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

Overview

Journal Molecules

Publisher MDPI

Specialty Biology

Date 2019 Aug 31

PMID 31466351

Authors

Danila A Iakovlev

Irina V Alekseeva

Yury N Vorobjev

Nikita A Kuznetsov

Olga S Fedorova

Affiliations

Soon will be listed here.

Abstract

Human SMUG1 (hSMUG1) hydrolyzes the -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.

References

Lazaridis T, Karplus M . Effective energy function for proteins in solution. Proteins. 1999; 35(2):133-52. DOI: 10.1002/(sici)1097-0134(19990501)35:2<133::aid-prot1>3.0.co;2-n. View

Kavli B, Sundheim O, Akbari M, Otterlei M, Nilsen H, Skorpen F . hUNG2 is the major repair enzyme for removal of uracil from U:A matches, U:G mismatches, and U in single-stranded DNA, with hSMUG1 as a broad specificity backup. J Biol Chem. 2002; 277(42):39926-36. DOI: 10.1074/jbc.M207107200. View

Wibley J, Waters T, Haushalter K, Verdine G, Pearl L . Structure and specificity of the vertebrate anti-mutator uracil-DNA glycosylase SMUG1. Mol Cell. 2003; 11(6):1647-59. DOI: 10.1016/s1097-2765(03)00235-1. View

Pettersen E, Goddard T, Huang C, Couch G, Greenblatt D, Meng E . UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13):1605-12. DOI: 10.1002/jcc.20084. View

Matsubara M, Tanaka T, Terato H, Ohmae E, Izumi S, Katayanagi K . Mutational analysis of the damage-recognition and catalytic mechanism of human SMUG1 DNA glycosylase. Nucleic Acids Res. 2004; 32(17):5291-302. PMC: 521670. DOI: 10.1093/nar/gkh859. View

Pettersen H, Sundheim O, Gilljam K, Slupphaug G, Krokan H, Kavli B . Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms. Nucleic Acids Res. 2007; 35(12):3879-92. PMC: 1919486. DOI: 10.1093/nar/gkm372. View

Popov A, Vorobev I . [GUI-BioPASED program for molecular dynamics modelling of biopolymers with a graphical user interface]. Mol Biol (Mosk). 2010; 44(4):735-42. View

Shapovalov M, Dunbrack Jr R . A smoothed backbone-dependent rotamer library for proteins derived from adaptive kernel density estimates and regressions. Structure. 2011; 19(6):844-58. PMC: 3118414. DOI: 10.1016/j.str.2011.03.019. View

Zhang W, Liu Z, Crombet L, Amaya M, Liu Y, Zhang X . Crystal structure of the mismatch-specific thymine glycosylase domain of human methyl-CpG-binding protein MBD4. Biochem Biophys Res Commun. 2011; 412(3):425-8. DOI: 10.1016/j.bbrc.2011.07.091. View

10.

Hashimoto H, Zhang X, Cheng X . Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation. Nucleic Acids Res. 2012; 40(17):8276-84. PMC: 3458566. DOI: 10.1093/nar/gks628. View

11.

Hashimoto H, Hong S, Bhagwat A, Zhang X, Cheng X . Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the thymine DNA glycosylase domain: its structural basis and implications for active DNA demethylation. Nucleic Acids Res. 2012; 40(20):10203-14. PMC: 3488261. DOI: 10.1093/nar/gks845. View

12.

Sjolund A, Senejani A, Sweasy J . MBD4 and TDG: multifaceted DNA glycosylases with ever expanding biological roles. Mutat Res. 2012; 743-744:12-25. PMC: 3661743. DOI: 10.1016/j.mrfmmm.2012.11.001. View

13.

Kuznetsova A, Kuznetsov N, Ishchenko A, Saparbaev M, Fedorova O . Step-by-step mechanism of DNA damage recognition by human 8-oxoguanine DNA glycosylase. Biochim Biophys Acta. 2013; 1840(1):387-95. DOI: 10.1016/j.bbagen.2013.09.035. View

14.

Nelson S, Dunn A, Kathe S, Warshaw D, Wallace S . Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases. Proc Natl Acad Sci U S A. 2014; 111(20):E2091-9. PMC: 4034194. DOI: 10.1073/pnas.1400386111. View

15.

Schormann N, Ricciardi R, Chattopadhyay D . Uracil-DNA glycosylases-structural and functional perspectives on an essential family of DNA repair enzymes. Protein Sci. 2014; 23(12):1667-85. PMC: 4253808. DOI: 10.1002/pro.2554. View

16.

Kuznetsov N, Bergonzo C, Campbell A, Li H, Mechetin G, de Los Santos C . Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition. Nucleic Acids Res. 2014; 43(1):272-81. PMC: 4288190. DOI: 10.1093/nar/gku1300. View

17.

Kuznetsov N, Kladova O, Kuznetsova A, Ishchenko A, Saparbaev M, Zharkov D . Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III. J Biol Chem. 2015; 290(23):14338-49. PMC: 4505503. DOI: 10.1074/jbc.M114.621128. View

18.

Ito S, Kuraoka I . Epigenetic modifications in DNA could mimic oxidative DNA damage: A double-edged sword. DNA Repair (Amst). 2015; 32:52-57. DOI: 10.1016/j.dnarep.2015.04.013. View

19.

Kuznetsov N, Kiryutin A, Kuznetsova A, Panov M, Barsukova M, Yurkovskaya A . The formation of catalytically competent enzyme-substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase. J Biomol Struct Dyn. 2016; 35(5):950-967. DOI: 10.1080/07391102.2016.1171800. View

20.

Miroshnikova A, Kuznetsova A, Vorobjev Y, Kuznetsov N, Fedorova O . Effects of mono- and divalent metal ions on DNA binding and catalysis of human apurinic/apyrimidinic endonuclease 1. Mol Biosyst. 2016; 12(5):1527-39. DOI: 10.1039/c6mb00128a. View