title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol.
abstract: PrimPol is a primase-polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol-/- cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, BN1 9RQ, UK.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Keen
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, BN1 9RQ, UK.
authors.forename: Stanislaw K
authors.initials: SK
authors.lastname: Jozwiakowski
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, BN1 9RQ, UK.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Bailey
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, BN1 9RQ, UK.
authors.forename: Julie
authors.initials: J
authors.lastname: Bianchi
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, BN1 9RQ, UK ajd21@sussex.ac.uk.
authors.forename: Aidan J
authors.initials: AJ
authors.lastname: Doherty
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D019915:DNA Primase / Q000737:chemistry* / Q000502:physiology; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008345:Manganese / Q000737:chemistry; D064251:Multifunctional Enzymes / Q000737:chemistry* / Q000502:physiology; D011485:Protein Binding; D029867:Xenopus Proteins / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
chemical_list: D064251:Multifunctional Enzymes; D029867:Xenopus Proteins; D008345:Manganese; D019915:DNA Primase; C585901:PrimPol protein, human; D004259:DNA-Directed DNA Polymerase; D015032:Zinc