Cellular and biochemical characterisation of PrimPol, a novel eukaryotic primase-polymerase involved in DNA damage tolerance

Genome stability is of upmost importance to life. DNA polymerases are essential for the duplication and maintenance of the genome but they cannot themselves begin synthesis of a DNA chain, and require the activity of specialised RNA polymerases called primases. In eukaryotic cells distinct enzymes catalyse these two essential processes. This thesis contains the characterisation of coiled-coil domain containing protein (CCDC)111, a previously uncharacterised protein conserved in a broad range of unicellular and multicellular eukaryotes including humans. CCDC111 is a member of the archaeaoeukaroytic primase (AEP) superfamily and uniquely for a eukaryotic enzyme possesses both primase and polymerase activities, and was thus renamed PrimPol. The work in this thesis implicates PrimPol in the process of DNA damage tolerance, a universal mechanism by which cells complete genome duplication in spite of potentially lethal DNA damage. The first results chapters detail the essential role of a PrimPol homologue (TbPrimPol2) in the important protozoan pathogen Trypanosoma brucei. A combination of molecular, cell biology, and biochemical analyses indicate a role for TbPrimPol2 in the post-replication tolerance of endogenously occurring DNA damage using its trans-lesion DNA synthesis activity. The remaining results chapters characterise PrimPol in human cultured cells, and demonstrate that this enzyme is present in both the nucleus and mitochondria. In the nucleus PrimPol functions in the cellular tolerance of ultraviolet (UV)- induced DNA damage, and is required to protect xeroderma pigmentosum variant (XP-V) cells, deficient in the UV lesion bypass polymerase Pol !, from the cytotoxic affects of UV radiation. Together, this thesis establishes the involvement of PrimPol in DNA damage tolerance from one of the earliest diverging eukaryotic organisms to man