The DNA Damage Response Pathway Contributes to the Stability of Chromosome III Derivatives Lacking Efficient Replicators

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 mrc1AQ 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（DNA replication）从多个复制起点（origin）起始。当多个相邻的复制起点无法被激活时，便会产生较大的复制起点间间隙（inter-origin gap）。目前对于细胞如何应对此类情况的认知仍较为有限。我们构建了缺失所有高效复制起点的酿酒酵母（Saccharomyces cerevisiae）三号染色体衍生片段5ORIΔ-ΔR，将其作为携带较大复制起点间间隙的染色体的研究模型。我们将该构建体应用于改良型合成遗传阵列（synthetic genetic array）筛选，以鉴定能够促进长段复制起点间间隙复制的基因及其编码产物。筛选得到的基因显著富集于DNA损伤（DNA damage）与复制应激（replication stress）信号通路的组分中。Mrc1p可被复制应激激活，并介导复制应激信号向下游蛋白的转导；然而，响应缺陷型mrc1AQ等位基因（allele）并未影响5ORIΔ-ΔR片段的维持，表明该通路并未参与该片段的稳定性维持。编码DNA损伤特异性介导因子Rad9p的基因缺失，以及两个信号通路共有的多个组分的缺失，会优先破坏5ORIΔ-ΔR片段的稳定性，表明DNA损伤应答（DNA damage response）通路参与了该片段的维持。我们发现，DNA损伤应答通路组分对ORIΔ染色体衍生体维持的作用，与其在DNA损伤修复（DNA repair）中的作用存在意外差异。在RAD53与CHK1编码的效应激酶（effector kinase）中，Chk1p在野生型细胞中对于降低由复制起点缺失引发的染色体不稳定性更为关键；而在缺失Chk1p的细胞中，Rad53p则发挥更为重要的作用。与之相反，在经DNA损伤试剂与羟基脲（hydroxyurea）处理后，RAD53在细胞存活与复制叉稳定性维持方面的作用比CHK1更为关键。ORIΔ染色体的维持并不依赖于同源重组（homologous recombination）。上述观察结果表明，存在一种不依赖于DNA损伤的机制，可增强ORIΔ染色体的稳定性。因此，DNA损伤应答通路的组分之所以能够维持基因组稳定性，并非仅通过识别并响应DNA模板损伤，还通过促进长段复制起点间间隙的复制来实现这一功能。