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.
Highlights
All cells require 50-nuclease or flap endonuclease (FEN) activity for DNA replication and repair processes [1]
The structure of Exonuclease IX (ExoIX) reveals a characteristic FEN fold with a central six-stranded mixed b-sheet surrounded by 10 a-helices, all linked by loops (Figure 1A and B)
In superpositions with the recent hFEN1 and human exonuclease 1 (hExo1) complex structures [9,10], the upper part of the loop clearly blocks the hole formed by the helical gateway
Summary
All cells require 50-nuclease or flap endonuclease (FEN) activity for DNA replication and repair processes [1]. FEN activity is involved in the removal of RNA from Okazaki fragments, which are formed on the lagging-strand during semi-discontinuous DNA replication. The b-sheet carries many of the conserved acidic residues, which cluster at the centre of the enzyme forming the Cat and Cat metal-binding sites responsible for the catalytic activity of these enzymes (Supplementary Figure S1A) [11]. These sites coordinate two divalent metal ions, which in prokaryotes have been observed to be separated by a distance of $8 A. Kinetic analyses of a mutant engineered so as to lack two conserved aspartate groups in Cat suggests that this site is involved in substrate binding rather than chemical catalysis [14]
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