Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae
Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae
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DOI：
10.1128/mcb.24.16.6891-6899.2004
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发表时间：
2004-08-01
影响因子：
5.3
通讯作者：
Haber, JE
中科院分区：
文献类型：
--
作者：
Wang, X;Ira, G;Haber, JE
有丝分裂双链断裂（DSB）诱导的基因转换涉及新的DNA合成。我们分析了几个必要的复制组件，Mcm蛋白，Cdc 45 p，和DNA连接酶1，在DNA合成的酿酒酵母HAT开关的要求。在mcm 7-td（温度诱导降解决定子）突变体中，当Mcm 7 p降解到检测水平以下时，HAT转换正常发生，表明在基因转换过程中缺乏Mcm 2 -7蛋白。cdc 45-td突变体也能够完成重组。令人惊讶的是，即使在消除酵母中的两种鉴定的DNA连接酶后，cdc 9 -1 dnl 4 Delta菌株也能够完成DSB修复。先前对携带PCNA、DNA聚合酶α（Polalpha）或引发酶的温度敏感等位基因的异步培养物的研究表明，这些突变抑制MAT转换（A. M.福尔摩斯和J. E. Haber，Cell 96：415-424，1999）。我们重新评估了这些蛋白在G（2）-停滞细胞中的作用。而PCNA仍然是MAT转换所必需的，Polalpha和引发酶都不需要。这些结果表明，在诱导DSB之前，使用Polalpha引发酶的ts等位基因将细胞阻滞在S期，隔离了修复所需的一些其他组分。我们的结论是，DNA合成过程中的基因转换是不同的S期复制，只涉及前导链聚合。
Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase 1, in the DNA synthesis of Saccharomyces cerevisiae HAT switching. In an mcm7-td (temperature-inducible degron) mutant, HAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Delta strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive allelles of PCNA, DNA polymerase alpha (Polalpha), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G(2)-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polalpha nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polalpha-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.