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.     

DNA annealing mediated by Rad52 and Rad59 proteins

In the budding yeast Saccharomyces cerevisiae, the RAD52 gene is essential for all homologous recombination events and its homologue, the RAD59 gene, is important for those that occur independently of RAD51. Both Rad52 and Rad59 proteins can anneal complementary single-stranded (ss) DNA. We quantitatively examined the ssDNA annealing activity of Rad52 and Rad59 proteins and found significant differences in their biochemical properties. First, and most importantly, they differ in their ability to anneal ssDNA that is complexed with replication protein A (RPA). Rad52 can anneal an RPA-ssDNA complex, but Rad59 cannot. Second, Rad59-promoted DNA annealing follows first-order reaction kinetics, whereas Rad52-promoted annealing follows second-order reaction kinetics. Last, Rad59 enhances Rad52-mediated DNA annealing at increased NaCl concentrations, both in the absence and presence of RPA. These results suggest that Rad59 performs different functions in the recombination process, and should be more accurately viewed as a Rad52 paralogue.     

Bringing Rad52 foci into focus

In 2007, we published the results of a genome-wide screen for ORFs that affect the frequency of Rad52 foci in yeast. That paper was published within the constraints of conventional online publishing tools, and it provided only a glimpse into the actual screen data. New tools in the JCB DataViewer now show how these data can—and should—be shared.

