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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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E296R gene expression-induced protection against drugs. (A) Drug sensitivity of E. coli strains carrying the E296R and Nfo genes. Graphs correspond to the sensitivity of E. coli BH110 (xth nfo) carrying pRSETA (black circle), pET11A-Nfo (white circle) and pRSETA-E296R (black square) to H2O2, t-BuO2H and MMS exposure. (B) Titers of BA71V wild type (black square) and vΔE296R (white circle) virus infections in the presence of H2O2, t-BuO2H and MMS.
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fig7: E296R gene expression-induced protection against drugs. (A) Drug sensitivity of E. coli strains carrying the E296R and Nfo genes. Graphs correspond to the sensitivity of E. coli BH110 (xth nfo) carrying pRSETA (black circle), pET11A-Nfo (white circle) and pRSETA-E296R (black square) to H2O2, t-BuO2H and MMS exposure. (B) Titers of BA71V wild type (black square) and vΔE296R (white circle) virus infections in the presence of H2O2, t-BuO2H and MMS.

Mentions: To address the biological role of the various activities of ASFV AP endonuclease, we used two different approaches. First, we carried out complementation assays for the E. coli xth nfo strain (BH110) exposed to various genotoxic agents. Among the oxidizing agents, H2O2 is produced by the host immune cells infected with certain viruses and therefore might generate oxidative lesions in the ASFV DNA (Israel and Gougerot-Pocidalo, 1997; Suzuki et al., 1997). It would thus be of particular interest to determine the capacity of the pE296R protein to confer protection to bacteria cells against H2O2 treatment. We used Nfo as a positive control for drug-sensitivity complementation of BH110 (Cunningham et al., 1986). As shown in Fig. 7A, the plasmids encoding pE296R and Nfo proteins, in contrast to empty vector, conferred resistance to BH110 cells when exposed to H2O2. Protection by the ASFV E296R gene against t-BuO2H and MMS was also observed (Fig. 7A), confirming previous results in E. coli (Lamarche and Tsai, 2006). Altogether, these data suggest that the viral AP endonuclease can repair 3′-blocking groups, oxidized bases and AP sites in vivo. Interestingly, the protection against H2O2 and MMS provided by both pE296R and Nfo endonucleases was very similar, suggesting highly efficient properties of the viral AP endonuclease to counteract DNA damage in vivo. It has been shown that expression of Nfo-G149D point mutant, that lacks the NIR activity but maintains the AP endonuclease activity in vitro, can complement double nfo xth mutant to the alkylating agent MMS challenge but not to t-BuO2H, suggesting that the NIR activity is involved in the removal of t-BuO2H-induced DNA damage (Ishchenko et al., 2006) Thereby, although in vitro the ASFV AP endonuclease-catalyzed NIR activity is weaker compared to other DNA repair functions, the protection of bacterial cells by pE296R against t-BuO2H exposure (Fig. 7A, central panel) suggest a potential role of pE296R in the repair of oxidative DNA base lesions via the DNA glycosylase-independent NIR pathway in vivo. These results suggest that the viral AP endonuclease possesses all the DNA repair functions of the host homologous AP endonuclease to remove damage to cellular DNA induced by various genotoxic agents.


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)

E296R gene expression-induced protection against drugs. (A) Drug sensitivity of E. coli strains carrying the E296R and Nfo genes. Graphs correspond to the sensitivity of E. coli BH110 (xth nfo) carrying pRSETA (black circle), pET11A-Nfo (white circle) and pRSETA-E296R (black square) to H2O2, t-BuO2H and MMS exposure. (B) Titers of BA71V wild type (black square) and vΔE296R (white circle) virus infections in the presence of H2O2, t-BuO2H and MMS.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958311&req=5

fig7: E296R gene expression-induced protection against drugs. (A) Drug sensitivity of E. coli strains carrying the E296R and Nfo genes. Graphs correspond to the sensitivity of E. coli BH110 (xth nfo) carrying pRSETA (black circle), pET11A-Nfo (white circle) and pRSETA-E296R (black square) to H2O2, t-BuO2H and MMS exposure. (B) Titers of BA71V wild type (black square) and vΔE296R (white circle) virus infections in the presence of H2O2, t-BuO2H and MMS.
Mentions: To address the biological role of the various activities of ASFV AP endonuclease, we used two different approaches. First, we carried out complementation assays for the E. coli xth nfo strain (BH110) exposed to various genotoxic agents. Among the oxidizing agents, H2O2 is produced by the host immune cells infected with certain viruses and therefore might generate oxidative lesions in the ASFV DNA (Israel and Gougerot-Pocidalo, 1997; Suzuki et al., 1997). It would thus be of particular interest to determine the capacity of the pE296R protein to confer protection to bacteria cells against H2O2 treatment. We used Nfo as a positive control for drug-sensitivity complementation of BH110 (Cunningham et al., 1986). As shown in Fig. 7A, the plasmids encoding pE296R and Nfo proteins, in contrast to empty vector, conferred resistance to BH110 cells when exposed to H2O2. Protection by the ASFV E296R gene against t-BuO2H and MMS was also observed (Fig. 7A), confirming previous results in E. coli (Lamarche and Tsai, 2006). Altogether, these data suggest that the viral AP endonuclease can repair 3′-blocking groups, oxidized bases and AP sites in vivo. Interestingly, the protection against H2O2 and MMS provided by both pE296R and Nfo endonucleases was very similar, suggesting highly efficient properties of the viral AP endonuclease to counteract DNA damage in vivo. It has been shown that expression of Nfo-G149D point mutant, that lacks the NIR activity but maintains the AP endonuclease activity in vitro, can complement double nfo xth mutant to the alkylating agent MMS challenge but not to t-BuO2H, suggesting that the NIR activity is involved in the removal of t-BuO2H-induced DNA damage (Ishchenko et al., 2006) Thereby, although in vitro the ASFV AP endonuclease-catalyzed NIR activity is weaker compared to other DNA repair functions, the protection of bacterial cells by pE296R against t-BuO2H exposure (Fig. 7A, central panel) suggest a potential role of pE296R in the repair of oxidative DNA base lesions via the DNA glycosylase-independent NIR pathway in vivo. These results suggest that the viral AP endonuclease possesses all the DNA repair functions of the host homologous AP endonuclease to remove damage to cellular DNA induced by various genotoxic agents.

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