The role of Mus101 in maintenance of genome stability

描述（由申请人提供）：细胞周期检查点对受损DNA的存在作出反应，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，基因组稳定性将面临风险，关键生长控制基因积累突变的可能性将显著增强。尽管我们对检查点如何调节细胞周期和组织DNA修复系统了解甚多，但相对而言，我们对检查点如何被受损的DNA激活知之甚少。检查点领域中出现的一种新观点是，在S阶段，复制分叉感知到某些形式的损伤。如果是这样，那么复制和检查点控制都需要的蛋白质代表了提供难以捉摸的损伤感知活动的良好候选者。裂变酵母中的Cut5蛋白（出芽酵母中的Dpb11）满足这一要求，因为它是DNA复制和对停滞复制分叉的检查点反应所必需的。此外，Dpb11已被证明在维持基因组稳定性方面发挥作用，因为即使细胞存活，Dpb11的次胚等位基因也会导致染色体重排的急剧增加。在脊椎动物中与Cut5/Dpb11最接近的是Mus101蛋白家族，人类TopBP1是其中的一员。尽管Mus101/TopBP1与酵母蛋白相似，在检查点控制中起着重要作用，但其特征仍然很差。为了进一步了解Mus101在复制和检查点控制中的作用，我的实验室在生化可处理的爪蟾卵提取系统和遗传可处理的线虫中表征了Mus101的活性。我们发现，非洲爪蟾卵提取物中Mus101的缺失阻断了DNA复制和DNA损伤检查点的激活。此外，秀丽隐杆线虫mus101同源基因的缺失导致胚胎致死性，并且在半胚条件下对DNA损伤敏感。这些发现表明Mus101是脊椎动物中与Cut5/Dpb11对应的基因。本研究的目的是将爪蟾卵提取物中Mus101的生化分析与秀丽隐杆线虫的遗传分析相结合，以全面描述Mus101在DNA复制、DNA损伤检查点激活和基因组稳定性维护中的功能。

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.