TY - JOUR

T1 - Mutational and kinetic analysis of lesion recognition by Escherichia coli endonuclease VIII

AU - Kladova, Olga A.

AU - Kuznetsova, Alexandra A.

AU - Fedorova, Olga S.

AU - Kuznetsov, Nikita A.

N1 - Publisher Copyright: © 2017 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2017/5/13

Y1 - 2017/5/13

N2 - Escherichia coli endonuclease VIII (Endo VIII) is a DNA glycosylase with substrate specificity for a wide range of oxidatively damaged pyrimidine bases. Endo VIII catalyzes hydrolysis of the N-glycosidic bond and β, δ-elimination of 3′ - and 5′ -phosphate groups of an apurinic/apyrimidinic site. Single mutants of Endo VIII L70S, L70W, Y71W, F121W, F230W, and P253W were analyzed here with the aim to elucidate the kinetic mechanism of protein conformational adjustment during damaged-nucleotide recognition and catalytic-complex formation. F121W substitution leads to a slight reduction of DNA binding and catalytic activity. F230W substitution slows the rate of the δ-elimination reaction indicating that interaction of Phe230 with a 5′ -phosphate group proceeds in the latest catalytic step. P253W Endo VIII has the same activity as the wild type (WT) enzyme. Y71W substitution slightly reduces the catalytic activity due to the effect on the later steps of catalytic-complex formation. Both L70S and L70W substitutions significantly decrease the catalytic activity, indicating that Leu70 plays an important role in the course of enzyme-DNA catalytic complex formation. Our data suggest that Leu70 forms contacts with DNA earlier than Tyr71 does. Therefore, most likely, Leu70 plays the role of a DNA lesion “sensor”, which is used by Endo VIII for recognition of a DNA damage site.

AB - Escherichia coli endonuclease VIII (Endo VIII) is a DNA glycosylase with substrate specificity for a wide range of oxidatively damaged pyrimidine bases. Endo VIII catalyzes hydrolysis of the N-glycosidic bond and β, δ-elimination of 3′ - and 5′ -phosphate groups of an apurinic/apyrimidinic site. Single mutants of Endo VIII L70S, L70W, Y71W, F121W, F230W, and P253W were analyzed here with the aim to elucidate the kinetic mechanism of protein conformational adjustment during damaged-nucleotide recognition and catalytic-complex formation. F121W substitution leads to a slight reduction of DNA binding and catalytic activity. F230W substitution slows the rate of the δ-elimination reaction indicating that interaction of Phe230 with a 5′ -phosphate group proceeds in the latest catalytic step. P253W Endo VIII has the same activity as the wild type (WT) enzyme. Y71W substitution slightly reduces the catalytic activity due to the effect on the later steps of catalytic-complex formation. Both L70S and L70W substitutions significantly decrease the catalytic activity, indicating that Leu70 plays an important role in the course of enzyme-DNA catalytic complex formation. Our data suggest that Leu70 forms contacts with DNA earlier than Tyr71 does. Therefore, most likely, Leu70 plays the role of a DNA lesion “sensor”, which is used by Endo VIII for recognition of a DNA damage site.

KW - Base excision repair

KW - Conformational dynamics

KW - DNA glycosylase

KW - Endonuclease VIII

KW - Stopped-flow enzyme kinetics

KW - ORTHOLOG

KW - FORMAMIDOPYRIMIDINE-DNA GLYCOSYLASE

KW - MECHANISM

KW - STEADY-STATE KINETICS

KW - SEARCH

KW - base excision repair

KW - stopped-flow enzyme kinetics

KW - CONFORMATIONAL DYNAMICS

KW - REPAIR

KW - DAMAGE RECOGNITION

KW - conformational dynamics

KW - FINGER

KW - endonuclease VIII

KW - REAL-TIME

UR - http://www.scopus.com/inward/record.url?scp=85019236248&partnerID=8YFLogxK

U2 - 10.3390/genes8050140

DO - 10.3390/genes8050140

M3 - Article

C2 - 28505099

AN - SCOPUS:85019236248

VL - 8

JO - Genes

JF - Genes

SN - 2073-4425

IS - 5

M1 - 140

ER -