Jozwiakowski, Stanislaw A, Keith, Brian J, Gilroy, Louise, Doherty, Aidan J and Connolly, Bernard A (2014) An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family. Nucleic Acids Research, 42 (15). pp. 9949-9963. ISSN 0305-1048

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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 zeta (a family-B translesion synthesis polymerase). Inactivation of the 3'–5' proofreading 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 amarked tendency tomake 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 errorprone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA.

Item Type:	Article
Schools and Departments:	School of Life Sciences > Biochemistry
Subjects:	Q Science Q Science > Q Science (General) > Q0300 Cybernetics Q Science > Q Science (General)
Depositing User:	Stanislaw Jozwiakowski
Date Deposited:	26 Aug 2014 13:43
Last Modified:	01 Apr 2017 09:18
URI:	http://srodev.sussex.ac.uk/id/eprint/49623

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