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

Olga A. Kladova, Alexandra A. Kuznetsova, Olga S. Fedorova, Nikita A. Kuznetsov

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article number140
Number of pages13
JournalGenes
Volume8
Issue number5
DOIs
Publication statusPublished - 13 May 2017

Keywords

  • Base excision repair
  • Conformational dynamics
  • DNA glycosylase
  • Endonuclease VIII
  • Stopped-flow enzyme kinetics
  • ORTHOLOG
  • FORMAMIDOPYRIMIDINE-DNA GLYCOSYLASE
  • MECHANISM
  • STEADY-STATE KINETICS
  • SEARCH
  • base excision repair
  • stopped-flow enzyme kinetics
  • CONFORMATIONAL DYNAMICS
  • REPAIR
  • DAMAGE RECOGNITION
  • conformational dynamics
  • FINGER
  • endonuclease VIII
  • REAL-TIME

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