Limits...
African swine fever virus AP endonuclease is a redox-sensitive enzyme that repairs alkylating and oxidative damage to DNA.

Redrejo-Rodríguez M, Ishchenko AA, Saparbaev MK, Salas ML, Salas J - Virology (2009)

Bottom Line: Protein pE296R contains one intramolecular disulfide bond, whose disruption by reducing agents might perturb the interaction of the viral AP endonuclease with the DNA substrate.The characterization of the 3'-->5' exonuclease and 3'-repair diesterase activities of pE296R indicates that it has strong preference for mispaired and oxidative base lesions at the 3'-termini of single-strand breaks.Finally, the viral protein protects against DNA damaging agents in both prokaryotic and eukaryotic cells, emphasizing its importance in vivo.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas/Universidad Autónoma de Madrid), Universidad Autónoma de Madrid, C/Nicolás Cabrera 1, 28049 Madrid, Spain.

ABSTRACT
African swine fever virus (ASFV) encodes an AP endonuclease (pE296R) which is essential for virus growth in swine macrophages. We show here that the DNA repair functions of pE296R (AP endonucleolytic, 3'-->5' exonuclease, 3'-diesterase and nucleotide incision repair (NIR) activities) and DNA binding are inhibited by reducing agents. Protein pE296R contains one intramolecular disulfide bond, whose disruption by reducing agents might perturb the interaction of the viral AP endonuclease with the DNA substrate. The characterization of the 3'-->5' exonuclease and 3'-repair diesterase activities of pE296R indicates that it has strong preference for mispaired and oxidative base lesions at the 3'-termini of single-strand breaks. Finally, the viral protein protects against DNA damaging agents in both prokaryotic and eukaryotic cells, emphasizing its importance in vivo. The biochemical and genetic properties of ASFV AP endonuclease are consistent with the repair of DNA damage generated by the genotoxic intracellular environment of the host macrophage.

Show MeSH

Related in: MedlinePlus

Redox-dependent modulation of ASFV AP endonucleolytic activity. AP endonuclease assays were carried out with 5′-[32P]-labelled RT-THF:CompT oligonucleotide duplex in the presence of 1 nM ASFV pE296R or 0.5 nM APE1, as indicated. The configuration of the substrate is indicated above (F stands for THF). (A) Effect of reducing agents. PAGE analysis of the reaction products. Lane 1, no enzyme; lanes 2–9, 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively; lanes 10–14, 0, 2, 10, 20 and 40 mM β-ME, respectively. (B) Graphic of inhibition of ASFV pE296R-catalyzed AP endonuclease activity by DTT and β-ME (inset). The 100% activity corresponds to the maximal enzyme activity obtained with 0.05 mM DTT or in absence of β-ME in the inset. Each point is the mean of two independent experiments. The error bar indicates standard deviation. (C) Reversion of DTT inhibition by H2O2. PAGE analysis of the reaction products. The proteins were first incubated with or without DTT (20 min, 4 °C) and then with or without H2O2 (10 min, 4 °C) before performing the incision experiment. Lanes 1 and 6, without DTT or H2O2; Lanes 2 and 7, 5 mM DTT; lanes 3 and 8, 10 mM H2O2;; lanes 4 and 5, 10 mM H2O2 and 1 and 10 mM DTT, respectively; lanes 9–13, 5 mM DTT and 1, 2, 5, 10 and 25 mM H2O2, respectively. (D) Graphic of reversion by H2O2 of the DTT inhibition of ASFV pE296R AP endonuclease activity. The 100% activity corresponds to the maximal enzyme activity obtained in the presence of 0.05 mM DTT.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2958311&req=5

