Functional cross-talk among Rad51, Rad54, and replication protein A in heteroduplex DNA joint formation

Saccharomyces cerevisiae Rad51, Rad54, and replication protein A (RPA) proteins work in concert to make heteroduplex DNA joints during homologous recombination. With plasmid length DNA substrates, maximal DNA joint formation is observed with amounts of Rad51 substantially below what is needed to saturate the initiating single-stranded DNA template, and, relative to Rad51, Rad54 is needed in only catalytic quantities. RPA is still indispensable for optimal reaction efficiency, but its role in this instance is to sequester free single-stranded DNA, which otherwise inhibits Rad51 and Rad54 functions. We also demonstrate that Rad54 helps overcome various reaction constraints in DNA joint formation. These results thus shed light on the function of Rad54 in the Rad51-mediated homologous DNA pairing reaction and also reveal a novel role of RPA in the presynaptic stage of this reaction.     

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.     

Characterization of strand exchange activity of yeast Rad51 protein.

The Saccharomyces cerevisiae RAD51 gene product takes part in genetic recombination and repair of DNA double strand breaks. Rad51, like Escherichia coli RecA, catalyzes strand exchange between homologous circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in the presence of ATP and ssDNA-binding protein. The formation of joint molecules between circular ssDNA and linear dsDNA is initiated at either the 5' or the 3' overhanging end of the complementary strand; joint molecules are formed only if the length of the overhanging end is more than 1 nucleotide. Linear dsDNAs with recessed complementary or blunt ends are not utilized. The polarity of strand exchange depends upon which end is used to initiate the formation of joint molecules. Joint molecules formed via the 5' end are processed by branch migration in the 3'-to-5' direction with respect to ssDNA, and joint molecules formed with a 3' end are processed in the opposite direction.     

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.     

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.     

The Rad52-Rad59 complex interacts with Rad51 and replication protein A

The RAD52 gene is essential for homology-dependent repair of double-strand breaks in Saccharomyces cerevisiae. Rad52 forms complexes with Rad51, replication protein A (RPA) or Rad59 and its presence is essential for the formation of Rad51-Rad52-Rad59 and RPA-Rad52-Rad59 complexes. The N-terminal region of Rad52, which is required for self-interaction to form a ring structure, is required for interaction with Rad59. Rad59 also shows self-interaction suggesting the formation of heteromeric and homomeric rings of Rad52 and Rad59. In wild-type cells, we propose the Rad51-Rad52-Rad59 complex is involved in conservative recombination events, including gene conversion and reciprocal recombination, whereas the Rad52-Rad59 complex participates in single-strand annealing.     

Physical interaction between replication protein A and Rad51 promotes exchange on single-stranded DNA

Replication protein A (RPA) is displaced from single-stranded DNA (ssDNA) by Rad51 during the initiation of homologous recombination. Interactions between these proteins have been reported, but the functional significance of the direct RPA-Rad51 interaction has yet to be elucidated. We have identified and characterized the interaction between DNA-binding domain A of RPA (RPA70A) and the N-terminal domain of Rad51 (Rad51N). NMR chemical shift mapping showed that Rad51N binds to the ssDNA-binding site of RPA70A, suggesting a competitive mechanism for the displacement of RPA from ssDNA by Rad51. A structure of the RPA70A-Rad51N complex was generated by experimentally guided modeling and then used to design mutations that disrupt the binding interface. Functional ATP hydrolysis assays were performed for wild-type Rad51 and a mutant defective in binding RPA. Rates of RPA displacement for the mutant were significantly below those of wild-type Rad51, suggesting that a direct RPA-Rad51 interaction is involved in displacing RPA in the initiation stage of genetic recombination.     

Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament

RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments.     

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     

Basis for avid homologous DNA strand exchange by human Rad51 and RPA

Human Rad51 (hRad51), a member of a conserved family of general recombinases, is shown here to have an avid capability to make DNA joints between homologous DNA molecules and promote highly efficient DNA strand exchange of the paired molecules over at least 5.4 kilobase pairs. Furthermore, maximal efficiency of homologous DNA pairing and strand exchange is strongly dependent on the heterotrimeric single-stranded DNA binding factor hRPA and requires conditions that lessen interactions of the homologous duplex with the hRad51-single-stranded DNA nucleoprotein filament. The homologous DNA pairing and strand exchange system described should be valuable for dissecting the action mechanism of hRad51 and for deciphering its functional interactions with other recombination factors.     

