The role of Mus101 in maintenance of genome stability

描述（由申请方提供）：细胞周期检查点对受损DNA的存在做出反应，并组织细胞对损伤的反应。如果没有完整的检查点控制系统，基因组的稳定性就处于危险之中，并且在关键生长控制基因中积累突变的可能性显著增强。尽管人们对检查点如何调节细胞周期和组织DNA修复系统知之甚少，但对检查点如何首先被受损的DNA激活知之甚少。检查点领域的一个新兴想法是，在S阶段，复制叉可以感知到某些形式的损坏。如果是这样，那么复制和检查点控制所需的蛋白质代表了提供难以捉摸的损伤传感活性的良好候选物。裂变酵母中的Cut 5蛋白（芽殖酵母中的Dpb 11）满足了这一要求，因为它是DNA复制和对停滞复制叉的检查点响应所必需的。此外，DPB 11已被证明在维持基因组稳定性方面发挥作用，因为DPB 11的亚纯型等位基因导致染色体重排的急剧增加，即使细胞是活的。在脊椎动物中与Cut 5/Dpb 11最接近的匹配是Mus 101蛋白家族，人类TopBP 1是其中的一员。尽管它与酵母蛋白相似，在检查点控制中具有已证实的作用，但Mus 101/TopBP 1的特征仍然很差。为了了解更多关于Mus 101在复制和检查点控制中的作用，我的实验室已经表征了Mus 101在生物化学上易处理的爪蟾卵提取物系统和遗传上易处理的线虫C.优雅我们已经发现，从非洲爪蟾卵提取物中耗尽Mus 101可以阻断DNA复制和DNA损伤检查点的激活。此外，C. elegans mus-101直系同源物导致胚胎致死，并且在亚形态条件下导致对DNA损伤的敏感性。这些发现证实了Mus 101是Cut 5/Dpb 11在脊椎动物中的对应物。本研究的目的是将非洲爪蟾卵提取物中Mus 101的联合收割机生化分析与C. elegans的研究，以充分描述Mus 101在DNA复制，DNA损伤检查点的激活和基因组稳定性的维持中的功能。

DESCRIPTION (provided by applicant): Cell cycle checkpoints react to the presence of damaged DNA, and organize the cellular response to the damage. Without intact checkpoint control systems, genome stability is at risk, and the potential for accumulating mutations in critical growth control genes is significantly enhanced. Although much is known about how checkpoints regulate the cell cycle and organize DNA repair systems, comparatively little is known about how checkpoints are activated by damaged DNA in the first place. An emerging idea in the checkpoint field is that, during S phase, some forms of damage are sensed by replication forks. If so, then a protein that is required for both replication and checkpoint control represents a good candidate to provide the elusive damage sensing activity. The Cut5 protein in fission yeast (Dpb11 in budding yeast) fulfills this requirement, as it is required for both DNA replication and the checkpoint response to stalled replication forks. Additionally, Dpb11 has been shown to play a role in maintenance of genome stability, as hypomorphic alleles of DPB11 cause a dramatic increase in chromosomal rearrangements even though the cells are viable. The closest match to Cut5/Dpb11 amongst vertebrates is the Mus101 protein family, of which the human TopBP1 is a member. Despite its similarity to a yeast protein with a proven role in checkpoint control, Mus101/TopBP1 remains poorly characterized. In order to understand more about the role of Mus101 in replication and checkpoint control, my laboratory has characterized Mus101 activity in both the biochemically tractable Xenopus egg extract system and the genetically tractable nematode C. elegans. We have found that depletion of Mus101 from Xenopus egg extracts blocks both DNA replication, and activation of the DNA damage checkpoint. Furthermore, depletion of the C. elegans mus-101 ortholog results in embryonic lethality and, under hypomorphic conditions, sensitivity to DNA damage. These findings establish that Mus101 is the vertebrate counterpart to Cut5/Dpb11. The goal of this proposal is to combine biochemical analysis of Mus101 in Xenopus egg extracts with genetic analysis in C. elegans to fully describe the Mus101 function in DNA replication, in activation of the DNA damage checkpoint, and in maintenance of genome stability.