title: An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family.
abstract: A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol ζ (a family-B translesion synthesis polymerase). Inactivation of the 3'-5' proof-reading exonuclease activity is also necessary. The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers. Tgo-Pol Z1 is also proficient at extending from primers that terminate opposite aberrant bases. The fidelity of Tgo-Pol Z1 is reduced, with a marked tendency to make changes at G:C base pairs. Together, these results suggest that the loop region of the fingers domain may play a critical role in determining whether a family-B enzyme falls into the accurate genome-replicating category or is an error-prone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA.
authors: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK Institute of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK s.k.jozwiakowski@sussex.ac.uk.
authors: Stanislaw K
authors: SK
authors: Jozwiakowski
authors: Institute of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK.
authors: Brian J
authors: BJ
authors: Keith
authors: Institute of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK.
authors: Louise
authors: L
authors: Gilroy
authors: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK.
authors: Aidan J
authors: AJ
authors: Doherty
authors: Institute of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK.
authors: Bernard A
authors: BA
authors: Connolly
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020137:Base Pair Mismatch; D004249:DNA Damage; D004257:DNA Polymerase II / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D009154:Mutation; D011740:Pyrimidine Dimers; D013698:Templates, Genetic; D019712:Thermococcus / Q000201:enzymology
chemical_list: D019843:Archaeal Proteins; D017931:DNA Primers; D011740:Pyrimidine Dimers; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase