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The Initiation of Eukaryotic DNA Replication: Mechanism, Regulation and Role in Genome Stability

Final Report Summary - EUKDNAREP (The Initiation of Eukaryotic DNA Replication: Mechanism, Regulation and Role in Genome Stability)

This project aimed to understand the mechanism and regulation of DNA replication, using primarily biochemical approaches. Work from this project culminated in our publication earlier this year describing the reconstitution of eukaryotic DNA replication origin firing with purified proteins (Yeeles et al. Nature 2015). This reconstitution required the expression and purification of 16 different budding yeast replication factors made from 42 polypeptides, and I am sure this would not have happened without ERC support. This was not only a pleasing ending to the EUKDNAREP grant, but also opens many new avenues to understand replication initiation in detail. Prior to establishing the fully reconstituted system, we showed that the MCM helicase loaded with purified proteins, could replicate in an S phase extract. Mass spectrometry of intermediates from these reactions was crucial for identifying the 16 factors we needed to purify (On et al. EMBO J. 2014).
We discovered a novel ATP hydrolysis-dependent proofreading mechanism that prevents accumulation of ‘dead-end complexes’ during the loading of the MCM replicative helicase (Frigola et al Nature 2013). We showed ATP hydrolysis by Cdc6 is required for this activity, whilst ATP hydrolysis by MCM subunits is required for helicase loading (Coster et al Mol Cell 2014). We showed that this proofreading activity disassembles intermediate complexes formed by origin recognition complex (ORC) that had been phosphorylated by cyclin dependent kinase (CDK), and thus plays a role in preventing re-replication during the cell cycle (Frigola et al. Nature 2013).
We showed that a human protein called Treslin/TICRR is the human orthologue of yeast Sld3 (Sanchez-Pulido et al. Curr. Biol. 2010), and we showed that the regulation of Sld3 by CDK, which we had previously discovered in yeast, is conserved in Treslin/TICRR (Boos et al. Curr. Biol. 2011). Moreover, we showed Sld3 is inhibited by the DNA damage checkpoint (Zegerman and Diffley Nature 2010), and this feature is also conserved in Treslin/TICRR (Boos et al. Curr. Biol. 2011).
Finally, we discovered a novel DNA replication factor (MTBP) which binds specifically to Treslin/TICRR (Boos et al. Science 2013). Because MTBP had previously been implicated in p53 degradation via Mdm2 interaction, this protein may link p53 to the initiation of DNA replication.