Mechanism of PCNA-Dependent Cdt1 Destruction in S phase

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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.