Homologous Recombination, but Not DNA Repair, Is Reduced in Vertebrate Cells Deficient in RAD52

ABSTRACT

Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generatedRAD52^−/− mutants of the chicken B-cell line DT40. Surprisingly, RAD52 ^−/− cells were not hypersensitive to DNA damages induced by γ-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52 ^−/− cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52 ^−/− cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.

ACKNOWLEDGMENTS

We would like to thank T. Shibata (Riken, Wako, Japan), T. Ogawa (Institute of Genetics, Mishima, Japan), and A. Pastink (University of Leiden, Leiden, The Netherlands) for critically reading the manuscript; Y. Kubota (Caltech, Pasadena, Calif.) for statistical analysis; and M. Hashishin, Y. Sato, O. Koga, and M. Hirao for their excellent technical assistance.

C.M. is the recipient of a JSPS Postdoctoral Fellowship. The Bayer-chair of the Department of Molecular Immunology and Allergology is supported by Bayer Yakuhin, Kyoto, Japan. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan and by a grant from The Mochida Memorial Foundation for Medical and Pharmaceutical Research. The Basel Institute for Immunology was founded and is supported by F. Hoffmann La-Roche Ltd., Basel, Switzerland.