Rad52 partially substitutes for the Rad51 paralog XRCC3 in maintaining chromosomal integrity in vertebrate cells

Article1 October 2001free access Rad52 partially substitutes for the Rad51 paralog XRCC3 in maintaining chromosomal integrity in vertebrate cells Akira Fujimori Akira Fujimori Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan Search for more papers by this author Seiji Tachiiri Seiji Tachiiri Department of Therapeutic Radiology and Oncology, Faculty of Medicine, Kyoto University, Shogoin-Kawaracho, Sakyo-ku, Kyoto, 606-8507 Japan Search for more papers by this author Eiichiro Sonoda Eiichiro Sonoda Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan CREST, JST (Japan Science and Technology), Saitama, Japan Search for more papers by this author Larry H. Thompson Larry H. Thompson BBR Program, Lawrence Livermore National Laboratory, PO Box 808, Livermore, CA, 94551-0808 USA Search for more papers by this author Pawan Kumar Dhar Pawan Kumar Dhar Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan CREST, JST (Japan Science and Technology), Saitama, Japan Search for more papers by this author Masahiro Hiraoka Masahiro Hiraoka Department of Therapeutic Radiology and Oncology, Faculty of Medicine, Kyoto University, Shogoin-Kawaracho, Sakyo-ku, Kyoto, 606-8507 Japan Search for more papers by this author Shunichi Takeda Shunichi Takeda Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan CREST, JST (Japan Science and Technology), Saitama, Japan Search for more papers by this author Yong Zhang Yong Zhang Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck Institute for Immunobiology, Stübeweg 51, D-79108 Freiburg, Germany Search for more papers by this author Michael Reth Michael Reth Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck Institute for Immunobiology, Stübeweg 51, D-79108 Freiburg, Germany Search for more papers by this author Minoru Takata Corresponding Author Minoru Takata Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan CREST, JST (Japan Science and Technology), Saitama, Japan Present address: Department of Immunology and Molecular Genetics, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama, 701-0192 Japan Search for more papers by this author Akira Fujimori Akira Fujimori Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan Search for more papers by this author Seiji Tachiiri Seiji Tachiiri Department of Therapeutic Radiology and Oncology, Faculty of Medicine, Kyoto University, Shogoin-Kawaracho, Sakyo-ku, Kyoto, 606-8507 Japan Search for more papers by this author Eiichiro Sonoda Eiichiro Sonoda Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan CREST, JST (Japan Science and Technology), Saitama, Japan Search for more papers by this author Larry H. Thompson Larry H. Thompson BBR Program, Lawrence Livermore National Laboratory, PO Box 808, Livermore, CA, 94551-0808 USA Search for more papers by this author Pawan Kumar Dhar Pawan Kumar Dhar Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan CREST, JST (Japan Science and Technology), Saitama, Japan Search for more papers by this author Masahiro Hiraoka Masahiro Hiraoka Department of Therapeutic Radiology and Oncology, Faculty of Medicine, Kyoto University, Shogoin-Kawaracho, Sakyo-ku, Kyoto, 606-8507 Japan Search for more papers by this author Shunichi Takeda Shunichi Takeda Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan CREST, JST (Japan Science and Technology), Saitama, Japan Search for more papers by this author Yong Zhang Yong Zhang Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck Institute for Immunobiology, Stübeweg 51, D-79108 Freiburg, Germany Search for more papers by this author Michael Reth Michael Reth Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck Institute for Immunobiology, Stübeweg 51, D-79108 Freiburg, Germany Search for more papers by this author Minoru Takata Corresponding Author Minoru Takata Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan CREST, JST (Japan Science and Technology), Saitama, Japan Present address: Department of