Department of Microbiology and Molecular Genetics, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, United States of America.
PLoS Genet. 2010 Dec 2;6(12):e1001227. doi: 10.1371/journal.pgen.1001227.
In eukaryotic chromosomes, DNA replication initiates at multiple origins. Large inter-origin gaps arise when several adjacent origins fail to fire. Little is known about how cells cope with this situation. We created a derivative of Saccharomyces cerevisiae chromosome III lacking all efficient origins, the 5ORIΔ-ΔR fragment, as a model for chromosomes with large inter-origin gaps. We used this construct in a modified synthetic genetic array screen to identify genes whose products facilitate replication of long inter-origin gaps. Genes identified are enriched in components of the DNA damage and replication stress signaling pathways. Mrc1p is activated by replication stress and mediates transduction of the replication stress signal to downstream proteins; however, the response-defective mrc1(AQ) allele did not affect 5ORIΔ-ΔR fragment maintenance, indicating that this pathway does not contribute to its stability. Deletions of genes encoding the DNA-damage-specific mediator, Rad9p, and several components shared between the two signaling pathways preferentially destabilized the 5ORIΔ-ΔR fragment, implicating the DNA damage response pathway in its maintenance. We found unexpected differences between contributions of components of the DNA damage response pathway to maintenance of ORIΔ chromosome derivatives and their contributions to DNA repair. Of the effector kinases encoded by RAD53 and CHK1, Chk1p appears to be more important in wild-type cells for reducing chromosomal instability caused by origin depletion, while Rad53p becomes important in the absence of Chk1p. In contrast, RAD53 plays a more important role than CHK1 in cell survival and replication fork stability following treatment with DNA damaging agents and hydroxyurea. Maintenance of ORIΔ chromosomes does not depend on homologous recombination. These observations suggest that a DNA-damage-independent mechanism enhances ORIΔ chromosome stability. Thus, components of the DNA damage response pathway contribute to genome stability, not simply by detecting and responding to DNA template damage, but also by facilitating replication of large inter-origin gaps.
在真核染色体中，DNA 复制从多个起点开始。当几个相邻的起点未能启动时，会产生较大的起点间间隙。目前尚不清楚细胞如何应对这种情况。我们创建了酿酒酵母染色体 III 的衍生物，该衍生物缺失了所有有效的起点，即 5ORIΔ-ΔR 片段，作为具有较大起点间间隙的染色体模型。我们使用该构建体在改良的合成遗传阵列筛选中鉴定了有助于复制长起点间间隙的基因。鉴定出的基因富含 DNA 损伤和复制应激信号通路的组成部分。Mrc1p 被复制应激激活，并介导复制应激信号向下游蛋白的转导；然而，反应缺陷的 mrc1(AQ)等位基因不会影响 5ORIΔ-ΔR 片段的维持，表明该途径不参与其稳定性。编码 DNA 损伤特异性介质 Rad9p 和两个信号通路之间共享的几种成分的基因缺失优先破坏 5ORIΔ-ΔR 片段，表明 DNA 损伤反应途径参与其维持。我们发现 DNA 损伤反应途径的组成部分对 ORIΔ 染色体衍生物的维持的贡献与它们对 DNA 修复的贡献之间存在意外差异。在 RAD53 和 CHK1 编码的效应激酶中，Chk1p 似乎在野生型细胞中对于减少由起点耗尽引起的染色体不稳定性更为重要，而 Rad53p 在缺乏 Chk1p 的情况下变得重要。相比之下，RAD53 在 DNA 损伤剂和羟基脲处理后对细胞存活和复制叉稳定性的重要性大于 CHK1。ORIΔ 染色体的维持不依赖于同源重组。这些观察结果表明，一种与 DNA 损伤无关的机制增强了 ORIΔ 染色体的稳定性。因此，DNA 损伤反应途径的组成部分通过检测和响应 DNA 模板损伤来提高基因组稳定性，还通过促进大的起点间间隙的复制来提高基因组稳定性。
