Exonuclease III is stimulated by the addition of Magnesium and Calcium ions.

<p>The 5′ end-labeled substrate subDS-AP (800 fmol) was incubated with 2 fmol of XthA with various metal ions at a concentration of 2 mM (lanes 2–9), with EDTA (lanes 10–13) and with DTT (lanes 14–19). Concentrations (mM) of EDTA and DTT are indicated on the figures. Substrate incubated with the enzyme without the addition of metal ion, EDTA or DTT was used as control (lane 1). Graphical representation of lanes 1–9, 10–13 and 14–19 of figure A is shown in B, C and D, respectively. The data in the graphs represent mean (±standard errors) of two independent experiments carried out in duplicates. The reaction was carried out for 15 min at 37°C and the products were analyzed on 20% polyacrylamide gels containing 8 M Urea and the DNA bands were visualized by autoradiography.</p

End is the major AP endonuclease in the mycobacterial cell-free protein extracts.

<p>(A) Schematic diagram of oligonucleotide substrate in which asterisk (*) and AP denote the radioactively labeled γ-³²P terminus and the abasic site in the substrate, respectively. The 5′ end-labeled substrate subDS-AP (20 fmol) was incubated with cell-free protein extracts (1 µg) of parental and mutant strains of <i>M.tuberculosis</i> for 30 min at 37°C. (B) The graph represents mean (±standard errors) of three independent experiments, of which one is shown in (A). (C) Influence of disruption of single or both the AP endonucleases on the ability of <i>M.tuberculosis</i> to withstand oxidative stress generated by cumene hydroperoxide (CHP). Graph depicts the range of the inhibition zones observed in the presence of various concentrations of cumene hydroperoxide. The values are represented as the mean (± standard errors) of two independent experiments carried out in triplicates. ∗,<i>P</i><0.05; ∗∗,<i>P</i><0.01; ∗∗∗,<i>P</i><0.001 (Two way ANOVA). NL, no lysates; NA, no activity; WT, <i>M.tuberculosis</i>; EK, MtbΔ<i>end</i>; EC, MtbΔ<i>end</i>Comp; XK, MtbΔ<i>xthA</i>; XC, MtbΔ<i>xthA</i>Comp; EX, MtbΔ<i>end</i>Δ<i>xthA</i>.</p

Endonuclease IV is stimulated by the addition of Magnesium and Calcium ions.

<p>The 5′ end-labeled substrate subDS-AP (800 fmol) was incubated with 2 fmol of End with various metal ions at a concentration of 2 mM (lanes 1–8), with EDTA (lanes 10–13) and with DTT (lanes 14–18). Concentrations (mM) of EDTA and DTT are indicated on the figures. Substrate incubated with the enzyme without the addition of metal ion, EDTA or DTT was used as control (lane 9). Graphical representation of lanes 1–9, 10–13 and 14–18 of figure A is shown in B, C and D, respectively. The data in the graphs represent mean (±standard errors) of two independent experiments carried out in duplicates. The reaction was carried out for 15 min at 37°C and the products were analyzed on 20% polyacrylamide gels containing 8 M Urea and the DNA bands were visualized by autoradiography.</p

Endonuclease IV Is the Major Apurinic/Apyrimidinic Endonuclease in <i>Mycobacterium tuberculosis</i> and Is Important for Protection against Oxidative Damage

<div><p>During the establishment of an infection, bacterial pathogens encounter oxidative stress resulting in the production of DNA lesions. Majority of these lesions are repaired by base excision repair (BER) pathway. Amongst these, abasic sites are the most frequent lesions in DNA. Class II apurinic/apyrimidinic (AP) endonucleases play a major role in BER of damaged DNA comprising of abasic sites. <i>Mycobacterium tuberculosis</i>, a deadly pathogen, resides in the human macrophages and is continually subjected to oxidative assaults. We have characterized for the first time two AP endonucleases namely Endonuclease IV (End) and Exonuclease III (XthA) that perform distinct functions in <i>M.tuberculosis</i>. We demonstrate that <i>M.tuberculosis</i> End is a typical AP endonuclease while XthA is predominantly a 3′→5′ exonuclease. The AP endonuclease activity of End and XthA was stimulated by Mg²⁺ and Ca²⁺ and displayed a preferential recognition for abasic site paired opposite to a cytosine residue in DNA. Moreover, End exhibited metal ion independent 3′→5′ exonuclease activity while in the case of XthA this activity was metal ion dependent. We demonstrate that End is not only a more efficient AP endonuclease than XthA but it also represents the major AP endonuclease activity in <i>M.tuberculosis</i> and plays a crucial role in defense against oxidative stress.</p></div

XthA displays a metal ion dependent 3′→5′ exonuclease activity on a large variety of substrates.