Immunology and Molecular Genetics, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama, 701-0192 Japan Search for more papers by this author Author Information Akira Fujimori1, Seiji Tachiiri2, Eiichiro Sonoda1,3, Larry H. Thompson4, Pawan Kumar Dhar1,3, Masahiro Hiraoka2, Shunichi Takeda1,3, Yong Zhang5, Michael Reth5 and Minoru Takata 1,3,6 1Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501 Japan 2Department of Therapeutic Radiology and Oncology, Faculty of Medicine, Kyoto University, Shogoin-Kawaracho, Sakyo-ku, Kyoto, 606-8507 Japan 3CREST, JST (Japan Science and Technology), Saitama, Japan 4BBR Program, Lawrence Livermore National Laboratory, PO Box 808, Livermore, CA, 94551-0808 USA 5Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck Institute for Immunobiology, Stübeweg 51, D-79108 Freiburg, Germany 6Present address: Department of Immunology and Molecular Genetics, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama, 701-0192 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5513-5520https://doi.org/10.1093/emboj/20.19.5513 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Yeast Rad52 DNA-repair mutants exhibit pronounced radiation sensitivity and a defect in homologous re combination (HR), whereas vertebrate cells lacking Rad52 exhibit a nearly normal phenotype. Bio chemical studies show that both yeast Rad52 and Rad55–57 (Rad51 paralogs) stimulate DNA-strand exchange mediated by Rad51. These findings raise the possibility that Rad51 paralogs may compensate for lack of Rad52 in vertebrate cells, explaining the absence of prominent phenotypes for Rad52-deficient cells. To test this hypothesis, using chicken DT40 cells, we generated conditional mutants deficient in both RAD52 and XRCC3, which is one of the five vertebrate RAD51 paralogs. Surprisingly, the rad52 xrcc3 double-mutant cells were non-viable and exhibited extensive chromosomal breaks, whereas rad52 and xrcc3 single mutants grew well. Our data reveal an overlapping (but non-reciprocal) role for Rad52 and XRCC3 in repairing DNA double-strand breaks. The present study shows that Rad52 can play an important role in HR repair by partially substituting for a Rad51 paralog. Introduction DNA double-strand breaks (DSBs) arise during DNA replication and from exposure to agents such as ionizing radiation (IR). A single DSB may cause cell death if left unrepaired. Non-homologous end joining (NHEJ) and homologous recombination (HR) are major DSB repair pathways, and both are conserved across eukaryotic cells (reviewed in Jeggo, 1998; Haber, 1999; Morrison and Takeda, 2000; Sonoda et al., 2001; Thompson and Schild, 2001). The genes involved in HR in the yeast Saccharo myces cerevisiae form the RAD52 epistasis group of genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2) (reviewed in Paques and Haber, 1999). Mutants of these genes are hypersensitive to IR and exhibit mitotic and meiotic recombination defects. Among the members of the Rad52 epistasis group, Rad51, a structural and functional homolog of Escherichia coli RecA, is conserved to the highest degree, exhibiting 69% amino acids sequence identity between S.cerevisiae and humans (Shinohara and Ogawa, 1995). This high degree of conservation suggests that the function of Rad51 is also conserved among eukaryotes. Defective Rad51 is lethal to higher eukaryotic cells, indicating a critical role for HR in repairing spontaneous DSBs arising during DNA replication (Tsuzuki et al., 1996; Sonoda et al., 1998). In vitro studies have shown that yeast and human Rad51 proteins form multimeric helical nucleoprotein filaments, similar to RecA proteins, which are assembled on single-stranded DNA (ssDNA) or on double-stranded DNA (dsDNA) containing either 5′ or 3′ single-stranded tails (Mazin et al., 2000; Sigurdsson et al., 2001). The nucleoprotein filaments mediate the search for homology, strand pairing and strand exchange (Baumann and West, 1998). Relatives of the Rad51 gene that probably arose by gene duplication and the evolution of new functions (paralogs) are