The Role of Active-Site Residues Phe98, His239, and Arg243 in DNA Binding and in the Catalysis of Human Uracil–DNA Glycosylase SMUG1

Abstract

Human SMUG1 (hSMUG1) hydrolyzes the N-glycosidic bond of uracil and some uracil lesions formed in the course of epigenetic regulation. Despite the functional importance of hSMUG1 in the DNA repair pathway, the damage recognition mechanism has been elusive to date. In the present study, our objective was to build a model structure of the enzyme–DNA complex of wild-type hSMUG1 and several hSMUG1 mutants containing substitution F98W, H239A, or R243A. Enzymatic activity of these mutant enzymes was examined by polyacrylamide gel electrophoresis analysis of the reaction product formation and pre-steady-state analysis of DNA conformational changes during enzyme–DNA complex formation. It was shown that substitutions F98W and H239A disrupt specific contacts generated by the respective wild-type residues, namely stacking with a flipped out Ura base in the damaged base-binding pocket or electrostatic interactions with DNA in cases of Phe98 and His239, respectively. A loss of the Arg side chain in the case of R243A reduced the rate of DNA bending and increased the enzyme turnover rate, indicating facilitation of the product release step.

Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1

“…SMUG1 H239A interaction with the uridine contain ing DNA. As reported previously [23], the activity of the SMUG1 H239A mutant toward U containing DNA is substantially lower than that of the WT enzyme or the R243A mutant. Nonetheless, formation of the enzyme-substrate complex between SMUG1 H239A and FRET U substrate was registered as changes in the FRET signal within a time interval of 100 s (Fig.…”

“…SMUG1 enzyme. Mutant SMUG1 R243A and H239A proteins were isolated from E. coli Rosetta 2 cells, transformed with the pET28c plasmid carrying the corre sponding human uracil DNA glycosylase gene, as described elsewhere [23,26]. All enzymatic assays were carried out at 25°C in 50 mM Tris HCl (pH 7.5) contain ing 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and…”

“…Currently, the structure of human DNA glycosylase SMUG1 remains unknown. Previously [23], we carried out homology modeling based on the structures of SMUG1 enzymes from Geobacter metallireducens (Protein Data Bank [PDB] ID 5H98 and 5H9I) [24] and Xenopus laevis (PDB 1OE4 and 10OE5) [10] and derived the structural model of the human enzyme in a free state and in a complex with uridine containing DNA ( Fig. 1).…”

“…Residues Arg243 (which occupies the space inside the DNA helix that is formed by uracil eversion) and His239 (which can make contacts with uracil located in the active site and DNA phosphate group) were proposed as functionally important amino acid residues of the intercalating loop. Mutational analy sis [23,25] has revealed that His239 is necessary for the catalytic hydrolysis of the N glycosidic bond.…”

“…Stage 2 involves intercalation of amino acid residues of the enzyme's intercalating loop. Previously [23], FRET study of the conformational dynamics of DNA substrate together with polyacrylamide gel elec trophoresis (PAGE) analysis of the kinetics of accumula tion of N glycosylase reaction products revealed that the SMUG1 H239A mutant is catalytically inactive, whereas the R243A mutant retains its full activity. To clarify the role of Arg243 and His239 in the specific recognition of damaged nucleotides and catalysis, here we performed comprehensive pre-steady state kinetic analysis of con formational changes in the SMUG1 mutants R243A and H239A, substrate DNA, and product of its transforma tion.…”