Chromosome Instability and Defective Recombinational Repair in Knockout Mutants of the Five Rad51 Paralogs

doi:10.1128/MCB.21.8.2858-2866.2001

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Molecular and Cellular Biology, April 2001, p. 2858-2866, Vol. 21, No. 8
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.8.2858-2866.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Chromosome Instability and Defective Recombinational Repair in Knockout Mutants of the Five Rad51 Paralogs

Minoru Takata,¹^,²^, Masao S. Sasaki,³ Seiji Tachiiri,¹ Toru Fukushima,¹ Eiichiro Sonoda,¹^,² David Schild,⁴ Larry H. Thompson,⁵ and Shunichi Takeda¹^,²^,^*

CREST Research Project, Radiation Genetics, Faculty of Medicine,¹ and Radiation Biology Center,³ Kyoto University, Sakyo-ku, Kyoto 606-8501, and CREST, JST (Japan Science and Technology), Kawaguchi,² Japan; Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720⁴; and Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 95441-0808⁵

Received 19 September 2000/Returned for modification 24 October 2000/Accepted 29 December 2000

The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologousDNA recombination (HR) in Saccharomyces cerevisiae and is essentialfor the proliferation of vertebrate cells. Five vertebrate genes,RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on thebasis of their sequence similarity to Rad51 (Rad51 paralogs).We generated mutants deficient in each of these proteins in thechicken B-lymphocyte DT40 cell line and report here the comparisonof four new mutants and their complemented derivatives with ourpreviously reported rad51b mutant. The Rad51 paralog mutationsall impair HR, as measured by targeted integration and sisterchromatid exchange. Remarkably, the mutant cell lines all exhibitvery similar phenotypes: spontaneous chromosomal aberrations,high sensitivity to killing by cross-linking agents (mitomycinC and cisplatin), mild sensitivity to gamma rays, and significantlyattenuated Rad51 focus formation during recombinational repairafter exposure to gamma rays. Moreover, all mutants show partialcorrection of resistance to DNA damage by overexpression of humanRad51. We conclude that the Rad51 paralogs participate in repairas a functional unit that facilitates the action of Rad51 inHR.

^* Corresponding author. Mailing 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: stakeda{at}rg1.rg.med.kyoto-u.ac.jp.

Present address: Department of Immunology and Molecular Genetics, Kawasaki Medical School, Kurashiki 701-0192,Japan.

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