present in yeast and higher eukaryotes (Thompson and Schild, 2001). These Rad51 paralogs include Rad55 and Rad57 (Johnson and Symington, 1995) in S.cerevisiae, and in vertebrates, XRCC2 (Cartwright et al., 1998b; Liu et al., 1998), XRCC3 (Tebbs et al., 1995; Liu et al., 1998), Rad51B/Rad51L1 (Albala et al., 1997; Rice et al., 1997; Cartwright et al., 1998a), Rad51C/Rad51L2 (Dosanjh et al., 1998) and Rad51D/Rad51L3 (Cartwright et al., 1998b; Kawabata and Saeki, 1998; Pittman et al., 1998). The five human Rad51 paralogs have only 20–30% identity to human Rad51, with each other, and with yeast Rad55 and Rad57 (reviewed in Thacker, 1999). Yeast two-hybrid studies of human Rad51 paralogs have shown that, unlike Rad51, none of them shows self-association while physical interactions occur between human Rad51 and XRCC3, XRCC3 and Rad51C, Rad51B and Rad51C, Rad51C and Rad51D, and Rad51D and XRCC2. Thus, each Rad51 paralog appears to have different interacting partners within the family (reviewed in Thompson and Schild, 2001). In analogy with the S.cerevisiae Rad55 and Rad57 proteins (Sung, 1997b), the vertebrate paralogs may provide Rad51 accessory functions. This notion is in agreement with the data from our previous genetic study in which all of the Rad51 paralog mutants derived from the chicken B lymphocyte DT40 line exhibited remarkably similar phenotypes (Takata et al., 2000, 2001). Thus, the Rad51 paralogs have non-overlapping roles, and they all participate in DNA repair mediated by HR. Moreover, all the Rad51-paralog mutants show defective Rad51 focus formation and partial correction of the sensitivity to DNA damage from overexpression of human Rad51. These observations suggest that one or more complexes involving Rad51 paralogs facilitate the action of Rad51 in HR. Rad52 appears to be essential for any type of HR during both meiotic and mitotic processes in S.cerevisiae (reviewed in Paques and Haber, 1999). In marked contrast, murine and chicken cells deficient in Rad52 show a minimal-deficiency phenotype with only a moderate decrease in gene targeting efficiency (Rijkers et al., 1998; Yamaguchi-Iwai et al., 1998) and no radiation sensitivity. Biochemical studies imply that Rad52 and Rad51 paralogs participate in HR in a similar way (Sung, 1997a,b; Benson et al., 1998; reviewed in Kanaar and Hoeijmakers, 1998; New et al., 1998; Shinohara and Ogawa, 1998). Purified mammalian and yeast Rad52 proteins facilitate the respective Rad51-mediated DNA-strand exchange in the presence of RPA protein, a factor binding to ssDNA. Similarly, the addition of Rad55–Rad57 heterodimer to Rad51 and RPA enhances Rad51-mediated strand exchange reaction in vitro (Sung, 1997b), whereas the biochemical activities of the five vertebrate Rad51 paralogs have just begun to be characterized (Braybrooke et al., 2000; Kurumizaka et al., 2001). The similar biochemical activities of yeast Rad55–Rad57 and Rad52 led us to investigate whether in vertebrate cells the Rad51 paralogs can substitute for Rad52, which would explain the nearly normal phenotype of Rad52-deficient cells. To test this hypothesis we generated a conditional RAD52−/−XRCC−/− mutant using an inducible Cre site-specific recombinase MerCreMer (Zhang et al., 1996, 1998). Our results show that Rad52 and Rad51 paralogs are indeed complementary in the maintenance of chromosomal integrity, as well as in HR-mediated repair of IR-induced DSBs in vertebrate cells. Results Experimental strategy To generate cells deficient in both XRCC3 and Rad52, we employed the tamoxifen (TAM)-inducible Cre–loxP system (Zhang et al., 1996, 1998). We generated a transgene containing the human XRCC3 (hXRCC3) and green fluorescent protein (GFP) genes flanked by loxP sequences on both sides (Figure 2A) and introduced this transgene, together with a gene encoding the chimeric Cre recombinase MerCreMer, into RAD52−/−XRCC3+/− cells by random integration to produce RAD52−/−XRCC3+/− hXRCC3+ cells. The intact XRCC3 allele in the transfected cells was subsequently disrupted by gene