Characterization of XCdc7/Dbf4 and Cdc7/Drf1 in DNA replication initiation

Complete and accurate DNA replication is essential for proper cell division and thus the genetic integrity of all organisms. Several mechanisms regulate the process of DNA replication particularly during the initiation stage. Initiation must be carefully regulated to ensure that each segment of the DNA is replicated at the appropriate time, and only once per cell cycle. Cells achieve this by having a distinct stage that occurs prior to S-phase when replication origins become licensed for replication. At the onset of S-phase, replication forks are initiated only at these licensed origins. Licensing corresponds to the ordered assembly of multiprotein complexes celled pre-replication complexes (pre-RCs) onto the DNA at replication origins. These complexes are composed of Orc, Cdc6, Cdt1, and Mcm proteins. Once assembled the pre-RCs are activated at the appropriate time to trigger DNA replication. Two major kinases are known to participate in this activation, Cdk2/Cyclin E and Cdc7/Dbf4, though their mechanism of action is unknown. How these kinases regulate the initiation of replication is an important question underlying the regulation of the cell cycle and the maintenance of genomic integrity in all organisms.

My study focuses on determining the role of the Cdc7 kinase in DNA replication initiation using an in vitro cell free system derived from Xenopus laevis eggs. Cdc7 kinase activity is reportedly dependent on a regulatory binding partner, called Dbf4. Members of our laboratory identified two potential Dbf4-related proteins in Xenopus laevis, XDbf4 and XDrf1. Since multiple Dbf4-related proteins within the same species had previously been reported in other organisms, I hypothesized that both of these proteins could interact with Xenopus Cdc7 to form active kinases that function during early embryonic DNA replication.

In this study I show that both XDbf4 and XDrf1 physically interact with XCdc7 in interphase egg extracts to form two separate active kinase complexes. Furthermore I suggest that these two complexes are developmentally regulated indicating the possibility that they could function during different stages of early Xenopus development. Interestingly my data indicate that only the XCdc7/Drf1 complex is required for efficient early embryonic DNA replication. Furthermore, this complex seems to function specifically at the point of pre-RC activation, and not during pre-RC assembly. I also examined the mechanisms that could contribute to the regulation of the XCdc7 kinase. Cell-cycle mediated phosphorylation of the XCdc7 complex did not appear to affect its kinase activity. However, pre-phosphorylation of its XMcm2 substrate by Cdk did enhance its phosohorylation by XCdc7. Therefore, this could indicate a direct relationship between S-phase Cdks and the Cdc7 kinase. In addition to these studies, I also analyzed the potential involvement of the XCdc7 kinases in replication checkpoints that ensure that the cell cycle does not proceed without proper DNA replication. My studies indicated that these two XCdc7 kinases are not targeted by the checkpoint pathway. These studies are important in determining the manner in which the Cdc7 kinase contributes to the regulation of eukaryotic DNA replication.