The Rad51 Paralog Rad51B Promotes Homologous Recombinational Repair

M Takata

M Sasaki

E Sonoda

T Fukushima

Bayer-Chair Department of Molecular Immunology and Allergy, Faculty of Medicine,^{CREST, JST (Japan Science and Technology),}^{and Radiation Biology Center,}^{Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan; Medical Genetics Center, Department of Cell Biology and Genetics, Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands}^{; and Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551-0808}⁵

^{Corresponding author. Present address: CREST Research Project, Radiation Genetics, Faculty of Medicine, Kyoto University, Konoe Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-4410. Fax: 81-75-753-4419. E-mail:}pj.ca.u-otoyk.dem.gr.1gr@adekats.

Received 1999 Sep 9; Revisions requested 1999 Oct 27; Accepted 2000 Jan 26.

Abstract

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out. RAD51B^{cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus.}RAD51B^{cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to γ-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in}RAD51B^{cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity.}

Abstract

Double-strand DNA breaks (DSBs) occur during DNA replication and are produced by ionizing radiation. Since DSBs are so deleterious to the cell, it is not surprising that there are two DSB repair pathways: nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). Repair of DSBs by HRR requires the presence of homologous duplex DNA elsewhere in the genome, i.e., either a homologous chromosome or, more likely, a sister chromatid. NHEJ simply acts to process and ligate broken ends without a requirement for extensive homology. These pathways are conserved from the yeast Saccharomyces cerevisiae to humans (5, 8, 9, 19, 49, 53, 64). While HRR is the primary mechanism of DSB repair in yeast, vertebrate cells use both the NHEJ and HRR pathways extensively (28, 34, 35, 44). The analysis of radiosensitive yeast mutants has revealed a number of genes involved in HRR, which comprise the RAD52 epistasis group (reviewed in references 4, 29, and 51).

Among the members of the RAD52 epistasis group, the structure and function of Rad51 have been conserved to a remarkable degree among all eukaryotes. Rad51 is structurally and functionally related to the Escherichia coli recombination protein RecA (reviewed in reference 32). The functional forms of both RecA and Rad51 are multimeric helical nucleoprotein filaments that form on single-stranded DNA ends produced at DSBs (41). These filaments are involved in the search for homologous sequence, DNA pairing, and strand exchange. Recombination intermediates produced in this way are then processed further in reactions that involve DNA synthesis, branch migration, resolution of Holliday junctions, and ligation (reviewed in reference 4). The conservation of the RAD52 epistasis group genes from yeast to vertebrate cells suggests that the basic mechanism of HRR is maintained during evolution. However, while S. cerevisiae RAD51 mutants are viable, Rad51 deficiency in vertebrate cells causes rapid chromosomal aberrations and cell death (54). One possible explanation for this lethality is that the larger size of the vertebrate genome requires more HRR activity for chromosome stability (22, 54).

RAD51 paralogs (genes related by duplication within a single genome) have been identified in many eukaryotes and constitute the Rad51-related gene family (63, 64). The completion of the S. cerevisiae genomic sequence established that four previously identified proteins, Rad51, Rad55, Rad57, and Dmc1, constitute the complete set of RecA relatives in this organism (1, 3, 7, 37, 50). Thus far, seven members of the Rad51 protein family have been identified in mammals. In human cells, Rad51 (49), Dmc1 (23), XRCC2 (15, 28, 36), XRCC3 (36, 44, 62), Rad51B (also called Rad51L1/hRec2) (2, 14, 47), Rad51C (Rad51L2) (18), and Rad51D (Rad51L3) (14, 30, 45) are highly conserved. While human Dmc1 is ∼50% identical to human Rad51, the other human Rad51 paralogs are 20 to 30% identical to human Rad51. These paralogs are less than 30% identical to each other and to yeast Rad55 and Rad57 (reviewed in reference 63). Overexpression of Rad51 in yeast partially suppresses the DNA repair defect of rad55 and rad57 mutant strains (25, 27), implying that Rad55 and Rad57 may functionally cooperate with Rad51. This idea is supported by physical interactions between Rad51 and Rad55 and between Rad55 and Rad57 (25, 27, 57). Similarly, physical interactions occur between human Rad51 and XRCC3, XRCC3 and Rad51C and between Rad51B and Rad51C (18, 36). These observations argue that Rad51 paralogs may function as Rad51 accessory factors, analogous to yeast Rad55 and Rad57. Just as Rad55 and Rad57 are important for HRR in yeast, in mammalian cells the Rad51 paralogs XRCC2 and XRCC3 have recently been shown to play an important role in radiation resistance through HRR (28, 44). Here we present evidence that the RAD51B gene is also important for HRR.

Very recently, a RAD51B knockout mutation was made in mice but embryonic lethality prevented analysis of the cellular phenotype (52). Transcription of RAD51B was induced following UV or γ irradiation, suggesting that it has a regulated response to DNA damage (42, 47). To investigate the role of Rad51B in vertebrate cells, we generated RAD51B^{cells from the hyperrecombinogenic chicken DT40 cell line (}11, 12). Comparison of the phenotype of this mutant with that of RAD54^{DT40 cells (}5) suggests that Rad51B and Rad54 have distinctly different roles in recombinational repair.

ACKNOWLEDGMENTS

We thank M. Hashishin, Y. Sato, O. Koga, and M. Hirao for excellent technical assistance and H. Kurumizaka and T. Shibata (Riken, Wako, Japan), S. C. West (Imperial Cancer Research Fund, South Mimms, United Kingdom), and D. Schild (Lawrence Berkeley National Laboratory, Berkeley, Calif.) for discussion and critical reading of the manuscript.

C. M. is the recipient of a JSPS postdoctoral fellowship. The Bayer-Chair Department of Molecular Immunology and Allergy is supported by Bayer Yakuhin, Kyoto, Japan. This work was supported in part by CREST, JST (Saitama, Japan); a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan; and grants from The Mochida Memorial Foundation for Medical and Pharmaceutical Research and from The Uehara Memorial Foundation. A portion of this work was prepared under the auspices of the U.S. Department of Energy under contract W-7405-ENG-48 (L.H.T.).

ACKNOWLEDGMENTS

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