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首页> 外文期刊>Molecular & cellular proteomics: MCP >Quantitative proteomic analysis of chromatin reveals that Ctf18 acts in the DNA replication checkpoint.
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Quantitative proteomic analysis of chromatin reveals that Ctf18 acts in the DNA replication checkpoint.

机译：染色质的定量蛋白质组学分析表明Ctf18在DNA复制检查点中起作用。

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摘要

Yeast cells lacking Ctf18, the major subunit of an alternative Replication Factor C complex, have multiple problems with genome stability. To understand the in vivo function of the Ctf18 complex, we analyzed chromatin composition in a ctf18Delta mutant using the quantitative proteomic technique of stable isotope labeling by amino acids in cell culture. Three hundred and seven of the 491 reported chromosomal proteins were quantitated. The most marked abnormalities occurred when cells were challenged with the replication inhibitor hydroxyurea. Compared with wild type, hydroxyurea-treated ctf18Delta cells exhibited increased chromatin association of replisome progression complex components including Cdc45, Ctf4, and GINS complex subunits, the polymerase processivity clamp PCNA and the single-stranded DNA-binding complex RPA. Chromatin composition abnormalities observed in ctf18Delta cells were very similar to those of an mrc1Delta mutant, which is defective in the activating the Rad53 checkpoint kinase in response to DNA replication stress. We found that ctf18Delta cells are also defective in Rad53 activation, revealing that the Ctf18 complex is required for engagement of the DNA replication checkpoint. Inappropriate initiation of replication at late origins, because of loss of the checkpoint, probably causes the elevated level of chromatin-bound replisome proteins in the ctf18Delta mutant. The role of Ctf18 in checkpoint activation is not shared by all Replication Factor C-like complexes, because proteomic analysis revealed that cells lacking Elg1 (the major subunit of a different Replication Factor C-like complex) display a different spectrum of chromatin abnormalities. Identification of Ctf18 as a checkpoint protein highlights the usefulness of chromatin proteomic analysis for understanding the in vivo function of proteins that mediate chromatin transactions.
机译：缺乏Ctf18（一种替代性复制因子C复合体的主要亚基）的酵母细胞在基因组稳定性方面存在多个问题。为了了解Ctf18复合体的体内功能，我们使用细胞培养物中氨基酸稳定同位素标记的定量蛋白质组学技术，分析了ctf18Delta突变体中的染色质组成。对491个报告的染色体蛋白中的307个进行了定量。当细胞受到复制抑制剂羟基脲攻击时，最明显的异常发生。与野生型相比，羟基脲处理的ctf18Delta细胞表现出增加的染色质缔合，包括Cdc45，Ctf4和GINS复杂亚基，聚合酶连续性钳位PCNA和单链DNA结合复杂RPA的复制体进展复杂组件。在ctf18Delta细胞中观察到的染色质组成异常与mrc1Delta突变体非常相似，该突变体在响应DNA复制压​​力而激活Rad53检查点激酶方面存在缺陷。我们发现ctf18Delta细胞在Rad53激活中也存在缺陷，这表明Ctf18复合物是DNA复制检查点参与所必需的。由于检查点的丢失，在较晚起点处复制的不适当启动可能导致ctf18Delta突变体中与染色质结合的复制体蛋白水平升高。 Ctf18在检查点激活中的作用并非所有复制因子C样复合物共有，因为蛋白质组学分析显示缺少Elg1（不同复制因子C样复合物的主要亚基）的细胞显示出不同的染色质异常谱。 Ctf18作为检查点蛋白的鉴定突出了染色质蛋白质组学分析对于理解介导染色质交易的蛋白质的体内功能的有用性。

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