ABSTRACT
Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9-2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism.
Subject(s)
DNA, Single-Stranded/chemistry , DNA, Viral/chemistry , Exodeoxyribonucleases/chemistry , Oligonucleotides/chemistry , Siphoviridae/chemistry , Viral Proteins/chemistry , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Gene Expression , Hydrogen Bonding , Models, Molecular , Mutagenesis, Site-Directed , Oligonucleotides/metabolism , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Siphoviridae/enzymology , Structure-Activity Relationship , Substrate Specificity , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
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.
