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| Subject Categories: Genome Stability & Dynamics |
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| The EMBO Journal
(2004) 23, 939–949, doi:10.1038/sj.emboj.7600091 Published online 5 February 2004 |
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| In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair |
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| Toshiko Miyazaki1, Debra A Bressan2, Miki Shinohara1, 5, James E Haber2 and Akira Shinohara1, 3, 4 |
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1 Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan 2 Rosenstiel Center, Brandeis University, Waltham, MA, USA 3 PRESTO, Japanese Science Technology Agency, Kawaguchi, Saitama, Japan 4 Institute for Protein Research, Osaka University, Suita, Osaka, Japan To whom correspondence should be addressed Akira Shinohara, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 6879 8624; Fax: +81 6879 8626; E-mail: ashino@protein.osaka-u.ac.jp 5 Present address: Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan Received 22 October 2003; Accepted 5 January 2004; Published online 5 February 2004. |
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| Abstract |
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Assembly and disassembly of Rad51 and Rad52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and Rad52 foci colocalize with a TetR–GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, we observe three distinct foci that colocalize with adjacent GFP chromosome marks. We compared the kinetics of focus formation with recombination intermediates and products when HO-cleaved MAT recombines with the donor, MATa. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATa. We present evidence for three distinct roles for Rad52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and Rad52. |
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| Keywords: mating-type switching, Rad51, Rad52, recombination |
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