targeting to generate rad52 xrcc3 hXRCC3+ cells (Figure 1A). (Hereafter we abbreviate the knockout nomenclature for simplification, e.g. rad52 = RAD52−/−.) MerCreMer carries two mutated hormone-binding domains of the murine estrogen receptor (Zhang et al., 1996, 1998), which binds the antagonist 4-hydroxytamoxifen (OH-TAM). Upon the addition of OH-TAM to the culture media, the chimeric Cre recombinase is transported into the nucleus, where it recognizes loxP sites and deletes both human hXRCC3 and GFP transgenes. The Cre-mediated recombination worked efficiently in rad52 xrcc3 hXRCC3+ DT40 cells. The human XRCC3 and GFP transgenes were deleted in virtually all of the cells within 24 h after the addition of OH-TAM, as verified by genomic PCR (Figure 2B) and flow cytometric analysis of GFP fluorescence (data not shown). Figure 1.Experimental strategy. (A) The functional analysis of Rad52 by comparing wild type and rad52, xrcc3 and rad52 xrcc3, and xrcc3 hRAD51+ and rad52 xrcc3 hRAD51+. (B) Western blot analysis of expression of endogenous chicken Rad51 and the human Rad51 transgene. Download figure Download PowerPoint Figure 2.Time course of MerCreMer-mediated deletion of the hXRCC3 transgene. (A) Schematic representation of the human XRCC3 transgene containing two LoxP recognition sequences (triangles), promoter sequences derived from cytomegalovirus (CMV), internal ribosomal entry site (IRES) and the EGFP gene encoding the enhanced GFP. Exposure of the cells to OH-TAM activates the chimeric Cre recombinase through nuclear localization, causing deletion of both XRCC3 and EGFP. The locations of the three PCR primers are indicated by arrows. (B) The extent of Cre-mediated deletion in the human XRCC3 transgene in rad52 xrcc3 hXRCC3+ hRAD51+ cells was determined by PCR using the indicated primer pairs shown in (A). Download figure Download PowerPoint Figure 1A summarizes the preparation of the DT40 clones employed in the present study. Each gene-targeting event was verified by Southern blotting as previously shown (Takata et al., 2001). The expression levels of human Rad51 transgene that is randomly integrated in xrcc3 hRAD51+ and rad52 xrcc3 hXRCC3+ hRAD51+ clones were measured by western blot analysis (Figure 1B). Two clones of each genotype, which expressed an ∼20-fold higher level of human Rad51 compared with that of endogenous Rad51, were used for subsequent analysis. Lethality of cells deficient in both XRCC3 and Rad52 The proliferative properties of clones were monitored by growth curves and plating efficiency. As previously observed (Yamaguchi-Iwai et al., 1998; Takata et al., 2001), wild-type and rad52 clones were indistinguishable in their growth properties, whereas xrcc3 cells proliferated significantly more slowly than the wild type (Figure 3A). Similarly, the plating efficiencies of cells in methylcellulose plates were 100% for wild-type and rad52 clones, whereas only ∼70% for xrcc3 cells (Figure 4). We showed previously that higher proportions of dead cells in xrcc3 cultures caused the slower growth rates as well as lower plating efficiencies (Takata et al., 2001). Figure 3.Defective proliferation of rad52 xrcc3 hXRCC3− cells. (A and C) Growth curves of the indicated cell cultures in the absence and presence of TAM. The data shown are the average results from two separate clones of each genotype. Standard errors are given by error bars. (B) Cell viability was assessed by flow cytometric analysis of PI uptake and forward scatter (FSC) representing the cell size. A fixed number of plastic beads was added before flow cytometric analysis to calibrate cell number. Cells falling in the R1 and R2 gates identify dead and viable cells, respectively, and numbers given show their percentages. The R3 gate was for the plastic beads, which are used as a reference to measure the cell number. Download figure Download PowerPoint Figure 4.No detectable colony formation of rad52 xrcc3 hXRCC3− cells. The histogram shows the percentage of metabolically viable cells that gave rise to colonies. The