Details

Autor(en) / Beteiligte

• 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, Jr, Virgil L

Titel

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

Ist Teil von

• Nucleic acids research, 2012-07, Vol.40 (13), p.6070-6081

Ort / Verlag

England

Erscheinungsjahr

2012

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.