Srs2 and Mus81-Mms4 Prevent Accumulation of Toxic Inter-Homolog Recombination Intermediates.
Srs2 and Mus81-Mms4 Prevent Accumulation of Toxic Inter-Homolog Recombination Intermediates.
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DOI：
10.1371/journal.pgen.1006136
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发表时间：
2016-07
期刊：
影响因子：
4.5
通讯作者：
Hishida T
中科院分区：
文献类型：
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作者：
Keyamura K;Arai K;Hishida T
同源重组是一种进化上保守的机制，它通过DNA中双链断裂和单链间隙的忠实修复以及停滞或崩溃的复制叉的恢复来促进基因组的稳定性。酿酒酵母atp依赖的DNA解旋酶Srs2（高度保守的UvrD解旋酶家族成员）在调控同源重组中具有多种作用。一个导致Srs2解旋酶死亡突变的突变（srs2K41A）被发现在二倍体细胞中是致命的，而在单倍体细胞中则不是。在二倍体细胞中，Srs2K41A引起同源间关节分子中间体的积累，增加自发Rad52病灶的水平，诱导染色体重排。灭活Rad51或删除Srs2的Rad51-相互作用结构域可抑制Srs2K41A的致死性和关节分子的积累，而Srs2的磷酸化和sumoylation及其与sumoylated增殖细胞核抗原（PCNA）的相互作用不是致死性所必需的。交叉连接内切酶Mus81和Mms4的结构特异性复合体也是二倍体生存所必需的，但单倍体、SRS2缺失突变体（srs2Δ）和二倍体srs2Δ mus81Δ突变体积累的关节分子中间体则不需要。我们的数据表明，Srs2和Mus81-Mms4在防止（或解决）有毒同源间连接分子的形成（或解决）中起关键作用，否则可能干扰染色体分离并导致遗传不稳定。同源重组（HR）是一种通常被认为是无错误的dna修复机制，因为它使用完整的姐妹染色单体作为模板。然而，在二倍体细胞中，HR也可能发生在同源染色体之间，这可能通过失去杂合性导致基因组不稳定。这种改变经常在遗传疾病和癌症中检测到，这表明需要严格控制这一过程以确保基因组的稳定性。酵母Srs2，从细菌到人类的保守，在HR的调控中起着多种作用。我们在这里证明了Srs2的解旋酶死亡突变体srs2K41A在二倍体细胞中是致命的，而在单倍体细胞中则不是。Srs2K41A在二倍体细胞中的表达导致同源间关节分子中间体的积累，并导致染色体重排。此外，srs2Δ mus81Δ双突变体具有严重的二倍体特异性生长缺陷，具有同源间关节分子的积累。这些数据表明，Srs2和Mus81-Mms4参与了防止同源间重组中间体积累的重要途径，从而降低了基因组不稳定的风险。
Homologous recombination is an evolutionally conserved mechanism that promotes genome stability through the faithful repair of double-strand breaks and single-strand gaps in DNA, and the recovery of stalled or collapsed replication forks. Saccharomyces cerevisiae ATP-dependent DNA helicase Srs2 (a member of the highly conserved UvrD family of helicases) has multiple roles in regulating homologous recombination. A mutation (srs2K41A) resulting in a helicase-dead mutant of Srs2 was found to be lethal in diploid, but not in haploid, cells. In diploid cells, Srs2K41A caused the accumulation of inter-homolog joint molecule intermediates, increased the levels of spontaneous Rad52 foci, and induced gross chromosomal rearrangements. Srs2K41A lethality and accumulation of joint molecules were suppressed by inactivating Rad51 or deleting the Rad51-interaction domain of Srs2, whereas phosphorylation and sumoylation of Srs2 and its interaction with sumoylated proliferating cell nuclear antigen (PCNA) were not required for lethality. The structure-specific complex of crossover junction endonucleases Mus81 and Mms4 was also required for viability of diploid, but not haploid, SRS2 deletion mutants (srs2Δ), and diploid srs2Δ mus81Δ mutants accumulated joint molecule intermediates. Our data suggest that Srs2 and Mus81–Mms4 have critical roles in preventing the formation of (or in resolving) toxic inter-homolog joint molecules, which could otherwise interfere with chromosome segregation and lead to genetic instability. Homologous recombination (HR) is a DNA-repair mechanism that is generally considered error free because it uses an intact sister chromatid as a template. However, in diploid cells, HR can also occur between homologous chromosomes, which can lead to genomic instability through loss of heterozygosity. This alteration is often detected in genetic disorders and cancer, suggesting that tight control of this process is required to ensure genome stability. Yeast Srs2, conserved from bacteria to humans, plays multiple roles in the regulation of HR. We show here that a helicase-dead mutant of Srs2, srs2K41A, is lethal in diploid cells but not in haploid cells. Expression of Srs2K41A in diploid cells causes inter-homolog joint molecule intermediates to accumulate, and leads to gross chromosomal rearrangements. Moreover, srs2Δ mus81Δ double mutants have a severe diploid-specific growth defect with accumulation of inter-homolog joint molecules. These data demonstrate that Srs2 and Mus81-Mms4 participate in essential pathways preventing accumulation of inter-homolog recombination intermediates, thereby reducing the risk of genome instability.