Single-molecule studies of the mechanism of eukaryotic helicase activation

Abstract

Eukaryotic DNA replication is a fundamental process that must occur accurately and only once per cell cycle. To ensure that origins only initiate once per cell cycle, the events of DNA replication initiation are temporally separated to different phases of the cell cycle. This regulation separates two key events that center the replicative DNA helicase Mcm2-7: helicase loading and helicase activation. During G1 phase, two inactive Mcm2-7 are loaded unto origin DNA. Upon entry into S phase, the association of multiple factors will promote helicase activity. Although loaded helicases mark all potential origins of replication, only the subset that is activated will promote origin initiation, and consequently DNA unwinding. After helicase activation the cell must duplicate its genome prior to chromosome segregation and cell division, making helicase activation the committed step of DNA replication. In my thesis, I describe a novel single-molecule reaction that recapitulates helicase activation in vitro with purified proteins. This single-molecule method allows real-time monitoring of protein associations and dissociations during helicase activation. Through these single-molecule reactions, I found that Cdc45 and GINS are recruited to Mcm2-7 in two stages. First, they are recruited to the unstructured N-terminal tails of Mcm2-7. DDK levels carefully control this initial recruitment, creating binding sites for these proteins that result in the formation of a previously unknown intermediate, which we call the Cdc45-tail-GINS (CtG) complex. Elevated DDK lead to increased numbers of CtG complexes formed on each Mcm2-7, which consequently increases the number of active Cdc45-Mcm2-7-GINS (CMG) helicases formed. This mechanism provides an explanation for the tight control of helicase activation by DDK activity during the cell cycle.