Regulation of Replication Checkpoint by Proteolysis

蛋白水解调节复制检查点

• 批准号: 6777947
• 负责人: HUI ZHANG
• 金额: $ 28.61万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-25 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6777947
• 关键词: DNA binding protein; DNA damage; DNA repair; DNA replication; biological signal transduction; cell cycle; cell cycle proteins; gamma radiation; laboratory rabbit; nucleic acid amplification techniques; protein kinase; proteolysis; ubiquitin

DESCRIPTION (provided by applicant): The broad and long term objectives of this proposal are to elucidate the molecular mechanisms that regulate genome stability in the cell cycle. Alteration of genome stability is a hallmark of human cancer. Cancer is often associated with polyploidy, aneuploidy and gene amplification. These genomic alterations allow cancer cells to proliferate under conditions that normal cells cannot. Recent studies suggest that replication is precisely regulated by the cell cycle to occur only once per cell cycle. Many lines of evidence indicate that replication licensing is critical for the control of re-replication and therefore genome reduplicatoin. In the cell cycle, the pre-replication complex (Pre-RC) assembles onto the replication origins at the end of mitosis and during G1 to potentiate chromatin duplication in S phase. The initial step for Pre-Rc assembly is the binding of the origin recognition complex (ORC) to the origins. CDC6 and CDT1 then associate with ORC to promote the loading of the MCM2-7 proteins. We have previously found that loss of either cyclin A or geminin, a replication inhibitor that binds to the replication licensing factor CDT1, induces accumulation of polyploid cells containing DNA content between 4N and 8N. In this application, we propose to investigate our new finding that CDT1 serves as a direct checkpoint target in response to DNA damage. We found that CDT1 is proteolyzed within minutes in response to gamma-irradiation. We have provided genetic and biochemical evidence indicating this checkpoint control represents a new checkpoint pathway independent of ATM/CHK2 and replication. We also present evidence for the involvement of an uncharacterized ubiquitin E3 ligase complex that targets CDT1 for ubiquitin-dependent degradation. We propose to investigate this new checkpoint. Our specific aims are: 1) To determine the signal that causes CDT1 degradation in response to DNA damage. 2) To isolate the new E3 ligase complex. 3) To recapitulate the CDT1 degradation in vitro. 4) To examine the cell cycle regulation of CDT1 by ubiquitin-dependent proteolysis. Since CDT1 is a critical regulator for replication licensing, understanding of its regulation should provide novel insight into the mechanism for genome stability.

描述（由申请人提供）：该提案的广泛和长期目标是阐明调节细胞周期基因组稳定性的分子机制。基因组稳定性的改变是人类癌症的标志。癌症通常与多倍体，非整倍性和基因扩增有关。这些基因组改变使癌细胞在正常细胞不能的条件下增殖。最近的研究表明，复制受细胞周期的精确调节，即每个细胞周期仅发生一次。许多证据表明，复制许可对于控制重新复制和因此基因组重复素至关重要。在细胞周期中，恢复复合物（PRE-RC）在有丝分裂结束的复制起源和G1期间组装到S相的染色质重复。 PRE-RC组装的初始步骤是原始识别复合物（ORC）与起源的结合。然后，CDC6和CDT1与ORC相关联，以促进MCM2-7蛋白的负载。我们先前已经发现，与复制许可因子CDT1结合的复制抑制剂的丧失，可诱导含有4N和8N之间的多倍体细胞的积累。在此应用程序中，我们建议研究CDT1作为响应DNA损伤的直接检查点目标的新发现。我们发现，CDT1在几分钟内响应γ-辐照蛋白水解。我们提供了遗传和生化证据，表明该检查点控制代表了独立于ATM/CHK2和复制的新检查点途径。我们还提供了证据表明，靶向CDT1的未表征的泛素E3连接酶复合物涉及泛素依赖性降解。我们建议研究这个新的检查点。我们的具体目的是：1）确定对DNA损伤响应CDT1降解的信号。 2）分离新的E3连接酶复合物。 3）在体外概括CDT1降解。 4）通过泛素依赖性蛋白水解检查CDT1的细胞周期调节。由于CDT1是复制许可的关键调节剂，因此对其调节的理解应提供对基因组稳定机制的新见解。