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Single-molecule view of coordination in a multi-functional DNA polymerase

Replication and repair of genomic DNA requires the actions of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here, we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA poly...

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Autores principales: Pauszek, Raymond F, Lamichhane, Rajan, Rajkarnikar Singh, Arishma, Millar, David P
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952088/
https://www.ncbi.nlm.nih.gov/pubmed/33704066
http://dx.doi.org/10.7554/eLife.62046
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author Pauszek, Raymond F
Lamichhane, Rajan
Rajkarnikar Singh, Arishma
Millar, David P
author_facet Pauszek, Raymond F
Lamichhane, Rajan
Rajkarnikar Singh, Arishma
Millar, David P
author_sort Pauszek, Raymond F
collection PubMed
description Replication and repair of genomic DNA requires the actions of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here, we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA polymerase I (Pol I) from Escherichia coli, a key enzyme involved in lagging-strand replication and base excision repair. Pol I contains active sites for template-directed DNA polymerization and 5’ flap processing in separate domains. We show that a DNA substrate can spontaneously transfer between polymerase and 5’ nuclease domains during a single encounter with Pol I. Additionally, we show that the flexibly tethered 5’ nuclease domain adopts different positions within Pol I-DNA complexes, depending on the nature of the DNA substrate. Our results reveal the structural dynamics that underlie functional coordination in Pol I and are likely relevant to other multi-functional DNA polymerases.
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spelling pubmed-79520882021-03-12 Single-molecule view of coordination in a multi-functional DNA polymerase Pauszek, Raymond F Lamichhane, Rajan Rajkarnikar Singh, Arishma Millar, David P eLife Structural Biology and Molecular Biophysics Replication and repair of genomic DNA requires the actions of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here, we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA polymerase I (Pol I) from Escherichia coli, a key enzyme involved in lagging-strand replication and base excision repair. Pol I contains active sites for template-directed DNA polymerization and 5’ flap processing in separate domains. We show that a DNA substrate can spontaneously transfer between polymerase and 5’ nuclease domains during a single encounter with Pol I. Additionally, we show that the flexibly tethered 5’ nuclease domain adopts different positions within Pol I-DNA complexes, depending on the nature of the DNA substrate. Our results reveal the structural dynamics that underlie functional coordination in Pol I and are likely relevant to other multi-functional DNA polymerases. eLife Sciences Publications, Ltd 2021-03-11 /pmc/articles/PMC7952088/ /pubmed/33704066 http://dx.doi.org/10.7554/eLife.62046 Text en © 2021, Pauszek et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Structural Biology and Molecular Biophysics
Pauszek, Raymond F
Lamichhane, Rajan
Rajkarnikar Singh, Arishma
Millar, David P
Single-molecule view of coordination in a multi-functional DNA polymerase
title Single-molecule view of coordination in a multi-functional DNA polymerase
title_full Single-molecule view of coordination in a multi-functional DNA polymerase
title_fullStr Single-molecule view of coordination in a multi-functional DNA polymerase
title_full_unstemmed Single-molecule view of coordination in a multi-functional DNA polymerase
title_short Single-molecule view of coordination in a multi-functional DNA polymerase
title_sort single-molecule view of coordination in a multi-functional dna polymerase
topic Structural Biology and Molecular Biophysics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952088/
https://www.ncbi.nlm.nih.gov/pubmed/33704066
http://dx.doi.org/10.7554/eLife.62046
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