Lee, A.J. orcid.org/0000-0003-2268-4645, Endo, M., Hobbs, J.K. orcid.org/0000-0002-5872-1404 et al. (1 more author) (2017) Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search. ACS Nano, 12 (1). pp. 272-278. ISSN 1936-0851
Abstract
Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. |
Keywords: | homologous recombination; DNA repair; RecA; recombinase A; high-speed AFM; DNA origami |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > Department of Physics and Astronomy (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 07 Feb 2018 16:32 |
Last Modified: | 06 Nov 2018 13:47 |
Published Version: | https://doi.org/10.1021/acsnano.7b06208 |
Status: | Published |
Publisher: | American Chemical Society |
Refereed: | Yes |
Identification Number: | 10.1021/acsnano.7b06208 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:127152 |