Miyazaki Toshiko, Bressan Debra A, Shinohara Miki, Haber James E, Shinohara Akira
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
EMBO J. 2004 Feb 25;23(4):939-49. doi: 10.1038/sj.emboj.7600091. Epub 2004 Feb 5.
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 MATalpha 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.
在MAT位点由HO核酸内切酶诱导的双链断裂（DSB）引发的同源重组过程中，通过免疫荧光监测Rad51和Rad52复合物的组装和解聚。DSB诱导的Rad51和Rad52焦点与MAT附近tetO序列处的TetR-GFP焦点共定位。在HO切割III号染色体上三个位点的菌株中，我们观察到三个不同的焦点，它们与相邻的GFP染色体标记共定位。当HO切割的MATα与供体MATa重组时，我们比较了焦点形成与重组中间体和产物的动力学。Rad51组装在HO切割后1小时发生。Rad51解聚发生在单链（ss）MAT DNA侵入MATa后开始新DNA合成的同时。我们提供了Rad52在重组中三个不同作用的证据：Rad51组装所必需的突触前作用、与Rad51细丝的突触作用以及Rad51解离后的突触后作用。额外的生化研究表明存在一种同时包含Rad51和Rad52的ssDNA复合物。
