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

PLoS One. 2018 Aug 15;13(8):e0202232. doi: 10.1371/journal.pone.0202232. eCollection 2018.

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-deficient 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 host-imposed factors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Catalytic Domain / genetics
  • DNA Damage
  • DNA Repair
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / chemistry*
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / genetics
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / metabolism*
  • Escherichia coli / drug effects
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Helicobacter pylori / drug effects
  • Helicobacter pylori / enzymology*
  • Helicobacter pylori / genetics
  • Hydrogen Peroxide / pharmacology
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Phylogeny
  • Sequence Homology, Amino Acid
  • Structural Homology, Protein
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Hydrogen Peroxide
  • DNA-(Apurinic or Apyrimidinic Site) Lyase

Grants and funding

This work was supported by grants to S.A. from the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan AP05130820 (www.biocenter.kz), to M.S. from la Ligue Nationale Française Contre le Cancer “Equipe Labellisée LIGUE 2016” grant EL2016.LNCC/MuS and Electricité de France RB 2017 (http://www.edf.fr), to A.A.I. from ERA.Net RUS Plus (www.eranet-rus.eu) [#306] and French National Center for Scientific Research (PRC CNRS/RFBR n1074 REDOBER), and to D.O.Z. from the Russian Ministry of Science and Education (6.5773.2017/6.7) and Russian Science Foundation (17-14-01190). A.T. was supported by Ph.D. fellowships from L.N. Gumilyov Eurasian National University (www.enu.kz), Kazakhstan. The sponsors played no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.