The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Corresponding author: Lorraine S. Symington, Institute of Cancer Research and Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 W. 168th St., New York, NY 10032., lss5{at}columbia.edu (E-mail)

Communicating editor: A. NICOLAS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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.

DNA double-strand breaks (DSBs) are potentially lethal lesions that are repaired by either homology-dependent recombinational repair or homology-independent mechanisms. The homology-dependent repair of DSBs requires genes of the RAD52 epistasis group (reviewed in PAQUES and HABER 1999 ). Most of these genes were identified by their requirement for the repair of ionizing radiation-induced DNA damage (GAME and MORTIMER 1974 ). RFA1, which encodes the largest subunit of the heterotrimeric DNA-binding complex, replication protein A (RPA), is also considered to be a member of the RAD52 group as certain non-null alleles show X-ray sensitivity, recombination deficiency, and genetic interaction with rad52 alleles (FIRMENICH et al. 1995 ; SMITH and ROTHSTEIN 1995 ; UMEZU et al. 1998 ). Although mutation of any of the genes in the RAD52 group results in sensitivity to ionizing radiation, there is considerable heterogeneity within the group when tested for recombination defects in various assays. In general, the genes within the RAD52 group fall into four subgroups based on the similarity of mutant phenotypes: MRE11, RAD50, and XRS2; RAD51 and RAD54 (and RDH54); RAD52 (and RAD59); and RAD55 and RAD57. The phenotype of rad59 mutants is most like rad52, but much less severe—hence the grouping with RAD52 (BAI and SYMINGTON 1996 ; BAI et al. 1999 ). Protein-protein interactions have been detected between members of most subgroups, as well as between some members of different groups. For example, Mre11, Rad50, and Xrs2 form a stable complex (USUI et al. 1998 ), Rad55 and Rad57 form a stable heterodimer (HAYS et al. 1995 ; JOHNSON and SYMINGTON 1995 ; SUNG 1997B ), and Rad54 interacts with Rad51 (JIANG et al. 1996 ; CLEVER et al. 1997 ). In addition, Rad51 interacts with Rad52 and Rad55 (SHINOHARA et al. 1992 ; MILNE and WEAVER 1993 ; HAYS et al. 1995 ; JOHNSON and SYMINGTON 1995 ).

Rad51, Rad52, Rad54, Rdh54, Rad55, Rad57, and RPA are required for efficient homologous pairing and strand exchange in vitro (SUNG 1994 ; SUNG 1997A , SUNG 1997B ; NEW et al. 1998 ; PETUKHOVA et al. 1998 , PETUKHOVA et al. 2000 ; SHINOHARA and OGAWA 1998 ; MAZIN et al. 2000 ; VAN KOMEN et al. 2000 ). Rad51 has significant homology to bacterial RecA proteins and is the key factor for homologous pairing and heteroduplex DNA formation in vitro. However, genetic assays have provided evidence for DSB repair that is independent of the RAD51, RAD54, RAD55, and RAD57 genes. These RAD51-independent events are generally detected using direct or inverted repeats and are thought to occur by a nonconservative mechanism that involves single-strand annealing (SSA; IVANOV et al. 1996 ; KANG and SYMINGTON 2000 ). Repair of a chromosomal DSB by strand invasion followed by replication to the end of the chromosome (break induced replication, or BIR) has also been observed in rad51, rad54, rad55, and rad57 mutants (MALKOVA et al. 1996 ; SIGNON et al. 2001 ). Even though BIR and gene conversion both require a common strand invasion step, BIR can occur in the absence of RAD51 and is considered to be nonconservative. These RAD51-independent events all require RAD52 and the annealing function of Rad52 is likely to play a central role in these events (MORTENSEN et al. 1996 ).

The RAD59 gene was identified in a screen for mutants that reduce the rate of RAD51-independent recombination between inverted repeats (BAI and SYMINGTON 1996 ). Synergism between rad51 and rad59 mutations is also observed for BIR (SIGNON et al. 2001 ) and for telomere maintenance in the absence of telomerase (CHEN et al. 2001 ). RAD59 encodes a 238-amino-acid protein with significant homology to the N-terminal region of Rad52, the region most highly conserved among Rad52 family members (BAI and SYMINGTON 1996 ). The C-terminal region of Rad52, which is absent from Rad59, is required for interaction with Rad51 (MILNE and WEAVER 1993 ). The observation that more than one copy of RAD52 suppresses the repair defect of rad59 mutants, and that certain non-null alleles of RAD52 have phenotypes similar to rad59 mutants, suggests that the two proteins have overlapping activities and/or form a complex (BAI and SYMINGTON 1996 ; BAI et al. 1999 ).

RAD52, RFA1, and RAD59 are the only members of the RAD52 epistasis group required for HO-induced deletion formation between direct repeats (SUGAWARA and HABER 1992 ; UMEZU et al. 1998 ; BAI et al. 1999 ; SUGAWARA et al. 2000 ). The requirement for RAD52 in this reaction is consistent with the observation that Rad52 promotes annealing of single-stranded DNA in vitro (MORTENSEN et al. 1996 ; SUGIYAMA et al. 1998 ). Purified Rad59 binds to single- and double-stranded DNA and also promotes annealing of complementary single-stranded DNA in vitro (PETUKHOVA et al. 1999 ). However, the annealing activity of Rad59 is not stimulated by RPA and Rad59 is unable to stimulate Rad51-promoted strand exchange in vitro (PETUKHOVA et al. 1999 ). This contrasts with Rad52, which is able to overcome the inhibition to strand exchange imposed by RPA, and shows RPA-stimulated strand annealing (SUNG 1997A ; BENSON et al. 1998 ; NEW et al. 1998 ; SHINOHARA and OGAWA 1998 ; SHINOHARA et al. 1998 ; SUGIYAMA et al. 1998 ). Rad52 interacts directly with RPA (PARK et al. 1996 ) and is thought to displace RPA from single-stranded DNA to promote annealing or load Rad51 in preparation for strand exchange. Thus Rad59 shares several in vitro activities with Rad52, except the ability to displace RPA to facilitate loading of Rad51. In this study we present evidence for interaction between Rad52 and Rad59 and suggest that the role of Rad59 in strand annealing is in the context of the Rad52-Rad59 complex.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media, growth conditions, and genetic methods:
Standard genetic methods were followed. Rich medium (YPD) and synthetic complete (SC) medium lacking the appropriate amino acid or nucleic acid base were prepared as described previously (SHERMAN et al. 1986 ). Selection for Ura- cells was performed on SC medium containing 5-fluoroorotic acid (5-FOA) at 1 mg/ml (BOEKE et al. 1987 ). To induce expression of Rad59, cultures were grown in synthetic medium containing 2% glucose prior to addition of galactose to 2%. To induce expression of HO endonuclease, cultures were grown in synthetic medium minus tryptophan containing 2% raffinose prior to addition of galactose to a final concentration of 2%. Yeast mating, sporulation, and tetrad dissection were performed as previously described (SHERMAN et al. 1986 ). Yeast cells were grown at 30°. Transformations were performed by the lithium acetate method (ITO et al. 1983 ).

