Mechanism of Eukaryotic Helicase Loading and Activation

Abstract

The Mcm2–7 helicase is the only eukaryotic DNA replication protein that participates in all steps of this essential process. Loading of the Mcm2–7 complex at origin DNA licenses replication origins during the G1 phase. Assembly of the Cdc45 and GINS helicase‐activating proteins onto the Mcm2–7 helicase is a central event during the replication origin activation. The resulting Cdc45/Mcm2–7/GINS (CMG) complex is the replicative DNA helicase during replication elongation.We are using a combination of genetic screens and biochemical assays that recapitulate the events of replication initiation to investigate the mechanism of Mcm2–7 loading and activation. Contrary to previous studies, we find that the majority of mutations predicted to inhibit Mcm2–7 helicase function (e.g. mutations in the ATP binding and hydrolysis motifs) are defective for helicase loading. This has led us to hypothesize that the process of Mcm2–7 helicase loading requires one or more activities that are also required for helicase activity of this complex. We will report on our latest studies investigating this connection. We have identified new lethal mutations in proteins predicted to activate the Mcm2–7 helicase. We will report on what we have learned from studying the corresponding mutant proteins using biochemical‐complementation assays for helicase activation and replication initiation.