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Building better polymerases: Engineering the replication of expanded genetic alphabets

Ouaray, Zahra, Benner, Steven A., Georgiadis, Millie M. and Richards, Nigel G. J. 2020. Building better polymerases: Engineering the replication of expanded genetic alphabets. Journal of Biological Chemistry 295 (50) , pp. 17046-17059. 10.1074/jbc.REV120.013745

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DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications” needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

Item Type: Article
Date Type: Published Online
Status: Published
Schools: Chemistry
Additional Information: Attribution 4.0 International (CC BY 4.0)
Publisher: American Society for Biochemistry and Molecular Biology
ISSN: 0021-9258
Funders: BBSRC
Date of First Compliant Deposit: 4 January 2021
Date of Acceptance: 30 September 2020
Last Modified: 05 Jan 2021 10:15

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