Complete screen data

https://doi.org/10.1083/jcb.201108095.dv The Rad52 protein has pivotal functions in double strand break repair and homologous recombination. The activity of Rad52 is often monitored by the subnuclear foci that it forms spontaneously in S phase or after DNA damage (Lisby et al., 2001). In mammals, the functions of yeast Rad52 may be divided between human RAD52 and the tumor suppressor BRCA2 (Feng et al., 2011). The full host of molecular players that govern Rad52 focus formation and maintenance was not well known when we initiated our screen. Using a high-content, image-based assay, we assessed the proportion of cells containing spontaneous Rad52-YFP foci in 4,805 viable Saccharomyces cerevisiae deletion strains (Alvaro et al., 2007). Starting with 96-well arrays of a deletion strain library, we created hybrid diploid strains (homozygous for the deletions) using systematic hybrid loss of heterozygosity (SHyLOH; Alvaro et al., 2006). We then manually and sequentially examined each strain using epifluorescence microscopy for the presence of Rad52-YFP foci. All of our image analysis was performed manually.As is often the case, our screen was published showing only a couple of representative images and providing data tables to summarize the findings. Tomes of data that could not be included in the published paper were relegated to supplemental Excel tables, typical of genome-wide screens. Also, the raw image data were sequestered in the laboratory on DVDs. With considerable help from JCB and Glencoe Software, we are delighted that the raw data from our Rad52 screen are now freely available online through the JCB DataViewer. A new interface within the JCB DataViewer brings presentation and preservation of high-content, multidimensional image-based screening data to a whole new level. To facilitate the development of this new interface, JCB required a dataset that was not time sensitive, and we were happy to provide our previously published Rad52 data. In the future, this new interface will be used to present high-content screening (HCS) datasets linked to published JCB papers. Indeed, the first publication of this sort appears in this issue of JCB (Rohn et al., 2011).The presentation of our data in the JCB DataViewer clearly shows the many benefits of this new publishing resource for the scientific community. Users now can view the complete collection of 3D image data across the entire screen, not just the two images in our original publication (Alvaro et al., 2007). Additionally, detailed information on image acquisition parameters, locus identities, and more is easily accessible (Fig. 1). Phenotypic scoring results can be visualized in interactive chart formats (Fig. 1), and search (Fig. 2) and database-linking tools (Fig. 1) allow extensive mining of the data for genes and phenotypes of interest. These tools provide an unprecedented view into HCS data in their entirety, as well as a means for authors to share and archive their data. This kind of accessibility to the direct visualization of the entire set of original screening data, on a scale previously only available to the scientists performing the screen, allows users to understand the full context of the image data analyzed in a screen. Furthermore, it is only through full access to the raw images and associated metadata that this information can be of maximum use to the community for large-scale data mining.Open in a separate windowFigure 1.The HCS interface of the JCB DataViewer provides interactive tools for the analysis of complete datasets from image-based screens. The miniviewer (top left) provides information for each gene in the screen through a zoomable and scrollable display of original multidimensional image data. It contains detailed metadata and a gene ontology (GO) summary, a link to a relevant external database (e.g., the Saccharomyces Genome Database [SGD]; top right), and a link to phenotypic scoring data for the complete screen in the chart view (bottom right). Within the chart view, hits designated by the screen authors are shown in blue, and the strain currently on display in the miniviewer is shown in red. The plate view (bottom left) shows the position of the strain of interest (red box) relative to other strains screened.Open in a separate windowFigure 2.The HCS interface of the JCB DataViewer provides search tools for the mining of complete datasets from image-based screens. (A) Users can search screen data by gene name or keywords (e.g., DNA repair). (B) Users can pick candidates for further analysis from the phenotypic scoring information in the chart view.As in all large-scale screens, the real data are variable; e.g., some strains provide a clear Rad52 focus phenotype, whereas others are more ambiguous. For our particular screen, images were not collected using automated technology but were acquired manually, strain by strain, over a period of months, leading to different levels of fluorescence intensity of Rad52-YFP as a result of, for example, changes in the intensity of our mercury arc lamp. Differences also exist in the number of fields and z stacks captured for each strain. In the absence of automated image collection, images from the primary screen in a few cases were not archived with the others and thus for all intents and purposes have been lost. In addition, our Rad52 screen only assayed nonessential genes, and some mutants are refractory to the SHyLOH methodology. Knowing all of this information allows users to view the data in a realistic manner and further highlights the importance of providing a central repository to archive HCS data.When published through conventional publication media, many important imaging details are known only to the original screeners. The new HCS interface of the JCB DataViewer shines a light on screening data as metadata become freely accessible, allowing any user to ask novel questions of the dataset. For example, the plate view for images (Fig. 1) allows users to assess whether neighboring colonies played any role in determining the phenotype and to delve deeper into why that might be. For example, are any “hits” a result of contamination from adjacent strains, resulting in clusters of positives? In the context of an automated screen, how were control and experimental samples arrayed across a plate during data collection? Did the controls on a particular plate behave as expected? Because our screen used a novel chromosome-specific loss of the heterozygosity method, users can ask whether mutations on specific chromosomes share features of Rad52 foci levels. The global resolution of the dataset provided through this new interface puts users of the dataset as close to the seat of the original screening scientist as possible, allowing them to ask, “what did the authors really see?”Presenting HCS data in the JCB DataViewer holds immense potential value to the scientific community. Through this new interface, users can access powerful interactive tools for analyzing scored phenotypes across the entire dataset (Fig. 1). Each gene ID can be charted against the phenotypic parameters scored in the original screen (e.g., the percentage of cells with Rad52 foci) and compared with all other loci (Fig. 1). Users can take our data and create their own list of hits based on their criteria, create a gallery of thumbnails for their selections (Fig. 2), and seamlessly move between their list of hits and the original data in the plate display format (Fig. 1). Users can also compare their candidates with our list (Fig. 2). The ability to visualize these data for comparative analyses creates a whole new perspective. The HCS interface of the JCB DataViewer allows users to look for their favorite gene, compare related genes, and discover new genes they never anticipated were involved in a given process.In summary, these new features of the JCB DataViewer will allow users to access the primary data from large-scale screens and to look at the full dataset to see what all of the images really look like. The ability to mine these data opens up whole new dimensions in data sharing and transparency. In the future, we anticipate that it will be possible to search many genome-wide screens, such as our Rad52 dataset, to identify commonalities in protein localization, concentration, cell morphology, etc. However, this will only occur if image data are archived and made freely available to the scientific community. We wholeheartedly support the efforts of JCB and hope that groups that use image-based HCS will increasingly make their images available using tools such as the JCB DataViewer.     

