| Each and every time a cell divides it must replicate millions of bases of DNA. In order to carry out this large feat, it initiates replication from thousands of sites along the DNA known as origins. Origins of DNA replication are licensed through the assembly of a chromatin-bound pre-replication complex consisting of ORC, Cdc6, Cdt1 and the MCM complex. Multiple regulatory mechanisms block new pre-replication complex assembly after the G1/S transition to prevent rereplication. The strict inhibition of licensing after the G1/S transition means that all potential origins used in S phase must have been licensed in the preceding G1. Therefore, it would seem crucial for the cell to sense whether enough origins have been licensed before entering S phase. This dissertation focuses on investigation of a novel "origin licensing checkpoint.";It is documented in this study that that depletion of either of two essential licensing factors, Cdc6 or Cdt1, in normal human fibroblasts induces a G1 arrest accompanied by inhibition of cyclin E/Cdk2 activity and hypophosphorylation of Rb. The Cdk2 inhibition is attributed to a reduction in the essential activating phosphorylation of T160 and an associated delay in Cdk2 nuclear accumulation. In contrast, licensing inhibition in the HeLa or U2OS cancer cell lines failed to down-regulate Cdk2 or Rb phosphorylation, and these cells died by apoptosis. Co-depletion of Cdc6 and p53 in normal cells restored Cdk2 activation and Rb phosphorylation, permitting them to enter S phase with a reduced rate of replication that was accompanied by markers of DNA damage. These results demonstrate dependence on origin licensing for multiple events required for G1 progression, and suggests a mechanism to prevent premature S phase entry that functions in normal cells. |
