Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes

Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA–D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB–DNA pre-incision complex at a site of damage, we have labeled UvrB a...

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Main Authors:	Hughes, Craig D., Wang, Hong, Ghodke, Harshad, Simons, Michelle, Towheed, Atif, Peng, Ye, Van Houten, Bennett, Kad, Neil M.
Format:	Online
Language:	English
Published:	Oxford University Press 2013
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3643590/

id	pubmed-3643590
recordtype	oai_dc
spelling	pubmed-36435902013-05-03 Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes Hughes, Craig D. Wang, Hong Ghodke, Harshad Simons, Michelle Towheed, Atif Peng, Ye Van Houten, Bennett Kad, Neil M. Genome Integrity, Repair and Replication Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA–D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB–DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC–DNA complex, we investigated three UvrB mutants: Y96A, a β-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell. Oxford University Press 2013-05 2013-03-19 /pmc/articles/PMC3643590/ /pubmed/23511970 http://dx.doi.org/10.1093/nar/gkt177 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Hughes, Craig D. Wang, Hong Ghodke, Harshad Simons, Michelle Towheed, Atif Peng, Ye Van Houten, Bennett Kad, Neil M.
spellingShingle	Hughes, Craig D. Wang, Hong Ghodke, Harshad Simons, Michelle Towheed, Atif Peng, Ye Van Houten, Bennett Kad, Neil M. Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes
author_facet	Hughes, Craig D. Wang, Hong Ghodke, Harshad Simons, Michelle Towheed, Atif Peng, Ye Van Houten, Bennett Kad, Neil M.
author_sort	Hughes, Craig D.
title	Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes
title_short	Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes
title_full	Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes
title_fullStr	Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes
title_full_unstemmed	Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes
title_sort	real-time single-molecule imaging reveals a direct interaction between uvrc and uvrb on dna tightropes
description	Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA–D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB–DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC–DNA complex, we investigated three UvrB mutants: Y96A, a β-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell.
publisher	Oxford University Press
publishDate	2013
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3643590/
_version_	1611974991899787264

Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes

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