An investigation into the role, recruitment, and regulation of PrimPol in DNA replication restart

DNA replication is one of life's fundamental processes. This delicate task is performed by a complex of molecular machines, known collectively as the replisome. At the heart of the replisome lie the replicative DNA polymerases which catalyse synthesis of daughter DNA strands with astonishing accuracy and efficiency. Nevertheless, these enzymes are prone to stalling upon encountering DNA damage lesions and secondary structures. In order to restart replication, DNA damage tolerance mechanisms are required. This published article-format thesis focusses on a recently discovered primase-polymerase, and member of the archaeo-eukaryotic primase (AEP) superfamily, involved in DNA damage tolerance, known as PrimPol. The work presented here builds on the initial characterisation of the enzyme, which identified potential roles in the bypass of DNA damage through translesion synthesis (TLS) and repriming of replication. The first presented article consists of a review of the AEP superfamily, functionally repositioning the group under the category of primase-polymerases. In the second paper, evidence is presented to suggest that PrimPol's activity is regulated by single-strand binding proteins, required due to the enzyme's error-prone nature. In the third chapter, a novel PrimPol binding protein, polymerase delta-interacting protein 2 (PolDIP2), is identified and characterised as a stimulatory factor for PrimPol's primer extension activities. Chapter 4 focusses on the development and use of a gel-based fluorescent primase assay to assess PrimPol's ability to reprime downstream of DNA damage lesions and secondary structures. The fifth presented paper identifies the molecular basis for PrimPol's interaction with replication protein A (RPA). Using biophysical, biochemical, and cellular approaches, this paper identifies the mechanism by which PrimPol is recruited to reprime replication. Lastly, in Chapter 6, a review of the presented articles in the context of the wider literature is included. Together, this work supports a role for PrimPol in repriming and restarting DNA replication following stalling at impediments, as well as identifying mechanisms involved in the recruitment and regulation of the enzyme.