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 hostimposed factors.
