Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells

Genetic studies in rodent and chicken mutant cell lines have suggested that Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3) play important roles in homologous recombinational repair of DNA double-strand breaks and in maintaining chromosome stability. Previous studies using yeast two- and three-hybrid systems have shown interactions among these proteins, but it is not clear whether these interactions occur simultaneously or sequentially in vivo. By utilizing immunoprecipitation with extracts of human cells expressing epitope-tagged Rad51 paralogs, we demonstrate that XRCC2 and Rad51D, while stably interacting with each other, co-precipitate with Rad51C but not with XRCC3. In contrast, Rad51C is pulled down with XRCC3, whereas XRCC2 and Rad51D are not. In addition, Rad51B could be pulled down with Rad51C and Rad51D, but not with XRCC3. These results suggest that Rad51C is involved in two distinct in vivo complexes: Rad51B–Rad51C–Rad51D–XRCC2 and Rad51C–XRCC3. In addition, we demonstrate that Rad51 co-precipitates with XRCC3 but not with XRCC2 or Rad51D, suggesting that Rad51 can be present in an XRCC3–Rad51C–Rad51 complex. These complexes may act as functional units and serve accessory roles for Rad51 in the presynapsis stage of homologous recombinational repair.     

Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells

Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require Rad51 and the five Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3), as has been shown in chicken and rodent cells. Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems. To test for interactions involving XRCC3 and Rad51C, stable human cell lines have been isolated that express (His)₆-tagged versions of XRCC3 or Rad51C. Ni²⁺-binding experiments demonstrate that XRCC3 and Rad51C interact in human cells. In addition, we find that Rad51C, but not XRCC3, interacts directly or indirectly with Rad51B, Rad51D and XRCC2. These results argue that there are at least two complexes of Rad51 paralogs in human cells (Rad51C–XRCC3 and Rad51B–Rad51C–Rad51D–XRCC2), both containing Rad51C. Moreover, Rad51 is not found in these complexes. X-ray treatment did not alter either the level of any Rad51 paralog or the observed interactions between paralogs. However, the endogenous level of Rad51C is moderately elevated in the XRCC3-overexpressing cell line, suggesting that dimerization between these proteins might help stabilize Rad51C.     

Roles of Rad51 paralogs for promoting homologous recombination in Leishmania infantum

To achieve drug resistance Leishmania parasite alters gene copy number by using its repeated sequences widely distributed through the genome. Even though homologous recombination (HR) is ascribed to maintain genome stability, this eukaryote exploits this potent mechanism driven by the Rad51 recombinase to form beneficial extrachromosomal circular amplicons. Here, we provide insights on the formation of these circular amplicons by analyzing the functions of the Rad51 paralogs. We purified three Leishmania infantum Rad51 paralogs homologs (LiRad51-3, LiRad51-4 and LiRad51-6) all of which directly interact with LiRad51. LiRad51-3, LiRad51-4 and LiRad51-6 show differences in DNA binding and annealing capacities. Moreover, it is also noteworthy that LiRad51-3 and LiRad51-4 are able to stimulate Rad51-mediated D-loop formation. In addition, we succeed to inactivate the LiRad51-4 gene and report a decrease of circular amplicons in this mutant. The LiRad51-3 gene was found to be essential for cell viability. Thus, we propose that the LiRad51 paralogs play crucial functions in extrachromosomal circular DNA amplification to circumvent drug actions and preserve survival.     

Ionizing radiation-induced foci formation of mammalian Rad51 and Rad54 depends on the Rad51 paralogs, but not on Rad52

Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response.     

Differing requirements for the Arabidopsis Rad51 paralogs in meiosis and DNA repair

In addition to the recombinase Rad51, vertebrates have five paralogs of Rad51, all members of the Rad51-dependent recombination pathway. These paralogs form two complexes (Rad51C/Xrcc3 and Rad51B/C/D/Xrcc2), which play roles in somatic recombination, DNA repair and chromosome stability. However, little is known of their possible involvement in meiosis, due to the inviability of the corresponding knockout mice. We have recently reported that the Arabidopsis homolog of one of these Rad51 paralogs (AtXrcc3) is involved in DNA repair and meiotic recombination and present here Arabidopsis lines carrying mutations in three other Rad51 paralogs (AtRad51B, AtRad51C and AtXrcc2). Disruption of any one of these paralogs confers hypersensitivity to the DNA cross-linking agent Mitomycin C, but not to gamma-irradiation. Moreover, the atrad51c-1 mutant is the only one of these to show meiotic defects similar to those of the atxrcc3 mutant, and thus only the Rad51C/Xrcc3 complex is required to achieve meiosis. These results support conservation of functions of the Rad51 paralogs between vertebrates and plants and differing requirements for the Rad51 paralogs in meiosis and DNA repair.     

