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首页> 外文期刊>Cell Division >The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence
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The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence

机译：S相检查点复合物Mrc1 / Tof1 / Csm3的亚基：动力学和相互依赖性

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Background The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. Findings This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. Conclusions In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.
机译：背景技术S期检查站旨在防止细胞生成容易导致基因组不稳定的大量单链DNA。当DNA合成被禁止时，酿酒酵母复合物Tof1 / Csm3 / Mrc1可抑制复制性MCM解旋酶。保持复制机制不变，可以在释放块时重新启动复制分支。尽管对Tof1 / Csm3 / Mrc1复合物的亚基进行了很好的研究，但仍需要确定每个单个亚基对三重复合物形成和功能的影响。研究结果当复合物完整且缺少亚基时，这项工作研究了带有GFP标签的亚基的细胞定位和染色质结合。我们证明复合物是在细胞核中形成，而不是在细胞质中形成的，因为Tof1，Csm3和Mrc1彼此独立地进入细胞核。通过原位染色质结合测定，我们表明需要Tof1-Csm3二聚体形成和染色质结合，以确保Mrc1与染色质的连接。我们的研究表明，向核内移位不是调节Mrc1染色质缔合时间的过程。我们还研究了Mrc1亚基在适应羟基脲存在过程中的核行为。我们的结果表明，长时间的HU孵育后，细胞会绕过S期检查点，并贯穿整个细胞周期。此过程伴随着Mrc1染色质脱离和Rad53脱磷酸化。结论在酿酒酵母中，S期检查点复合体Mrc1 / Tof1 / Csm3的亚基独立进入细胞核，并在其中形成Tof1-Csm3二聚体，以确保Mrc1的染色质结合并有利于DNA复制和S期检查点叉。逮捕。在适应羟基脲存在的过程中，Mrc1从染色质脱离，Rad53检查点的活性降低，以允许S期检查点逃逸并完成细胞周期。

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