Yeast strains and plasmids:
Saccharomyces cerevisiae strains used in this study are listed in Table 1. Strains T334 and PJ69-4A have been described previously (JAMES et al. 1996 ; LEWIS et al. 1998 ). Briefly, PJ69-4A is a strain designed for detecting protein-protein interactions by the two-hybrid assay and contains HIS3, ADE2, and lacZ reporters driven by the GAL1, GAL2, and GAL7 promoters, respectively. T334 contains the reg1-501 mutation, which allows galactose induction in the presence of glucose (HOVLAND et al. 1989 ; LEWIS et al. 1998 ). All other strains are derivatives of strains W303-1A or W303-1B (THOMAS and ROTHSTEIN 1989 ). To construct strain LSY959, W1479-80B (SMITH and ROTHSTEIN 1999 ) was first transformed with plasmid pRS414:MAT to allow mating to LSY836. Haploid progeny derived from this cross were grown nonselectively and then screened on SC-Trp to identify plasmid-free segregants. Segregation of rad52::HIS5 was scored by streaking progeny onto solid YPD medium and irradiating with 50 krad in a Gammacell 220 ⁶⁰Co irradiator. Progeny that failed to grow were scored as rad52::HIS5. Segregation of rfa1-D228Y was scored by restriction length polymorphism analysis, as described previously (SMITH and ROTHSTEIN 1995 ). To construct LSY997 and LSY999, LSY959-15D and LSY959-23C, respectively, were plated on SC medium containing 5-FOA to select for cells that have undergone a pop-out event, resulting in loss of the URA3 marker.

To construct pGAD10-RAD59 and pGBD-RAD59, a 951-bp fragment containing the RAD59 open reading frame (ORF) plus 275 bp of 3' noncoding sequence and a BglII site at the 5' end was generated by PCR using genomic DNA as the template and the following primers: 5'-GGGGAAGATCTTAATGACGTACAAGCGAAGCC and 5'-TTCGTTACCTTGGAATGGTATGT. To construct pGAD10-RAD59, the PCR fragment was digested with BglII and cloned into the BglII site of pGAD10 (gift from S. Fields). To construct pGBD-RAD59, the PCR fragment was digested with BglII and subcloned into the BglII site of pNotA/T7 (5 Prime) to generate pNotA/T7:RAD59. pNotA/T7:RAD59 was digested with BglII and the 918-bp fragment was cloned into the BamHI site of pGBD-C2 (JAMES et al. 1996 ) to generate pGBD-RAD59. To construct pRAD59-CAD and pRAD59-CDBD, a 736-bp fragment containing BamHI sites on the 5' and 3' ends and the RAD59 ORF minus the stop codon was generated by PCR using genomic DNA as the template and the following primers: 5'-GGATCCAAGTCTTATGACGATACAAGCGAAGCCC and 5'-GGATCCGCGGTTTGATATGCGTGCCTTTAGC. The PCR fragment was subcloned into the pGEM-T Easy (Promega, Madison, WI) vector system to generate pGEM-T:RAD59. pGEM-T:RAD59 was digested with BamHI and the 736-bp fragment was cloned into the BamHI site of pCAD2 (PRINTEN and SPRAGUE 1994 ) to generate pRAD59-CAD and into the BamHI site of pCDBD-1e (gift from R. Brazas) to generate pRAD59-CDBD. Prior to use, the structures of pGAD10-RAD59, pGBD-RAD59, pRAD59-CAD, and pRAD59-CDBD were confirmed by DNA sequencing and in a functional assay by complementation of the gamma ray sensitivity of a rad59 null strain (data not shown). The RAD52 two-hybrid plasmids were generous gifts from D. Weaver and P. Berg (MILNE and WEAVER 1993 ; HAYS et al. 1998 ). Plasmid YDL059 contains the RAD59 gene tagged at the C terminus with the V5 epitope and six histidine residues and is regulated by the GAL1 promoter (Invitrogen, San Diego). Rad52 was overexpressed using plasmid p52.1 (SUNG 1997A ). pFH800 contains the HO gene regulated by the GAL1 promoter on a CEN4 TRP1 vector (NICKOLOFF et al. 1989 ). To construct pADW6, which was used to express Rad59 tagged at its carboxy terminus with the V5 epitope (Rad59-V5) from its native promoter, a 1800-bp fragment containing RAD59 tagged at its C terminus with the V5 epitope tag was amplified using the following primers: 5'-AAGCTTGATGATCCACTAGTACGG and 5'-GGATCCGATTCATTAATGCAGGG with plasmid YDL059 as a DNA template. The PCR fragment was subcloned into the pGEM-T Easy (Promega) vector system to generate pGEM-T:GAL-RAD59-V5. pGEM-T:GAL-RAD59-V5 was digested with SpeI and the resulting 757-bp fragment containing the 3' end of RAD59 tagged with the V5 epitope was cloned into the SpeI sites of pRS416:RAD59, replacing a 1200-bp fragment containing the 3' end of RAD59. PET14b:RAD59 was made by cloning a RAD59-containing fragment, digested with NdeI and BglII, between the NdeI and BamHI sites of pET14b. The RAD59 fragment was made by PCR using primers BP59c: 5'-GGATAAACAGACAAACATATGACGATACAAGCGAAGCCCAG and BP59d: 5'-TTCGTTACCTTGGAATGGTATGT with the plasmid pRS416:RAD59 as the DNA template.

Two-hybrid analysis:
ß-Galactosidase activity was assayed quantitatively in permeabilized cells as described previously (ADAMS et al. 1998 ). At least four individual transformants of each category were assayed.

Co-immunoprecipitation:
For immunoprecipitations from cells overexpressing Rad52 and Rad59-V5, T334 cells overexpressing Rad52, a V5 epitope-tagged Rad59 (Rad59-V5), or both Rad52 and Rad59-V5 were grown to mid-log phase in selective synthetic medium. Galactose was added to a final concentration of 2% and incubation was continued for 16 hr. For immunoprecipitations from cells expressing Rad59-V5 in single copy, LSY999 cells (rad52 rad59) and LSY997 cells (rad59) carrying pRS416 or pRS416:RAD59-V5 were grown to mid-log phase in selective synthetic medium. For both sets of immunoprecipitations, cells (50 ml) were harvested, washed twice with 20 mM Tris at pH 7.4, 200 mM NaCl and stored at -70°. Extracts were prepared and immunoprecipitation performed as described previously (STRAHL-BOLSINGER et al. 1997 ). -V5 monoclonal antibody (Invitrogen) was used to immunoprecipitate Rad59 and -Rad52 crude serum (kindly provided by R. Rothstein) was used to precipitate Rad52. The V5 immunoprecipitation was probed for Rad52 using affinity-purified -Rad52 antibody (kindly provided by R. Rothstein) and affinity-purified -Rad51 antibody (kindly provided by P. Sung). The Rad52 immunoprecipitation was probed for Rad59-V5 using -V5-HRP monoclonal antibody (Invitrogen).

Physical analysis of HO-induced single-strand annealing:
Strains containing pFH800 (NICKOLOFF et al. 1989 ) were grown for 24 hr in 20 ml SC (raffinose) -Trp -Ura medium, which selects for retention of pFH800 and the leu2 duplication, respectively. Cells were then used to inoculate 350 ml SC (raffinose) -Trp. Cultures were grown to an OD₆₀₀ of 0.3–0.5. A 50-ml sample was removed for the 0-hr time point and then galactose was added to a final concentration of 2%. Incubation was continued and 50-ml samples were removed at the given times. For the overnight time point samples, the final aliquot was removed, centrifuged, resuspended in sterile water, and incubated overnight at 30°. For all samples, cells were harvested by centrifugation and washed with water. Cell pellets were then frozen in liquid nitrogen. DNA was extracted from the thawed cell pellets and digested with SpeI. DNA fragments were separated by electrophoresis through 0.8% agarose gels, transferred to nylon membranes, and hybridized with a 400-bp PCR fragment generated by amplification of sequences from the YCL017 ORF, which is adjacent to LEU2. Quantitation of the HO-cut and product bands was done using a Molecular Dynamics (Sunnyvale, CA) Storm 445 SI phosphorimager and IMAGE-QUANT software.

