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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.

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Inhibition of ASFV pE296R 3′ → 5′ exonuclease and NIR activities by DTT. Assays for 3′ → 5′ exonuclease were carried out with 5′-[32P]-labelled ExoC/RexA oligonucleotide duplex, while NIR activity was analyzed with 5′-[32P]-labelled RT-DHU/CompG. The configuration of the substrate is shown above (D stands for DHU) Enzyme concentration is shown for each group of samples. (A) PAGE analysis of 3′ → 5′ exonuclease degradation and NIR cleavage products. Lanes 1 and 10, no enzyme; lanes 2–9 and 11–18, 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively. Note that an empty lane separates lanes 13 and 14. (B) Graphic representation of inhibition of exonuclease and NIR activities by DTT. The 100% activity corresponds to the maximal activity obtained in each experiment. Each point is the mean of two independent experiments. The error bar indicates standard deviation.
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fig2: Inhibition of ASFV pE296R 3′ → 5′ exonuclease and NIR activities by DTT. Assays for 3′ → 5′ exonuclease were carried out with 5′-[32P]-labelled ExoC/RexA oligonucleotide duplex, while NIR activity was analyzed with 5′-[32P]-labelled RT-DHU/CompG. The configuration of the substrate is shown above (D stands for DHU) Enzyme concentration is shown for each group of samples. (A) PAGE analysis of 3′ → 5′ exonuclease degradation and NIR cleavage products. Lanes 1 and 10, no enzyme; lanes 2–9 and 11–18, 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively. Note that an empty lane separates lanes 13 and 14. (B) Graphic representation of inhibition of exonuclease and NIR activities by DTT. The 100% activity corresponds to the maximal activity obtained in each experiment. Each point is the mean of two independent experiments. The error bar indicates standard deviation.

Mentions: To further examine the redox modulation of protein pE296R in vitro, we also measured the effect of DTT on the 3′ → 5′ exonuclease, NIR, 3′-phosphatase and 3′-phosphodiesterase activities of the ASFV protein. DTT inhibited the exonuclease activity of pE296R (Fig. 2). Moreover, incubation with H2O2 turned back the DTT effect (data not shown).


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)

Inhibition of ASFV pE296R 3′ → 5′ exonuclease and NIR activities by DTT. Assays for 3′ → 5′ exonuclease were carried out with 5′-[32P]-labelled ExoC/RexA oligonucleotide duplex, while NIR activity was analyzed with 5′-[32P]-labelled RT-DHU/CompG. The configuration of the substrate is shown above (D stands for DHU) Enzyme concentration is shown for each group of samples. (A) PAGE analysis of 3′ → 5′ exonuclease degradation and NIR cleavage products. Lanes 1 and 10, no enzyme; lanes 2–9 and 11–18, 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively. Note that an empty lane separates lanes 13 and 14. (B) Graphic representation of inhibition of exonuclease and NIR activities by DTT. The 100% activity corresponds to the maximal activity obtained in each experiment. Each point is the mean of two independent experiments. The error bar indicates standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Inhibition of ASFV pE296R 3′ → 5′ exonuclease and NIR activities by DTT. Assays for 3′ → 5′ exonuclease were carried out with 5′-[32P]-labelled ExoC/RexA oligonucleotide duplex, while NIR activity was analyzed with 5′-[32P]-labelled RT-DHU/CompG. The configuration of the substrate is shown above (D stands for DHU) Enzyme concentration is shown for each group of samples. (A) PAGE analysis of 3′ → 5′ exonuclease degradation and NIR cleavage products. Lanes 1 and 10, no enzyme; lanes 2–9 and 11–18, 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively. Note that an empty lane separates lanes 13 and 14. (B) Graphic representation of inhibition of exonuclease and NIR activities by DTT. The 100% activity corresponds to the maximal activity obtained in each experiment. Each point is the mean of two independent experiments. The error bar indicates standard deviation.
Mentions: To further examine the redox modulation of protein pE296R in vitro, we also measured the effect of DTT on the 3′ → 5′ exonuclease, NIR, 3′-phosphatase and 3′-phosphodiesterase activities of the ASFV protein. DTT inhibited the exonuclease activity of pE296R (Fig. 2). Moreover, incubation with H2O2 turned back the DTT effect (data not shown).

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