TY - JOUR

T1 - Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1

AU - Iakovlev, D. A.

AU - Alekseeva, I. V.

AU - Kuznetsov, N. A.

AU - Fedorova, O. S.

PY - 2020/5/1

Y1 - 2020/5/1

N2 - Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme’s tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.

AB - Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme’s tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.

KW - conformational dynamics

KW - DNA repair

KW - fluorescence

KW - human uracil-DNA glycosylase SMUG1

KW - stopped-flow kinetics

UR - http://www.scopus.com/inward/record.url?scp=85085036230&partnerID=8YFLogxK

UR - https://www.elibrary.ru/item.asp?id=43281062

U2 - 10.1134/S0006297920050089

DO - 10.1134/S0006297920050089

M3 - Article

C2 - 32571189

AN - SCOPUS:85085036230

VL - 85

SP - 594

EP - 603

JO - Biochemistry (Moscow)

JF - Biochemistry (Moscow)

SN - 0006-2979

IS - 5

ER -