number of viable cells was measured by flow cytometry as shown in Figure 3B before plating cells in methylcellulose plates. Histograms of TAM+ (3 days) show the plating efficiency of cells that were treated with OH-TAM in liquid media for 3 days. No colonies was obtained from the wells plated with 10 000 cells of rad52 xrcc3 hXRCC3− (asterisk). Download figure Download PowerPoint As OH-TAM covalently binds to DNA (White, 1999), we examined proliferative properties of cells that were exposed to OH-TAM. However, addition of OH-TAM did not affect the proliferation of wild-type and rad52 cells (growth curve in Figure 3A and cloning efficiency in Figure 4). Similarly, exposure of xrcc3 cells to OH-TAM for 3 days did not significantly reduce their plating efficiency. Thus, we used this condition to conduct the following experiments. While rad52 xrcc3 hXRCC3+ cells multiplied with the same kinetics as wild-type and rad52 cells (Figure 3A, left panel), 3 days after the addition of OH-TAM, rad52 xrcc3 hXRCC3− cells stopped proliferating (Figure 3A, right panel) and began to die (Figure 3B, right panel). These findings were in agreement with the absence of any surviving rad52 xrcc3 hXRCC3− cells after exposure to OH-TAM for 3 days (Figure 4). These observations are in marked contrast to the survival of rad52 and xrcc3 cell populations even after continuous exposure of the cells to OH-TAM (data not shown). From these results, we conclude that cells deficient in both Rad52 and XRCC3 are unable to proliferate. To investigate the cause of cell death, we analyzed chromosomal breaks in mitotic cells. In agreement with previous findings (Yamaguchi-Iwai et al., 1998; Takata et al., 2001), we found ∼0.1 aberrations per cell in the xrcc3 culture, whereas wild-type and rad52 cells showed few chromosomal aberrations (Figure 5A; Table I). These values were not dependent on OH-TAM. Remarkably, rad52 xrcc3 hXRCC3− cells had 0.08 and 0.38 aberrations per cell at days 2 and 3, respectively, after adding OH-TAM (Tables I and II). We previously showed that the level of spontaneous chromosomal breaks of various HR-deficient DT40 clones is closely correlated with the rate of cell death during the cell cycle (reviewed in Morrison and Takeda, 2000). These observations indicate that the increased chromosomal breaks may account for the massive cell death of rad52 xrcc3 hXRCC3− cells. It should be noted that cells growing with normal kinetics occasionally appeared at <10−5 frequency (Figure 3C). Such clones retained the GFP and XRCC3 transgenes in the continuous presence of OH-TAMs. Figure 5.Spontaneous and IR-induced chromosomal aberrations. (A) Spontaneously occurring chromosomal breaks are shown. OH-TAM+ indicates cells that were exposed to OH-TAM for 3 days. One hundred mitotic cells were analyzed in each case. (B) IR-induced chromosomal breaks were determined by subtracting spontaneously occurring breaks from the number of breaks after IR treatment. OH-TAM+ indicates cells that were exposed to OH-TAM for 2 days. The numbers of spontaneous chromosomal breaks are shown in Table I, while IR-induced breaks are shown in Table II. Download figure Download PowerPoint Table 1. Spontaneous chromosomal breaks Cell clone TAMa Chromatid type Chromosome type Total aberrations (per cell ±SE)c Breaksb Gapsb Breaksb Gapsb Wild type − 0 0 0 1 0.01 ± 0.010 + 0 1 0 0 0.01 ± 0.010 rad52 − 1 1 1 0 0.03 ± 0.017 + 0 0 2 2 0.04 ± 0.020 xrcc3 − 1 1 6 4 0.12 ± 0.035 + 0 3 3 7 0.13 ± 0.036 rad52 xrcc3 hXRCC3 − 2 2 0 2 0.06 ± 0.024 + 7 2 25 4 0.38 ± 0.062 xrcc3 hRAD51+ − 0 1 0 1 0.02 ± 0.014 + 0 0 0 2 0.02 ± 0.014 rad52 xrcc3 hXRCC3 hRAD51+ − 0 0 0 1 0.01 ± 0.010 + 1 4 2 13 0.20 ± 0.045 a Cells were treated with colcemid after 3 days of culture in the presence or absence of OH-TAM. b Data are presented as macrochromosomal (1–5 and Z) aberrations per 100 metaphase spreads. c If the numbers of cells analyzed and total chromosomal aberrations are defined as N and x, respectively, the number of total aberrations per cell ±SE is calculated as x/N ± √x/N, based on the Poisson distribution of spontaneous chromosomal aberrations we observed previously (Sonoda et al., 1998). Table 2. IR-induced chromosomal breaks Cell clone IR (Gy)a Chromatid type Chromosome type Total aberrations (per cell ±SE) Breaksb Gapsb Breaksb Gapsb Wild type 0 0 0 0 1 0.01 ± 0.010 0.3 2 0 1 2 0.05 ± 0.022 2.0 5 2 5 8 0.20 ± 0.045 rad52 0 1 1 1 0 0.03 ± 0.017 0.3 2 0 4 1 0.07 ± 0.026 2.0 7 3 4 2 0.16 ± 0.040 xrcc3 0 3 0 3 6 0.12 ± 0.035 0.3 8 3 7 12 0.30 ± 0.055 2.0 28 5 4 29 0.66 ± 0.081 rad52 xrcc3 hXRCC3+ 0 0 2 0 2 0.04 ± 0.020 0.3 1 1 4 0 0.06 ± 0.024 2.0 7 4 0 13 0.24 ± 0.049 rad52 xrcc3 hXRCC3− 0 0 0 4 4 0.08 ± 0.028 0.3 16 1 4 13 0.34 ± 0.058 2.0 26 19 11 24 0.80 ± 0.089 rad52 xrcc3 hXRCC3− hRAD51+ 0 0 2 1 7 0.10 ± 0.032 0.3 2 6 0 16 0.24 ± 0.049 2.0 0 2 2 52 0.56 ± 0.075 Data were calculated and are presented as described for Table I. a Cells were treated with colcemid for 3 h after γ-irradiation. Cells were exposed to OH-TAM for 2 days. At least 100 cells were analyzed. b The number of aberrations per 100 cells is presented. Importance of Rad52 in DSB repair in XRCC3-deficient cells In order to assess the role of Rad52 in DSB repair, we tried to rescue rad52 xrcc3 cells by expressing the hRad51 cDNA transgene. We previously found that overexpression of hRad51 partially suppressed mutant phenotypes of all Rad51-paralog mutants (Takata et al., 2001). hRad51 overexpression rescued rad52 xrcc3 hXRCC3− cells, although rad52 xrcc3 hXRCC3− hRAD51+ cells proliferated at a significantly slower rate than wild type (Figure 3C). Likewise, only 5–10% of the cells that were exposed to OH-TAM for 3 days gave rise to colonies in methylcellulose plates (Figure 4). The overexpression of hRad51 appears to enhance the repair of spontaneously-occurring DNA damage in rad52 xrcc3 hXRCC3− hRAD51+ cells because their chromosomal breaks were significantly reduced when compared with rad52 xrcc3 hXRCC3− cells (Figure 5A). While this observation is in agreement with the suppression of a mutant phenotype of xrcc3 cells by overexpression of hRad51 (Takata et al., 2001), it is not clear whether this overproduction also substitutes for the lack of Rad52. Rad51 overexpression reduced the plating efficiency of rad52 xrcc3 hXRCC3+ cells but not that of wild-type (data not shown) or xrcc3 cells, implying that Rad51 overexpression is rather toxic in the absence of Rad52. To analyze the involvement of Rad52 in IR-induced DSB repair, we measured chromosomal breaks following IR by comparing the genotypes of wild type and rad52, and of xrcc3 and rad52 xrcc3. Because of the massive cell death in rad52 xrcc3 cells at day 3 after addition of OH-TAM, we examined the effect of IR at day 2 (Figure 5B; Table II). To evaluate HR-mediated DSB repair capability, we measured chromosomal aberrations in cells that were irradiated in the late S to G2 phases when HR is preferentially used for DSB repair over NHEJ in DT40 cells (Takata et al., 1998). As most cells irradiated in the late S to G2 phases are expected to reach the M phase within 3 h after IR, we measured chromosomal breaks in cells entering mitosis between 0 and 3 h. As expected, rad52 xrcc3 hXRCC3− exhibited greater levels of IR-induced chromosomal aberrations in comparison with xrcc3 (Figure 5B). These results indicate the involvement of Rad52 in repairing IR-induced DSBs in the absence of the XRCC3 gene. This conclusion led us to analyze whether or not introduction of a RAD52 transgene into a xrcc3 clone can suppress its mutant phenotype. However, Rad52 overexpression appears to be toxic to the transfectants because their cloning efficiencies were consistently decreased to 15 to 31% from ∼70% of the parental xrcc3 clone. Furthermore, these Rad52 overexpressing clones were more sensitive to cisplatin than xrcc3 cells by colony formation assay (data not shown). In contrast, Rad51 overexpression in rad52 xrcc3 (Figure 5B) as well as xrcc3 clone (Takata et al., 2001) suppressed their elevated sensitivities to γ-rays. Discussion Rad52 and the Rad51 