USP9X controls DNA replication fork stability and the replication stress checkpoint through Claspin

Cells possess checkpoint pathways which are important for maintaining genome stability and preventing cancer. These signalling pathways include the ATR and CHK1 kinases which are activated in response to DNA damage or replication stress. Activation of CHK1 by ATR requires the mediator protein Claspin. Claspin also regulates the rate of fork progression during DNA replication. The levels of Claspin are cell cycle regulated and importantly Claspin is stabilised during S-phase. A growing number of ubiquitin ligases and deubiquitylating enzymes (DUBs) have been shown to affect Claspin stability, however how they cooperate in regulating Claspin levels remains unclear. In this thesis, I have characterised an affinity tagged overexpression system allowing for the induction of Claspin. I extensively optimised a purification protocol for this protein and using, SILAC, a quantitative proteomic approach, I identified several proteins that differentially co-purify with Claspin compared to control cells. Among these, I found a novel DUB, USP9X. I have confirmed by reciprocal immunoprecipitation experiments that it binds to Claspin. Using siRNA depletion and pharmacological inhibition strategies, I found that USP9X controls Claspin stability in an S-phase specific manner. I have also demonstrated that USP9X protects proteasome-dependent Claspin degradation. Using a DNA fiber spreading approach I have established that USP9X contributes to DNA replication fork stability. I have also established that USP9X contributes to the intra S-phase replication checkpoint response and the ability of a replication fork to recover following replication stress. Further still I have demonstrated that the depletion of USP9X leads to the spontaneous accumulation of DNA damage; however this damage can be significantly rescued with the simultaneous overexpression of Claspin. Therefore I propose that USP9X, through its role in the regulation of Claspin, is a novel player involved in the maintenance of genomic stability and in the DNA replication stress response pathway.2020-02-0