Mechanism of PCNA-Dependent Cdt1 Destruction in S phase

描述（由申请人提供）：在真核细胞中，每个复制起点的DMA复制起始被限制为每个S期一次，确保基因组在每个细胞周期中精确复制一次。如果不能适当地限制起始点的再激发，就会导致基因组不稳定，并可能导致癌症。起源在G1期由ORC、Cdc6和Cdt1授权，它们协同募集MCM2-7解旋酶。一旦起点在S期开始复制，它就失去活性。起始后，由于Cdt1活性通过双胞蛋白和泛素介导的Cdt1蛋白水解而受到抑制，直到下一个G1期，起源才能重新获得许可。使用来自爪蟾卵提取物的无细胞系统，我的宿主实验室发现Cdt1在S期被Cul4-Ddb1-Cdt2泛素连接酶泛素化，这种方式需要Cdt1与DMA复制叉上的PCNA相互作用。然而，关于Cdt1破坏的机制，许多问题仍未得到解答。例如，Cdt1选择性结合到PCNA染色质结合池的基础是未知的，这对于确保S期特异性Cdt1破坏是重要的。此外，Cdt1内介导其破坏的序列还没有很好地定义。在Aim中，我将使用重组策略来概括Cdt1与dna结合的PCNA的选择性结合，从而阐明其机制。目的2将进行Cdt1的突变分析，以确定在爪蟾卵提取物的DNA复制过程中Cdt1破坏所需的特定氨基酸。此外，我将询问在dna结合的PCNA中，这些氨基酸是否介导Cdt1与Cul4-Ddb1-Cdt2的结合。最后，为了测试所提出的Cdt1破坏机制，我将尝试在体外使用纯化的组分重建PCNA-和cul4 - ddb1 - cdt2依赖的Cdt1泛素化。Cdt1在几种肿瘤细胞中过表达，在小鼠中人工Cdt1过表达增加了致癌潜能。因此，了解Cdt1破坏的分子机制对癌症生物学具有重要意义。

DESCRIPTION (provided by applicant): In eukaryotic cells, initiation of DMA replication at each origin of replication is restricted to once per S phase, insuring that the genome is duplicated precisely once during each cell cycle. Failure to properly restrict origin re-firing results in genomic instability and probably causes cancer. Origins are licensed in G1 by ORC, Cdc6, and Cdt1 which cooperate to recruit the MCM2-7 helicase. Once the origin initiates replication in S phase, it is inactivated. After initiation, origins cannot be re-licensed until the following G1 phase due to inhibition of Cdt1 activity through Geminin and ubiquitin-mediated proteolysis of Cdt1. Using a cell-free system derived from Xenopus egg extracts, my host laboratory fond that Cdt1 is ubiquitylated during S phase by the Cul4-Ddb1-Cdt2 ubiquitin ligase in a manner that requires interaction of Cdt1 with PCNA at the DMA replication fork. However, many questions remain unanswered with regard to the mechanism of Cdt1 destruction. For example, the basis for selective binding of Cdt1 to the chromatin-bound pool of PCNA, which is important to insure S phase-specific Cdt1 destruction, is unknown. In addition, the sequences within Cdt1 which mediate its destruction are not well defined. In Aim 1 will use reconstitution strategies to recapitulate the selective binding of Cdt1 to DNA-bound PCNA and thereby elucidate its mechanism. In Aim 2 will perform a mutational analysis of Cdt1 to identify specific amino acids which are required for Cdt1 destruction during DNA replication in Xenopus egg extracts. In addition, I will ask whether these amino acids mediate binding of Cdt1 to Cul4-Ddb1-Cdt2 in the context of DNA-bound PCNA. Finally, to test the proposed mechanism of Cdt1 destruction, I will attempt to reconstitute PCNA- and Cul4-Ddb1-Cdt2-dependent ubiquitylation of Cdt1 in vitro using purified components. Cdt1 is overexpressed in several tumor cells, and artificial Cdt1 over expression in mice increases oncogenic potential. Therefore, understanding the molecular mechanism of Cdt1 destruction is important for cancer biology. To avoid diseases such as cancer, our cells must create a precise copy of their genomes before each cell division. To limit genome duplication to a single round, a DNA replication factor called Cdt1 is normally destroyed after the first round has occurred. I propose to study in molecular detail how this destruction process takes place. Because Cdt1 over expression can cause cancer, the work is highly relevant for human health.