Functional analysis of DNA replication initiation

Abstract

Initiation of DNA replication is highly conserved from yeast to human. A crucial factor during this process is the replicative helicase MCM2-7, a ring-shaped hetero-hexameric protein assembly, which unwinds DNA at the replication fork. In a multi-step process, ORC, Cdc6 and Cdt1 promote the loading of two MCM2-7 hexamer into a head-to-head double hexamer around double stranded DNA. The recent cryo-EM structure of an ORC/Cdc6/Cdt1/MCM2-7 (OCCM) intermediate has revealed that the MCM2-7 hexamer and ORC/Cdc6 are connected by C-terminal winged helix domains (WHDs). However, how these WHDs function for helicase loading and MCM2-7 double-hexamer formation has been largely unknown. Using a combination of biochemical and structural approaches, we have investigated the function of the different Mcm C-termini during pre-RC formation. In this study, we developed a new concept, explaining how distinct functions of the individual WHDs drive the multi-step pre-RC formation process. In particular, we discovered that the Mcm5 WHD serves as a key factor in order to signal helicase loading defects and to induce quality control during pre-RC formation. In addition, it was found that the Mcm5 WHD is also required for the OCCM to double hexamer transition. While exploring the Mcm7 WHD, we discovered new rules and requirements for ATP hydrolysis dependent Cdt1 release. Finally, we identified novel mechanism in CDK dependent control of helicase loading, which are important for genome stability. The work performed as part of this thesis has resulted in the discovery of several new mechanisms, which together provide fundamental insights into the function and regulation of the highly complex eukaryotic helicase loading process.