Establishing DNA combing to investigate DNA replication stress in vitro

Tollitt, James and Copeland, Nikki (2017) Establishing DNA combing to investigate DNA replication stress in vitro. Masters thesis, Lancaster University.

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During the mitotic mammalian cell cycle cells faithfully replicate their DNA utilizing multiple DNA replication sites known as origins of replication. DNA is replicated to provide each daughter cell a complete copy of the genome. Replication proceeds bi-directionally from a minority of potential origins licensed for replication by a variety of replication factor proteins. Replication is catalysed by processive replication enzymes known as DNA polymerases and is limited to the synthesis phase (S phase) of the cell cycle. Changes in the timing of replication, origin usage and replication rate are indicative of DNA replication stress, a proposed hallmark of cancer that causes genome instability. Cell cycle progression is largely controlled by the activity of cyclin dependent kinases (CDKs) and their cyclin binding partners. Here using an in vitro cell-free DNA replication system we analyse the interplay between Ciz1 and cyclin A/CDK2 in regulation of the initiation phase of DNA replication. This demonstrates that Ciz1 modulates and enhances the activity of cyclin A-CDK2 in cell free DNA replication assays and that Ciz1 increases the permissive CDK range that can promote DNA replication. Next the inhibitory effect of Ap4A in cell free DNA replication assays is studied. These data suggest that Ap4A inhibits initiation by reducing loading of the replicative helicase MCM2-7 and the DNA polymerase sliding clamp PCNA. These data suggest that Ap4A can inhibit the firing of replication origins through disruption of replication complex assembly. Finally, DNA combing is established to measure replication parameters. Here we find that the replication fork progresses at 1.3kbp/min in mouse fibroblast cells, consistent with other studies, and quantify replication fork stalling by replication inhibitor aphidicolin. These data demonstrate the potential for cell free DNA replication assays to be combined with DNA combing to dissect replication parameters and characterise DNA replication stress in future studies.

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Thesis (Masters)
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28 Apr 2017 15:46
Last Modified:
16 Jul 2024 05:38