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Identification of a residue critical for the excision of 3'-blocking ends in apurinic/apyrimidinic endonucleases of the Xth family.

Castillo-Acosta VM, Ruiz-Pérez LM, Yang W, González-Pacanowska D, Vidal AE - Nucleic Acids Res. (2009)

Bottom Line: Among the resistant clones, we isolated a mutant in the nuclease domain of APE1 (D70A) with an increased capacity to remove 3'-blocking ends in vitro.D70 of APE1 aligns with A138 of LMAP and mutation of the latter to aspartate significantly reduces its 3'-phosphodiesterase activity.Kinetic analysis shows a novel role of residue D70 in the excision rate of 3'-blocking ends.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Armilla (Granada), Spain.

ABSTRACT
DNA single-strand breaks containing 3'-blocking groups are generated from attack of the sugar backbone by reactive oxygen species or after base excision by DNA glycosylase/apurinic/apyrimidinic (AP) lyases. In human cells, APE1 excises sugar fragments that block the 3'-ends thus facilitating DNA repair synthesis. In Leishmania major, the causal agent of leishmaniasis, the APE1 homolog is the class II AP endonuclease LMAP. Expression of LMAP but not of APE1 reverts the hypersensitivity of a xth nfo repair-deficient Escherichia coli strain to the oxidative compound hydrogen peroxide (H(2)O(2)). To identify the residues specifically involved in the repair of oxidative DNA damage, we generated random mutations in the ape1 gene and selected those variants that conferred protection against H(2)O(2). Among the resistant clones, we isolated a mutant in the nuclease domain of APE1 (D70A) with an increased capacity to remove 3'-blocking ends in vitro. D70 of APE1 aligns with A138 of LMAP and mutation of the latter to aspartate significantly reduces its 3'-phosphodiesterase activity. Kinetic analysis shows a novel role of residue D70 in the excision rate of 3'-blocking ends. The functional and structural differences between the parasite and human enzymes probably reflect a divergent molecular evolution of their DNA repair responses to oxidative damage.

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Comparison of the activities of wild-type and mutant AP endonucleases at different magnesium concentrations. (A and B) The 3′-phosphodiesterase and (C and D) AP endonuclease activity assays were carried out in standard buffer with increasing Mg2+ concentrations (1–40 mM) by incubating the enzymes with 500 nM of the 3′-PG or THF substrate, respectively. Specific activities (left) and relative activities (right) are represented for each of the enzymes. Relative activities were calculated assigning a value of 100% to the maximal activity reached for an enzyme at a given Mg2+ concentration. To monitor the excision of phosphoglycolate and AP site incision in the absence of magnesium in the reaction mixture, a reaction with 20 mM EDTA was included. Enzymes are represented with the following symbols: APE1 (filled circle), APE1D70A (open circle), LMAP (filled square) and LMAPA138D (open square). Units are defined as picomoles of reaction product generated per minute at 37°C.
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Figure 6: Comparison of the activities of wild-type and mutant AP endonucleases at different magnesium concentrations. (A and B) The 3′-phosphodiesterase and (C and D) AP endonuclease activity assays were carried out in standard buffer with increasing Mg2+ concentrations (1–40 mM) by incubating the enzymes with 500 nM of the 3′-PG or THF substrate, respectively. Specific activities (left) and relative activities (right) are represented for each of the enzymes. Relative activities were calculated assigning a value of 100% to the maximal activity reached for an enzyme at a given Mg2+ concentration. To monitor the excision of phosphoglycolate and AP site incision in the absence of magnesium in the reaction mixture, a reaction with 20 mM EDTA was included. Enzymes are represented with the following symbols: APE1 (filled circle), APE1D70A (open circle), LMAP (filled square) and LMAPA138D (open square). Units are defined as picomoles of reaction product generated per minute at 37°C.

Mentions: Structural and mutagenesis data have led to postulate the participation of the carboxylate group of D70 in the coordination of one metal ion during the catalytical process that leads to AP site cleavage. However, the function of D70 appears to be restricted to mammalian AP endonucleases since this amino acid residue is absent in the catalytic sites of protozoan and prokaryotic enzymes. To investigate the role of D70 in metal ion coordination, we compared the activities of APE1, LMAP and their respective mutant forms over a range of Mg2+ concentrations. Irrespectively of the concentration tested, APE1D70A and LMAP showed higher 3′-phosphodiesterase activity than APE1 and LMAPA138D (Figure 6A and B, left). In agreement with previous studies, APE1 (and LMAPA138D) reached a maximum of 3′-PG excision activity at Mg2+ concentrations around 2.5 mM, the excision being severely inhibited (below 25%) at 10 mM and higher ion concentrations (Figure 6A and B, right) (45,46). In contrast, APE1D70A and LMAP exhibited a different pattern of inhibition by magnesium. Both enzymes reached maximum activity at higher metal concentrations and were more refractory to Mg2+ inhibition, thus still retaining about 50% of activity at 20 mM (Figure 6A and B). These results confirm that amino acid changes D70A in APE1 and A138D in LMAP have opposing effects on the 3′-phosphodiesterase activity and show that the residue D70 has an important role in magnesium-mediated inhibition. Thus, when a certain metal concentration is reached, D70 may bind to Mg2+ which results in inhibition of the enzyme activity.Figure 6.


