TY - JOUR

T1 - Search for Modified DNA Sites with the Human Methyl-CpG-Binding Enzyme MBD4

AU - Yakovlev, D. A.

AU - Kuznetsova, A. A.

AU - Fedorova, O. S.

AU - Kuznetsov, N. A.

PY - 2017

Y1 - 2017

N2 - The MBD4 enzyme initiates the process of DNA demethylation by the excision of modified DNA bases, resulting in the formation of apurinic/apyrimidinic sites. MBD4 contains a methyl-CpG-binding domain which provides the localization of the enzyme at the CpG sites, and a DNA glycosylase domain that is responsible for the catalytic activity. The aim of this work was to clarify the mechanisms of specific site recognition and formation of catalytically active complexes between model DNA substrates and the catalytic N-glycosylase domain MBD4cat. The conformational changes in MBD4cat and DNA substrates during their interaction were recorded in real time by stopped-flow detection of the fluorescence of tryptophan residues in the enzyme and fluorophores in DNA. A kinetic scheme of MBD4cat interaction with DNA was proposed, and the rate constants for the formation and decomposition of transient reaction intermediates were calculated. Using DNA substrates of different lengths, the formation of the catalytically active complex was shown to follow the primary DNA binding step which is responsible for the search and recognition of the modified base. The results reveal that in the primary complex of MBD4cat with DNA containing modified nucleotides, local melting and bending of the DNA strand occur. On the next step, when the catalytically competent conformation of the enzyme-substrate complex is formed, the modified nucleotide is everted from the double DNA helix into the active center and the void in the helix is filled by the enzyme's amino acids.

AB - The MBD4 enzyme initiates the process of DNA demethylation by the excision of modified DNA bases, resulting in the formation of apurinic/apyrimidinic sites. MBD4 contains a methyl-CpG-binding domain which provides the localization of the enzyme at the CpG sites, and a DNA glycosylase domain that is responsible for the catalytic activity. The aim of this work was to clarify the mechanisms of specific site recognition and formation of catalytically active complexes between model DNA substrates and the catalytic N-glycosylase domain MBD4cat. The conformational changes in MBD4cat and DNA substrates during their interaction were recorded in real time by stopped-flow detection of the fluorescence of tryptophan residues in the enzyme and fluorophores in DNA. A kinetic scheme of MBD4cat interaction with DNA was proposed, and the rate constants for the formation and decomposition of transient reaction intermediates were calculated. Using DNA substrates of different lengths, the formation of the catalytically active complex was shown to follow the primary DNA binding step which is responsible for the search and recognition of the modified base. The results reveal that in the primary complex of MBD4cat with DNA containing modified nucleotides, local melting and bending of the DNA strand occur. On the next step, when the catalytically competent conformation of the enzyme-substrate complex is formed, the modified nucleotide is everted from the double DNA helix into the active center and the void in the helix is filled by the enzyme's amino acids.

KW - conformational dynamics

KW - DNA demethylation

KW - DNA repair

KW - MBD4

KW - Pre-steady-state kinetics

KW - RECOGNITION

KW - MECHANISM

KW - CRYSTAL-STRUCTURE

KW - THYMINE

KW - CONFORMATIONAL DYNAMICS

KW - 2-AMINOPURINE

KW - DAMAGE

KW - MED1 MBD4

KW - REPAIR

KW - pre-steady-state kinetics

KW - GLYCOSYLASE DOMAIN

UR - http://www.scopus.com/inward/record.url?scp=85017565865&partnerID=8YFLogxK

U2 - 10.32607/20758251-2017-9-1-88-98

DO - 10.32607/20758251-2017-9-1-88-98

M3 - Article

AN - SCOPUS:85017565865

VL - 9

SP - 26

EP - 37

JO - Acta Naturae

JF - Acta Naturae

SN - 2075-8251

IS - 1

ER -