Postreplication gaps at UV lesions are signals for checkpoint activation
Postreplication gaps at UV lesions are signals for checkpoint activation
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DOI：
10.1073/pnas.1003449107
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发表时间：
2010-05-04
影响因子：
11.1
通讯作者：
Kelly, Thomas J.
中科院分区：
文献类型：
--
作者：
Callegari, A. John;Clark, Emily;Kelly, Thomas J.
真核细胞暴露于紫外线下会诱导检查点反应，从而在细胞进入 S 期后延迟细胞周期进程。据推测，当复制叉遇到紫外线诱导的 DNA 损伤时，这种检查点反应会发出信号，产生结构的存在，从而提供修复时间。为了深入了解信号结构的本质，我们使用延时显微镜来确定跨损伤 DNA 聚合酶缺陷对裂殖酵母裂殖酵母检查点反应的影响。我们发现，编码跨损伤 DNA 聚合酶 Pol kappa 和 Pol eta 的基因的破坏显着延长了检查点反应，表明这些酶的底物是检查点激活的信号。令人惊讶的是，我们没有发现任何证据表明跨损伤聚合酶 Rev1 和 Pol zeta 修复了检查点识别的结构，尽管它们在紫外线照射后维持活力方面发挥着作用。定量流式细胞术显示，缺乏跨损伤聚合酶的细胞以与 WT 细胞相同的速率复制紫外线损伤的 DNA，这表明缺乏 Pol kappa 和 Pol eta 的细胞检查点反应的增强并不是复制叉停滞的结果。这些观察结果支持了一个模型，其中模板链中具有未修复的 UV 损伤的复制后 DNA 缺口既充当跨损伤聚合酶的底物，又充当检查点激活的信号。
Exposure of eukaryotic cells to UV light induces a checkpoint response that delays cell-cycle progression after cells enter S phase. It has been hypothesized that this checkpoint response provides time for repair by signaling the presence of structures generated when the replication fork encounters UV-induced DNA damage. To gain insight into the nature of the signaling structures, we used time-lapse microscopy to determine the effects of deficiencies in translesion DNA polymerases on the checkpoint response of the fission yeast Schizosaccharomyces pombe. We found that disruption of the genes encoding translesion DNA polymerases Pol kappa and Pol eta significantly prolonged the checkpoint response, indicating that the substrates of these enzymes are signals for checkpoint activation. Surprisingly, we found no evidence that the translesion polymerases Rev1 and Pol zeta repair structures that are recognized by the checkpoint despite their role in maintaining viability after UV irradiation. Quantitative flow cytometry revealed that cells lacking translesion polymerases replicate UV-damaged DNA at the same rate at WT cells, indicating that the enhanced checkpoint response of cells lacking Pol kappa and Pol eta is not the result of stalled replication forks. These observations support a model in which postreplication DNA gaps with unrepaired UV lesions in the template strand act both as substrates for translesion polymerases and as signals for checkpoint activation.