Novel Phosphorylation Sites on Cdt1 Regulate Its Activity (2016)

Undergraduates: Yasemin Cole, Pedro Pozo Professor Jeanette Cook

Faculty Advisor: Jeanette Cook
Department: Biology

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Origin licensing prepares the genome for replication during the cell cycle. The overexpression of Cdt1, an origin licensing protein, is toxic to cells because it induces re-replication of DNA. The phosphorylation regulation of Cdt1 is not fully understood. This study investigates how phosphorylation of two newly identified phosphorylation sites (S93 and T152) regulates Cdt1 function. We generated three human U2OS cell lines harboring doxycycline inducible mutationally altered versions of Cdt1 [S93A, T152A, and a combination of the two (¿¿¿2A¿¿¿)]. To determine the cellular phenotypes of each mutant cell line, colony forming assays were performed by overexpressing ectopic Cdt1. Non-phosphorylatable Cdt1 proteins, especially 2A, cause reduced cellular toxicity compared to WT, indicating hypomorphic activity. DNA damage markers, such as phospho-Chk1, are highly expressed in cells with overexpressed Cdt1-WT and Cdt1-S93A. Further tests will confirm low levels of phospho-Chk1 expression in cells with overexpressed Cdt1-T152A and Cdt1-2A. Overexpressed Cdt1-WT and Cdt1-S93A are predicted to induce higher incidence of re-replication than overexpressed Cdt1-T152A and Cdt1-2A, via flow cytometry. These results suggest phosphorylation at S93 and T152 both contribute to Cdt1 function in origin licensing. Future studies of how phosphorylation of Cdt1 residues S93 and T152 affects Cdt1 protein dynamics and cell growth will lead to a better understanding of the control mechanisms of Cdt1.