Rad52 and Rad59 exhibit both overlapping and distinct functions

Homologous recombination is an important pathway for the repair of DNA double-strand breaks (DSBs). In the yeast Saccharomyces cerevisiae, Rad52 is a central recombination protein, whereas its paralogue, Rad59, plays a more subtle role in homologous recombination. Both proteins can mediate annealing of complementary single-stranded DNA in vitro, but only Rad52 interacts with replication protein A and the Rad51 recombinase. We have studied the functional overlap between Rad52 and Rad59 in living cells using chimeras of the two proteins and site-directed mutagenesis. We find that Rad52 and Rad59 have both overlapping as well as separate functions in DSB repair. Importantly, the N-terminus of Rad52 possesses functions not supplied by Rad59, which may account for its central role in homologous recombination.     

Human Rad51 amino acid residues required for Rad52 binding.

The Rad51 protein, a homologue of the bacterial RecA protein, is an essential factor for both meiotic and mitotic recombination. The N-terminal domain of the human Rad51 protein (HsRad51) directly interacts with DNA. Based on a yeast two-hybrid analysis, it has been reported that the N-terminal region of the Saccharomyces cerevisiae Rad51 protein binds Rad52;S. cerevisiae Rad51 and Rad52 both activate the homologous pairing and strand exchange reactions. Here, we show that the HsRad51 N-terminal region, which corresponds to the Rad52-binding region of ScRad51, does not exhibit strong binding to the human Rad52 protein (HsRad52). To investigate its function, the C-terminal region of HsRad51 was randomly mutagenized. Although this region includes the two segments corresponding to the putative DNA-binding sites of RecA, all seven of the mutants did not decrease, but instead slightly increased, the DNA binding. In contrast, we found that some of these HsRad51 mutations significantly decreased the HsRad52 binding. Therefore, we conclude that these amino acid residues are required for the HsRad51.HsRad52 binding. HsRad52, as well as S. cerevisiae Rad52, promoted homologous pairing between ssDNA and dsDNA, and higher homologous pairing activity was observed in the presence of both HsRad51 and HsRad52 than with either HsRad51 or HsRad52 alone. The HsRad51 F259V mutation, which strongly impaired the HsRad52 binding, decreased the homologous pairing in the presence of both HsRad51 and HsRad52, without affecting the homologous pairing by HsRad51 alone. This result suggests the importance of the HsRad51.HsRad52 interaction in homologous pairing.     

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.     

A species-specific interaction of Rad51 and Rad52 proteins in eukaryotes

The structures and properties of the Rad51 and Rad52 proteins in eukaryotes are described. Both proteins form a complex and are responsible for recombination and repair reactions. The N-terminal region of the Rad51 protein interacts with the C-terminal region of the Rad52 protein. Species-specific interaction is probably essential for the functioning of these genes.     

In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination

Repairing a double-strand break by homologous recombination requires binding of the strand exchange protein Rad51p to ssDNA, followed by synapsis with a homologous donor. Here we used chromatin immunoprecipitation to monitor the in vivo association of Saccharomyces cerevisiae Rad51p with both the cleaved MATa locus and the HML alpha donor. Localization of Rad51p to MAT precedes its association with HML, providing evidence of the time needed for the Rad51 filament to search the genome for a homologous sequence. Rad51p binding to ssDNA requires Rad52p. The absence of Rad55p delays Rad51p binding to ssDNA and prevents strand invasion and localization of Rad51p to HML alpha. Lack of Rad54p does not significantly impair Rad51p recruitment to MAT or its initial association with HML alpha; however, Rad54p is required at or before the initiation of DNA synthesis after synapsis has occurred at the 3' end of the invading strand.     

