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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 pE296R DNA binding by DTT. Electrophoretic mobility shift assay (EMSA) was carried out with 5′-[32P]-labelled RT-S/CompT oligonucleotide duplex. The configuration of the substrate is indicated above (S stands for a 5′-phosphothioate-bound AP site). (A) Non-denaturing gel electrophoresis. Lane 1, no enzyme; lanes 2–9, in the presence of 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively. The bands corresponding to free DNA are indicated with an open arrow and bound DNA with a black arrow. (B) Graphic representation of the DNA binding inhibition by DTT. The 100% binding corresponds to the maximal band shift obtained in the presence of 0.05 mM DTT. Each point is the mean of three independent experiments. The error bar indicates standard deviation.
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fig3: Inhibition of pE296R DNA binding by DTT. Electrophoretic mobility shift assay (EMSA) was carried out with 5′-[32P]-labelled RT-S/CompT oligonucleotide duplex. The configuration of the substrate is indicated above (S stands for a 5′-phosphothioate-bound AP site). (A) Non-denaturing gel electrophoresis. Lane 1, no enzyme; lanes 2–9, in the presence of 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively. The bands corresponding to free DNA are indicated with an open arrow and bound DNA with a black arrow. (B) Graphic representation of the DNA binding inhibition by DTT. The 100% binding corresponds to the maximal band shift obtained in the presence of 0.05 mM DTT. Each point is the mean of three independent experiments. The error bar indicates standard deviation.

Mentions: To examine the mechanism of inhibition of DNA repair activities of the viral protein by reducing agents, we measured pE296R binding to an AP site-containing DNA duplex using electrophoretic mobility shift assay (EMSA) (Fig. 3). We used an oligonucleotide with a 5′-phosphothioate bound to the abasic sugar, to block AP endonuclease incision activity. About 45% of the substrate (open head arrow) in the absence of DTT was in complex with the enzyme and produced a shifted band (black head arrow) (Fig. 3A, lane 2 and B). The increase of DTT concentration produced a decrease of protein–DNA complex formation (Fig. 3A, lanes 3–9 and B). Moreover, the patterns of inhibition of endonuclease activity and DNA binding are similar, suggesting that the inhibition of catalytic activity of the viral protein by reducing agents is due to impairment of the recognition/binding step. This is further supported by the difference in KM values at 0 and 0.5 mM DTT.


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 pE296R DNA binding by DTT. Electrophoretic mobility shift assay (EMSA) was carried out with 5′-[32P]-labelled RT-S/CompT oligonucleotide duplex. The configuration of the substrate is indicated above (S stands for a 5′-phosphothioate-bound AP site). (A) Non-denaturing gel electrophoresis. Lane 1, no enzyme; lanes 2–9, in the presence of 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively. The bands corresponding to free DNA are indicated with an open arrow and bound DNA with a black arrow. (B) Graphic representation of the DNA binding inhibition by DTT. The 100% binding corresponds to the maximal band shift obtained in the presence of 0.05 mM DTT. Each point is the mean of three independent experiments. The error bar indicates standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Inhibition of pE296R DNA binding by DTT. Electrophoretic mobility shift assay (EMSA) was carried out with 5′-[32P]-labelled RT-S/CompT oligonucleotide duplex. The configuration of the substrate is indicated above (S stands for a 5′-phosphothioate-bound AP site). (A) Non-denaturing gel electrophoresis. Lane 1, no enzyme; lanes 2–9, in the presence of 0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM DTT, respectively. The bands corresponding to free DNA are indicated with an open arrow and bound DNA with a black arrow. (B) Graphic representation of the DNA binding inhibition by DTT. The 100% binding corresponds to the maximal band shift obtained in the presence of 0.05 mM DTT. Each point is the mean of three independent experiments. The error bar indicates standard deviation.
Mentions: To examine the mechanism of inhibition of DNA repair activities of the viral protein by reducing agents, we measured pE296R binding to an AP site-containing DNA duplex using electrophoretic mobility shift assay (EMSA) (Fig. 3). We used an oligonucleotide with a 5′-phosphothioate bound to the abasic sugar, to block AP endonuclease incision activity. About 45% of the substrate (open head arrow) in the absence of DTT was in complex with the enzyme and produced a shifted band (black head arrow) (Fig. 3A, lane 2 and B). The increase of DTT concentration produced a decrease of protein–DNA complex formation (Fig. 3A, lanes 3–9 and B). Moreover, the patterns of inhibition of endonuclease activity and DNA binding are similar, suggesting that the inhibition of catalytic activity of the viral protein by reducing agents is due to impairment of the recognition/binding step. This is further supported by the difference in KM values at 0 and 0.5 mM DTT.

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