The interaction between endonuclease V, the cyclobutane pyrimidine dimer-specific N-glycosylase/abasic lyase from bacteriophage T4, and DNA was investigated by DNase I footprinting methods. The catalytically inactive mutant E23Q was found to interact with a smaller region of DNA at the abasic site analog, tetrahydrofuran, than at a thymine dimer site. Like the wild-type enzyme, the mutant contacted the DNA substrates primarily on the strand opposite the damage. The various complexes examined by footprinting techniques represent distinct points along the catalytic pathway of endonuclease V: before catalysis at a dimer, after N-glycosylase action but before abasic lyase action, and before catalysis at an abasic site. The differences between the footprints of the mutant and wild-type enzymes on both DNA substrates likely represent subtly different conformations within these complexes.Endonuclease V is a 16-kDa N-glycosylase/ abasic (AP) lyase from bacteriophage T4 specific for UV-induced cyclobutane pyrimidine dimers. Mechanistic studies have demonstrated that the N-terminal (Thr-2) ␣NH 2 group of the enzyme initiates the glycosylase action by forming an imino intermediate with the C-1Ј of the 5Ј sugar within the pyrimidine dimer (3,14,15). Phosphodiester bond scission occurs by a -elimination mechanism to produce a cleaved site with 5Ј phosphate and 3Ј ␣,-unsaturated aldehyde termini (2,6,8). In addition to the catalytic amino group at the N terminus, the acidic residue Glu-23 is essential for both activities (4, 5, 9). The structure of endonuclease V has been determined to a 1.6-Å (1 Å ϭ 0.1 nm) resolution by X-ray crystallography (12), but little is known about the overall conformation of the protein-DNA complex. In a previous study, DNase I, 1,10-phenanthrolinecopper, and methylation protection assays were used to determine the contacts made between wild-type endonuclease V and a thymine dimer-containing oligonucleotide. To perform these experiments, the imino intermediate was reduced by the addition of NaBH 4 , which results in a dead-end covalent complex (7). The results of the DNase I footprinting of this complex demonstrated that the enzyme interacted with the substrate primarily on the strand opposite the thymine dimer and in a very asymmetric manner. To extend our knowledge about endonuclease V-DNA interactions, in the current study we describe DNase I protection assays using the catalytically inactive endonuclease V mutant, E23Q, bound to both thymine dimer-containing and tetrahydrofuran (THF [an AP site analog])-containing DNA. With the protection patterns displayed by wild-type enzyme covalently trapped to dimer-containing DNA (7), these results allow us to compare the protein-DNA interactions of endonuclease V at different points along its catalytic pathway: before catalysis (E23Q-dimer-containing DNA complex), after the N-glycosylase but before AP lyase action (wild-type enzyme-dimer-containing DNA covalent complex), and at an abasic site (wild-type enzyme-and E23Q-THFcontaining DNA complexes).
DNa...