Identification of a residue critical for the excision of 3'-blocking ends in apurinic/apyrimidinic endonucleases of the Xth family.

Castillo-Acosta VM, Ruiz-Pérez LM, Yang W, González-Pacanowska D, Vidal AE - Nucleic Acids Res. (2009)

Comparison of the activities of wild-type and mutant AP endonucleases at different magnesium concentrations. (A and B) The 3′-phosphodiesterase and (C and D) AP endonuclease activity assays were carried out in standard buffer with increasing Mg2+ concentrations (1–40 mM) by incubating the enzymes with 500 nM of the 3′-PG or THF substrate, respectively. Specific activities (left) and relative activities (right) are represented for each of the enzymes. Relative activities were calculated assigning a value of 100% to the maximal activity reached for an enzyme at a given Mg2+ concentration. To monitor the excision of phosphoglycolate and AP site incision in the absence of magnesium in the reaction mixture, a reaction with 20 mM EDTA was included. Enzymes are represented with the following symbols: APE1 (filled circle), APE1D70A (open circle), LMAP (filled square) and LMAPA138D (open square). Units are defined as picomoles of reaction product generated per minute at 37°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: Comparison of the activities of wild-type and mutant AP endonucleases at different magnesium concentrations. (A and B) The 3′-phosphodiesterase and (C and D) AP endonuclease activity assays were carried out in standard buffer with increasing Mg2+ concentrations (1–40 mM) by incubating the enzymes with 500 nM of the 3′-PG or THF substrate, respectively. Specific activities (left) and relative activities (right) are represented for each of the enzymes. Relative activities were calculated assigning a value of 100% to the maximal activity reached for an enzyme at a given Mg2+ concentration. To monitor the excision of phosphoglycolate and AP site incision in the absence of magnesium in the reaction mixture, a reaction with 20 mM EDTA was included. Enzymes are represented with the following symbols: APE1 (filled circle), APE1D70A (open circle), LMAP (filled square) and LMAPA138D (open square). Units are defined as picomoles of reaction product generated per minute at 37°C.
Mentions: Structural and mutagenesis data have led to postulate the participation of the carboxylate group of D70 in the coordination of one metal ion during the catalytical process that leads to AP site cleavage. However, the function of D70 appears to be restricted to mammalian AP endonucleases since this amino acid residue is absent in the catalytic sites of protozoan and prokaryotic enzymes. To investigate the role of D70 in metal ion coordination, we compared the activities of APE1, LMAP and their respective mutant forms over a range of Mg2+ concentrations. Irrespectively of the concentration tested, APE1D70A and LMAP showed higher 3′-phosphodiesterase activity than APE1 and LMAPA138D (Figure 6A and B, left). In agreement with previous studies, APE1 (and LMAPA138D) reached a maximum of 3′-PG excision activity at Mg2+ concentrations around 2.5 mM, the excision being severely inhibited (below 25%) at 10 mM and higher ion concentrations (Figure 6A and B, right) (45,46). In contrast, APE1D70A and LMAP exhibited a different pattern of inhibition by magnesium. Both enzymes reached maximum activity at higher metal concentrations and were more refractory to Mg2+ inhibition, thus still retaining about 50% of activity at 20 mM (Figure 6A and B). These results confirm that amino acid changes D70A in APE1 and A138D in LMAP have opposing effects on the 3′-phosphodiesterase activity and show that the residue D70 has an important role in magnesium-mediated inhibition. Thus, when a certain metal concentration is reached, D70 may bind to Mg2+ which results in inhibition of the enzyme activity.Figure 6.

Bottom Line: Among the resistant clones, we isolated a mutant in the nuclease domain of APE1 (D70A) with an increased capacity to remove 3'-blocking ends in vitro.D70 of APE1 aligns with A138 of LMAP and mutation of the latter to aspartate significantly reduces its 3'-phosphodiesterase activity.Kinetic analysis shows a novel role of residue D70 in the excision rate of 3'-blocking ends.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Armilla (Granada), Spain.

ABSTRACT
DNA single-strand breaks containing 3'-blocking groups are generated from attack of the sugar backbone by reactive oxygen species or after base excision by DNA glycosylase/apurinic/apyrimidinic (AP) lyases. In human cells, APE1 excises sugar fragments that block the 3'-ends thus facilitating DNA repair synthesis. In Leishmania major, the causal agent of leishmaniasis, the APE1 homolog is the class II AP endonuclease LMAP. Expression of LMAP but not of APE1 reverts the hypersensitivity of a xth nfo repair-deficient Escherichia coli strain to the oxidative compound hydrogen peroxide (H(2)O(2)). To identify the residues specifically involved in the repair of oxidative DNA damage, we generated random mutations in the ape1 gene and selected those variants that conferred protection against H(2)O(2). Among the resistant clones, we isolated a mutant in the nuclease domain of APE1 (D70A) with an increased capacity to remove 3'-blocking ends in vitro. D70 of APE1 aligns with A138 of LMAP and mutation of the latter to aspartate significantly reduces its 3'-phosphodiesterase activity. Kinetic analysis shows a novel role of residue D70 in the excision rate of 3'-blocking ends. The functional and structural differences between the parasite and human enzymes probably reflect a divergent molecular evolution of their DNA repair responses to oxidative damage.

Show MeSH
Related in: MedlinePlus