| Summary: | <p>The human genome continually undergoes DNA damage that is endogenous and exogenous in origin. To protect the genome from this DNA damage, cells have evolved a plethora of signalling pathways, known as the DNA damage response (DDR). The poly(ADP-ribose) polymerase (PARP) enzyme family can regulate a variety of DDR pathways by modifying substrates with ADP-ribose, a type of chemical post-translational modification. However, our understanding of these pathways, and the factors involved in them, remains in their infancy. Chemical inhibitors of PARPs (PARPi) have emerged as key therapeutic tools to combat human diseases, thereby underlining the importance of understanding the pathways controlled by this enzyme family.</p>
<p>To investigate whether additional PARP enzymes might function in the absence of PARP1 and PARP2 in response to DNA damage, a genetic approach using a small-scale siRNA screen was taken. From this, I found that loss of PARP6 or PARP14 may rescue the PARPi-induced further sensitisation of PARP1/PARP2 knockout cells to methyl methanesulfonate (MMS). This suggests that PARP6 or PARP14 may be trapped to DNA by olaparib and supports the notion that either of these PARPs may participate in the repair of MMS-induced DNA damage in the absence of PARP1 and PARP2.</p>
<p>To reveal novel DDR proteins that are synthetic lethal with the combined loss of PARP1 and PARP2, a genome-wide CRISPR dropout screen was performed. From this, I identified the poorly characterised protein C16orf72 as being synthetic lethal with the combined loss of PARP1 and PARP2. Genetic deletion of C16orf72 sensitises cells to the DNA replication stress-inducing agents, hydroxyurea (HU) and aphidicolin (APH) indicating a function for C16orf72 in the DDR pathways that respond to DNA damage induced by these agents. Indeed, I find that C16orf72 is recruited to chromatin in response to exogenous replication stress, thereby localising it to damaged chromatin. In addition, I find that C16orf72 is required to limit replication catastrophe under conditions of prolonged replication stress. Together, these findings suggest that C16orf72 is a novel DDR factor that functions in response to DNA replication stress.</p>
<p>Collectively, my findings represent two major advances. Firstly, I identify PARP6 or PARP14 as novel PARP family members that may participate in the repair of MMS-induced DNA damage in the absence of PARP1 and PARP2. Secondly, I identify C16orf72 as a novel component of the DDR and show that it functions in response to DNA replication stress. Taken together, my findings expand our understanding of two important aspects of the DDR: ADP-ribosylation signalling and the DNA replication stress response.</p>
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