In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair
In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair
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DOI：
10.1038/sj.emboj.7600091
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发表时间：
2004-02-25
期刊：
影响因子：
11.4
通讯作者：
Shinohara, A
中科院分区：
文献类型：
--
作者：
Miyazaki, T;Bressan, DA;Shinohara, A
Rad 51和Rad 52复合物的组装和拆卸进行了监测，在同源重组过程中发起的HO内切核酸酶诱导的双链断裂（DSB）在MAT位点的免疫荧光。DSB诱导的Rad 51和Rad 52焦点与TetR-GFP焦点共定位在与MAT相邻的tetO序列处。在菌株中，HO切割染色体III上的三个位点，我们观察到三个不同的病灶，与相邻的GFP染色体标记共定位。我们比较了焦点形成的动力学与重组中间体和产品时，HO-切割MAT α与供体，MAT α重组。Rad 51组装发生在HO切割后1小时。Rad 51解体发生在单链（ss）MAT DNA侵入MAT α后启动新DNA合成的同时。我们提出的证据表明，Rad 52在重组中的三个不同的角色：Rad 51组装所需的突触前作用，Rad 51丝的突触作用，以及Rad 51解离后的突触后作用。另外的生物化学研究表明存在含有Rad 51和Rad 52的ssDNA复合物。
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, MATalpha. 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 MATalpha. 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.