Role of the Saccharomyces cerevisiae Rad51 paralogs in sister chromatid recombination

Rad51 requires a number of other proteins, including the Rad51 paralogs, for efficient recombination in vivo. Current evidence suggests that the yeast Rad51 paralogs, Rad55 and Rad57, are important in formation or stabilization of the Rad51 nucleoprotein filament. To gain further insights into the function of the Rad51 paralogs, reporters were designed to measure spontaneous or double-strand break (DSB)-induced sister or nonsister recombination. Spontaneous sister chromatid recombination (SCR) was reduced 6000-fold in the rad57 mutant, significantly more than in the rad51 mutant. Although the DSB-induced recombination defect of rad57 was suppressed by overexpression of Rad51, elevated temperature, or expression of both mating-type alleles, the rad57 defect in spontaneous SCR was not strongly suppressed by these same factors. In addition, the UV sensitivity of the rad57 mutant was not strongly suppressed by MAT heterozygosity, even though Rad51 foci were restored under these conditions. This lack of suppression suggests that Rad55 and Rad57 have different roles in the recombinational repair of stalled replication forks compared with DSB repair. Furthermore, these data suggest that most spontaneous SCR initiates from single-stranded gaps formed at stalled replication forks rather than DSBs.     

Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins.

Rad51p is a eukaryotic homolog of RecA, the central homologous pairing and strand exchange protein in Escherichia coli. Rad54p belongs to the Swi2p/Snf2p family of DNA-stimulated ATPases. Both proteins are also important members of the RAD52 group which controls recombinational DNA damage repair of double-strand breaks and other DNA lesions in Saccharomyces cerevisiae. Here we demonstrate by genetic, molecular and biochemical criteria that Rad51 and Rad54 proteins interact. Strikingly, overexpression of Rad54p can functionally suppress the UV and methyl methanesulfonate sensitivity caused by a deletion of the RAD51 gene. However, no suppression was observed for the defects of rad51 cells in the repair of gamma-ray-induced DNA damage, mating type switching or spontaneous hetero-allelic recombination. This suppression is genetically dependent on the presence of two other members of the recombinational repair group, RAD55 and RAD57. Our data provide compelling evidence that Rad51 and Rad54 proteins interact in vivo and that this interaction is functionally important for recombinational DNA damage repair. As both proteins are conserved throughout evolution from yeasts to humans, a similar protein-protein interaction may be expected in other organisms.     

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.     

Rad51 protein stimulates the branch migration activity of Rad54 protein

The Rad51 and Rad54 proteins play important roles during homologous recombination in eukaryotes. Rad51 forms a nucleoprotein filament on single-stranded DNA and performs the initial steps of double strand break repair. Rad54 belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that Rad54 promotes branch migration of Holliday junctions. Here we find that human Rad51 (hRad51) significantly stimulates the branch migration activity of hRad54. The stimulation appears to be evolutionarily conserved, as yeast Rad51 also stimulates the branch migration activity of yeast Rad54. We further investigated the mechanism of this stimulation. Our results demonstrate that the stimulation of hRad54-promoted branch migration by hRad51 is driven by specific protein-protein interactions, and the active form of the hRad51 filament is more stimulatory than the inactive one. The current results support the hypothesis that the hRad51 conformation state has a strong effect on interaction with hRad54 and ultimately on the function of hRad54 in homologous recombination.     

Inter-subunit interactions that coordinate Rad51's activities

Rad51 is the central catalyst of homologous recombination in eukaryotes and is thus critical for maintaining genomic integrity. Recent crystal structures of filaments formed by Rad51 and the closely related archeal RadA and eubacterial RecA proteins place the ATPase site at the protomeric interface. To test the relevance of this feature, we mutated conserved residues at this interface and examined their effects on key activities of Rad51: ssDNA-stimulated ATP hydrolysis, DNA binding, polymerization on DNA substrates and catalysis of strand-exchange reactions. Our results show that the interface seen in the crystal structures is very important for nucleoprotein filament formation. H352 and R357 of yeast Rad51 are essential for assembling the catalytically competent form of the enzyme on DNA substrates and coordinating its activities. However, contrary to some previous suggestions, neither of these residues is critical for ATP hydrolysis.     

Preferential binding to branched DNA strands and strand-annealing activity of the human Rad51B, Rad51C, Rad51D and Xrcc2 protein complex

The Rad51B, Rad51C, Rad51D and Xrcc2 proteins are Rad51 paralogs, and form a complex (BCDX2 complex) in mammalian cells. Mutant cells defective in any one of the Rad51-paralog genes exhibit spontaneous genomic instability and extreme sensitivity to DNA-damaging agents, due to inefficient recombinational repair. Therefore, the Rad51 paralogs play important roles in the maintenance of genomic integrity through recombinational repair. In the present study, we examined the DNA-binding preference of the human BCDX2 complex. Competitive DNA-binding assays using seven types of DNA substrates, single-stranded DNA (ssDNA), double-stranded DNA, 5′- and 3′-tailed duplexes, nicked duplex DNA, Y-shaped DNA and a synthetic Holliday junction, revealed that the BCDX2 complex preferentially bound to the two DNA substrates with branched structures (the Y-shaped DNA and the synthetic Holliday junction). Furthermore, the BCDX2 complex catalyzed the strand-annealing reaction between a long linear ssDNA (1.2 kb in length) and its complementary circular ssDNA. These properties of the BCDX2 complex may be important for its roles in the maintenance of chromosomal integrity.     

