction. Here, we examine the functional significance of the Rad51/Rad52 interaction. Through a series of deletions, we have identified residues 409-420 of Rad52 as being indispensable and likely sufficient for its interaction with Rad51. We have constructed a four-amino acid deletion mutation within this region of Rad52 to ablate its interaction with Rad51. We show that the rad52delta409-412 mutant protein is defective in the mediator function in vitro even though none of the other Rad52 activities, namely, DNA binding, ssDNA annealing, and protein oligomerization, are affected. We also show that the sensitivity of the rad52delta409-412 mutant to ionizing radiation can be complemented by overexpression of Rad51. These results thus demonstrate the significance of the Rad51-Rad52 interaction in homologous recombination.     

The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 protein

Replication protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination. Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication. We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)). This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11).ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPA.ssDNA complex. RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 protein.ssDNA nucleoprotein complex is assembled prior to introduction of the mutant RPA. However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 protein more slowly than wild-type RPA and, as a consequence, Rad51 protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11). Rad52 protein can stimulate displacement of RPA(rfa1-t11) from ssDNA by Rad51 protein, but the rate of displacement remains slow compared with wild-type RPA. These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 protein and that RPA displacement by Rad51 protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation.     

Rad51 protein controls Rad52-mediated DNA annealing

In Saccharomyces cerevisiae, Rad52 protein plays an essential role in the repair of DNA double-stranded breaks (DSBs). Rad52 and its orthologs possess the unique capacity to anneal single-stranded DNA (ssDNA) complexed with its cognate ssDNA-binding protein, RPA. This annealing activity is used in multiple mechanisms of DSB repair: single-stranded annealing, synthesis-dependent strand annealing, and cross-over formation. Here we report that the S. cerevisiae DNA strand exchange protein, Rad51, prevents Rad52-mediated annealing of complementary ssDNA. Efficient inhibition is ATP-dependent and involves a specific interaction between Rad51 and Rad52. Free Rad51 can limit DNA annealing by Rad52, but the Rad51 nucleoprotein filament is even more effective. We also discovered that the budding yeast Rad52 paralog, Rad59 protein, partially restores Rad52-dependent DNA annealing in the presence of Rad51, suggesting that Rad52 and Rad59 function coordinately to enhance recombinational DNA repair either by directing the processed DSBs to repair by DNA strand annealing or by promoting second end capture to form a double Holliday junction. This regulation of Rad52-mediated annealing suggests a control function for Rad51 in deciding the recombination path taken for a processed DNA break; the ssDNA can be directed to either Rad51-mediated DNA strand invasion or to Rad52-mediated DNA annealing. This channeling determines the nature of the subsequent repair process and is consistent with the observed competition between these pathways in vivo.     

Rad52 protein has a second stimulatory role in DNA strand exchange that complements replication protein-A function

Rad52 protein plays a central role in double strand break repair and homologous recombination in Saccharomyces cerevisiae. We have identified a new mechanism by which Rad52 protein stimulates Rad51 protein-promoted DNA strand exchange. This function of Rad52 protein is revealed when subsaturating amounts (relative to the single-stranded DNA concentration) of replication protein-A (RPA) are used. Under these conditions, Rad52 protein is needed for extensive DNA strand exchange. Interestingly, in this new role, Rad52 protein neither acts simply as a single strand DNA-binding protein per se nor, in contrast to its previously identified stimulatory roles, does it require physical interaction with RPA because it can be substituted by the Escherichia coli single strand DNA-binding protein. We propose that Rad52 protein acts by stabilizing the Rad51 presynaptic filament.     

ATP-dependent and ATP-independent roles for the Rad54 chromatin remodeling enzyme during recombinational repair of a DNA double strand break

The efficient and accurate repair of DNA double strand breaks (DSBs) is critical to cell survival, and defects in this process can lead to genome instability and cancers. In eukaryotes, the Rad52 group of proteins dictates the repair of DSBs by the error-free process of homologous recombination (HR). A critical step in eukaryotic HR is the formation of the initial Rad51-single-stranded DNA presynaptic nucleoprotein filament. This presynaptic filament participates in a homology search process that leads to the formation of a DNA joint molecule and recombinational repair of the DSB. Recently, we showed that the Rad54 protein functions as a mediator of Rad51 binding to single-stranded DNA, and here, we find that this activity does not require ATP hydrolysis. We also identify a novel Rad54-dependent chromatin remodeling event that occurs in vivo during the DNA strand invasion step of HR. This ATP-dependent remodeling activity of Rad54 appears to control subsequent steps in the HR process.     

