Regulation of Replication Checkpoint by Proteolysis

描述(由申请人提供)：这项建议的广泛和长期目标是阐明在细胞周期中调节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一个标志。癌症通常与多倍体、非整倍体和基因扩增有关。这些基因组变化允许癌细胞在正常细胞不能的条件下增殖。最近的研究表明，复制精确地受到细胞周期的调节，每个细胞周期只发生一次。许多证据表明，复制许可对于控制再复制以及基因组复制至关重要。在细胞周期中，复制前复合体(Pre-RC)在有丝分裂末期和G1期聚集在复制起点上，以增强S期的染色质复制。Pre-RC组装的第一步是将起始点识别复合体(ORC)与起始点结合。然后CDC6和CDT1与ORC结合，促进MCM2-7蛋白的负载。我们之前已经发现，细胞周期蛋白A或与复制许可因子CDT1结合的复制抑制因子gminin的缺失，会诱导DNA含量在4N到8N之间的多倍体细胞的积累。在本申请中，我们建议调查我们的新发现，即CDT1作为DNA损伤的直接检查点目标。我们发现，CDT1在几分钟内就会对伽马射线做出反应而被蛋白质降解。我们已经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和复制的新的检查点途径。我们还提出了证据，证明参与了一个未确定的泛素E3连接酶复合体，它针对CDT1进行泛素依赖的降解。我们建议调查这个新的检查站。我们的具体目标是：1)确定导致CDT1降解的信号，以响应DNA损伤。2)分离新的E3连接酶复合体。3)概述CDT1在体外的降解情况。4)检测泛素依赖的蛋白水解物对CDT1细胞周期的调节。由于CDT1是复制许可的关键调控因子，对其调控的了解将为基因组稳定性的机制提供新的见解。

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.