Building better polymerases: Engineering the replication of expanded genetic alphabets |
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Authors: | Zahra Ouaray Steven A. Benner Millie M. Georgiadis Nigel G. J. Richards |
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Affiliation: | 1.School of Chemistry, Cardiff University, Park Place, Cardiff, United Kingdom;2.Foundation for Applied Molecular Evolution, Alachua, Florida, USA;3.Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA |
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Abstract: | 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. |
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Keywords: | biotechnology computer modeling directed evolution DNA polymerase enzyme mechanism enzyme structure molecular dynamics nucleoside/nucleotide analogue protein-DNA interaction synthetic biology X-ray crystallography expanded genetic alphabets |
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