DNA-interacting proteins have roles in multiple processes, many operating as molecular machines which undergo dynamic meta-stable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner, fast enough to sample the molecular transitions across the free-energy landscape. Progress has been made over the past decade in utilizing cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection signal-to-noise of molecular signatures and transition events. In the present paper, we discuss a range of techniques we have developed to monitor DNA–protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our laboratory made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new super-resolution fluorescence microscope.
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April 2015
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Conference Article|
April 07 2015
Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer
Adam J. M. Wollman;
Adam J. M. Wollman
*Biological Physical Sciences Institute (BPSI), Departments of Physics and Biology, University of York, York YO10 5DD, U.K.
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Helen Miller;
Helen Miller
*Biological Physical Sciences Institute (BPSI), Departments of Physics and Biology, University of York, York YO10 5DD, U.K.
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Zhaokun Zhou;
Zhaokun Zhou
*Biological Physical Sciences Institute (BPSI), Departments of Physics and Biology, University of York, York YO10 5DD, U.K.
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Mark C. Leake
Mark C. Leake
1
*Biological Physical Sciences Institute (BPSI), Departments of Physics and Biology, University of York, York YO10 5DD, U.K.
1To whom correspondence should be addressed (emailmark.leake@york.ac.uk).
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Publisher: Portland Press Ltd
Received:
September 19 2014
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© The Authors Journal compilation © 2015 Biochemical Society
2015
Biochem Soc Trans (2015) 43 (2): 139–145.
Article history
Received:
September 19 2014
Citation
Adam J. M. Wollman, Helen Miller, Zhaokun Zhou, Mark C. Leake; Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer. Biochem Soc Trans 1 April 2015; 43 (2): 139–145. doi: https://doi.org/10.1042/BST20140253
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