Identification and characterisation of post-translational modifications that regulate human PrimPol

DNA replication is hindered by lesions and obstacles arising both endogenously and exogenously, which stall the replication machinery, leading to DNA replication fork stalling or collapse. DNA damage tolerance (DDT) pathways allow the replisome to bypass impediments without impairing replication. One such DDT pathway involves repriming DNA synthesis and is mediated by Primase- Polymerase (PrimPol) in human cells. How this pathway is regulated and deployed during the cell cycle or following damage is poorly understood.

This thesis investigates the regulation of PrimPol by post-translational modifications. We establish that PrimPol is phosphorylated at key amino acid residues in PrimPol’s C-terminus by cell-cycle kinases. Chapter 3 investigates the role of serine 538 phosphorylation. We establish that this modification is performed by Polo-like kinase 1, with increasing phosphorylation occurring in G2 and mitosis and its delay or reversal in response to replication stress. When this residue was mutated to prevent phosphorylation, cells exhibited increased sensitivity to genotoxic agents, aberrant recruitment of PrimPol to chromatin, and increased genomic instability.

Chapter 4 investigates serine 499 phosphorylation, establishing that this modification is performed by cyclin-dependent kinase 1 (CDK1) and regulated during the cell cycle. S499 phosphorylated and unphosphorylated PrimPol is maintained across G1 and S phase and prevention of this modification induces sensitivity to UV-C damage and replication stress induced by aphidicolin, camptothecin and olaparib.

Finally, Chapter 5 describes the generation of a cell line, expressing endogenous levels of PrimPol tagged with a fluorescent label, as a tool for enabling a better understanding of the localisation and recruitment of PrimPol protein.

Together, these studies establish the critical role of post-translational modifications in the regulation of PrimPol’s activities and recruitment during the cell cycle and in response to DNA damage. This study defines important regulatory pathways and reveals the deleterious consequences that deregulated repriming has on cell survival and genome stability.