Determination of rates of spontaneous deletion formation:
At least two independent isolates of each strain were used to determine the rates of spontaneous deletion formation in three to seven trials. Mean rates from at least three trials of each strain are presented. Single colonies of each isolate were grown on YPD for 2–4 days. Nine individual colonies of each strain were suspended in water and appropriate dilutions were plated on SC to determine total cell number and on 5-FOA plates to determine the number of Ura- cells. Median recombination frequencies (5-FOA-resistant cells/total cells) were determined and rates (events/cell/generation) were calculated according to the following formula: rate = , where N is the total number of cells in the colony and N₀ (number of initial cells) = 1 (DRAKE 1970 ).

Proteins:
Rad59 was purified as the fusion protein with the N-terminal His₆-affinity tag from the Escherichia coli strain BL21/DE3 carrying plasmids pET14b-RAD59 and pLysS. Cells were grown in Luria broth medium containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol at 37° to OD₆₀₀ = 0.3–0.4. The cultures were cooled to 18° and isopropyl-1-thio-ß-D-galactopyranoside was added to 0.4 mM. Cells were cultured at 18° for an additional 16 hr. Cells were harvested and resuspended in 40 ml buffer A [20 mM Tris, pH 8.0, 300 mM NaCl, 10% glycerol, 7 mM mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.1% Triton, 1 mg/liter pepstatin A, and 0.5 mg/liter leupeptin]/liter culture volume. Cells were lysed by three freeze/thaw cycles and then briefly sonicated. The lysate was centrifuged at 15 K for 45 min and the supernatant removed to fresh tubes containing 1 ml Talon resin (CLONTECH, Palo Alto, CA)/liter culture volume equilibrated with buffer A. After mixing at 4° for 20 min the solution was transferred to an empty column and washed extensively with buffer A and then with 20 ml buffer A + 10 mM imidazole. Rad59 was eluted with buffer A containing 100 mM imidazole. Fractions containing Rad59 were dialyzed against 20 mM Tris, pH 8.0, 10% glycerol, 10 mM mercaptoethanol, 1 mM EDTA, and 0.1 mM PMSF until the conductivity was equivalent to 60 mM NaCl and then applied to a 1-ml Q-sepharose column (Pharmacia, Piscataway, NJ). Fifty percent of Rad59 was in the unbound fraction and directly applied to a 1-ml heparin agarose column (Pharmacia). Rad59 was eluted with a gradient of 100–800 mM NaCl in 20 mM Tris, pH 8.0, 10% glycerol, 10 mM mercaptoethanol, 1 mM EDTA, and 0.1 mM PMSF. Fractions containing Rad59 were stored at -80°. Purified Rad52 and RPA were generous gifts from S. Kowlaczykowski.

DNA annealing:
Annealing of a ³²P-labeled 48-mer oligonucleotide (oligo-25) and a complementary unlabeled 48-mer oligonucleotide (oligo-26) was performed as described in SUGIYAMA et al. 1998 except that the reaction buffer contained 30 mM Tris-Cl (pH 7.5), 5 mM MgCl₂, and 1 mM dithiothreitol. For the reaction containing RPA, Rad52, and Rad59, Rad52 and Rad59 were incubated together at room temperature for 1 min before addition to the preformed RPA-oligonucleotide complexes. In all reactions, DNA concentrations were 200 nM, Rad59 concentration was 50 nM, Rad52 concentration was 20 nM, and RPA concentration was 30 nM. Annealing was monitored by separation through 10% polyacrylamide gels in TAE buffer and quantified with a Molecular Dynamics Storm 445 SI phosphorimager and IMAGE-QUANT software. Numbers presented are the mean of three to six trials.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Rad59 and Rad52 interact in the two-hybrid system:
RAD52 present in more than one copy partially suppresses the gamma ray sensitivity of rad59 mutants, which suggests a physical interaction between Rad59 and Rad52 or overlapping functions (BAI and SYMINGTON 1996 ). The yeast two-hybrid system was used to determine whether Rad52 and Rad59 interact. Previous studies indicated that Rad52 homomeric interactions could be detected only by the two-hybrid system using fusion proteins in which the carboxy terminus of Rad52 was fused to either the Gal4 DNA-binding domain (GBD) or the Gal4 transactivating domain (GAD), but not by using amino-terminal fusion proteins (HAYS et al. 1998 ). We reasoned that interaction between Rad59 and Rad52 may also require the use of Rad52 carboxy-terminal fusions. The two-hybrid strain PJ69-4A (JAMES et al. 1996 ), expressing a carboxy-terminal Rad52-GAD fusion protein and either an amino-terminal or carboxy-terminal GBD-Rad59 fusion protein, activated expression of the GAL7-lacZ reporter gene, as determined by ß-galactosidase activity (Table 2). However, there was no activation if an amino-terminal GAD-Rad52 fusion protein was used. Activation required the presence of both Rad52 and Rad59 because no activation was detected when fusion proteins were coexpressed with the GBD- or GAD-encoding plasmids (Table 2).

We were unable to detect an interaction between Rad59 and Rad52 using a Rad52-GBD carboxy-terminal fusion protein and either an amino-terminal or carboxy-terminal Rad59 fusion to GAD (Table 2). We were also unable to detect a Rad59 homomeric interaction using any combination of fusion proteins. Both the amino- and carboxy-terminal Rad59 fusions to the Gal4 activation are functional as assayed by complementation of the gamma ray sensitivity of a rad59 null strain (data not shown), but fusion of GAD to Rad59 may interfere with the ability of GAD to activate transcription.

Rad59 and Rad52 co-immunoprecipitate from yeast extracts:
To obtain biochemical evidence for physical interaction between Rad59 and Rad52, we introduced plasmids overexpressing Rad52 and Rad59 tagged at the carboxy terminus with the V5 epitope (Rad59-V5) into strain T334, a trp1 derivative of strain 334 (HOVLAND et al. 1989 ; LEWIS et al. 1998 ). Rad52 was found to co-immunoprecipitate with Rad59-V5, and Rad59-V5 co-immunoprecipitated with Rad52 (Fig 1A). In control experiments, Rad52 did not co-immunoprecipitate when Rad59-V5 was not expressed and Rad59-V5 did not co-immunoprecipitate well when Rad52 was not overexpressed (Fig 1A). The interaction was unaffected by DNaseI or ethidium bromide, indicating that it was not due to independent association of the proteins with DNA present in the extract (data not shown). These results indicate that the interaction between Rad59 and Rad52 is specific.

View larger version (42K): In this window In a new window Download PPT slide   Figure 1. Co-immunoprecipitation of Rad59-V5 and Rad52. (A) Protein extracts (1 mg) were prepared from T334 cells overexpressing the indicated proteins. Rad52 and Rad59-V5 were immunoprecipitated (IP) with -Rad52 polyclonal antibody and -V5 monoclonal antibody, respectively. (B) Protein extracts (5–14 mg) were prepared from LSY999 cells (rad59 rad52) and LSY997 cells (rad59) carrying pRS416 or pRS416:RAD59-V5. Rad52 and Rad59-V5 were immunoprecipitated as in A. (A and B) The proteins were separated by SDS-PAGE and immunoblotted with -V5 monoclonal antibody to detect co-immunoprecipitation of Rad59-V5 with Rad52 and -Rad52 affinity-purified polyclonal antibody or -Rad51 affinity-purified polyclonal antibody to detect co-immunoprecipitation of Rad52 or Rad51 with Rad59-V5, respectively. (C) Proteins in the input crude extracts (200 µg) from cells overexpressing Rad52 and Rad59-V5 were immunoblotted with -Rad52 affinity-purified polyclonal antibody and -V5 monoclonal antibody. (D) Proteins in the input crude extracts (100–350 µg) from cells expressing Rad52 and Rad59-V5 from the native promoters were immunoblotted with -Rad52 affinity-purified polyclonal antibody, -V5 monoclonal antibody, and -Rad51 affinity-purified polyclonal antibody. Higher- and lower-molecular-weight proteins in the Rad52 immunoblot are cross-reacting species that are present in extracts from strains that lack RAD52.

