Regulation of p97 segregase recruitment to chromatin during DNA replication

Tarcan, Zeynep (2022). Regulation of p97 segregase recruitment to chromatin during DNA replication. University of Birmingham. Ph.D.

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Abstract

Every step of chromosomal replication must be executed in an error free manner to protect genome stability. Ubiquitylation is a fundamental regulatory post-translational protein modification that controls intracellular signalling events including regulation of DNA replication. p97 segregase works through binding proteins that are marked by ubiquitin and extracting them from membranes, cellular surfaces or protein complexes that they are interacting with.

Little is known about how p97 with its cofactors regulates unperturbed DNA replication. Our group has shown over the last few years that p97 segregase plays a key role in unloading of the replicative helicase from chromatin at termination of DNA replication. To prime it for unloading during S-phase, the terminated helicase is first ubiquitylated by Cul2\(^{Lrr1}\) ubiquitin ligase.

Using the well-established cell-free system of Xenopus leavis egg extract, which is ideal for studying DNA replication at the biochemical level, we have now analysed the mode of p97 recruitment to chromatin during S-phase. Our data suggest that the p97 core complex is primarily brought to S-phase chromatin not through direct binding to its substrate: the polyubiquitylated proteins, but by its cofactor Ubxn7, which in turn binds to active (neddylated) Cul2 ubiquitin ligase. Immunodepletion of Ubxn7 from the egg extract leads to a delay in unloading of terminated helicase despite an accumulation of active Cul2\(^{Lrr1}\) ligase. In conclusion, we have identified Ubxn7 as a p97 cofactor playing a role specifically during S-phase unloading of terminated helicase by interacting with Cul2\(^{Lrr1}\) ubiquitin ligase and p97. Ubxn7 also interplays with Faf1 for recruitment of p97 to polyubiquitylated CMG helicase in S phase and mitosis. Furthermore, I also identified a number of posttranslational modifications of p97 in S phase and mitosis, which may regulate p97 interactions with cofactors in higher-order complex assemblies and allow for chain type specificity of p97 recognition. Finally, I also identified that RPA could be a potential substrate of p97 in the S phase and mitosis, whilst p97 might be itself a possible substrate of ATR kinase in mitosis.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):