Cell cycle execution point analysis of ORC function and characterization of the checkpoint response to ORC inactivation in Saccharomyces cerevisiae
Cell cycle execution point analysis of ORC function and characterization of the checkpoint response to ORC inactivation in Saccharomyces cerevisiae
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DOI：
10.1111/j.1365-2443.2006.00967.x
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发表时间：
2006-06-01
期刊：
影响因子：
2.1
通讯作者：
Aparicio, Oscar M.
中科院分区：
文献类型：
--
作者：
Gibson, Daniel G.;Bell, Stephen P.;Aparicio, Oscar M.
染色体复制通过在G1期的单个复制起点组装复制前复合物（pre-RC）开始，随后在S期激活这些复合物。在酿酒酵母中，起点识别复合物（ORC）在整个细胞周期中结合复制起点并参与前RC组装。ORC是否在复制起始或任何其他必要的细胞周期过程中在前RC组装之后发挥额外的作用尚不清楚。为了研究ORC在确定的细胞周期期间的功能，我们使用在ORC的ORC 1、ORC 2或ORC 5亚基中含有条件突变的菌株进行细胞周期执行点分析。我们发现ORC对于复制起始是必不可少的，但是对于复制延长或以后的细胞周期事件是不可缺少的。ORC突变细胞中的缺陷启动导致由DNA损伤和纺锤体组装检查点途径强制执行的不完全复制和有丝分裂停滞。纺锤体组装检查点的参与意味着由于不完全复制和/或DNA损伤导致的着丝粒-纺锤体附着或姐妹染色单体凝聚力的缺陷。值得注意的是，在ORC 1功能的半允许条件下，单独的纺锤体检查点就足以阻止增殖，这表明该检查点对复制起始缺陷高度敏感。我们讨论了这些重叠的检查点的潜在意义，以及我们的研究结果对先前假设的ORC在其他细胞周期功能中的作用的影响。
Chromosomal replication initiates through the assembly of a prereplicative complex (pre-RC) at individual replication origins in the G1-phase, followed by activation of these complexes in the S-phase. In Saccharomyces cerevisiae, the origin recognition complex (ORC) binds replication origins throughout the cell cycle and participates in pre-RC assembly. Whether the ORC plays an additional role subsequent to pre-RC assembly in replication initiation or any other essential cell cycle process is not clear. To study the function of the ORC during defined cell cycle periods, we performed cell cycle execution point analyses with strains containing a conditional mutation in the ORC1, ORC2 or ORC5 subunit of ORC. We found that the ORC is essential for replication initiation, but is dispensable for replication elongation or later cell cycle events. Defective initiation in ORC mutant cells results in incomplete replication and mitotic arrest enforced by the DNA damage and spindle assembly checkpoint pathways. The involvement of the spindle assembly checkpoint implies a defect in kinetochore-spindle attachment or sister chromatid cohesion due to incomplete replication and/or DNA damage. Remarkably, under semipermissive conditions for ORC1 function, the spindle checkpoint alone suffices to block proliferation, suggesting this checkpoint is highly sensitive to replication initiation defects. We discuss the potential significance of these overlapping checkpoints and the impact of our findings on previously postulated role(s) of ORCs in other cell cycle functions.