Investigation of Cdc45 function during helicase activation

Abstract

Eukaryotic DNA replication requires the stepwise loading of Mcm2-7 helicase complexes at origins of replication. These loaded helicases need to be activated during DNA replication initiation to unwind the DNA double helix and provide a ssDNA template for replicative DNA polymerases. Eukaryotic cells segregate helicase loading and activation events to ensure a single round of DNA replication per cell cycle. Upon entry into S-phase, Cdc45 and GINS associate with the Mcm2-7 complex to form the CMG complex, which is the active eukaryotic replicative helicase. Consistent with a role in stimulating helicase activity, in addition to its role during DNA replication initiation, Cdc45 is also required during DNA elongation. In this thesis, I have investigated how Cdc45 functions during DNA replication. I characterized four lethal and three temperature- sensitive point mutations in CDC45. Intriguingly, the temperature-sensitive mutants were specifically defective for DNA replication initiation and not for elongation. This suggests that the requirements for Cdc45 function during DNA replication initiation are distinct from those involved in replication elongation.