Elucidation and characterisation of checkpoint kinase interactions with the replisome

Change log
Hadjicharalambous, Andreas 

DNA must be accurately and faithfully copied in each cell cycle by the process of DNA replication. During replication, cells must endure assaults from a myriad of exogenous and endogenous agents and events which can interfere with replication. Cells have evolved a surveillance mechanism called the DNA replication checkpoint (DRC) which ensures that replication is successful, even under conditions which perturb replication (also called replication stress). Checkpoint kinases are the major actor of DRC activation, coordinating multiple cellular events with resolution of these replication stresses, partly by being recruited to the replisome, the machinery that conducts DNA replication. Regulation of replisome stability is one of the essential functions of these checkpoint kinases. Since replication stress is a cellular feature of many tumours and is a major driver of precancerous lesion formation, it is important to understand how checkpoint kinases stabilise the replisome.

It is however unclear what the role of these checkpoint kinases are at the replisome; how they interact with it and how they regulate its stability. During my thesis I optimised and used an in vivo interaction identification technique called biotin proximity labelling to elucidate novel interactors of the budding yeast checkpoint kinase Rad53 with the replisome. Using this technique, Pol1, a polymerase involved in DNA replication, was found to be a putative Rad53 interactor. Pol1 interacts with the FHA1 domain of Rad53 and abrogation of this interaction leads to loss of viability when cells are grown under replication stress. Furthermore, I demonstrate that this interaction is important for efficient replisome restart.

Altogether, work presented in this thesis furthers our understanding of the way checkpoint kinases interact and regulate the replisome during DRC activation.

Zegerman, Philip
BioID, Checkpoint kinases, DNA replication, Proximity Labelling, Rad53, Replication Stress, TurboID
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Medical Research Council (2496663)