fig1: Redox-dependent modulation of ASFV AP endonucleolytic activity. AP endonuclease assays were carried out with 5′-[32P]-labelled RT-THF:CompT oligonucleotide duplex in the presence of 1 nM ASFV pE296R or 0.5 nM APE1, as indicated. The configuration of the substrate is indicated above (F stands for THF). (A) Effect of reducing agents. PAGE analysis of the reaction products. Lane 1, no enzyme; lanes 2–9, 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively; lanes 10–14, 0, 2, 10, 20 and 40 mM β-ME, respectively. (B) Graphic of inhibition of ASFV pE296R-catalyzed AP endonuclease activity by DTT and β-ME (inset). The 100% activity corresponds to the maximal enzyme activity obtained with 0.05 mM DTT or in absence of β-ME in the inset. Each point is the mean of two independent experiments. The error bar indicates standard deviation. (C) Reversion of DTT inhibition by H2O2. PAGE analysis of the reaction products. The proteins were first incubated with or without DTT (20 min, 4 °C) and then with or without H2O2 (10 min, 4 °C) before performing the incision experiment. Lanes 1 and 6, without DTT or H2O2; Lanes 2 and 7, 5 mM DTT; lanes 3 and 8, 10 mM H2O2;; lanes 4 and 5, 10 mM H2O2 and 1 and 10 mM DTT, respectively; lanes 9–13, 5 mM DTT and 1, 2, 5, 10 and 25 mM H2O2, respectively. (D) Graphic of reversion by H2O2 of the DTT inhibition of ASFV pE296R AP endonuclease activity. The 100% activity corresponds to the maximal enzyme activity obtained in the presence of 0.05 mM DTT.

Mentions: In previous studies, dithiothreitol (DTT) has been frequently used in reaction buffers for various AP endonucleases of different origins (Levin et al., 1988; Salas-Pacheco et al., 2003; Wilson, 2005), including ASFV AP endonuclease (Lamarche and Tsai, 2006; Redrejo-Rodríguez et al., 2006). However, it has been shown that DTT strongly inhibits the 3′ → 5′ exonuclease activity of Nfo (Kerins et al., 2003). Since it was possible that reducing agents might also inhibit any of the different repair activities of pE296R protein, we performed, as a first approach, AP endonuclease assays with the viral enzyme. The assays were carried out with an AP site-containing duplex oligonucleotide, in the presence of different concentrations of DTT and β-mercaptoethanol (β-ME) (Fig. 1). The AP endonucleolytic activity of pE296R shows a slight increase in the presence of a low DTT concentration (50 μM, Fig. 1A, lane 3 and Fig. 1B), but at 1 mM and higher DTT concentrations AP site incision was strongly inhibited with less than 10% of the activity remaining (Fig. 1A, lanes 7–9, and Fig. 1B). With β-ME, almost complete inhibition is observed at concentrations of 10 mM and higher (Fig. 1A, lanes 12–14 and Fig. 1B, inset).


African swine fever virus AP endonuclease is a redox-sensitive enzyme that repairs alkylating and oxidative damage to DNA.

Redrejo-Rodríguez M, Ishchenko AA, Saparbaev MK, Salas ML, Salas J - Virology (2009)

Redox-dependent modulation of ASFV AP endonucleolytic activity. AP endonuclease assays were carried out with 5′-[32P]-labelled RT-THF:CompT oligonucleotide duplex in the presence of 1 nM ASFV pE296R or 0.5 nM APE1, as indicated. The configuration of the substrate is indicated above (F stands for THF). (A) Effect of reducing agents. PAGE analysis of the reaction products. Lane 1, no enzyme; lanes 2–9, 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively; lanes 10–14, 0, 2, 10, 20 and 40 mM β-ME, respectively. (B) Graphic of inhibition of ASFV pE296R-catalyzed AP endonuclease activity by DTT and β-ME (inset). The 100% activity corresponds to the maximal enzyme activity obtained with 0.05 mM DTT or in absence of β-ME in the inset. Each point is the mean of two independent experiments. The error bar indicates standard deviation. (C) Reversion of DTT inhibition by H2O2. PAGE analysis of the reaction products. The proteins were first incubated with or without DTT (20 min, 4 °C) and then with or without H2O2 (10 min, 4 °C) before performing the incision experiment. Lanes 1 and 6, without DTT or H2O2; Lanes 2 and 7, 5 mM DTT; lanes 3 and 8, 10 mM H2O2;; lanes 4 and 5, 10 mM H2O2 and 1 and 10 mM DTT, respectively; lanes 9–13, 5 mM DTT and 1, 2, 5, 10 and 25 mM H2O2, respectively. (D) Graphic of reversion by H2O2 of the DTT inhibition of ASFV pE296R AP endonuclease activity. The 100% activity corresponds to the maximal enzyme activity obtained in the presence of 0.05 mM DTT.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2958311&req=5

