Exploring the roles and mechanisms of Cdc18 in the cell cycle of fission yeast
The replication factor Cdc18 (Cdc6 in other organisms) is required to initiate DNA replication and the S phase checkpoint, which restrains mitosis until replication is faithfully completed. In this thesis I have investigated these roles within the cell cycle of the single-celled eukaryote Schizosaccharomyces pombe (fission yeast), and in addition I have identified putative new roles for this replication factor. The Cdc18 ATPase loads the MCM complex, a putative DNA helicase, onto chromatin in late mitosis and early G1 phases in preparation for DNA replication. I have shown that the association of Cdc18 with chromatin does not require ATP binding, whilst ATP binding is necessary to initiate an S phase checkpoint signal. ATP binding and hydrolysis are required for MCM loading activity and hence DNA replication. I also present evidence indicating that Cdc18 can restrain mitosis from G1 in the absence of DNA replication, perhaps as part of a complex with the replication factors Cdtl and the ORC complex. From my work it appears that Cdc18 is required during S phase for at least two further roles. Firstly, Cdc18 is needed to maintain the S phase checkpoint when DNA replication is affected by perturbations that are likely to involve stalled forks or DNA damage. Secondly, Cdc18 may be required in an unperturbed S phase to prepare cells for entry into or progression through mitosis. This latter role is unlikely to involve DNA metabolism, and may instead be related to chromosome preparation or dynamics.