To verify that interaction occurs when the proteins are expressed at normal levels, these experiments were repeated using rad59 or rad52 rad59 strains containing a CEN plasmid with RAD59 tagged at the carboxy terminus with the V5 epitope (Rad59-V5) and regulated by the native promoter. Rad52 was found to co-immunoprecipitate with Rad59-V5, and Rad59-V5 co-immunoprecipitated with Rad52 (Fig 1B), indicating that overexpression of the proteins is not required to detect their interaction. In control experiments, Rad52 did not co-immunoprecipitate when Rad59-V5 was not expressed and Rad59-V5 did not co-immunoprecipitate when Rad52 was not expressed (Fig 1B).

Previous studies have shown that Rad51 and Rad52 co-immunoprecipitate from yeast extracts (SUNG 1997A ). We were unable to detect Rad51 with Rad59 in immunoprecipitations from the RAD52 strain using the -V5 antibody, even though it was present in crude extracts (Fig 1B and Fig D). This result suggests that the Rad52/Rad59 complex is distinct from the Rad51/Rad52 complex.

In Fig 1C and Fig D, equal amounts of total protein were loaded into the lanes containing crude protein extracts. When both RAD59-V5 and RAD52 were overexpressed, a greater amount of Rad59-V5 was detected than when RAD59-V5 was overexpressed alone (Fig 1C). Likewise, when both RAD59-V5 and RAD52 were expressed from their native promoters, a greater amount of Rad59-V5 was detected than when RAD59-V5 was expressed alone (Fig 1D). These results suggest that Rad52 may play a role in stabilizing the Rad59 protein.

Rad59 promotes the annealing of complementary oligonucleotides and RPA-oligonucleotide complexes:
Several groups have demonstrated in vitro annealing activity by Rad52 using either plasmid or oligonucleotide substrates (MORTENSEN et al. 1996 ; SHINOHARA et al. 1998 ; SUGIYAMA et al. 1998 ). The annealing of plasmid length DNA molecules to form complex networks is promoted by Rad59, but unlike Rad52 is not stimulated by the addition of RPA (PETUKHOVA et al. 1999 ). We wished to determine if Rad59 mediates the annealing of complementary oligonucleotides to form duplex products and if this reaction is inhibited by RPA.

Complementary 48-mer oligonucleotides were incubated together with or without the protein of interest, and annealed products were monitored by gel electrophoresis (Fig 2; SUGIYAMA et al. 1998 ). In the absence of protein, 29% of the ³²P-labeled oligonucleotide annealed to the unlabeled complementary oligonucleotide within 2 min (Fig 2A and Fig C). Consistent with the results of SUGIYAMA et al. 1998 , 100% of the DNA was annealed by 2 min when 20 nM Rad52 was included in the reaction. When 50 nM Rad59 was included in the reaction, 91% of the DNA was annealed at 2 min and 100% annealed at 4 min (Fig 2A and Fig C). This result shows that, like Rad52, Rad59 mediates annealing of complementary oligonucleotides in vitro.

View larger version (49K): In this window In a new window Download PPT slide   Figure 2. Rad59 stimulation of oligonucleotide annealing. A 32P-labeled 48-mer oligonucleotide (oligo 25) was incubated with a complementary unlabeled 48-mer oligonucleotide (oligo 26) in 100 µl reaction buffer in the presence of the proteins indicated, and the products were monitored by PAGE. The final concentrations of Rad59, Rad52, and RPA were 50 nM, 20 nM, and 30 nM, respectively. (A) Reactions were started by the addition of oligo 26. (B) Reactions were started by the addition of the indicated proteins to preformed RPA-oligonucleotide complexes. (C–D) The percentages of DNA annealing in A and B, respectively, are plotted as a fraction of total radioactivity per lane.

The RPA complex plays both a stimulatory and inhibitory role in in vitro DNA annealing of plasmid length DNA molecules. RPA binds ssDNA, competing with Rad52 for ssDNA-binding sites, thereby inhibiting DNA annealing. RPA also removes secondary structure, which hinders annealing, thereby stimulating the DNA annealing reaction (SHINOHARA and OGAWA 1998 ; SUGIYAMA et al. 1998 ). This stimulation requires specific protein-protein interaction between Rad52 and RPA (SUGIYAMA et al. 1998 ). When using short oligonucleotide substrates, which have little or no secondary structure, RPA has only an inhibitory effect (SUGIYAMA et al. 1998 ). Consistent with the results of SUGIYAMA et al. 1998 , RPA was found to inhibit spontaneous annealing with just 20% of the DNA annealed at 8 min (Fig 2B and Fig D). Rad52 promotes the annealing of the oligonucleotides when they have been preincubated with RPA with 47% of the DNA annealed at 1 min, 64% annealed at 2 min, and 95% annealed at 8 min (Fig 2B and Fig D). When 50 nM Rad59 was added to the complementary RPA-oligonucleotide complexes, 38% of the DNA was annealed at 1 min, 50% was annealed at 2 min, and 73% was annealed at 8 min (Fig 2B and Fig D), indicating that Rad59, like Rad52, mediates the annealing of complementary RPA-coated oligonucleotides. When a mixture of Rad52 and Rad59 was added to the complementary RPA-oligonucleotide complexes, the DNA was annealed in an additive manner, with 55% of the DNA annealed at 1 min, 75% annealed at 2 min, and 97% annealed at 8 min. Thus, under these conditions, Rad59 stimulates Rad52 in the annealing of complementary RPA-oligonucleotide complexes.

RAD59 is not responsible for SSA in the absence of RAD52:
Repair of a DSB made within nonhomologous sequences between direct repeats occurs primarily by the SSA pathway. The ends of the break are processed by a 5' to 3' exonuclease, exposing complementary single-strand regions of the direct repeats that can anneal, resulting in a deletion of the unique DNA between the direct repeats (HABER 1995 ). This process is almost completely dependent on RAD52 (SUGAWARA and HABER 1992 ) and partially dependent on RAD59 (BAI et al. 1999 ; SUGAWARA et al. 2000 ), but it is independent of RAD51, RAD54, RAD55, and RAD57 (IVANOV et al. 1996 ). The defect in SSA caused by rad52 is partially suppressed by the rfa1-D228Y allele, which decreases the cellular levels of RPA (SMITH and ROTHSTEIN 1995 , SMITH and ROTHSTEIN 1999 ). Rad52 is thought to be required for the removal of RPA from single-stranded DNA tails during strand invasion and SSA (SUNG 1997A ; NEW et al. 1998 ; SHINOHARA and OGAWA 1998 ; SMITH and ROTHSTEIN 1999 ). SMITH and ROTHSTEIN 1999 suggest that an annealing factor that does not normally have access to single-stranded DNA can gain access to the DNA to promote annealing when cellular levels of RPA are limiting, as they are in rfa1-D228Y mutants, thereby annealing the DNA in the absence of Rad52. Given the SSA defects of rad59 mutants and the in vitro strand annealing activity of Rad59 protein (Fig 2; PETUKHOVA et al. 1999 ), we hypothesized that Rad59 is responsible for the SSA in rad52 rfa1-D228Y mutants and in rad52 mutants. Strains containing 2.4-kb direct repeats of the leu2 gene, separated by 5.7 kb of plasmid sequences containing a copy of the URA3 gene and an HO endonuclease cut site (Fig 3A; SMITH and ROTHSTEIN 1999 ), were used to monitor SSA. Induction of HO results in the formation of a DSB between the leu2 repeats and deletions are detected by the appearance of a novel 5.7-kb SpeI fragment. As described previously, the rad59 strain showed a delay in formation of the deletion product, but the final product level was reduced only two- to threefold compared with the wild-type strain (Fig 3B). The kinetics and efficiency of SSA in the rad59 rfa1-D288Y strain were similar to that observed for the rad59 strain. Deletion products were barely detectable in both the rad52 and rad52 rad59 strains. Analysis of the rad52 rfa1-D288Y and rad52 rfa1-D288Y rad59 strains revealed similar levels of deletion products, indicating that RAD59 is not required for SSA in the absence of RAD52. The reduced level of Rad59 protein observed in rad52 mutants could possibly account for the inability of Rad59 to substitute for Rad52 in vivo (Fig 1). However, even when Rad59 was overexpressed in a rad52 strain we were unable to suppress the defect in deletion formation (data not shown).

