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An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family

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posted on 2023-06-08, 18:08 authored by Stanislaw A Jozwiakowski, Brian J Keith, Louise Gilroy, Aidan DohertyAidan Doherty, Bernard A Connolly
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.

History

Publication status

  • Published

File Version

  • Published version

Journal

Nucleic Acids Research

ISSN

0305-1048

Publisher

Oxford University Press

Issue

15

Volume

42

Page range

9949-9963

Department affiliated with

  • Biochemistry Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2014-08-26

First Open Access (FOA) Date

2014-08-26

First Compliant Deposit (FCD) Date

2014-08-22

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