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.