View larger version (106K): In this window In a new window Download PPT slide   Figure 3. Kinetics of HO-induced deletion formation. (A) Schematic representation of the leu2 direct-repeat substrate showing the locations of the SpeI sites, the HO cleavage site, and hybridization probe. After cleavage by HO, an 8.3-kb fragment is produced from the 13.4-kb SpeI fragment. The single-stranded tails formed after resection from the DSB site can anneal to form a deletion product that is detected as a 5.7-kb SpeI fragment. (B–I) Deletion formation was monitored in eight backgrounds: (B) wild type; (C) rad52; (D) rad59 rad52; (E) rad59; (F) rfa1-D228Y; (G) rad52 rfa1-D228Y; (H) rad59 rad52 rfa1-D228Y; and (I) rad59 rfa1-D228Y. DNA was extracted at the times shown and digested with SpeI. The positions of parental, HO-cut fragment, and deletion products are shown to the right of the autoradiograms. O/N, overnight.

Elevation of rates of spontaneous deletion formation in rfa1-D228Y mutants is independent of RAD59:
Rates of spontaneous deletion formation between 2.4-kb direct repeats were determined using the construct depicted in Fig 3A. Spontaneous deletions can occur by a variety of mechanisms, including SSA, intrachromatid reciprocal exchange, sister chromatid conversion or exchange, replication slippage, or sister strand exchange (reviewed in KLEIN 1995 ). Collectively, these events are identified as Ura^- (5-FOA^R) colonies in this assay. The rate of deletion formation in a rad59 mutant was not significantly different from the wild-type strain (Table 3). The rate of spontaneous deletion formation in rad52 mutants was reduced 4.8-fold as compared to wild type, and the rate was reduced an additional 2.3-fold in rad59 rad52 double mutants, consistent with a previous study (JABLONOVICH et al. 1999 ). The rate of spontaneous deletion formation in the rfa1-D228Y mutant was elevated >22-fold as compared to wild type, and this elevation was partially dependent on RAD52, consistent with the findings of SMITH and ROTHSTEIN 1999 . The rate in the rad59 rfa1-D228Y mutant was not significantly changed from the rate in rfa1-D228Y, and the rate in the rad59 rad52 rfa1-D228Y mutant was not significantly different from the rate in rad52 rfa1-D228Y (Table 3). These results indicate that the elevation of the rate of spontaneous deletion formation in rfa1-D228Y mutants is independent of RAD59.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genes in the RAD52 epistasis group are required for the repair of ionizing radiation-induced DNA damage, but the mutants show considerable heterogeneity in assays for spontaneous or double-strand break-induced recombination (PAQUES and HABER 1999 ). This heterogeneity is due to the variety of pathways used for the repair of double-strand breaks. RAD52 is required for all pathways of homology-dependent repair, including gene conversion, break-induced replication, and single-strand annealing (WHITE and HABER 1990 ; SUGAWARA and HABER 1992 ; RATTRAY and SYMINGTON 1994 ; MALKOVA et al. 1996 ). In contrast, RAD51, RAD54, RAD55, and RAD57 are essential for gene conversion, but dispensable for BIR and SSA (IVANOV et al. 1996 ; SIGNON et al. 2001 ). The RAD59 gene encodes a protein with significant homology to Rad52 and appears to function in the same pathways of homology-dependent double-strand break repair (DSBR) as RAD52. However, rad59 mutants show much less severe phenotypes in these assays than rad52 mutants (BAI and SYMINGTON 1996 ; BAI et al. 1999 ; JABLONOVICH et al. 1999 ; BARTSCH et al. 2000 ; SUGAWARA et al. 2000 ; SIGNON et al. 2001 ). Although Rad52 and Rad59 catalyze similar reactions in vitro, strand annealing shows greater dependence on RAD52 than RAD59 in vivo (Fig 3). We propose that Rad59 functions primarily in the context of Rad52 to enhance the activity of Rad52 in gene conversion, BIR, and SSA.

We demonstrated physical interaction between Rad52 and Rad59 by the two-hybrid system and co-immunoprecipitation, which is central to this hypothesis. These results extend and solidify conclusions drawn from suggestive data showing suppression of the rad59 radiation sensitivity by more than one copy of RAD52 and the isolation of a non-null rad52 allele with a phenotype similar to rad59 that acts synergistically with rad59 (BAI and SYMINGTON 1996 ; BAI et al. 1999 ). Rad52 is also known to form stable complexes with Rad51 and to interact with RPA (PARK et al. 1996 ; HAYS et al. 1998 ; SONG and SUNG 2000 ). The complex containing Rad52 and Rad59 appears not to contain Rad51, but could possibly include RPA. Rad52 forms ring structures when visualized by electron microscopy and binds to the ends of single-stranded DNA with the DNA apparently wrapped around the outside of the heptameric ring (SHINOHARA et al. 1998 ; PARSONS et al. 2000 ; STASIAK et al. 2000 ). We currently have no evidence for an oligomeric form of Rad59. The GBD-Rad59 and GAD-Rad59 fusions failed to interact in the two-hybrid assay, and the GAD-Rad59 fusions were also defective for interaction with Rad52. Since the GAD-Rad59 and Rad59-GAD fusions complement the ionizing radiation sensitivity of rad59 strains, and therefore retain Rad59 function, the failure in the two-hybrid assay could be due to the inability of GAD to activate transcription, the metric for interaction.

