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Building better polymerases: Engineering the replication of expanded genetic alphabets

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 transf...

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Detalles Bibliográficos
Autores principales: Ouaray, Zahra, Benner, Steven A., Georgiadis, Millie M., Richards, Nigel G. J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863901/
https://www.ncbi.nlm.nih.gov/pubmed/33004440
http://dx.doi.org/10.1074/jbc.REV120.013745
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author Ouaray, Zahra
Benner, Steven A.
Georgiadis, Millie M.
Richards, Nigel G. J.
author_facet Ouaray, Zahra
Benner, Steven A.
Georgiadis, Millie M.
Richards, Nigel G. J.
author_sort Ouaray, Zahra
collection PubMed
description 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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spelling pubmed-78639012021-03-23 Building better polymerases: Engineering the replication of expanded genetic alphabets Ouaray, Zahra Benner, Steven A. Georgiadis, Millie M. Richards, Nigel G. J. J Biol Chem JBC Reviews 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. American Society for Biochemistry and Molecular Biology 2020-12-11 2020-10-01 /pmc/articles/PMC7863901/ /pubmed/33004440 http://dx.doi.org/10.1074/jbc.REV120.013745 Text en © 2020 Ouaray et al. Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0) .
spellingShingle JBC Reviews
Ouaray, Zahra
Benner, Steven A.
Georgiadis, Millie M.
Richards, Nigel G. J.
Building better polymerases: Engineering the replication of expanded genetic alphabets
title Building better polymerases: Engineering the replication of expanded genetic alphabets
title_full Building better polymerases: Engineering the replication of expanded genetic alphabets
title_fullStr Building better polymerases: Engineering the replication of expanded genetic alphabets
title_full_unstemmed Building better polymerases: Engineering the replication of expanded genetic alphabets
title_short Building better polymerases: Engineering the replication of expanded genetic alphabets
title_sort building better polymerases: engineering the replication of expanded genetic alphabets
topic JBC Reviews
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863901/
https://www.ncbi.nlm.nih.gov/pubmed/33004440
http://dx.doi.org/10.1074/jbc.REV120.013745
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