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中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、非整倍体和基因扩增有关。这些基因组改变使癌细胞能够在正常细胞无法增殖的条件下增殖。最近的研究表明，复制受到细胞周期的精确调节，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再复制以及基因组重复至关重要。在细胞周期中，复制前复合物 (Pre-RC) 在有丝分裂末期和 G1 期间组装到复制起点上，以增强 S 期的染色质复制。 Pre-Rc 组装的第一步是将起点识别复合物 (ORC) 与起点结合。然后 CDC6 和 CDT1 与 ORC 结合以促进 MCM2-7 蛋白的装载。我们之前发现，细胞周期蛋白 A 或 Geminin（一种与复制许可因子 CDT1 结合的复制抑制剂）的丢失会诱导含有 4N 至 8N 之间 DNA 含量的多倍体细胞的积累。在此应用中，我们建议研究我们的新发现，即 CDT1 作为响应 DNA 损伤的直接检查点目标。我们发现 CDT1 在伽马射线照射下几分钟内就会被蛋白水解。我们提供了遗传和生化证据，表明这种检查点控制代表了一种独立于 ATM/CHK2 和复制的新检查点途径。我们还提供了证据表明，一种未表征的泛素 E3 连接酶复合物参与了靶向 CDT1 的泛素依赖性降解。我们建议调查这个新的检查点。我们的具体目标是：1) 确定导致 CDT1 因 DNA 损伤而降解的信号。 2) 分离新的E3连接酶复合物。 3）重现CDT1体外降解。 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因子，因此了解其调节应该为基因组稳定性机制提供新的见解。