The high copy suppression of the rad59 repair defect by RAD52 could also be interpreted as evidence for overlapping functions of the two proteins. Biochemical characterization of Rad59 reveals several activities in common with Rad52, including DNA binding and annealing of complementary single-stranded DNA (Fig 2; PETUKHOVA et al. 1999 ). Rad52 can overcome the inhibitory effect of RPA to annealing of complementary oligonucleotides. Similarly, Rad59 is able to partially overcome the inhibition to annealing by RPA (Fig 2), but the annealing of long molecules by Rad59 is not enhanced by RPA (PETUKHOVA et al. 1999 ). In combined reactions we found a slight stimulation of Rad52-promoted annealing of RPA-coated oligonucleotides by Rad59. This result is consistent with the idea that Rad59 acts to enhance the activity of Rad52 in strand annealing.

rad52 mutants are highly defective in SSA in vivo, suggesting that Rad59 is unable to substitute for the annealing activity of Rad52. Rad52 is known to interact with RPA and is thought to displace RPA from single-stranded DNA to promote strand annealing or to recruit Rad51 (PARK et al. 1996 ; HAYS et al. 1998 ; SMITH and ROTHSTEIN 1999 ). The SSA defect of rad52 strains is suppressed by the rfa1-D288Y mutation, which results in lower cellular levels of RPA (SMITH and ROTHSTEIN 1995 , SMITH and ROTHSTEIN 1999 ). However, even in the rad52 rfa1-D288Y strain, in vivo annealing was independent of RAD59. This suggests that another factor promotes annealing in the rad52 rad59 rfa1-D288Y strain or that, when RPA levels are low, spontaneous annealing can occur with greater efficiency, eliminating the requirement for a dedicated annealing protein. The rate of spontaneous deletion formation between 2.4-kb direct repeats was unaffected by the rad59 mutation, which is in contrast to a study by JABLONOVICH et al. 1999 , who found a 1.5- to 3-fold reduction in rate. This may be due to the difference in the length of the repeats within the direct-repeat substrates. The direct repeats used by JABLONOVICH et al. 1999 are 0.3 kb long and the direct repeats used in this study are 2.4 kb long. Results from the Haber lab (SUGAWARA et al. 2000 ) suggest that the rad59 defect in SSA and DSB-induced gene conversion becomes less severe as the length of homology is increased. Thus, the weak effect of the rad59 mutation on spontaneous and DSB-induced deletion formation in this study may be due to the use of long repeats. One interpretation of these results is that Rad52 alone can efficiently anneal long single-stranded regions, but when the sequences are short Rad52 becomes more dependent on Rad59. In rad52 rad59 double mutants the rate of spontaneous deletion formation was slightly lower than observed for the rad52 strain, consistent with the results of JABLONOVICH et al. 1999 . We were unable to detect a similar decrease in the physical assay for SSA in the rad52 rad59 double mutant. This could be due to the limitation of the method used (quantitation of weak bands by phosphorimaging) or could reflect the requirement for RAD59 in a different pathway of spontaneous deletion formation.

In addition to SSA, RAD52 and RAD59 are both implicated in BIR. Diploid cells can repair a DSB induced at the MAT locus on one chromosome III homolog by gene conversion (the preferred mode of repair) or BIR. In rad52 mutants only chromosome loss events occur due to the absolute requirement for RAD52 in both repair processes (MALKOVA et al. 1996 ). By contrast, in rad51 mutants where gene conversion is eliminated, repair can occur by BIR, albeit inefficiently (MALKOVA et al. 1996 ). Mutation of RAD59 has no effect on gene conversion in a RAD51 strain, but the rad51 rad59 double mutant shows a sevenfold reduction in BIR events with a concomitant increase in chromosome loss events (SIGNON et al. 2001 ). The requirement for both RAD52 and RAD59 in BIR suggests that the initial strand invasion event to prime DNA synthesis is promoted by Rad52 or the Rad52/Rad59 complex. This could occur by annealing between the invading single strand and a region of the donor duplex that is transiently unwound, either by replication or transcription. If the initial annealing step is confined to short regions of DNA, this might explain the requirement for Rad59 acting together with Rad52. Alternatively, Rad59 or the Rad52/Rad59 complex might be important for recruiting components of the replication apparatus.

RAD52 is essential for homology-dependent repair of double-strand breaks in yeast, but in mouse and chicken bursal cells it appears to play a less important role in repair. Deletion of MmRAD52 does not cause embryonic lethality, unlike RAD51 (LIM and HASTY 1996 ; TSUZUKI et al. 1996 ), and cell lines deficient for mouse or chicken RAD52 are resistant to ionizing radiation and exhibit only a slight decrease in homologous recombination (RIJKERS et al. 1998 ; YAMAGUCHI-IWAI et al. 1998 ). The yeasts S. cerevisiae, Kluyveromyces lactis, and Schizosaccharomyces pombe each have two Rad52-like proteins (BAI and SYMINGTON 1996 ; SUTO et al. 1999 ; VAN DEN BOSCH et al. 2001A , VAN DEN BOSCH et al. 2001B ). In S. cerevisiae, RAD52 is essential for DSBR and RAD59 plays an auxiliary role (BAI and SYMINGTON 1996 ; BAI et al. 1999 ). The fission yeast Rad52-like proteins show more of a division of labor with rad22A playing the major role, but synergistic defects in radiation sensitivity and meiosis are found in rad22A rad22B/rti1 double mutants (SUTO et al. 1999 ; VAN DEN BOSCH et al. 2001). We suggest higher eukaryotes will have other Rad52-like proteins and these will contribute to the repair of DSBs with the known Rad52 homologs.

	ACKNOWLEDGMENTS

We thank P. Berg, R. Brazas, M. Carlson, S. Fields, R. Rothstein, and P. Sung for gifts of antibodies, plasmids, and yeast strains, and S. Kowalczykowski for the generous gift of Rad52 and RPA proteins. We thank members of the Symington laboratory and W. K. Holloman for critical reading of the manuscript. The research described in the article was supported by grants from the National Institutes of Health (GM41784, T32 CA09503, and T32 AI07161)

Manuscript received May 2, 2001; Accepted for publication July 23, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ADAMS, A., D. E. GOTTSCHLING, C. A. KAISER and T. STEARNS, 1998 Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

BAI, Y. and L. S. SYMINGTON, 1996  A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 10:2025-2037[Abstract/Free Full Text].

BAI, Y., A. P. DAVIS, and L. S. SYMINGTON, 1999  A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59. Genetics 153:1117-1130[Abstract/Free Full Text].

BARTSCH, S., L. E. KANG, and L. S. SYMINGTON, 2000  RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates. Mol. Cell. Biol. 20:1194-1205[Abstract/Free Full Text].

BENSON, F. E., P. BAUMANN, and S. C. WEST, 1998  Synergistic actions of Rad51 and Rad52 in recombination and DNA repair. Nature 391:401-404[Medline].

BOEKE, J. D., J. TRUEHEART, G. NATSOULIS, and G. R. FINK, 1987  5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154:164-175[Medline].

CHEN, Q., A. IJPMA, and C. W. GREIDER, 2001  Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events. Mol. Cell. Biol. 21:1819-1827[Abstract/Free Full Text].

CLEVER, B., H. INTERTHAL, J. SCHMUCKLI-MAURER, J. KING, and M. SIGRIST et al., 1997  Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins. EMBO J. 16:2535-2544[Medline].

DRAKE, J. W., 1970 The Molecular Basis of Mutation. Holden-Day, San Francisco.

FIRMENICH, A. A., M. ELIAS-ARNANZ, and P. BERG, 1995  A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52. Mol. Cell. Biol. 15:1620-1631[Abstract].

GAME, J. C. and R. K. MORTIMER, 1974  A genetic study of x-ray sensitive mutants in yeast. Mutat. Res. 24:281-292[Medline].

HABER, J. E., 1995  In vivo biochemistry: physical monitoring of recombination induced by site-specific endonucleases. Bioessays 17:609-620[Medline].

HAYS, S. L., A. A. FIRMENICH, and P. BERG, 1995  Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins. Proc. Natl. Acad. Sci. USA 92:6925-6929[Abstract/Free Full Text].

HAYS, S. L., A. A. FIRMENICH, P. MASSEY, R. BANERJEE, and P. BERG, 1998  Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA. Mol. Cell. Biol. 18:4400-4406[Abstract/Free Full Text].

HOVLAND, P., J. FLICK, M. JOHNSTON, and R. A. SCLAFANI, 1989  Galactose as a gratuitous inducer of GAL gene expression in yeasts growing on glucose. Gene 83:57-64[Medline].

