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Snapshots of a modified nucleotide moving through the confines of a DNA polymerase

DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucle...

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Autores principales: Kropp, Heike Maria, Dürr, Simon Leonard, Peter, Christine, Diederichs, Kay, Marx, Andreas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176618/
https://www.ncbi.nlm.nih.gov/pubmed/30224478
http://dx.doi.org/10.1073/pnas.1811518115
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author Kropp, Heike Maria
Dürr, Simon Leonard
Peter, Christine
Diederichs, Kay
Marx, Andreas
author_facet Kropp, Heike Maria
Dürr, Simon Leonard
Peter, Christine
Diederichs, Kay
Marx, Andreas
author_sort Kropp, Heike Maria
collection PubMed
description DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucleotide and postincorporation elongation to proceed with the synthesis of the nascent DNA strand. The structural basis for postincorporation elongation is currently unknown. We addressed this issue and successfully crystallized KlenTaq DNA polymerase in six closed ternary complexes containing the enzyme, the modified DNA substrate, and the incoming nucleotide. Each structure shows a high-resolution snapshot of the elongation of a modified primer, where the modification “moves” from the 3′-primer terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity significantly. The study highlights the plasticity of the system as origin of the broad substrate properties of DNA polymerases and facilitates the design of improved systems.
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spelling pubmed-61766182018-10-11 Snapshots of a modified nucleotide moving through the confines of a DNA polymerase Kropp, Heike Maria Dürr, Simon Leonard Peter, Christine Diederichs, Kay Marx, Andreas Proc Natl Acad Sci U S A Biological Sciences DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucleotide and postincorporation elongation to proceed with the synthesis of the nascent DNA strand. The structural basis for postincorporation elongation is currently unknown. We addressed this issue and successfully crystallized KlenTaq DNA polymerase in six closed ternary complexes containing the enzyme, the modified DNA substrate, and the incoming nucleotide. Each structure shows a high-resolution snapshot of the elongation of a modified primer, where the modification “moves” from the 3′-primer terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity significantly. The study highlights the plasticity of the system as origin of the broad substrate properties of DNA polymerases and facilitates the design of improved systems. National Academy of Sciences 2018-10-02 2018-09-17 /pmc/articles/PMC6176618/ /pubmed/30224478 http://dx.doi.org/10.1073/pnas.1811518115 Text en Copyright © 2018 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/ This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Kropp, Heike Maria
Dürr, Simon Leonard
Peter, Christine
Diederichs, Kay
Marx, Andreas
Snapshots of a modified nucleotide moving through the confines of a DNA polymerase
title Snapshots of a modified nucleotide moving through the confines of a DNA polymerase
title_full Snapshots of a modified nucleotide moving through the confines of a DNA polymerase
title_fullStr Snapshots of a modified nucleotide moving through the confines of a DNA polymerase
title_full_unstemmed Snapshots of a modified nucleotide moving through the confines of a DNA polymerase
title_short Snapshots of a modified nucleotide moving through the confines of a DNA polymerase
title_sort snapshots of a modified nucleotide moving through the confines of a dna polymerase
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176618/
https://www.ncbi.nlm.nih.gov/pubmed/30224478
http://dx.doi.org/10.1073/pnas.1811518115
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