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.     

Homomeric interaction of the mouse Rad52 protein

The Rad52 protein plays a crucial role in repairing DNA damage and homologous recombination, possibly by virtue of its ability to catalyze annealing of single-stranded DNA. In agreement with recent genetic data, we here present results based on the two-hybrid system, demonstrating that mouse Rad52p is able to form homomeric complexes. A small domain necessary and sufficient for the self-interaction is located in the conserved N-terminus of the protein. These data contribute to the important insights into the architecture of the multi-protein complex involved in recombinational DNA repair.     

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.     

Radiation-induced assembly of Rad51 and Rad52 recombination complex requires ATM and c-Abl

Cells from individuals with the recessive cancer-prone disorder ataxia telangiectasia (A-T) are hypersensitive to ionizing radiation (I-R). ATM (mutated in A-T) is a protein kinase whose activity is stimulated by I-R. c-Abl, a nonreceptor tyrosine kinase, interacts with ATM and is activated by ATM following I-R. Rad51 is a homologue of bacterial RecA protein required for DNA recombination and repair. Here we demonstrate that there is an I-R-induced Rad51 tyrosine phosphorylation, and this induction is dependent on both ATM and c-Abl. ATM, c-Abl, and Rad51 can be co-immunoprecipitated from cell extracts. Consistent with the physical interaction, c-Abl phosphorylates Rad51 in vitro and in vivo. In assays using purified components, phosphorylation of Rad51 by c-Abl enhances complex formation between Rad51 and Rad52, which cooperates with Rad51 in recombination and repair. After I-R, an increase in association between Rad51 and Rad52 occurs in wild-type cells but not in cells with mutations that compromise ATM or c-Abl. Our data suggest signaling mediated through ATM, and c-Abl is required for the correct post-translational modification of Rad51, which is critical for the assembly of Rad51 repair protein complex following I-R.     

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.     

The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing

The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.     

Mgm101: A double-duty Rad52-like protein

Mgm101 has well-characterized activity for the repair and replication of the mitochondrial genome. Recent work has demonstrated a further role for Mgm101 in nuclear DNA metabolism, contributing to an S-phase specific DNA interstrand cross-link repair pathway that acts redundantly with a pathway controlled by Pso2 exonuclease. Due to involvement of FANCM, FANCJ and FANCP homologues (Mph1, Chl1 and Slx4), this pathway has been described as a Fanconi anemia-like pathway. In this pathway, Mgm101 physically interacts with the DNA helicase Mph1 and the MutSα (Msh2/Msh6) heterodimer, but its precise role is yet to be elucidated. Data presented here suggests that Mgm101 functionally overlaps with Rad52, supporting previous suggestions that, based on protein structure and biochemical properties, Mgm101 and Rad52 belong to a family of proteins with similar function. In addition, our data shows that this overlap extends to the function of both proteins at telomeres, where Mgm101 is required for telomere elongation during chromosome replication in rad52 defective cells. We hypothesize that Mgm101 could, in Rad52-like manner, preferentially bind single-stranded DNAs (such as at stalled replication forks, broken chromosomes and natural chromosome ends), stabilize them and mediate single-strand annealing-like homologous recombination event to prevent them from converting into toxic structures.     

Rad51-independent interchromosomal double-strand break repair by gene conversion requires Rad52 but not Rad55, Rad57, or Dmc1

Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.     