ITO, H., Y. FUKUDA, K. MURATA, and A. KIMURA, 1983  Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163-168[Abstract/Free Full Text].

IVANOV, E. L., N. SUGAWARA, J. FISHMAN-LOBELL, and J. E. HABER, 1996  Genetic requirements for the single-strand annealing pathway of double-strand break repair in Saccharomyces cerevisiae.. Genetics 142:693-704[Abstract].

JABLONOVICH, Z., B. LIEFSHITZ, R. STEINLAUF, and M. KUPIEC, 1999  Characterization of the role played by the RAD59 gene of Saccharomyces cerevisiae in ectopic recombination. Curr. Genet. 36:13-20[Medline].

JAMES, P., J. HALLADAY, and E. A. CRAIG, 1996  Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144:1425-1436[Abstract].

JIANG, H., Y. XIE, P. HOUSTON, K. STEMKE-HALE, and U. H. MORTENSEN et al., 1996  Direct association between the yeast Rad51 and Rad54 recombination proteins. J. Biol. Chem. 271:33181-33186[Abstract/Free Full Text].

JOHNSON, R. D. and L. S. SYMINGTON, 1995  Functional differences and interactions among the putative RecA homologs Rad51, Rad55, and Rad57. Mol. Cell. Biol. 15:4843-4850[Abstract].

KANG, L. E. and L. S. SYMINGTON, 2000  Aberrant double-strand break repair in rad51 mutants of Saccharomyces cerevisiae. Mol. Cell. Biol. 20:9162-9172[Abstract/Free Full Text].

KLEIN, H. L., 1995  Genetic control of intrachromosomal recombination. Bioessays 17:147-159[Medline].

LEWIS, L. K., J. M. KIRCHNER, and M. A. RESNICK, 1998  Requirement for end-joining and checkpoint functions, but not RAD52-mediated recombination, after EcoRI endonuclease cleavage of Saccharomyces cerevisiae DNA. Mol. Cell. Biol. 18:1891-1902[Abstract/Free Full Text].

LIM, D. S. and P. HASTY, 1996  A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol. Cell. Biol. 16:7133-7143[Abstract].

MALKOVA, A., E. L. IVANOV, and J. E. HABER, 1996  Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication. Proc. Natl. Acad. Sci. USA 93:7131-7136[Abstract/Free Full Text].

MAZIN, A. V., C. J. BORNARTH, J. A. SOLINGER, W. D. HEYER, and S. C. KOWALCZYKOWSKI, 2000  Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament. Mol. Cell 6:583-592[Medline].

MILNE, G. T. and D. T. WEAVER, 1993  Dominant negative alleles of RAD52 reveal a DNA repair/recombination complex including Rad51 and Rad52. Genes Dev. 7:1755-1765[Abstract/Free Full Text].

MORTENSEN, U. H., C. BENDIXEN, I. SUNJEVARIC, and R. ROTHSTEIN, 1996  DNA strand annealing is promoted by the yeast Rad52 protein. Proc. Natl. Acad. Sci. USA 93:10729-10734[Abstract/Free Full Text].

NEW, J. H., T. SUGIYAMA, E. ZAITSEVA, and S. C. KOWALCZYKOWSKI, 1998  Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature 391:407-410[Medline].

NICKOLOFF, J. A., J. D. SINGER, M. F. HOEKSTRA, and F. HEFFRON, 1989  Double-strand breaks stimulate alternative mechanisms of recombination repair. J. Mol. Biol. 207:527-541[Medline].

PAQUES, F. and J. E. HABER, 1999  Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63:349-404[Abstract/Free Full Text].

PARK, M. S., D. L. LUDWIG, E. STIGGER, and S. H. LEE, 1996  Physical interaction between human RAD52 and RPA is required for homologous recombination in mammalian cells. J. Biol. Chem. 271:18996-19000[Abstract/Free Full Text].

PARSONS, C. A., P. BAUMANN, E. VAN DYCK, and S. C. WEST, 2000  Precise binding of single-stranded DNA termini by human RAD52 protein. EMBO J. 19:4175-4181[Medline].

PETUKHOVA, G., S. STRATTON, and P. SUNG, 1998  Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393:91-94[Medline].

PETUKHOVA, G., S. A. STRATTON, and P. SUNG, 1999  Single strand DNA binding and annealing activities in the yeast recombination factor Rad59. J. Biol. Chem. 274:33839-33842[Abstract/Free Full Text].

PETUKHOVA, G., P. SUNG, and H. KLEIN, 2000  Promotion of Rad51-dependent D-loop formation by yeast recombination factor Rdh54/Tid1. Genes Dev. 14:2206-2215[Abstract/Free Full Text].

PRINTEN, J. A. and G. F. SPRAGUE, JR., 1994  Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. Genetics 138:609-619[Abstract].

RATTRAY, A. J. and L. S. SYMINGTON, 1994  Use of a chromosomal inverted repeat to demonstrate that the RAD51 and RAD52 genes of Saccharomyces cerevisiae have different roles in mitotic recombination. Genetics 138:587-595[Abstract].

RIJKERS, T., J. VAN DEN OUWELAND, B. MOROLLI, A. G. ROLINK, and W. M. BAARENDS et al., 1998  Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation. Mol. Cell. Biol. 18:6423-6429[Abstract/Free Full Text].

SHERMAN, F., G. FINK and J. HICKS, 1986 Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SHINOHARA, A., H. OGAWA, and T. OGAWA, 1992  Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69:457-470[Medline].

SHINOHARA, A. and T. OGAWA, 1998  Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature 391:404-407[Medline].

SHINOHARA, A., M. SHINOHARA, T. OHTA, S. MATSUDA, and T. OGAWA, 1998  Rad52 forms ring structures and co-operates with RPA in single-strand DNA annealing. Genes Cells 3:145-156[Abstract].

SIGNON, L., A. MALKOVA, M. L. NAYLOR, H. KLEIN, and J. E. HABER, 2001  Genetic requirements for RAD51- and RAD54-independent break-induced replication repair of a chromosomal double-strand break. Mol. Cell. Biol. 21:2048-2056[Abstract/Free Full Text].

SMITH, J. and R. ROTHSTEIN, 1995  A mutation in the gene encoding the Saccharomyces cerevisiae single-stranded DNA-binding protein Rfa1 stimulates a RAD52-independent pathway for direct-repeat recombination. Mol. Cell. Biol. 15:1632-1641[Abstract].

SMITH, J. and R. ROTHSTEIN, 1999  An allele of RFA1 suppresses RAD52-dependent double-strand break repair in Saccharomyces cerevisiae. Genetics 151:447-458[Abstract/Free Full Text].

SONG, B. and P. SUNG, 2000  Functional interactions among yeast Rad51 recombinase, Rad52 mediator, and replication protein A in DNA strand exchange. J. Biol. Chem. 275:15895-15904[Abstract/Free Full Text].

STASIAK, A. Z., E. LARQUET, A. STASIAK, S. MULLER, and A. ENGEL et al., 2000  The human Rad52 protein exists as a heptameric ring. Curr. Biol. 10:337-340[Medline].

STRAHL-BOLSINGER, S., A. HECHT, K. LUO, and M. GRUNSTEIN, 1997  SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev. 11:83-93[Abstract/Free Full Text].

SUGAWARA, N. and J. E. HABER, 1992  Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation. Mol. Cell. Biol. 12:563-575[Abstract/Free Full Text].

SUGAWARA, N., G. IRA, and J. E. HABER, 2000  DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair. Mol. Cell. Biol. 20:5300-5309[Abstract/Free Full Text].

