The interaction of T4 endonuclease V E23Q mutant with thymine dimer- and tetrahydrofuran-containing DNA

Abstract

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...

Chemistry of Glycosylases and Endonucleases Involved in Base-Excision Repair

“…The participation of Glu 23 as a general base in catalyzing the AP-lyase activity is also consistent with the pH optimum of 5.5 for the lyase catalysis . Glu 23 has also been shown to be necessary for the subsequent δ-elimination reactions catalyzed by T4-endo V. The replacement of Glu 23 with other amino acids does not adversely affect specific DNA binding, further supporting its general role in catalysis rather than damage recognition …”

“…248 Glu 23 has also been shown to be necessary for the subsequent δ-elimination reactions catalyzed by T4-endo V. The replacement of Glu 23 with other amino acids does not adversely affect specific DNA binding, further supporting its general role in catalysis rather than damage recognition. 249 In the cocrystal structure of the E23Q mutant T4endo V bound to a pyrimidine dimer-containing duplex, 216 the R-amino group lies approximately 3.8 Å from the C1′ atom of the 5′-deoxyribose of the thymine dimer making it capable of participating as the nucleophile. The amide of Gln 23 makes three polar contacts with the base and sugar of the 5′-T of the T]T dimer, suggesting that the corresponding carboxyl group (Glu 23) of the native enzyme is positioned favorably for participation in the glycosylase activity.…”

Uncoupling of Nucleotide Flipping and DNA Bending by the T4 Pyrimidine Dimer DNA Glycosylase

“…Additionally, the footprint of T4-Pdg is estimated to be 11 bases, which argues against multiple enzyme molecules binding to the same oligonucleotide substrate (48). Therefore, the flipping and bending signals are measuring different intermediates in dissociation of a common complex and the 2.5-fold difference in off rates for flipping and bending indicates that flipping and bending are not simultaneous events.…”

Structure of T4 Pyrimidine Dimer Glycosylase in a Reduced Imine Covalent Complex with Abasic Site-containing DNA

“…A mutation of Glu23 to glutamine (E23Q) completely abolishes the catalytic activity of T4-Pdg 36,37 and changes its mode of DNA binding, as shown by footprinting experiments with an E23Q mutant. 42 The results of mutation of E23 to an aspartic acid residue differ, with one laboratory showing no activity 36 while another group reported both CPD and AP site DNA nicking. 37 A similar role for a catalytic carboxylic acid residue was found in DNA glycosylases/AP-lyases that belong to other structural families, such as Glu2 in E. coli Fpg and Nei proteins [43][44][45][46] and Asp139 in Bacillus stearothermophilus Nth.…”