Unrepaired Heteroduplex DNA in Saccharomyces Cerevisiae Is Decreased in Rad1 Rad52-Independent Recombination

A direct repeat recombination assay between SUP4 heteroalleles detects unrepaired heteroduplex DNA (hDNA) as sectored colonies. The frequency of unrepaired heteroduplex is dependent on the mismatch and is highest in a construct that generates C:C or G:G mispairs and lowest in one that generates T:G or C:A mispairs. In addition, unrepaired hDNA increases for all mismatches tested in pms1 mismatch repair-deficient strains. These results support the notion that hDNA is formed across the SUP4 repeats during the recombination event and is then subject to mismatch repair. The effects of various repair and recombination defective mutations on this assay were examined. Unrepaired heteroduplex increases significantly only in rad52 mutant strains. In addition, direct repeat recombination is reduced 2-fold in rad52 mutant strains, while in rad51, rad54, rad55 and rad57 mutants direct repeat recombination is increased 3-4-fold. Mutations in the excision repair gene, RAD1, do not affect the frequency of direct repeat recombination. However, the level of unrepaired heteroduplex is slightly decreased in rad1 mutant strains. Similar to previous studies, rad1 rad52 double mutants show a synergistic reduction in direct repeat recombination (35-fold). Interestingly, unrepaired heteroduplex is reduced 4-fold in the double mutants. Experiments with shortened repeats suggest that the reduction in unrepaired heteroduplex is due to decreased hDNA tract length in the double mutant strain.     

Rad52 promotes postinvasion steps of meiotic double-strand-break repair

During DNA double-strand-break (DSB) repair by recombination, the broken chromosome uses a homologous chromosome as a repair template. Early steps of recombination are well characterized: DSB ends assemble filaments of RecA-family proteins that catalyze homologous pairing and strand-invasion reactions. By contrast, the postinvasion steps of recombination are poorly characterized. Rad52 plays an essential role during early steps of recombination by mediating assembly of a RecA homolog, Rad51, into nucleoprotein filaments. The meiosis-specific RecA-homolog Dmc1 does not show this dependence, however. By exploiting the Rad52 independence of Dmc1, we reveal that Rad52 promotes postinvasion steps of both crossover and noncrossover pathways of meiotic recombination in Saccharomyces cerevisiae. This activity resides in the N-terminal region of Rad52, which can anneal complementary DNA strands, and is independent of its Rad51-assembly function. Our findings show that Rad52 functions in temporally and biochemically distinct reactions and suggest a general annealing mechanism for reuniting DSB ends during recombination.     

In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair

Assembly and disassembly of Rad51 and Rad52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and Rad52 foci colocalize with a TetR-GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, we observe three distinct foci that colocalize with adjacent GFP chromosome marks. We compared the kinetics of focus formation with recombination intermediates and products when HO-cleaved MATalpha recombines with the donor, MATa. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATa. We present evidence for three distinct roles for Rad52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and Rad52.     

Heteroduplex joint formation by a stoichiometric complex of Rad51 and Rad52 of Saccharomyces cerevisiae

Both Rad51 and Rad52 are required for homologous genetic recombination in Saccharomyces cerevisiae. Rad51 promotes heteroduplex joint formation, a general step in homologous recombination. Rad52 facilitates the binding of Rad51 to replication protein A (RPA)-coated single-stranded DNA. The requirement of RPA can be avoided in vitro, if the single-stranded DNA is short. Using short single-stranded DNA and homologous double-stranded DNA, in the absence of RPA, we found that Rad52 (optimal at three per Rad51) was still required for Rad51-promoted heteroduplex joint formation in vitro, as assayed by the formation of D-loops, suggesting another role for Rad52. Rad51 has to bind to the single-stranded DNA before the addition of double-stranded DNA for efficient D-loop formation. Immunoprecipitation and single-stranded DNA-bead precipitation analyses revealed the presence of the free and DNA-bound complexes of Rad51 and Rad52 at a 1 to 2 stoichiometry. In the presence of single-stranded DNA, in addition to Rad51, Rad52 was required for extensive untwisting that is an intermediate step toward D-loop formation. Thus, these results suggest that the formation of the stoichiometric complex of Rad52 with Rad51 on single-stranded DNA is required for the functional binding of the protein-single-stranded DNA complex to the double-stranded DNA to form D-loops.