Fig 1.
Purified H. pylori AP endonuclease, HpXth, has an AP site cleavage activity.
In brief, 10 nM 5’-[32P]labeled 30mer THF•T duplex was incubated with various concentrations of HpXth for 0–5 min at 30 °C in the standard reaction buffer. Denaturing PAGE analysis of products of the reaction. Lane 1, control THF•T, no enzyme; lanes 2–5, as in lane 1 but 0.1, 0.2, 0.5, and 1 nM HpXth with incubation for 5 min, respectively; lanes 6–11, as in lane 1 but incubated with 0.5 nM HpXth for 0, 0.5, 1, 2, 3, or 5 min, respectively. For details, see Materials and methods.
Fig 2.
Divalent-metal ion dependence of HpXth’s AP site cleavage activity.
In brief, 10 nM 5’-[32P]labeled 30mer THF•T duplex was incubated with 0.2 nM HpXth for 5 min at 37 °C in the standard reaction buffer containing various concentrations of metal cations. (A) Denaturing PAGE analysis of products of the reaction. Lane 1, THF•T and 0.1 nM APE1 in buffer BER; lane 2, control THF•T, no enzyme; lane 3, as in lane 2 but HpXth in the standard reaction buffer but without cations; lanes 4 and 5, as in lane 3 but 1 and 0.1 mM EDTA, respectively; lanes 6–9, as in lane 3 but 1, 5, 10, and 20 mM MgCl2, respectively; lanes 10–13, as in lanes 6–9 but 1–20 mM MnCl2; lanes 14–17, as in lanes 6–9 but 1–20 mM CaCl2. The arrows denote the position of the 30mer substrate and 10mer cleavage product. (B) Graphical representation of data from panel A. The statistical significance of the differences among the mean values were evaluated using two-tailed Student’s test (***P < 0.001; **P < 0.01; *P < 0.05 and nsP > 0.05). For details, see Materials and methods.
Fig 3.
Dependence of HpXth’s AP site cleavage activity on reaction conditions.
Briefly, 10 nM 5’-[32P]labeled 30mer THF•T duplex was incubated with 0.2 nM HpXth for 5 min in the standard reaction buffer but with varying temperature, pH, concentrations of Mg2+ and salt. Products of the reaction were resolved by denaturing PAGE, then visualized by phosphorimaging and quantified in ImageQuant. (A) Graphical representation of Mg2+ concentration dependence. (B) Graphical representation of pH dependence. (C) Graphical representation of temperature dependence. (D) Graphical representation of ionic-strength dependence. The statistical significance of the differences among the mean values were evaluated using two-tailed Student’s test (***P < 0.001; **P < 0.01; *P < 0.05 and nsP > 0.05). For details, see Materials and methods.
Fig 4.
Comparative characterization of AP site cleavage and 3’→5’ exonuclease activities of wild-type HpXth and mutant HpXth-D144N.
In brief, 10 nM 5’-[32P]labeled THF•T and Exo20•RexTRec were incubated at 30 °C with increasing amounts of the HpXth-WT and HpXth-D144N proteins for 5 min at 30 °C in the standard reaction buffer. Lanes 1–7, THF•T; lane 1, control, no enzyme; lanes 2–4, 0.5, 2, and 10 nM HpXth-WT, respectively; lanes 5–7, 2, 10, and 100 nM HpXth-D144N, respectively; lanes 8–14, same as in lanes 1–7 but with Exo20•RexTRec. Products of the reaction were analyzed by denaturing PAGE. The arrows denote the position of the 30mer and 20mer substrates and 10mer and 9mer cleavage products. For details, see Materials and methods.
Fig 5.
3’-Repair phosphodiesterase and 3’-phosphatase activities of the HpXth protein.