<p>Schematic diagram of oligonucleotide substrates (refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071535#pone-0071535-t001" target="_blank">Table 1</a> for details) in which asterisk (*) denotes the radioactively labeled γ-³²P terminus. (A) 20 fmol of 5′ end-labeled substrate subSS-Exo was incubated without protein (np,lane 1), with 2 fmol of either End (lane 2) or XthA (lane 3) in a buffer containing 10 mM MgCl₂ for 15 min at 37°C. (B) 20 fmol of 5′ end-labeled substrate subDS-REC (3′ recessed substrate) was incubated without protein (lane 1), with 2 fmol of XthA without any metal (lane 2), XthA in a buffer containing 10 mM MgCl₂ (lane 3) or XthA in a buffer containing 10 mM MnCl₂ (lane 4). (C) 20 fmol of each of the 5′ end-labeled DNA substrates (subDS-REC, subDS-BLUNT and subDS-NICK) were incubated in the absence (–) or presence (+) of 10 fmol of End (lanes 1–6) and XthA (lanes 7–12).</p

End displays stronger AP endonuclease activity in comparison to XthA.

<p>Schematic diagram of oligonucleotide substrates in which asterisk (*) and AP denote the radioactively labeled γ-³²P terminus and the abasic site in the substrate, respectively. 5′ end-labeled substrate (200 fmol) was incubated with 5 fmol of either End or XthA at 37°C. (A) Lanes 1–3, subDS-AP was incubated without protein (np), with End or with XthA, respectively, for 30 min. (B) Lanes 1–3, subSS-AP was incubated without protein, with XthA or with End, respectively, for 30 min. (C) Time dependence of End and XthA on AP endonuclease activity. The reaction was carried out for different intervals of time (0–15 min). The products were analyzed by denaturing PAGE on 20% polyacrylamide gels containing 8 M Urea, and the DNA bands were visualized by autoradiography. The position of 29 mer substrate and 18 mer incision product is indicated. (D) Graphical representation of results in C. The data in the graph represent mean (±standard errors) of two independent experiments carried out in duplicates.</p

Sequence alignments of XthA with other AP endonuclease homologs.

<p>Alignment of the C-terminal 127 residues of MtbXthA (Rv0427c), with non-functional AP endonuclease paralogue of <i>N.meningitidis</i> (Nexo) and functional AP endonucleases of <i>E.coli</i> (ECExoIII), <i>N.meningitidis</i> (Nape) and <i>Homo sapiens</i> (Hap1) indicates the plausible role of histidine (H201) for low AP endonuclease activity. The residues involved in abasic ribose binding and catalysis are indicated in brown and green color, respectively. Absolutely conserved residues in the alignment are colored in dark blue and light blue for partial identity. Secondary structure elements are shown in red for α-helices and green for β-strands. CLUSTALW was employed for the initial multiple sequence alignment of proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071535#pone.0071535-Thompson1" target="_blank">[71]</a>. This was followed by using the programme JalView <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071535#pone.0071535-Clamp1" target="_blank">[72]</a> for the prediction of conserved residues and secondary structure.</p

Preferential recognition of cytosine opposite the abasic site by End and XthA.

<p>Each of the four 5′ end-labeled substrates subAP·T/A/G/C (800 fmol) were separately incubated with 2 fmol of either End (A) or XthA (B) for 25 min at 37°C. The AP endonuclease activity was measured under standard assay conditions and plotted as relative activities when acting on different duplex substrates. The data represents mean (±standard errors) of two independent experiments carried out in duplicates.</p

DNA substrates employed in the study.

<p>SS is single-strand; DS is double-strand; nt is nucleotides; AP is the abasic site; asteriks (*) denotes the γ-³²P radioactively labelled terminus.</p

3′→5′ exonuclease activity of End and XthA on AP substrate.

<p>Schematic diagram of oligonucleotide substrates in which asterisk (*) and AP denote the radioactively labeled γ-³²P terminus and the abasic site in the substrate, respectively. 5′ end-labeled substrate subDS-AP (20 fmol) was incubated with 10 fmol of either End (A) or XthA (B) for variable time at 37°C. (A) Lanes 1–5, incubation of substrate with End in standard buffer for 0, 1, 2, 5 and 15 min, respectively. (B) Lanes 1–4, incubation of substrate with XthA in standard buffer containing 10 mM MgCl₂ for 0, 1, 2 and 15 min, respectively. Lane 5, XthA was incubated with the substrate for 15 min in standard buffer without Magnesium. The position of 29 mer substrate and 18 mer incision product is indicated.</p