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The structure of Escherichia coli ExoIX--implications for DNA binding and catalysis in flap endonucleases

DOI: 10.1093/nar/gkt591 DOI Help
PMID: 23821668 PMID Help

Authors: Christopher S. Anstey-gilbert , Glyn R. Hemsworth (University of Leeds) , Claudia S. Flemming (University of Sheffield) , Michael R. G. Hodskinson (University of SheUniversity of Sheffield Medical Schoolffield) , Jing Zhang (University of Sheffield Medical School) , Svetlana E. Sedelnikova (The University of Sheffield) , Timothy J. Stillman (University of Sheffield) , Jon R. Sayers (University of Sheffield Medical School) , Peter J. Artymiuk (University of Sheffield)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nucleic Acids Research

State: Published (Approved)
Published: June 2013
Diamond Proposal Number(s): 300 , 1218

Open Access Open Access

Abstract: Escherichia coli Exonuclease IX (ExoIX), encoded by the xni gene, was the first identified member of a novel subfamily of ubiquitous flap endonucleases (FENs), which possess only one of the two catalytic metal-binding sites characteristic of other FENs. We have solved the first structure of one of these enzymes, that of ExoIX itself, at high resolution in DNA-bound and DNA-free forms. In the enzyme–DNA cocrystal, the single catalytic site binds two magnesium ions. The structures also reveal a binding site in the C-terminal domain where a potassium ion is directly coordinated by five main chain carbonyl groups, and we show this site is essential for DNA binding. This site resembles structurally and functionally the potassium sites in the human FEN1 and exonuclease 1 enzymes. Fluorescence anisotropy measurements and the crystal structures of the ExoIX:DNA complexes show that this potassium ion interacts directly with a phosphate diester in the substrate DNA.

Journal Keywords: Calcium; DNA; Exodeoxyribonucleases; Flap; Humans; Magnesium; Models; Molecular; Phosphoric; Potassium

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography

Other Facilities: PX14.1 at the former Daresbury Synchrotron