Holliday junction binding activity of the human Rad51B protein

The human Rad51B protein is involved in the recombinational repair of damaged DNA. Chromosomal rearrangements of the Rad51B gene have been found in uterine leiomyoma patients, suggesting that the Rad51B gene suppresses tumorigenesis. In the present study, we found that the purified Rad51B protein bound to single-stranded DNA and double-stranded DNA in the presence of ATP and either Mg(2+) or Mn(2+) and hydrolyzed ATP in a DNA-dependent manner. When the synthetic Holliday junction was present along with the half-cruciform and double-stranded oligonucleotides, the Rad51B protein only bound to the synthetic Holliday junction, which mimics a key intermediate in homologous recombination. In contrast, the human Rad51 protein bound to all three DNA substrates with no obvious preference. Therefore, the Rad51B protein may have a specific function in Holliday junction processing in the homologous recombinational repair pathway in humans.     

Molecular dissection of interactions between Rad51 and members of the recombination-repair group

Recombination is important for the repair of DNA damage and for chromosome segregation during meiosis; it has also been shown to participate in the regulation of cell proliferation. In the yeast Saccharomyces cerevisiae, recombination requires products of the RAD52 epistasis group. The Rad51 protein associates with the Rad51, Rad52, Rad54, and Rad55 proteins to form a dynamic complex. We describe a new strategy to screen for mutations which cause specific disruption of the interaction between certain proteins in the complex, leaving other interactions intact. This approach defines distinct protein interaction domains and protein relationships within the Rad51 complex. Alignment of the mutations onto the constructed three-dimensional model of the Rad51 protein reveal possible partially overlapping interfaces for the Rad51-Rad52 and the Rad51-Rad54 interactions. Rad51-Rad55 and Rad51-Rad51 interactions are affected by the same spectrum of mutations, indicating similarity between the two modes of binding. Finally, the detection of a subset of mutations within Rad51 which disrupt the interaction with mutant Rad52 protein but activate the interaction with Rad54 suggests that dynamic changes within the Rad51 protein may contribute to an ordered reaction process.     

Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs

The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.     

Probing Rad51-DNA interactions by changing DNA twist

In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.     

Cellular localization of human Rad51C and regulation of ubiquitin-mediated proteolysis of Rad51

Rad51-catalyzed homologous recombination is an important pathway for repair of DNA double strand breaks and maintenance of genome integrity in vertebrate cells. Five proteins referred to as Rad51 paralogs promote Rad51 activity and are proposed to act at various, and in some cases, multiple stages in the recombination pathway. Imaging studies of native Rad51 have revealed its cellular response to DNA damage, yet visualization of the paralog proteins has met with limited success. In this study, we are able to detect endogenous Rad51C and Xrcc3 in human cells. In an effort to determine how Rad51, Rad51C, and Xrcc3 influence the pattern of localization of each other over the time course of DNA damage and repair, we have made the unexpected observation that Rad51 degradation via the ubiquitin-mediated proteasome pathway occurs as a natural part of recombinational DNA repair. Additionally, we find that Rad51C plays an important role in regulating this process. This article contains supplementary material, which may be viewed at the Journal of Cellular Biochemistry website at http://www.interscience.wiley.com/jpages/0730-2312/suppmat/index.html.     

Interaction of human recombination proteins Rad51 and Rad54.

The cDNA for human protein HsRad54, which is a structural homolog of Saccharomyces cerevisiae recombination/repair protein Rad54, was cloned and expressed in Escherichia coli. As demonstrated by analysis in vitro and in vivo, HsRad54 protein interacts with human Rad51 recombinase. The interaction is mediated by the N-terminal domain of HsRad54 protein, which interacts with both free and DNA-bound HsRad51 protein.     

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.     

Heterology tolerance and recognition of mismatched base pairs by human Rad51 protein

Human Rad51 (hRad51) promoted homology recognition and subsequent strand exchange are the key steps in human homologous recombination mediated repair of DNA double-strand breaks. However, it is still not clear how hRad51 deals with sequence heterology between the two homologous chromosomes in eukaryotic cells, which would lead to mismatched base pairs after strand exchange. Excessive tolerance of sequence heterology may compromise the fidelity of repair of DNA double-strand breaks. In this study, fluorescence resonance energy transfer (FRET) was used to monitor the heterology tolerance of human Rad51 mediated strand exchange reactions, in real time, by introducing either G-T or I-C mismatched base pairs between the two homologous DNA strands. The strand exchange reactions were much more sensitive to G-T than to I-C base pairs. These results imply that the recognition of homology and the tolerance of heterology by hRad51 may depend on the local structural motif adopted by the base pairs participating in strand exchange. AnhRad51 mutant protein (hRad51K133R), deficient in ATP hydrolysis, showed greater heterology tolerance to both types of mismatch base pairing, suggesting that ATPase activity may be important for maintenance of high fidelity homologous recombination DNA repair.