(A) Enzyme concentration and time-dependent cleavage of the 5’-[32P]labeled Exo20THF•RexTNick oligonucleotide duplex by HpXth. In brief, 10 nM Exo20THF•RexTNick was incubated with increasing amounts of HpXth in the standard reaction buffer at 30 °C for various periods. Lanes 1–6, time-dependent cleavage; lanes 7–12, enzyme concentration—dependent cleavage. The arrows denote the position of substrate “20THFmer” and cleavage product “20mer.” (B) Enzyme concentration—dependent cleavage of the 3’-[32P]labeled Exo20P•RexTNick oligonucleotide duplex by HpXth. In brief, 10 nM Exo20P•RexTNick was incubated with various concentrations of HpXth in the standard reaction buffer for 5 min at 30 °C. Lane 1, Exo20P•RexTNick incubated with 1 U of calf Intestinal alkaline phosphatase, CIP; lanes 2–3, control, no enzyme; lanes 4–9, as in lane 2 but 0.5, 1, 2, 5, 10 and 20 nM HpXth. The arrows denote the position of the 20mer substrate (20mer) and 3’-terminal phosphate product (*p). Products of the reaction were analyzed by denaturing PAGE. For details see Materials and methods.
Fig 6.
An assay of the nucleotide incision activity of H. pylori HpXth toward a 30mer oligonucleotide duplex containing a modified base.
In brief, 10 nM 3’-[α-32P]-dAMP-labeled 31mer oligonucleotide duplexes containing various DNA base lesions were incubated with increasing amounts of the purified HpXth protein in the buffer NIR for 10 min at 30 °C. Products of the reaction were analyzed by denaturing PAGE. Lanes 1–5, αdA•T; lane 1, 2 nM APE1; lane 2, control no enzyme; lanes 3–5, 5, 20, and 100 nM HpXth, respectively; lanes 6–10, same as in lanes 1–5 but DHU•G; lanes 11–15, same as in lanes 1–5 but 5ohC•G. The arrows denote the position of the 31mer substrate and 21mer cleavage product. The statistical significance of the differences among the mean values were evaluated using two-tailed Student’s test (***P < 0.001; **P < 0.01; *P < 0.05 and nsP > 0.05). For details see Materials and methods.
Table 1.
A comparison of kinetic parameters of AP endonucleases from E. coli, H. pylori, and humans.
Fig 7.
Phylogenetic analysis and structural models of H. pylori AP endonuclease.
(A) An unrooted phylogenetic tree of the EEP superfamily AP endonucleases (230 sequences, one species per taxonomic class). Different families are indicated by colored arcs: yellow, ExoIII-like; green, Nape-like; blue, Mth212-like; magenta, Ape1-like; and red, Ape2-like. The clade combining HpXth, B. subtilis ExoA, and the related bacterial and archaeal sequences is labeled “Archaea+Bacteria.” The known NIR-proficient and NIR-deficient enzymes are indicated. (B) Overlay of the structure of human APE1 (1DEW, cyan) and the HpXth model (green). All active-site residues are shown but left unlabeled for clarity. The DNA groove—contacting residues possibly involved in the NIR function are indicated by red carbon atoms. (C) Alignment of HpXth and four core members of AP endonuclease families (human APE1 catalytic domain, E. coli Xth, N. meningitidis NApe, and M. thermautotrophicus Mth212). Metal-binding sites A and B are red and blue, respectively, non—metal-binding active site residues are green; other DNA-binding residues are yellow.
Fig 8.
Differential drug sensitivity of AP endonuclease—deficient E. coli strain BH110 (DE3) carrying a plasmid coding for the H. pylori AP endonuclease.
The strains are represented as follows: the BH110 (DE3) strain carrying control empty vector pBluescript II SK+ (pBSK) (□), strain BH110 (DE3) carrying pBW21-Nfo (■), or pBSK-HpXth (▼), or pBSK-HpXth-D144N (△). Each survival curve represents at least three independent experiments. (A) The survival of the MMS-treated E. coli AP endonuclease—deficient strains. (B) The survival of the E. coli AP endonuclease—deficient strains under oxidative stress. The statistical significance of the differences among the mean values were evaluated using two-tailed Student’s test (***P < 0.001; **P < 0.01; *P < 0.05 and nsP > 0.05). For details, see Materials and methods.