Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking.
Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate de...
Main Authors: | , , |
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Format: | Journal article |
Language: | English |
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2014
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_version_ | 1797059831312416768 |
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author | Uphoff, S Sherratt, D Kapanidis, A |
author_facet | Uphoff, S Sherratt, D Kapanidis, A |
author_sort | Uphoff, S |
collection | OXFORD |
description | Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures. |
first_indexed | 2024-03-06T20:09:42Z |
format | Journal article |
id | oxford-uuid:2a24e103-abb1-41c5-8bd1-9639729c8cc9 |
institution | University of Oxford |
language | English |
last_indexed | 2024-03-06T20:09:42Z |
publishDate | 2014 |
record_format | dspace |
spelling | oxford-uuid:2a24e103-abb1-41c5-8bd1-9639729c8cc92022-03-26T12:23:17ZVisualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:2a24e103-abb1-41c5-8bd1-9639729c8cc9EnglishSymplectic Elements at Oxford2014Uphoff, SSherratt, DKapanidis, AProtein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures. |
spellingShingle | Uphoff, S Sherratt, D Kapanidis, A Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking. |
title | Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking. |
title_full | Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking. |
title_fullStr | Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking. |
title_full_unstemmed | Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking. |
title_short | Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking. |
title_sort | visualizing protein dna interactions in live bacterial cells using photoactivated single molecule tracking |
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