Characterization of biochemical properties of an apurinic/apyrimidinic endonuclease from Helicobacter pylori

Aigerim Turgimbayeva, Sailau Abeldenov, Dmitry O. Zharkov, Alexander A. Ishchenko, Yerlan Ramankulov, Murat Saparbaev, Bekbolat Khassenov

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Apurinic/apyrimidinic (AP) endonucleases play critical roles in the repair of abasic sites and strand breaks in DNA. Complete genome sequences of Helicobacter pylori reveal that this bacterial specie has a single AP endonuclease. An H. pylori homolog of Xth (HpXth) is a member of exonuclease III family, which is represented by Escherichia coli Xth. Currently, it remains unknown whether this single AP endonuclease has DNA repair activities similar to those of its counterpart in E. coli and other bacteria. We report that HpXth possesses efficient AP site cleavage, 3′-repair phosphodiesterase, and 3′-phosphatase activities but not the nucleotide incision repair function. Optimal reaction conditions for HpXth's AP endonuclease activity are low ionic strength, high Mg2+ concentration, pH in the range 7-8, and temperature 30°C. The kinetic parameters measured under steady-state conditions showed that HpXth removes the AP site, 3′-blocking sugar-phosphate, and 3′-terminal phosphate in DNA strand breaks with good efficiency (kcat/KM = 1240, 44, and 5,4 μM-1·min-1, respectively), similar to that of E. coli Xth. As expected, the presence of HpXth protein in AP endonuclease Ddeficient E. coli xth nfo strain significantly reduced the sensitivity to an alkylating agent and H2O2. Mutation of active site residue D144 in HpXth predicted to be essential for catalysis resulted in a complete loss of enzyme activities. Several important structural features of HpXth were uncovered by homology modeling and phylogenetic analysis. Our data show the DNA substrate specificity of H. pylori AP endonuclease and suggest that HpXth counteracts the genotoxic effects of DNA damage generated by endogenous and hostimposed factors.

Original languageEnglish
Article numbere0202232
Pages (from-to)e0202232
Number of pages26
JournalPLoS ONE
Volume13
Issue number8
DOIs
Publication statusPublished - 15 Aug 2018

Keywords

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Bacterial Proteins/chemistry
  • Catalytic Domain/genetics
  • DNA Damage
  • DNA Repair
  • DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry
  • Escherichia coli Proteins/chemistry
  • Escherichia coli/drug effects
  • Helicobacter pylori/drug effects
  • Hydrogen Peroxide/pharmacology
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Phylogeny
  • Sequence Homology, Amino Acid
  • Structural Homology, Protein
  • Substrate Specificity
  • SUBSTRATE SPECIFICITIES
  • ESCHERICHIA-COLI
  • BASE EXCISION-REPAIR
  • PATHWAY
  • AP ENDONUCLEASE
  • OXIDATIVE DNA-DAMAGE
  • EXONUCLEASE-III
  • CRYSTAL-STRUCTURE
  • IV
  • NUCLEOTIDE INCISION REPAIR

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