TY - JOUR

T1 - Characterization of biochemical properties of an apurinic/apyrimidinic endonuclease from Helicobacter pylori

AU - Turgimbayeva, Aigerim

AU - Abeldenov, Sailau

AU - Zharkov, Dmitry O.

AU - Ishchenko, Alexander A.

AU - Ramankulov, Yerlan

AU - Saparbaev, Murat

AU - Khassenov, Bekbolat

N1 - Publisher Copyright: Copyright © 2018 Turgimbayeva et al.

PY - 2018/8/15

Y1 - 2018/8/15

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Amino Acid Substitution

KW - Bacterial Proteins/chemistry

KW - Catalytic Domain/genetics

KW - DNA Damage

KW - DNA Repair

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry

KW - Escherichia coli Proteins/chemistry

KW - Escherichia coli/drug effects

KW - Helicobacter pylori/drug effects

KW - Hydrogen Peroxide/pharmacology

KW - Kinetics

KW - Models, Molecular

KW - Mutagenesis, Site-Directed

KW - Phylogeny

KW - Sequence Homology, Amino Acid

KW - Structural Homology, Protein

KW - Substrate Specificity

KW - SUBSTRATE SPECIFICITIES

KW - ESCHERICHIA-COLI

KW - BASE EXCISION-REPAIR

KW - PATHWAY

KW - AP ENDONUCLEASE

KW - OXIDATIVE DNA-DAMAGE

KW - EXONUCLEASE-III

KW - CRYSTAL-STRUCTURE

KW - IV

KW - NUCLEOTIDE INCISION REPAIR

UR - http://www.scopus.com/inward/record.url?scp=85053703584&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0202232

DO - 10.1371/journal.pone.0202232

M3 - Article

C2 - 30110394

AN - SCOPUS:85053703584

VL - 13

SP - e0202232

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 8

M1 - e0202232

ER -