The mechanism of Mus81-Mms4 cleavage site selection distinguishes it from the homologous endonuclease Rad1-Rad10
The mechanism of Mus81-Mms4 cleavage site selection distinguishes it from the homologous endonuclease Rad1-Rad10
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DOI：
10.1128/mcb.23.10.3487-3496.2003
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发表时间：
2003-05-01
影响因子：
5.3
通讯作者：
Brill, SJ
中科院分区：
文献类型：
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作者：
Bastin-Shanower, SA;Fricke, WM;Brill, SJ
Mus 81-Mms 4和Rad 1-Rad 10是同源的结构特异性核酸内切酶，其从不同底物切割3'分支，并且分别是酿酒酵母中复制叉稳定性和核苷酸切除修复所需的。我们探索了这种生物化学和遗传特异性的基础。Mus 81-Mms 4切割位点，即瓣5'的5个核苷酸（nt）的切口，不像Rad 1-Rad 10那样由分支点决定，而是由瓣连接处的DNA链的5'末端决定。结果，核酸内切酶显示出反向底物特异性;在瓣的4 nt内缺少5'端的底物被Mus 81-Mms 4切割得很差，但被Rad 1 -10切割得很好。遗传学上，我们表明，mus 81和sgs 1突变体是敏感的喜树碱诱导的DNA损伤。此外，mus 81 sgs 1合成致死需要同源重组，如通过RusA表达抑制突变体表型。这些数据最容易通过一个模型来解释，其中Mus 81-Mms 4和Sgs 1-Top3的体内底物是复制叉塌陷下游的3'侧翼重组中间体。
Mus81-Mms4 and Rad1-Rad10 are homologous structure-specific endonucleases that cleave 3' branches from distinct substrates and are required for replication fork stability and nucleotide excision repair, respectively, in the yeast Saccharomyces cerevisiae. We explored the basis of this biochemical and genetic specificity. The Mus81-Mms4 cleavage site, a nick 5 nucleotides (nt) 5' of the flap, is determined not by the branch point, like Rad1-Rad10, but by the 5' end of the DNA strand at the flap junction. As a result, the endonucleases show inverse substrate specificity; substrates lacking a 5' end within 4 nt of the flap are cleaved poorly by Mus81-Mms4 but are cleaved well by Rad1-10. Genetically, we show that both mus81 and sgs1 mutants are sensitive to camptothecin-induced DNA damage. Further, mus81 sgs1 synthetic lethality requires homologous recombination, as does suppression of mutant phenotypes by RusA expression. These data are most easily explained by a model in which the in vivo substrate of Mus81-Mms4 and Sgs1-Top3 is a 3' flap recombination intermediate downstream of replication fork collapse.