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Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion

The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chai...

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Autores principales: Hohlbein, Johannes, Aigrain, Louise, Craggs, Timothy D., Bermek, Oya, Potapova, Olga, Shoolizadeh, Pouya, Grindley, Nigel D. F., Joyce, Catherine M., Kapanidis, Achillefs N.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Pub. Group 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3715850/
https://www.ncbi.nlm.nih.gov/pubmed/23831915
http://dx.doi.org/10.1038/ncomms3131
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author Hohlbein, Johannes
Aigrain, Louise
Craggs, Timothy D.
Bermek, Oya
Potapova, Olga
Shoolizadeh, Pouya
Grindley, Nigel D. F.
Joyce, Catherine M.
Kapanidis, Achillefs N.
author_facet Hohlbein, Johannes
Aigrain, Louise
Craggs, Timothy D.
Bermek, Oya
Potapova, Olga
Shoolizadeh, Pouya
Grindley, Nigel D. F.
Joyce, Catherine M.
Kapanidis, Achillefs N.
author_sort Hohlbein, Johannes
collection PubMed
description The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.
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spelling pubmed-37158502013-07-19 Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion Hohlbein, Johannes Aigrain, Louise Craggs, Timothy D. Bermek, Oya Potapova, Olga Shoolizadeh, Pouya Grindley, Nigel D. F. Joyce, Catherine M. Kapanidis, Achillefs N. Nat Commun Article The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection. Nature Pub. Group 2013-07-08 /pmc/articles/PMC3715850/ /pubmed/23831915 http://dx.doi.org/10.1038/ncomms3131 Text en Copyright © 2013, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. http://creativecommons.org/licenses/by-nc-sa/3.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
spellingShingle Article
Hohlbein, Johannes
Aigrain, Louise
Craggs, Timothy D.
Bermek, Oya
Potapova, Olga
Shoolizadeh, Pouya
Grindley, Nigel D. F.
Joyce, Catherine M.
Kapanidis, Achillefs N.
Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion
title Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion
title_full Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion
title_fullStr Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion
title_full_unstemmed Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion
title_short Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion
title_sort conformational landscapes of dna polymerase i and mutator derivatives establish fidelity checkpoints for nucleotide insertion
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3715850/
https://www.ncbi.nlm.nih.gov/pubmed/23831915
http://dx.doi.org/10.1038/ncomms3131
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