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

Danila A Iakovlev,Olga S Fedorova,Nikita A Kuznetsov,Yury N Vorobjev,Irina V Alekseeva

doi:10.3390/molecules24173133

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.

Highlights

Human SMUG1 is one of four human uracil–DNA glycosylases together with thymine–DNA glycosylase, uracil–DNA glycosylase (UNG), and methyl-CpG-binding domain
To verify the damage recognition process, in the present study, we modeled the structures of Human SMUG1 (hSMUG1)–DNA complexes with the WT enzyme and a few mutant forms of the enzyme containing substitution of catalytic amino acid residue His239 with Ala, a substitution of Phe98, which directly interacts with the damaged base, with Trp or a substitution of Arg243 in the intercalating loop with Ala
Substrate approach was chosen to model the structures of free WT hSMUG1 and free mutants F98W, H239A, Because the structures of human SMUG1 have not yet been obtained yet, the molecular dynamics (MD) simulation and R243A

Summary

Introduction

Human SMUG1 (hSMUG1; single-strand selective monofunctional uracil–DNA glycosylase) is one of four human uracil–DNA glycosylases together with thymine–DNA glycosylase (hTDG), uracil–DNA glycosylase (UNG), and methyl-CpG-binding domain (hMBD4). HUNG, hTDG, and hSMUG1 belong to different families (I, II, and III, respectively) of the large uracil–DNA glycosylase structural superfamily, [7] whereas hMBD4 belongs to structural superfamily HhH (helix–hairpin–helix) of DNA-binding proteins [8]. C-terminal motif and asparagine-243, which is found in family II in the N-terminal catalytic motif [7] (Figure 1A). Molecules 2019, 24, 3133 terminal motif and asparagine-243, which is found in family II in the N-terminal catalytic motif [7] Currently, the structure of hSMUG1 is unknown, the detailed structure of this enzyme . The structure of hSMUG1 is unknown, the detailed structure of this enzyme was predicted [9]currently, by homology modeling via structure-based alignment with GmeSMUG1 from was predicted [9] by homology modeling via structure-based alignment with GmeSMUG1 from

Objectives

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Conclusion