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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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Effect of the amino acid substitutions on the removal of different 3′-blocking groups. (A) The 3′-phosphodiesterase activity of APE1, APE1D70A (upper gel), LMAP and LMAPA138D (lower gel) was tested on 21-mer DNA duplex containing a nick with a 3′-dRP residue and labeled at the 5′ of the lesion-carrying strand. Increasing amounts of the AP endonucleases (0–2.5 nM) were incubated for 10 min at 37°C with the oligonucleotide substrate (5 nM) under standard reaction conditions. (B) The 3′-phosphatase activity was tested on a 32P-labeled 21-mer DNA duplex containing a nick with a 3′-P residue produced as described in Materials and Methods section. Two concentrations of enzyme (1 nM and 2 nM) were incubated 10 min at 37°C with the double-stranded oligonucleotide substrate (5 nM) under standard reaction conditions. The products of the reactions were separated by denaturing 20% PAGE. Positions in the gel of the substrates and the 3′-OH product of the reaction are indicated with arrows.
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Figure 3: Effect of the amino acid substitutions on the removal of different 3′-blocking groups. (A) The 3′-phosphodiesterase activity of APE1, APE1D70A (upper gel), LMAP and LMAPA138D (lower gel) was tested on 21-mer DNA duplex containing a nick with a 3′-dRP residue and labeled at the 5′ of the lesion-carrying strand. Increasing amounts of the AP endonucleases (0–2.5 nM) were incubated for 10 min at 37°C with the oligonucleotide substrate (5 nM) under standard reaction conditions. (B) The 3′-phosphatase activity was tested on a 32P-labeled 21-mer DNA duplex containing a nick with a 3′-P residue produced as described in Materials and Methods section. Two concentrations of enzyme (1 nM and 2 nM) were incubated 10 min at 37°C with the double-stranded oligonucleotide substrate (5 nM) under standard reaction conditions. The products of the reactions were separated by denaturing 20% PAGE. Positions in the gel of the substrates and the 3′-OH product of the reaction are indicated with arrows.

Mentions: Similar differences were also found for the 3′-phosphodiesterase activity measured on a 3′-dRP substrate (38). Damaged 3′-ends consisting in a nicked DNA with a 3′-dRP residue can arise in the course of BER repair by the action of bifunctional DNA glycosylases such as NTH and OGG1, which are endowed with AP lyase activity and catalyze a β-elimination reaction. Figure 3A shows that the enzymes lacking the aspartate at position 70 (APE1D70A) or 138 (LMAP) have a higher 3′-phosphodiesterase activity also on this type of lesion. Relatively low amounts (0.5 nM) of APE1D70A or LMAP catalyze the excision of all of the 3′-dRP substrate used in the assay. For APE1 and LMAPA138D, the complete excision of the fragmented 3′-phosphoribose residues requires an enzyme concentration five times higher.Figure 3.


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)

Effect of the amino acid substitutions on the removal of different 3′-blocking groups. (A) The 3′-phosphodiesterase activity of APE1, APE1D70A (upper gel), LMAP and LMAPA138D (lower gel) was tested on 21-mer DNA duplex containing a nick with a 3′-dRP residue and labeled at the 5′ of the lesion-carrying strand. Increasing amounts of the AP endonucleases (0–2.5 nM) were incubated for 10 min at 37°C with the oligonucleotide substrate (5 nM) under standard reaction conditions. (B) The 3′-phosphatase activity was tested on a 32P-labeled 21-mer DNA duplex containing a nick with a 3′-P residue produced as described in Materials and Methods section. Two concentrations of enzyme (1 nM and 2 nM) were incubated 10 min at 37°C with the double-stranded oligonucleotide substrate (5 nM) under standard reaction conditions. The products of the reactions were separated by denaturing 20% PAGE. Positions in the gel of the substrates and the 3′-OH product of the reaction are indicated with arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665217&req=5

Figure 3: Effect of the amino acid substitutions on the removal of different 3′-blocking groups. (A) The 3′-phosphodiesterase activity of APE1, APE1D70A (upper gel), LMAP and LMAPA138D (lower gel) was tested on 21-mer DNA duplex containing a nick with a 3′-dRP residue and labeled at the 5′ of the lesion-carrying strand. Increasing amounts of the AP endonucleases (0–2.5 nM) were incubated for 10 min at 37°C with the oligonucleotide substrate (5 nM) under standard reaction conditions. (B) The 3′-phosphatase activity was tested on a 32P-labeled 21-mer DNA duplex containing a nick with a 3′-P residue produced as described in Materials and Methods section. Two concentrations of enzyme (1 nM and 2 nM) were incubated 10 min at 37°C with the double-stranded oligonucleotide substrate (5 nM) under standard reaction conditions. The products of the reactions were separated by denaturing 20% PAGE. Positions in the gel of the substrates and the 3′-OH product of the reaction are indicated with arrows.
Mentions: Similar differences were also found for the 3′-phosphodiesterase activity measured on a 3′-dRP substrate (38). Damaged 3′-ends consisting in a nicked DNA with a 3′-dRP residue can arise in the course of BER repair by the action of bifunctional DNA glycosylases such as NTH and OGG1, which are endowed with AP lyase activity and catalyze a β-elimination reaction. Figure 3A shows that the enzymes lacking the aspartate at position 70 (APE1D70A) or 138 (LMAP) have a higher 3′-phosphodiesterase activity also on this type of lesion. Relatively low amounts (0.5 nM) of APE1D70A or LMAP catalyze the excision of all of the 3′-dRP substrate used in the assay. For APE1 and LMAPA138D, the complete excision of the fragmented 3′-phosphoribose residues requires an enzyme concentration five times higher.Figure 3.

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