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，基因组稳定性就会受到威胁，关键生长控制基因积累突变的可能性会显著增强。虽然关于检查点如何调节细胞周期和组织DNA修复系统已经知道很多，但相对较少的人知道检查点是如何被受损的DNA激活的。检查点领域的一个新想法是，在S阶段，复制叉子可以感知到某些形式的损害。如果是这样，那么复制和检查点控制都需要的蛋白质是提供难以捉摸的损伤感知活动的很好的候选者。分裂酵母中的Cut5蛋白(萌芽酵母中的Dpb11)满足了这一要求，因为它是DNA复制和对停滞的复制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DPB11的亚型等位基因导致染色体重排急剧增加，即使细胞是活的。脊椎动物中与Cut5/Dpb11最匹配的是Mus101蛋白家族，人类TopBP1是该家族的成员。尽管Mus101/TopBP1与酵母蛋白相似，但它在检查点控制中的作用已被证实，但它的特征仍然很差。为了更多地了解Mus101在复制和检查点控制中的作用，我的实验室已经确定了Mus101在生物化学上易处理的非洲爪哇卵子提取系统和遗传上易处理的线虫线虫中的活性。我们发现，耗尽非洲爪哇卵子提取物中的Mus101既阻止了DNA复制，也阻止了DNA损伤检查点的激活。此外，线虫MUS-101同源基因的耗尽会导致胚胎死亡，在亚形条件下，还会对DNA损伤敏感。这些发现证实了Mus101是Cut5/Dpb11的脊椎动物对应基因。这项建议的目的是将非洲爪哇卵提取液中Mus101的生化分析与线虫的遗传分析相结合，以全面描述Mus101在DNA复制、激活DNA损伤检查点和维持基因组稳定性方面的功能。

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.