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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2021
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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. |
format | Online Article Text |
id | pubmed-7952088 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
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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