paralogs are complementary in repairing DSB This is the first genetic study that clearly shows a role of Rad52 in DSB repair in vertebrates. Our results are in agreement with the important role of Rad52 in yeast strains, as well as with co-localization of Rad52 with Rad51 following IR in mammalian cells (Liu and Maizels, 2000). These observations are in marked contrast with no obvious phenotype of rad52 DT40 cells (Yamaguchi-Iwai et al., 1998), murine ES cells and mice (Rijkers et al., 1998). Thus, Rad52 may play an important role in repairing DSBs in XRCC3-deficient cells but not in the wild-type cells. XRCC3 almost fully substitutes for lack of Rad52 while Rad52 can only partially substitute for lack of XRCC3. As cells deficient in the other four Rad51 paralogs have similar phenotypes as XRCC3-deficient cells (Takata et al., 2001), it seems reasonable to expect that the other Rad51 paralogs and Rad52 may also have complementary functions in maintaining chromosomal integrity and repairing IR-induced DSBs. Although Rad52 is required for virtually every mitotic recombination event in S.cerevisiae (Paques and Haber, 1999), its ortholog in vertebrates appears to be dispensable. There are four possible explanations for this species difference. First, Rad52 may not be involved in conventional HR but in other DNA metabolism pathways in vertebrate cells. However, this possibility is unlikely because immunocytochemical experiments suggest a coordinated response of mammalian Rad52 and Rad51 to DNA damage (Liu and Maizels, 2000). Moreover, both the yeast and human Rad52 proteins form ring structures (Van Dyck et al., 1999; Stasiak et al., 2000) and stimulate DNA-strand exchange promoted by Rad51 protein in vitro (Benson et al., 1998; New et al., 1998; Shinohara and Ogawa, 1998), further emphasizing the conservation of the roles of human and yeast Rad52 in HR. Secondly, there might be an as yet undescribed Rad52 homolog in vertebrates (Kanaar and Hoeijmakers, 1998), although the human genome does not seem to contain other Rad52-like genes (Wood et al., 2001). Thirdly, the relative importance of HR and end joining differs between vertebrates and yeast, such that during evolution the Ku proteins in vertebrate cells may have assumed a portion of the function of Rad52 in yeast DSB repair. The end-joining pathway plays a much more important role in vertebrate cells compared with yeast (Milne et al., 1996; reviewed in Jeggo, 1998; Essers et al., 2000). Biochemical studies suggest that Ku proteins protect DSB ends from exonuclease activity, as does the Rad52 protein (Van Dyck et al., 1999). These results have led to the proposal that recognition of DSB by either Rad52 or Ku protein directs repair by Rad52-dependent HR or Ku-dependent NHEJ pathways, respectively (Van Dyck et al., 1999). To investigate the possibility that the Ku proteins substitute for Rad52 in DSB repair, we generated a DT40 mutant deficient in both Ku70 and Rad52 (rad52 ku70). This double mutant exhibited radiosensitivity very similar to that of ku70 cells (data not shown), although rad54 ku70 cells exhibited much higher sensitivity than either single mutant (Takata et al., 1998). Thus, while the HR and end-joining pathways are complementary to each other in DSB repair, the nearly normal phenotype of Rad52-deficient cells is not explained by an overlapping, compensatory function of the Ku proteins. Fourthly, the precise mechanism of HR likely differs between vertebrates and yeasts so that analogous molecules may compensate for the lack of Rad52 in vertebrate cells. The present data show that a Rad51 paralog (XRCC3) indeed complements defective Rad52 in DSB repair in DT40 cells. This genetic study, combined with biochemical studies (Sung, 1997a,b; Benson et al., 1998; New et al., 1998; Shinohara and Ogawa, 1998) showing that mammalian and yeast Rad52 proteins, as well as yeast Rad55–57, facilitate Rad51-mediated DNA-strand exchange, suggests that Rad52 and the Rad51 paralogs share overlapping roles as co-factors of Rad51 in HR repai