Functional interactions among yeast Rad51 recombinase, Rad52 mediator, and replication protein A in DNA strand exchange

Rad51-catalyzed DNA strand exchange is greatly enhanced by the single-stranded (ss) DNA binding factor RPA if the latter is introduced after Rad51 has already nucleated onto the initiating ssDNA substrate. Paradoxically, co-addition of RPA with Rad51 to the ssDNA to mimic the in vivo situation diminishes the level of strand exchange, revealing competition between RPA and Rad51 for binding sites on ssDNA. Rad52 promotes strand exchange but only when there is a need for Rad51 to compete with RPA for loading onto ssDNA. Rad52 is multimeric, binds ssDNA, and targets Rad51 to ssDNA. Maximal restoration of pairing and strand exchange requires amounts of Rad52 substoichiometric to Rad51 and involves a stable, equimolar complex between Rad51 and Rad52. The Rad51-Rad52 complex efficiently utilizes a ssDNA template saturated with RPA for homologous pairing but does not appear to be more active than Rad51 when an RPA-free ssDNA template is used. Rad52 does not substitute for RPA in the pairing and strand exchange reaction nor does it lower the dependence of the reaction on Rad51 or RPA.     

The F-Box DNA helicase Fbh1 prevents Rhp51-dependent recombination without mediator proteins

A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1(-) mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.     

Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators

In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog) and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog)–mediated homologous recombination (HR) and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA), which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA) precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1–mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA     

Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase

Homologous recombination represents an important means for the error-free elimination of DNA double-strand breaks and other deleterious DNA lesions from chromosomes. The Rad51 recombinase, a member of the RAD52 group of recombination proteins, catalyzes the homologous recombination reaction in the context of a helical protein polymer assembled on single-stranded DNA (ssDNA) that is derived from the nucleolytic processing of a primary lesion. The assembly of the Rad51-ssDNA nucleoprotein filament, often referred to as the presynaptic filament, is prone to interference by the single-strand DNA-binding factor replication protein A (RPA). The Saccharomyces cerevisiae Rad52 protein facilitates presynaptic filament assembly by helping to mediate the displacement of RPA from ssDNA. On the other hand, disruption of the presynaptic filament by the Srs2 helicase leads to a net exchange of Rad51 for RPA. To understand the significance of protein-protein interactions in the control of Rad52- or Srs2-mediated presynaptic filament assembly or disassembly, we have examined two rad51 mutants, rad51 Y388H and rad51 G393D, that are simultaneously ablated for Rad52 and Srs2 interactions and one, rad51 A320V, that is differentially inactivated for Rad52 binding for their biochemical properties and also for functional interactions with Rad52 or Srs2. We show that these mutant rad51 proteins are impervious to the mediator activity of Rad52 or the disruptive function of Srs2 in concordance with their protein interaction defects. Our results thus provide insights into the functional significance of the Rad51-Rad52 and Rad51-Srs2 complexes in the control of presynaptic filament assembly and disassembly. Moreover, our biochemical studies have helped identify A320V as a separation-of-function mutation in Rad51 with regards to a differential ablation of Rad52 interaction.Homologous recombination (HR)³ helps maintain genomic stability by eliminating DNA double-strand breaks induced by ionizing radiation and chemical reagents, by restarting damaged or collapsed DNA replication forks, and by elongating shortened telomeres especially when telomerase is dysfunctional (1–3). Accordingly, defects in HR invariably lead to enhanced sensitivity to genotoxic agents, chromosome aberrations, and tumor development (4, 5). In meiosis also, HR helps mediate the linkage of homologous chromosome pairs via arm cross-overs, thus ensuring the proper segregation of chromosomes at the first meiotic division (6). Accordingly, HR mutants exhibit a plethora of meiotic defects, including early meiotic cell cycle arrest, aneuploidy, and inviability.Much of the knowledge regarding the mechanistic basis of HR has been derived from studies of model organisms, such as the budding yeast Saccharomyces cerevisiae. Genetic analyses in S. cerevisiae have led to the identification of the RAD52 group of genes, namely, RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54, MRE11, and XRS2 (1), that are needed for the successful execution of HR. Each member of the RAD52 group of genes has an orthologue in higher eukaryotes, including humans, and mutations in any of these genes cause defects in HR and repair of double-strand breaks.The DNA pairing and strand invasion step of the HR reaction is mediated by RAD51-encoded protein, which is orthologous to the Escherichia coli recombinase RecA (2). Like RecA, Rad51 polymerizes on ssDNA, derived from the nucleolytic processing of a primary lesion such as a double-strand break, to form a right-handed nucleoprotein filament, often referred to as the presynaptic filament (3, 7). The presynaptic filament engages dsDNA, conducts a search for homology in the latter, and catalyzes DNA joint formation between the recombining ssDNA and dsDNA partners upon the location of homology (1, 3). As such, the timely and efficient assembly of the presynaptic filament is indispensable for the successful execution of HR.Because the nucleation of Rad51 onto ssDNA is a rate-limiting process, presynaptic filament assembly is prone to interference by the single-strand DNA-binding protein replication protein A (RPA) (1, 3, 7). In reconstituted biochemical systems, the addition of Rad52 counteracts the inhibitory action of RPA (8, 9). Consistent with the biochemical results, in both mitotic and meiotic cells, the recruitment of Rad51 to double-strand breaks is strongly dependent on Rad52 (10–12). This effect of Rad52 on Rad51 presynaptic filament assembly has been termed a “recombination mediator” function (13).Interestingly, genetic studies have shown that the Srs2 helicase fulfills the role of an anti-recombinase. Specifically, mutations in Srs2 often engender a hyper-recombinational phenotype and can also help suppress the DNA damage sensitivity of rad6 and rad18 mutants, because of the heightened HR proficiency being able to substitute for the post-replicative DNA repair defects of these mutant cells (2, 14). Importantly, in reconstituted systems, Srs2 exerts a strong inhibitory effect on Rad51-mediated reactions in a manner that is potentiated by RPA. Biochemical and electron microscopic analyses have provided compelling evidence that Srs2 acts by disassembling the presynaptic filament, to effect the replacement of Rad51 by RPA (15, 16). The ability of Srs2 to dissociate the presynaptic filament relies on its ATPase activity, revealed using mutant variants, K41A and K41R, that harbor changes in the Walker type A motif involved in ATP engagement. Accordingly, the srs2 K41A and srs2 K41R mutants are biologically inactive (17).In both yeast two-hybrid and biochemical analyses, a complex of Rad51 with either Rad52 or Srs2 can be captured (1, 16). Using yeast two-hybrid-based mutagenesis, several rad51 mutant alleles, A320V, Y388H, and G393D, that engender a defect in the yeast two-hybrid association with Rad52 have been found (18). Here we document our biochemical studies demonstrating the inability of these rad51 mutant proteins to physically and functionally interact with Rad52. Interestingly, we find that two of these rad51 mutants, namely, Y388H and G393D, are also defective in Srs2 interaction. Accordingly, these mutant rad51 proteins form presynaptic filaments that are resistant to the disruptive action of Srs2. Our results thus emphasize the role of Rad51-Rad52 and Rad51-Srs2 interactions in the regulation of Rad51 presynaptic filament assembly and maintenance, and they also reveal the presence of overlapping Rad52 and Srs2 interaction motifs in Rad51. In these regards, our biochemical studies have identified the A320V change as a separation-of-function mutation in Rad51.     

