Journal Article

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

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

2018

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Alkylation / Analysis / AP endonuclease / Bacteriology / Biochemistry / Biochemistry, Molecular Biology / Biology and Life Sciences / Biotechnology / Cancer / Catalysis / Data processing / Deoxyribonucleic acid / DNA / DNA damage / DNA repair / E coli / Endonuclease / Enzymatic activity / Enzymes / Escherichia coli / Exonuclease / Gene mutation / Gene sequencing / Genetic aspects / Genomes / Genotoxicity / Helicobacter pylori / Homology / Hydrogen peroxide / Ionic strength / Life Sciences / Magnesium / Medicine and Health Sciences / Microbiology and Parasitology / Mutation / Nucleotide sequence / Phosphatase / Phosphates / Phosphodiesterase / Phylogeny / Proteins / Repair / Research and Analysis Methods / Substrate specificity / Substrates / Sugar