Functional differences and interactions among the putative RecA homologs Rad51, Rad55, and Rad57.

The genes of the Saccharomyces cerevisiae RAD52 epistasis group are required for the repair of ionizing radiation-induced DNA damage. Three of these genes, RAD51, RAD55, and RAD57, have been identified as putative RecA homologs. An important feature of RecA is its ability to bind and hydrolyze ATP. RAD55 and RAD57 contain putative nucleotide binding motifs, and the importance of these motifs was determined by constructing site-directed mutations of the conserved lysine residue within the Walker A-box. Changing the lysine residue to arginine or alanine resulted in a mutant phenotype in DNA repair and sporulation for Rad55 but not for Rad57. Protein-protein interactions among Rad51, Rad55, and Rad57 were tested for by the two-hybrid system. Rad55 was shown to interact with Rad51 and Rad57 but not with itself. Additionally, no interaction between Rad57 and Rad51 or between Rad57 and itself was detected. Consistent with the hypothesis that Rad55 and Rad57 may function within, or stabilize, a protein complex, we found that RAD51 expressed from a high-copy-number plasmid suppresses the DNA repair defect of strains carrying rad55 and rad57 mutations. These data, in conjunction with other reports, demonstrate the importance of protein-protein interactions in the process of DNA repair.     

Rad51 uses one mechanism to drive DNA strand exchange in both directions

The Rad51 protein of Saccharomyces cerevisiae, like its bacterial counterpart RecA, promotes strand exchange between circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in vitro. However, the two proteins differ in the requirement for initiating joint molecules and in the polarity of branch migration. Whereas RecA initiates joint molecules from any type of ends on the dsDNA and branch migration proceeds exclusively in the 5'- to 3'-direction with respect to the single strand DNA substrate, initiation mediated by Rad51 requires a complementary 3' or 5' overhanging end of the linear dsDNA and branch migration proceeds in either direction. Here we report that the rates of Rad51-mediated branch migration in either the 5'- to 3'- or 3'- to 5'-directions are affected to the same extent by temperature and MgCl(2). Furthermore, branch migration in both directions is equally impeded by insertions of non-homologous sequences in the dsDNA, inserts of 6 base pairs or more being completely inhibitory. We have also found that the preference of strand exchange in the 5'- to 3'-direction does not change if RPA is replaced by Escherichia coli SSB or T4 gene 32 proteins, suggesting that the preference for the direction of strand exchange is intrinsic to Rad51. Based on these results, we conclude that Rad51-promoted branch migration in either direction occurs fundamentally by the same mechanism, quite probably by stabilizing successively formed heteroduplex base pair.     

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.     

Identification of a novel human Rad51 variant that promotes DNA strand exchange

Rad51 plays a key role in the repair of DNA double-strand breaks through homologous recombination, which is the central process in the maintenance of genomic integrity. Five paralogs of the human Rad51 gene (hRad51) have been identified to date, including hRad51B, hRad51C, hRad51D, Xrcc2 and Xrcc3. In searches of additional hRad51 paralogs, we identified a novel hRad51 variant that lacked the sequence corresponding to exon 9 (hRad51-Δex9). The expected amino acid sequence of hRad51-Δex9 showed a frame-shift at codon 259, which resulted in a truncated C-terminus. RT-PCR analysis revealed that both hRad51 and hRad51-Δex9 were prominently expressed in the testis, but that there were subtle differences in tissue specificity. The hRad51-Δex9 protein was detected as a 31-kDa protein in the testis and localized at the nucleus. In addition, the hRad51-Δex9 protein showed a DNA-strand exchange activity comparable to that of hRad51. Taken together, these results indicate that hRad51-Δex9 promotes homologous pairing and DNA strand exchange in the nucleus, suggesting that alternative pathways in hRad51- or hRad51-Δex9-dependent manners exist for DNA recombination and repair.     

