Publication Date:
2014-09-02
Description:
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 (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.
Print ISSN:
0305-1048
Electronic ISSN:
1362-4962
Topics:
Biology
