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...

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Main Authors: Uphoff, S, Sherratt, D, Kapanidis, A
Format: Journal article
Language:English
Published: 2014
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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.
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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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AT sherrattd visualizingproteindnainteractionsinlivebacterialcellsusingphotoactivatedsinglemoleculetracking
AT kapanidisa visualizingproteindnainteractionsinlivebacterialcellsusingphotoactivatedsinglemoleculetracking