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Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase

X-ray crystallography provides excellent structural data on protein–DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein–DNA interactions even in extensively s...

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Autores principales: Roberts, Victoria A., Pique, Michael E., Hsu, Simon, Li, Sheng, Slupphaug, Geir, Rambo, Robert P., Jamison, Jonathan W., Liu, Tong, Lee, Jun H., Tainer, John A., Ten Eyck, Lynn F., Woods, Virgil L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401472/
https://www.ncbi.nlm.nih.gov/pubmed/22492624
http://dx.doi.org/10.1093/nar/gks291
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author Roberts, Victoria A.
Pique, Michael E.
Hsu, Simon
Li, Sheng
Slupphaug, Geir
Rambo, Robert P.
Jamison, Jonathan W.
Liu, Tong
Lee, Jun H.
Tainer, John A.
Ten Eyck, Lynn F.
Woods, Virgil L.
author_facet Roberts, Victoria A.
Pique, Michael E.
Hsu, Simon
Li, Sheng
Slupphaug, Geir
Rambo, Robert P.
Jamison, Jonathan W.
Liu, Tong
Lee, Jun H.
Tainer, John A.
Ten Eyck, Lynn F.
Woods, Virgil L.
author_sort Roberts, Victoria A.
collection PubMed
description X-ray crystallography provides excellent structural data on protein–DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein–DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein–DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG–DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210–220 and 251–264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG–DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.
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spelling pubmed-34014722012-07-23 Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase Roberts, Victoria A. Pique, Michael E. Hsu, Simon Li, Sheng Slupphaug, Geir Rambo, Robert P. Jamison, Jonathan W. Liu, Tong Lee, Jun H. Tainer, John A. Ten Eyck, Lynn F. Woods, Virgil L. Nucleic Acids Res Genome Integrity, Repair and Replication X-ray crystallography provides excellent structural data on protein–DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein–DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein–DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG–DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210–220 and 251–264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG–DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway. Oxford University Press 2012-07 2012-04-06 /pmc/articles/PMC3401472/ /pubmed/22492624 http://dx.doi.org/10.1093/nar/gks291 Text en © The Author(s) 2012. 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.
spellingShingle Genome Integrity, Repair and Replication
Roberts, Victoria A.
Pique, Michael E.
Hsu, Simon
Li, Sheng
Slupphaug, Geir
Rambo, Robert P.
Jamison, Jonathan W.
Liu, Tong
Lee, Jun H.
Tainer, John A.
Ten Eyck, Lynn F.
Woods, Virgil L.
Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase
title Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase
title_full Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase
title_fullStr Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase
title_full_unstemmed Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase
title_short Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase
title_sort combining h/d exchange mass spectroscopy and computational docking reveals extended dna-binding surface on uracil-dna glycosylase
topic Genome Integrity, Repair and Replication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401472/
https://www.ncbi.nlm.nih.gov/pubmed/22492624
http://dx.doi.org/10.1093/nar/gks291
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