The role of active-site residues Phe98, HiS239, and Arg243 in DNA binding and in the catalysis of human uracil–DNA glycosylase SMUG1

Standard

The role of active-site residues Phe98, HiS239, and Arg243 in DNA binding and in the catalysis of human uracil–DNA glycosylase SMUG1. / Iakovlev, Danila A.; Alekseeva, Irina V.; Vorobjev, Yury N. et al.

In: Molecules, Vol. 24, No. 17, 3133, 28.08.2019.

Research output: Contribution to journal › Article › peer-review

BibTeX

@article{104ef7ce9b3649d8968e959c7bf3180c,

title = "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.",

keywords = "DNA repair, Fluorescence, Homology modeling, Human uracil–DNA glycosylase, Molecular dynamics simulation, Mutant, SMUG1, Stopped-flow kinetics, Structure, DOMAIN, molecular dynamics simulation, THYMINE, REPAIR, homology modeling, fluorescence, STRUCTURAL BASIS, EXCISION, human uracil-DNA glycosylase, SPECIFICITY, structure, DAMAGE-RECOGNITION, 5-HYDROXYMETHYLURACIL, ENZYME, stopped-flow kinetics, mutant, PROTEINS",

author = "Iakovlev, {Danila A.} and Alekseeva, {Irina V.} and Vorobjev, {Yury N.} and Kuznetsov, {Nikita A.} and Fedorova, {Olga S.}",

year = "2019",

month = aug,

day = "28",

doi = "10.3390/molecules24173133",

language = "English",

volume = "24",

journal = "Molecules",

issn = "1420-3049",

publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",

number = "17",

}

RIS

TY - JOUR

T1 - The role of active-site residues Phe98, HiS239, and Arg243 in DNA binding and in the catalysis of human uracil–DNA glycosylase SMUG1

AU - Iakovlev, Danila A.

AU - Alekseeva, Irina V.

AU - Vorobjev, Yury N.

AU - Kuznetsov, Nikita A.

AU - Fedorova, Olga S.

PY - 2019/8/28

Y1 - 2019/8/28

N2 - 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.

AB - 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.

KW - DNA repair

KW - Fluorescence

KW - Homology modeling

KW - Human uracil–DNA glycosylase

KW - Molecular dynamics simulation

KW - Mutant

KW - SMUG1

KW - Stopped-flow kinetics

KW - Structure

KW - DOMAIN

KW - molecular dynamics simulation

KW - THYMINE

KW - REPAIR

KW - homology modeling

KW - fluorescence

KW - STRUCTURAL BASIS

KW - EXCISION

KW - human uracil-DNA glycosylase

KW - SPECIFICITY

KW - structure

KW - DAMAGE-RECOGNITION

KW - 5-HYDROXYMETHYLURACIL

KW - ENZYME

KW - stopped-flow kinetics

KW - mutant

KW - PROTEINS

UR - http://www.scopus.com/inward/record.url?scp=85071402677&partnerID=8YFLogxK

U2 - 10.3390/molecules24173133

DO - 10.3390/molecules24173133

M3 - Article

C2 - 31466351

AN - SCOPUS:85071402677

VL - 24

JO - Molecules

JF - Molecules

SN - 1420-3049

IS - 17

M1 - 3133

ER -

ID: 21346256