Deegan, TD; (2015) Investigating the Mechanism of Activation of the Mcm2-7 Replicative Helicase. Doctoral thesis , UCL (University College London).

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Abstract

DNA replication initiation is co-ordinated with progression through the cell cycle via a two-step mechanism. The first step, termed origin licensing, involves the assembly of a pre-replicative complex (pre-RC) at origins of replication, in which the Mcm2-7 replicative helicase is loaded onto DNA in an inactive form. Origin unwinding and DNA synthesis is only initiated during the second step of this process, origin firing, which requires the recruitment of multiple ‘firing factors’, such as Sld3/7 and Cdc45, as well as the activity of two essential cell cycle regulated kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). Whilst studies of the mechanism of origin licensing have been greatly furthered by the availability of an in vitro pre-RC assembly assay, the subsequent activation of the replicative helicase has not been well characterised, and how the firing factor proteins catalyse the complex topological changes required for Mcm2-7 helicase activation is unknown. In this study, I used an in vitro biochemical approach to investigate the function of DDK and Sld3/7 in origin firing. I reconstituted the recruitment of Sld3/7 to the pre- RC in vitro, which is thought to be the first step during Mcm2-7 helicase activation. I observed recruitment of Sld3/7 to be dependent on phosphorylation of the loaded Mcm2-7 complex by DDK, and was subsequently able to map the Mcm2-7 binding activity of Sld3/7 to a central domain of Sld3. By isolating a number of point mutants in Sld3 that were specifically defective in Mcm2-7 binding, I showed that the Sld3-Mcm2-7 interaction is essential for replicative helicase activation. Furthermore, I showed that the central portion of Sld3 contains a Cdc45 interacting site, which is also required for efficient replication initiation. Subsequently, I showed that Sld3 can interact with both Mcm6 and Mcm4 in a phosphorylation-dependent manner. Sld3 itself was shown to contain a novel phosphopeptide binding activity, and can interact with numerous phosphorylated residues throughout the N-terminal half of Mcm6. Elimination of these phosphorylation sites resulted in defects in both Sld3/7 recruitment to the pre-RC and replicative helicase activation. Thus, the novel DDK-dependent Sld3-Mcm2-7 interaction described in this study helps to explain the function of DDK during the early stages of origin firing. Additionally, the observation that Sld3 can interact directly with phosphorylated residues on Mcm2-7 indicates that Sld3 is a reader of DDK activity. As both an essential CDK substrate and a DDK reader, Sld3 thus functions as a point of intersection for the activities of CDK and DDK during Mcm2-7 helicase activation.

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