Mechanism of Mcm10 function during DNA replication

Abstract

All life needs to replicate its genome completely and do so with limited errors. In eukaryotic cells, DNA replication is accomplished by a multi-stage process involving numerous protein assemblies. The core component of this process is the replicative DNA helicase, Mcm2-7. Mcm2-7 complexes are loaded around origins of DNA replication during G1 phase. As cells transition from G1 to S phase, the Mcm2-7 helicases are activated resulting in the recruitment of DNA polymerases and accessory proteins to begin DNA synthesis. Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Mcm2-7 helicase-activation requires binding of the helicase-activating proteins, Cdc45 and GINS (forming the CMG complex). DNA unwinding cannot proceed, however, until an additional protein, Mcm10, associates with the CMG. Mcm10 continues to travel with the replication fork after stimulating initial DNA unwinding but a function for Mcm10 during DNA replication elongation had not been established. Using a combination of molecular genetics and reconstituted biochemical assays, this thesis will outline the function of Mcm10 throughout DNA replication. I show that Mcm10 binds a conserved motif located between the OB-fold and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. In addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding is identified. These findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication.