TY - JOUR

T1 - Pre-steady-state kinetic analysis of damage recognition by human single-strand selective monofunctional uracil-DNA glycosylase SMUG1

AU - Kuznetsova, Alexandra A.

AU - Iakovlev, Danila A.

AU - Misovets, Inna V.

AU - Ishchenko, Alexander A.

AU - Saparbaev, Murat K.

AU - Kuznetsov, Nikita A.

AU - Fedorova, Olga S.

N1 - Publisher Copyright: © 2017 The Royal Society of Chemistry.

PY - 2017/11/21

Y1 - 2017/11/21

N2 - In all organisms, DNA glycosylases initiate base excision repair pathways resulting in removal of aberrant bases from DNA. Human SMUG1 belongs to the superfamily of uracil-DNA glycosylases catalyzing the hydrolysis of the N-glycosidic bond of uridine and uridine lesions bearing oxidized groups at C5: 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), and 5-hydroxyuridine (5hoU). An apurinic/apyrimidinic (AP) site formed as the product of an N-glycosylase reaction is tightly bound to hSMUG1, thus inhibiting the downstream action of AP-endonuclease APE1. The steady-state kinetic parameters (kcat and KM; obtained from the literature) correspond to the enzyme turnover process limited by the release of hSMUG1 from the complex with the AP-site. In the present study, our objective was to carry out a stopped-flow fluorescence analysis of the interaction of hSMUG1 with a DNA substrate containing a dU:dG base pair to follow the pre-steady-state kinetics of conformational changes in both molecules. A comparison of kinetic data obtained by means of Trp and 2-aminopurine fluorescence and Förster resonance energy transfer (FRET) detection allowed us to elucidate the stages of specific and nonspecific DNA binding, to propose the mechanism of damaged base recognition by hSMUG1, and to determine the true rate of the catalytic step. Our results shed light on the kinetic mechanism underlying the initiation of base excision repair by hSMUG1 using the "wedge" strategy for DNA lesion search.

AB - In all organisms, DNA glycosylases initiate base excision repair pathways resulting in removal of aberrant bases from DNA. Human SMUG1 belongs to the superfamily of uracil-DNA glycosylases catalyzing the hydrolysis of the N-glycosidic bond of uridine and uridine lesions bearing oxidized groups at C5: 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), and 5-hydroxyuridine (5hoU). An apurinic/apyrimidinic (AP) site formed as the product of an N-glycosylase reaction is tightly bound to hSMUG1, thus inhibiting the downstream action of AP-endonuclease APE1. The steady-state kinetic parameters (kcat and KM; obtained from the literature) correspond to the enzyme turnover process limited by the release of hSMUG1 from the complex with the AP-site. In the present study, our objective was to carry out a stopped-flow fluorescence analysis of the interaction of hSMUG1 with a DNA substrate containing a dU:dG base pair to follow the pre-steady-state kinetics of conformational changes in both molecules. A comparison of kinetic data obtained by means of Trp and 2-aminopurine fluorescence and Förster resonance energy transfer (FRET) detection allowed us to elucidate the stages of specific and nonspecific DNA binding, to propose the mechanism of damaged base recognition by hSMUG1, and to determine the true rate of the catalytic step. Our results shed light on the kinetic mechanism underlying the initiation of base excision repair by hSMUG1 using the "wedge" strategy for DNA lesion search.

KW - BASE EXCISION-REPAIR

KW - ESCHERICHIA-COLI

KW - CONFORMATIONAL DYNAMICS

KW - SUBSTRATE-SPECIFICITY

KW - DEAMINATED CYTOSINE

KW - AMINO-ACID

KW - FLUORESCENCE

KW - ENZYME

KW - 2-AMINOPURINE

KW - MECHANISM

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism

KW - Humans

KW - Uridine/analogs & derivatives

KW - Fluorescence Resonance Energy Transfer

KW - Kinetics

KW - Uracil-DNA Glycosidase/metabolism

UR - http://www.scopus.com/inward/record.url?scp=85035000729&partnerID=8YFLogxK

U2 - 10.1039/c7mb00457e

DO - 10.1039/c7mb00457e

M3 - Article

C2 - 29051947

AN - SCOPUS:85035000729

VL - 13

SP - 2638

EP - 2649

JO - Molecular BioSystems

JF - Molecular BioSystems

SN - 1742-206X

IS - 12

ER -