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

Academic Article
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Overview

authors

  • Roberts, V. A.
  • Pique, M. E.
  • Hsu, S.
  • Li, S.
  • Slupphaug, G.
  • Rambo, R. P.
  • Jamison, J. W.
  • Liu, T.
  • Lee, J. H.
  • Tainer, John
  • Ten Eyck, L. F.
  • Woods, V. L.

publication date

  • July 2012

journal

  • Nucleic Acids Research  Journal

abstract

  • 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.

subject areas

  • Algorithms
  • Catalytic Domain
  • Computational Biology
  • Crystallography, X-Ray
  • DNA
  • DNA-Binding Proteins
  • Deuterium Exchange Measurement
  • Mass Spectrometry
  • Models, Molecular
  • Uracil-DNA Glycosidase
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Identity

PubMed Central ID

  • PMC3401472

International Standard Serial Number (ISSN)

  • 0305-1048

Digital Object Identifier (DOI)

  • 10.1093/nar/gks291

PubMed ID

  • 22492624
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Additional Document Info

start page

  • 6070

end page

  • 6081

volume

  • 40

issue

  • 13

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