SUGIYAMA, T., J. H. NEW, and S. C. KOWALCZYKOWSKI, 1998  DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA. Proc. Natl. Acad. Sci. USA 95:6049-6054[Abstract/Free Full Text].

SUNG, P., 1994  Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science 265:1241-1243[Abstract/Free Full Text].

SUNG, P., 1997a  Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase. J. Biol. Chem. 272:28194-28197[Abstract/Free Full Text].

SUNG, P., 1997b  Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev. 11:1111-1121[Abstract/Free Full Text].

SUTO, K., A. NAGATA, H. MURAKAMI, and H. OKAYAMA, 1999  A double-strand break repair component is essential for S phase completion in fission yeast cell cycling. Mol. Biol. Cell 10:3331-3343[Abstract/Free Full Text].

THOMAS, B. J. and R. ROTHSTEIN, 1989  The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene. Genetics 123:725-738[Abstract/Free Full Text].

TSUZUKI, T., Y. FUJII, K. SAKUMI, Y. TOMINAGA, and K. NAKAO et al., 1996  Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. USA 93:6236-6240[Abstract/Free Full Text].

UMEZU, K., N. SUGAWARA, C. CHEN, J. E. HABER, and R. D. KOLODNER, 1998  Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism. Genetics 148:989-1005[Abstract/Free Full Text].

USUI, T., T. OHTA, H. OSHIUMI, J. TOMIZAWA, and H. OGAWA et al., 1998  Complex formation and functional versatility of Mre11 of budding yeast in recombination. Cell 95:705-716[Medline].

VAN DEN BOSCH, M., K. VREEKEN, J. B. ZONNEVELD, J. A. BRANDSMA, and M. LOMBAERTS et al., 2001a  Characterization of RAD52 homologs in the fission yeast Schizosaccharomyces pombe. Mutat. Res. 461:311-323[Medline].

VAN DEN BOSCH, M., J. B. ZONNEVELD, P. H. LOHMAN, and A. PASTINK, 2001b  Isolation and characterization of the RAD59 homologue of Kluyveromyces lactis. Curr. Genet. 39:305-310[Medline].

VAN KOMEN, S., G. PETUKHOVA, S. SIGURDSSON, S. STRATTON, and P. SUNG, 2000  Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54. Mol. Cell 6:563-572[Medline].

WHITE, C. I. and J. E. HABER, 1990  Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. EMBO J. 9:663-673[Medline].

YAMAGUCHI-IWAI, Y., E. SONODA, J. M. BUERSTEDDE, O. BEZZUBOVA, and C. MORRISON et al., 1998  Homologous recombination, but not DNA repair, is reduced in vertebrate cells deficient in RAD52. Mol. Cell. Biol. 18:6430-6435[Abstract/Free Full Text].

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				D. S. Wei and Y. S. Rong A Genetic Screen For DNA Double-Strand Break Repair Mutations in Drosophila Genetics, September 1, 2007; 177(1): 63 - 77. [Abstract] [Full Text] [PDF]

				A. Decottignies Microhomology-Mediated End Joining in Fission Yeast Is Repressed by Pku70 and Relies on Genes Involved in Homologous Recombination Genetics, July 1, 2007; 176(3): 1403 - 1415. [Abstract] [Full Text] [PDF]

				Y. Wu, J. S. Siino, T. Sugiyama, and S. C. Kowalczykowski The DNA Binding Preference of RAD52 and RAD59 Proteins: IMPLICATIONS FOR RAD52 AND RAD59 PROTEIN FUNCTION IN HOMOLOGOUS RECOMBINATION J. Biol. Chem., December 29, 2006; 281(52): 40001 - 40009. [Abstract] [Full Text] [PDF]

				Y. Wu, T. Sugiyama, and S. C. Kowalczykowski DNA Annealing Mediated by Rad52 and Rad59 Proteins J. Biol. Chem., June 2, 2006; 281(22): 15441 - 15449. [Abstract] [Full Text] [PDF]

				F. Cortes-Ledesma, F. Malagon, and A. Aguilera A Novel Yeast Mutation, rad52-L89F, Causes a Specific Defect in Rad51-Independent Recombination That Correlates With a Reduced Ability of Rad52-L89F to Interact With Rad59 Genetics, September 1, 2004; 168(1): 553 - 557. [Abstract] [Full Text] [PDF]

				E. Haghnazari and W.-D. Heyer The DNA damage checkpoint pathways exert multiple controls on the efficiency and outcome of the repair of a double-stranded DNA gap Nucleic Acids Res., August 10, 2004; 32(14): 4257 - 4268. [Abstract] [Full Text] [PDF]

				J. Z. Torres, S. L. Schnakenberg, and V. A. Zakian Saccharomyces cerevisiae Rrm3p DNA Helicase Promotes Genome Integrity by Preventing Replication Fork Stalling: Viability of rrm3 Cells Requires the Intra-S-Phase Checkpoint and Fork Restart Activities Mol. Cell. Biol., April 15, 2004; 24(8): 3198 - 3212. [Abstract] [Full Text] [PDF]

				J. Yu, K. Marshall, M. Yamaguchi, J. E. Haber, and C. F. Weil Microhomology-Dependent End Joining and Repair of Transposon-Induced DNA Hairpins by Host Factors in Saccharomyces cerevisiae Mol. Cell. Biol., February 1, 2004; 24(3): 1351 - 1364. [Abstract] [Full Text] [PDF]

				M. Tsukamoto, K. Yamashita, T. Miyazaki, M. Shinohara, and A. Shinohara The N-Terminal DNA-Binding Domain of Rad52 Promotes RAD51-Independent Recombination in Saccharomyces cerevisiae Genetics, December 1, 2003; 165(4): 1703 - 1715. [Abstract] [Full Text] [PDF]

				X. Yu and A. Gabriel Ku-Dependent and Ku-Independent End-Joining Pathways Lead to Chromosomal Rearrangements During Double-Strand Break Repair in Saccharomyces cerevisiae Genetics, March 1, 2003; 163(3): 843 - 856. [Abstract] [Full Text] [PDF]

				L. S. Symington Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair Microbiol. Mol. Biol. Rev., December 1, 2002; 66(4): 630 - 670. [Abstract] [Full Text] [PDF]

				M. R. Singleton, L. M. Wentzell, Y. Liu, S. C. West, and D. B. Wigley Structure of the single-strand annealing domain of human RAD52 protein PNAS, October 15, 2002; 99(21): 13492 - 13497. [Abstract] [Full Text] [PDF]

				S. Gonzalez-Barrera, M. Garcia-Rubio, and A. Aguilera Transcription and Double-Strand Breaks Induce Similar Mitotic Recombination Events in Saccharomyces cerevisiae Genetics, October 1, 2002; 162(2): 603 - 614. [Abstract] [Full Text] [PDF]

				T. E. Wilson A Genomics-Based Screen for Yeast Mutants With an Altered Recombination/End-Joining Repair Ratio Genetics, October 1, 2002; 162(2): 677 - 688. [Abstract] [Full Text] [PDF]

				G. Ira and J. E. Haber Characterization of RAD51-Independent Break-Induced Replication That Acts Preferentially with Short Homologous Sequences Mol. Cell. Biol., September 15, 2002; 22(18): 6384 - 6392. [Abstract] [Full Text] [PDF]

				J. A. Freedman and S. Jinks-Robertson Genetic Requirements for Spontaneous and Transcription-Stimulated Mitotic Recombination in Saccharomyces cerevisiae Genetics, September 1, 2002; 162(1): 15 - 27. [Abstract] [Full Text] [PDF]