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，基因组稳定性就会面临风险，关键生长控制基因中积累突变的可能性也会显着增强。尽管人们对检查点如何调节细胞周期和组织 DNA 修复系统了解很多，但对于检查点如何被受损 DNA 激活的了解相对较少。检查点领域的一个新兴想法是，在 S 阶段，复制叉会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就是提供难以捉摸的损伤感知活性的良好候选者。裂殖酵母中的 Cut5 蛋白（芽殖酵母中的 Dpb11）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，Dpb11 已被证明在维持基因组稳定性方面发挥着作用，因为 DPB11 的亚等位基因会导致染色体重排急剧增加，即使细胞是有活力的。脊椎动物中与 Cut5/Dpb11 最接近的匹配是 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，并在检查点控制中发挥了已被证明的作用，但其特征仍然很少。为了更多地了解 Mus101 在复制和检查点控制中的作用，我的实验室在生物化学上易处理的非洲爪蟾卵提取物系统和遗传上易处理的线虫秀丽隐杆线虫中表征了 Mus101 的活性。我们发现，从非洲爪蟾卵提取物中去除 Mus101 会阻断 DNA 复制和 DNA 损伤检查点的激活。此外，秀丽隐杆线虫mus-101直系同源物的消耗会导致胚胎致死，并且在低等态条件下，会导致对DNA损伤的敏感性。这些发现证实 Mus101 是 Cut5/Dpb11 的脊椎动物对应物。该提案的目标是将非洲爪蟾卵提取物中 Mus101 的生化分析与秀丽隐杆线虫的遗传分析相结合，以充分描述 Mus101 在 DNA 复制、DNA 损伤检查点激活和维持基因组稳定性中的功能。

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.