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

Anstey-Gilbert, Christopher S., Hemsworth, Glyn R., Flemming, Claudia S., Hodskinson, Michael R., Zhang, Jing, Sedelnikova, Svetlana E., Stillman, Timothy J., Sayers, Jon R., and Artymiuk, Peter J. (2013) The structure of Escherichia coli ExoIX: implications for DNA binding and catalysis in flap endonucleases. Nucleic Acids Research, 41 (17). pp. 8357-8367.

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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.

Item ID: 34848
Item Type: Article (Research - C1)
ISSN: 1362-4962
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© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Funders: Biotechnology and Biological Sciences Research Council (BBSCR), University of Sheffield, University Publications Fund
Projects and Grants: BBSRC grant 50/D16001, BBSCR grant B19466
Date Deposited: 20 Aug 2014 23:21
FoR Codes: 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060112 Structural Biology (incl Macromolecular Modelling) @ 100%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
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