fig1: Redox-dependent modulation of ASFV AP endonucleolytic activity. AP endonuclease assays were carried out with 5′-[32P]-labelled RT-THF:CompT oligonucleotide duplex in the presence of 1 nM ASFV pE296R or 0.5 nM APE1, as indicated. The configuration of the substrate is indicated above (F stands for THF). (A) Effect of reducing agents. PAGE analysis of the reaction products. Lane 1, no enzyme; lanes 2–9, 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively; lanes 10–14, 0, 2, 10, 20 and 40 mM β-ME, respectively. (B) Graphic of inhibition of ASFV pE296R-catalyzed AP endonuclease activity by DTT and β-ME (inset). The 100% activity corresponds to the maximal enzyme activity obtained with 0.05 mM DTT or in absence of β-ME in the inset. Each point is the mean of two independent experiments. The error bar indicates standard deviation. (C) Reversion of DTT inhibition by H2O2. PAGE analysis of the reaction products. The proteins were first incubated with or without DTT (20 min, 4 °C) and then with or without H2O2 (10 min, 4 °C) before performing the incision experiment. Lanes 1 and 6, without DTT or H2O2; Lanes 2 and 7, 5 mM DTT; lanes 3 and 8, 10 mM H2O2;; lanes 4 and 5, 10 mM H2O2 and 1 and 10 mM DTT, respectively; lanes 9–13, 5 mM DTT and 1, 2, 5, 10 and 25 mM H2O2, respectively. (D) Graphic of reversion by H2O2 of the DTT inhibition of ASFV pE296R AP endonuclease activity. The 100% activity corresponds to the maximal enzyme activity obtained in the presence of 0.05 mM DTT.
Mentions: In previous studies, dithiothreitol (DTT) has been frequently used in reaction buffers for various AP endonucleases of different origins (Levin et al., 1988; Salas-Pacheco et al., 2003; Wilson, 2005), including ASFV AP endonuclease (Lamarche and Tsai, 2006; Redrejo-Rodríguez et al., 2006). However, it has been shown that DTT strongly inhibits the 3′ → 5′ exonuclease activity of Nfo (Kerins et al., 2003). Since it was possible that reducing agents might also inhibit any of the different repair activities of pE296R protein, we performed, as a first approach, AP endonuclease assays with the viral enzyme. The assays were carried out with an AP site-containing duplex oligonucleotide, in the presence of different concentrations of DTT and β-mercaptoethanol (β-ME) (Fig. 1). The AP endonucleolytic activity of pE296R shows a slight increase in the presence of a low DTT concentration (50 μM, Fig. 1A, lane 3 and Fig. 1B), but at 1 mM and higher DTT concentrations AP site incision was strongly inhibited with less than 10% of the activity remaining (Fig. 1A, lanes 7–9, and Fig. 1B). With β-ME, almost complete inhibition is observed at concentrations of 10 mM and higher (Fig. 1A, lanes 12–14 and Fig. 1B, inset).

Bottom Line: Protein pE296R contains one intramolecular disulfide bond, whose disruption by reducing agents might perturb the interaction of the viral AP endonuclease with the DNA substrate.The characterization of the 3'-->5' exonuclease and 3'-repair diesterase activities of pE296R indicates that it has strong preference for mispaired and oxidative base lesions at the 3'-termini of single-strand breaks.Finally, the viral protein protects against DNA damaging agents in both prokaryotic and eukaryotic cells, emphasizing its importance in vivo.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas/Universidad Autónoma de Madrid), Universidad Autónoma de Madrid, C/Nicolás Cabrera 1, 28049 Madrid, Spain.

ABSTRACT
African swine fever virus (ASFV) encodes an AP endonuclease (pE296R) which is essential for virus growth in swine macrophages. We show here that the DNA repair functions of pE296R (AP endonucleolytic, 3'-->5' exonuclease, 3'-diesterase and nucleotide incision repair (NIR) activities) and DNA binding are inhibited by reducing agents. Protein pE296R contains one intramolecular disulfide bond, whose disruption by reducing agents might perturb the interaction of the viral AP endonuclease with the DNA substrate. The characterization of the 3'-->5' exonuclease and 3'-repair diesterase activities of pE296R indicates that it has strong preference for mispaired and oxidative base lesions at the 3'-termini of single-strand breaks. Finally, the viral protein protects against DNA damaging agents in both prokaryotic and eukaryotic cells, emphasizing its importance in vivo. The biochemical and genetic properties of ASFV AP endonuclease are consistent with the repair of DNA damage generated by the genotoxic intracellular environment of the host macrophage.

Show MeSH
Related in: MedlinePlus