Browse by author
Lookup NU author(s): Tom Killelea, Professor Bernard Connolly
Full text for this publication is not currently held within this repository. Alternative links are provided below where available.
The family-B DNA polymerases obtained from the order Thermococcales, for example, Pyrococcus furiosus (Pfu-Pol) are commonly used in the polymerase chain reaction (PCR) because of their high thermostability and low error rates. Most of these polymerases contain four cysteines, arranged as two disulfide bridges. With Pfu-Pol C429-C443 forms one of the disulfides (DB1) and C507-C510 (DB2) the other. Although the disulfides are well conserved in the enzymes from the hyperthermophilic Thermococcales, they are less prevalent in euryarchaeal polymerases from other orders, and tend to be only found in other hyperthermophiles. Here, we report on the effects of deleting the disulfide bridges by mutating the relevant cysteines to serines. A variety of techniques, including differential scanning calorimetry and differential scanning fluorimetry, have shown that both disulfides make a contribution to thermostability, with DB1 being more important than DB2. However, even when both disulfides are removed, sufficient thermostability remains for normal (identical to the wild type) performance in PCR and quantitative (real-time) PCR. Therefore, polymerases totally lacking cysteine are fully compatible with most PCR-based applications. This observation opens the way to further engineering of polymerases by introduction of a single cysteine followed by appropriate chemical modification.
Author(s): Killelea T, Connolly BA
Publication type: Article
Publication status: Published
Journal: ChemBioChem
Year: 2011
Volume: 12
Issue: 9
Pages: 1330-1336
Print publication date: 20/05/2011
ISSN (print): 1439-4227
ISSN (electronic): 1439-7633
Publisher: Wiley - V C H Verlag GmbH & Co. KGaA
URL: http://dx.doi.org/10.1002/cbic.201100145
DOI: 10.1002/cbic.201100145
Altmetrics provided by Altmetric
