Regulation of DNA replication machinery by Mrc1 in fission yeast
Regulation of DNA replication machinery by Mrc1 in fission yeast
复制标题
DOI：
10.1534/genetics.106.060053
复制
发表时间：
2006-09-01
期刊：
影响因子：
3.3
通讯作者：
Nakagawa, Takuro
中科院分区：
文献类型：
--
作者：
Nitani, Naoki;Nakamura, Ken-ichi;Nakagawa, Takuro
染色体的忠实复制对于基因组完整性至关重要。在酵母中，ORC 在整个细胞周期中结合复制起点。然而，Cdc45 在 S 期之前结合这些，并且在复制过程中，它通过 MCM 解旋酶沿着 DNA 移动。当复制进程受到抑制时，检查点调节被认为可以稳定复制叉；然而，详细机制仍不清楚。为了检查复制起始和延伸缺陷之间的关系以及对复制延伸阻断的反应，我们分别使用了 Orc1 和 Cdc45-orp1-4 和 sna41-928 的裂变酵母突变体，在它们各自的 BrdU 掺入半允许温度下。 orp1和sna41细胞在缺乏Chk1（一种DNA损伤检查点激酶）的情况下表现出HU超敏反应，并且在完全激活Cds1（一种复制检查点激酶）方面存在缺陷，表明正常复制是Cds1激活所必需的。 Mrc1 需要激活 Cds1 并防止复制机制从 DNA 合成中解偶联。我们观察到，虽然 orp1 或 sna41 突变部分抑制了 cds1 细胞的 HU 敏感性，但 sna41 特异性抑制了 mrc1 细胞的 HU 敏感性。有趣的是，sna41 缓解了从 HU 逮捕中恢复的缺陷，而没有增加 Cds1 活性。除了 sna41 之外，MCM 的特定突变也抑制了 mrc1 细胞的 HU 敏感性。因此，在延伸过程中，Mrc1 可能会负向调节 Cdc45 和 MCM 解旋酶，以使停滞的分叉能够恢复复制。
Faithful replication of chromosomes is crucial to genome integrity. In yeast, the ORC binds replication origins throughout the cell cycle. However, Cdc45 binds these before S-phase, and, during replication, it moves along the DNA with MCM helicase. When replication progression is inhibited, checkpoint regulation is believed to stabilize the replication fork; the detailed mechanism, however, remains unclear. To examine the relationship between replication initiation and elongation defects and the response to replication elongation block, we used fission yeast mutants of Orc1 and Cdc45-orp1-4 and sna41-928, respectively-at their respective semipermissive temperatures with regard to BrdU incorporation. Both orp1 and sna41 cells exhibited HU hypersensitivity in the absence of Chk1, a DNA damage checkpoint kinase, and were defective in full activation of Cds1, a replication checkpoint kinase, indicating that normal replication is required for Cds1 activation. Mrc1 is required to activate Cds1 and prevent the replication machinery from uncoupling from DNA synthesis. We observed that, while either the orp1 or the sna41 mutation partially suppressed HU sensitivity of cds1 cells, sna41 specifically suppressed that of mrc1 cells. Interestingly, sna41 alleviated the defect in recovery from HU arrest without increasing Cds1 activity. In addition to sna41, specific mutations of MCM suppressed the HU sensitivity of mrc1 cells. Thus, during elongation, Mrc1 may negatively regulate Cdc45 and MCM helicase to render stalled forks capable of resuming replication.