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.
Highlights
Cellular DNA is continuously and inevitably assaulted by a variety of endogenous and environmental genotoxic agents
base excision repair (BER) can handle the majority of oxidized DNA bases, some lesions, such as α-anomers of deoxynucleosides generated by free radicals under anoxic conditions, are not repaired by DNA glycosylases but rather by AP endonucleases via the nucleotide incision repair (NIR) pathway [11,12,13]
The cDNA coding for AP endonuclease H. pylori homolog of Escherichia coli exonuclease III (Xth) (HpXth) was prepared by PCR from the DNA of H. pylori, and oligonucleotide primers as described in Materials and Methods
Summary
Cellular DNA is continuously and inevitably assaulted by a variety of endogenous and environmental genotoxic agents. BER can handle the majority of oxidized DNA bases, some lesions, such as α-anomers of deoxynucleosides (αdN) generated by free radicals under anoxic conditions, are not repaired by DNA glycosylases but rather by AP endonucleases via the NIR pathway [11,12,13]. In this glycosylase-independent pathway, an AP endonuclease directly cleaves DNA 5’ to the lesion and generates a single-strand break with a 5’-dangling damaged nucleotide [5, 14]. Human major AP endonuclease 1, APE1, in addition to NIR and BER functions contains abasic RNA cleavage activity, including the ability to process abasic ribonucleotides embedded in DNA [17]
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