RAD51 variant proteins from human lung and kidney tumors exhibit DNA strand exchange defects

In human cells, error-free repair of DNA double-strand breaks requires the DNA pairing and strand exchange activities of RAD51 recombinase. Activation of RAD51 recombination activities requires the assembly of RAD51 presynaptic filaments on the single-stranded DNA that forms at resected DSB ends. Mutations in proteins that control presynaptic filament assembly, such as BRCA2, and in RAD51 itself, are associated with human breast cancer. Here we describe the properties of two mutations in RAD51 protein that derive from human lung and kidney tumors, respectively. Sequence variants Q268P and Q272L both map to the DNA binding loop 2 (L2) region of RAD51, a motif that is involved in DNA binding and in the allosteric activation of ATP hydrolysis and DNA strand exchange activities. Both mutations alter the thermal stability, DNA binding, and ATPase properties of RAD51, however both variants retain intrinsic DNA strand exchange activity towards oligonucleotide substrates under optimized conditions. In contrast, both Q268P and Q272L variants exhibit drastically reduced DNA strand exchange activity in reaction mixtures containing long homologous ssDNA and dsDNA substrates and human RPA protein. Mixtures of wild-type and variant proteins also exhibit reduced DNA strand exchange activity, suggesting that heterozygous mutations could negatively affect DNA recombination and repair processes in vivo. Together, the findings of this study suggest that hypomorphic missense mutations in RAD51 protein could be drivers of genomic instability in cancer cells, and thereby contribute to the etiology of metastatic disease.