The role of Mus101 in maintenance of genome stability

Mus101 在维持基因组稳定性中的作用

• 批准号: 7056801
• 负责人: MATTHEW MICHAEL
• 金额: $ 28.03万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7056801
• 关键词: Caenorhabditis elegans; DNA damage; DNA repair; DNA replication; DNA replication origin; Xenopus oocyte; affinity chromatography; cell cycle; gene expression; genetic screening; genome; immunoprecipitation; mass spectrometry; molecular genetics; polymerase chain reaction; protein structure function

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.

描述（申请人提供）：细胞周期检查点对受损的DNA的存在反应，并组织细胞对损伤的反应。没有完整的检查点控制系统，基因组稳定性就有风险，并且在关键生长控制基因中积累突变的潜力显着增强。尽管对检查点如何调节细胞周期并组织DNA修复系统知之甚少，但首先，关于检查点如何被损坏的DNA激活的情况相对较少。检查点字段中的一个新兴想法是，在S阶段，通过复制叉来感知某些形式的损坏。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害感应活动的好候选者。裂变酵母中的Cut5蛋白（在发芽酵母中的DPB11）满足了这一要求，因为DNA复制和对停滞的复制叉的检查点响应都需要。此外，DPB11已被证明在维持基因组稳定性中起着作用，因为DPB11的肌科会导致染色体重排的急剧增加，即使细胞的生存也是如此。在脊椎动物中，最接近Cut5/dpb11的匹配是MUS101蛋白家族，其中人TopBP1是成员。尽管MUS101/TOPBP1在检查点控制中具有相似的酵母蛋白相似，但表征仍然很差。为了更多地了解MUS101在复制和检查点控制中的作用，我的实验室已经表征了MUS101在生物化学上可触及的Xenopus卵提取系统和遗传性障碍线虫C.秀拉斯秀丽隐杆线虫中的活性。我们发现，从爪蟾卵提取物中耗尽MUS101会阻止DNA复制和激活DNA损伤检查点。此外，秀丽隐杆线虫MUS-101直系同源物的耗竭会导致胚胎致死性，在下型条件下，对DNA损伤的敏感性。这些发现表明，MUS101是切割5/dpb11的脊椎动物对应物。该建议的目的是将Xenopus卵提取物中MUS101的生物化学分析与秀丽隐杆线虫中的遗传分析相结合，以充分描述DNA复制中的MUS101功能，激活DNA损伤检查点以及维持基因组稳定性。