Human Rad54 protein stimulates DNA strand exchange activity of hRad51 protein in the presence of Ca2+

Rad51 and Rad54 proteins play a key role in homologous recombination in eukaryotes. Recently, we reported that Ca2+ is required in vitro for human Rad51 protein to form an active nucleoprotein filament that is important for the search of homologous DNA and for DNA strand exchange, two critical steps of homologous recombination. Here we find that Ca2+ is also required for hRad54 protein to effectively stimulate DNA strand exchange activity of hRad51 protein. This finding identifies Ca2+ as a universal cofactor of DNA strand exchange promoted by mammalian homologous recombination proteins in vitro. We further investigated the hRad54-dependent stimulation of DNA strand exchange. The mechanism of stimulation appeared to include specific interaction of hRad54 protein with the hRad51 nucleoprotein filament. Our results show that hRad54 protein significantly stimulates homology-independent coaggregation of dsDNA with the filament, which represents an essential step of the search for homologous DNA. The results obtained indicate that hRad54 protein serves as a dsDNA gateway for the hRad51-ssDNA filament, promoting binding and an ATP hydrolysis-dependent translocation of dsDNA during the search for homologous sequences.     

An archaeal Rad54 protein remodels DNA and stimulates DNA strand exchange by RadA

Rad54 protein is a key member of the RAD52 epistasis group required for homologous recombination in eukaryotes. Rad54 is a duplex DNA translocase that remodels both DNA and protein–DNA complexes, and functions at multiple steps in the recombination process. Here we use biochemical criteria to demonstrate the existence of this important protein in a prokaryotic organism. The Sulfolobus solfataricus Rad54 (SsoRad54) protein is a double-strand DNA-dependent ATPase that can alter the topology of duplex DNA. Like its eukaryotic homolog, it interacts directly with the S. solfataricus Rad51 homologue, SsoRadA, to stimulate DNA strand exchange. Confirmation of this protein as an authentic Rad54 homolog establishes an essential phylogenetic bridge for identifying Rad54 homologs in the archaeal and bacterial domains.     

Coordinated response of mammalian Rad51 and Rad52 to DNA damage

Biochemical analysis has shown that mammalian Rad51 and Rad52 interact and synergize in DNA recombination reactions in vitro, but these proteins have not been shown to function together in response to DNA damage in vivo. By analysis of murine cells expressing murine Rad52 tagged with green fluorescent protein (GFP)–Rad52, we now show that DNA damage causes Rad51 and GFP–Rad52 to colocalize in distinct nuclear foci. Cells expressing GFP–Rad52 show both increased survival and an increased number of Rad51 foci, raising the possibility that Rad52 is limiting for repair. These observations provide evidence of coordinated function of Rad51 and Rad52 in vivo and support the hypothesis that Rad52 plays an important role in the DNA damage response in mammalian cells.     

Stability of Rad51 recombinase and persistence of Rad51 DNA repair foci depends on post-translational modifiers,ubiquitin and SUMO

The DNA double-strand breaks are particularly deleterious, especially when an error-free repair pathway is unavailable, enforcing the error-prone recombination pathways to repair the lesion. Cells can resume the cell cycle but at the expense of decreased viability due to genome rearrangements. One of the major players involved in recombinational repair of DNA damage is Rad51 recombinase, a protein responsible for presynaptic complex formation. We previously showed that an increased level of this protein promotes the usage of illegitimate recombination. Here we show that the level of Rad51 is regulated via the ubiquitin-dependent proteolytic pathway. The ubiquitination of Rad51 depends on multiple E3 enzymes, including SUMO-targeted ubiquitin ligases. We also demonstrate that Rad51 can be modified by both ubiquitin and SUMO. Moreover, its modification with ubiquitin may lead to opposite effects: degradation dependent on Rad6, Rad18, Slx8, Dia2, and the anaphase-promoting complex, or stabilization dependent on Rsp5. We also show that post-translational modifications with SUMO and ubiquitin affect Rad51's ability to form and disassemble DNA repair foci, respectively, influencing cell cycle progression and cell viability in genotoxic stress conditions. Our data suggest the existence of a complex E3 ligases network that regulates Rad51 recombinase's turnover, its molecular activity, and access to DNA, limiting it to the proportions optimal for the actual cell cycle stage and growth conditions, e.g., stress. Dysregulation of this network would result in a drop in cell viability due to uncontrolled genome rearrangement in the yeast cells. In mammals would promote the development of genetic diseases and cancer.