Cameron, George; (2022) Single-molecule visualisation of eukaryotic replisome collisions with pre-replication and cohesin complexes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Eukaryotic replisomes must copy the sequence of chromatinised DNA whilst co-ordinating a number of replication associated processes. DNA replication is carefully co-ordinated by the cell cycle. The origin recognition complex (ORC) licenses DNAs with an inactive double hexamer of Mcm2-7 hexamers to form pre-replication complexes (pre-RCs) strictly in G1-phase. Chromosomes are licensed with an excess of pre-RCs, not all of which are routinely used for replication. During S-phase, origin firing occurs and a proportion of pre-RCs are remodelled into the Cdc45-Mcm2-7-GINS (CMG) helicase. Parental duplex DNA is unwound by CMG and the rest of the replisome is assembled around CMG. The fate of dormant pre-RCs during replication is poorly understood. CMG encircles single stranded DNA whilst pre-RCs encircle double stranded DNA, so pre-RCs are a potential barrier to CMG progression. Reconstituted in vitro reactions have shown that CMG activity alone is insufficient to remove pre-RCs, and that pre-RCs can slide ahead of CMG. Pre-RCs must be removed at some point during replication to ensure proper termination of DNA replication can occur and that no pre-RCs are bound to newly replicated DNA. Pre-RC fate during replication in a cell-free system of Xenopus laevis egg extracts is investigated here. I developed a system to visualise CMG collision with pre-RCs in real-time using single-molecule imaging of labelled Mcm2-7 complexes on surface tethered DNAs. Using this sensitive assay, I show that pre-RCs are mostly removed ahead of CMG upon collision. By depleting factors in egg extracts used for the same assay, a role for the RTEL1 and FANCJ helicases in removing pre-RCs after CMG collision is demonstrated. This adds to evidence showing general roles of accessory helicases at replication forks. Sister chromatid cohesion must be co-ordinated with DNA replication. For cohesion, the cohesin complex tethers newly synthesised nascent DNAs together in preparation for mitosis. Cohesin is a large ring-shaped complex that can topologically entrap DNA(s). One pathway for sister chromatid cohesion in yeast involves cohesin complexes loaded onto DNA in G1-phase being converted into cohesive structures during DNA replication. By loading fluorescently labelled Xenopus cohesin on surface tethered DNAs and then replicating DNAs with labelled CMG, cohesin conversion is investigated in extracts. I show that cohesin is pushed ahead of CMG to sites where replication termination occurs, where cohesin remains during CMG disassembly. I provide evidence that cohesin associates with both new sister chromatids of replicated surface tethered DNAs. Together this evidence points towards a model of cohesion establishment during DNA replication termination.

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