Limits...
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.

Show MeSH

Related in: MedlinePlus

Michaelis–Menten plots for the calculation of the Km and Vmax values of the AP endonuclease activity. (A) APE1 (filled circle), APE1D70A (open circle); (B) LMAP (filled square), LMAPA138D (open square). The kcat and Km values represent the average from three independent determinations. Standard deviations did not exceed 20% for any of the values calculated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2665217&req=5

Figure 5: Michaelis–Menten plots for the calculation of the Km and Vmax values of the AP endonuclease activity. (A) APE1 (filled circle), APE1D70A (open circle); (B) LMAP (filled square), LMAPA138D (open square). The kcat and Km values represent the average from three independent determinations. Standard deviations did not exceed 20% for any of the values calculated.

Mentions: Mutagenesis studies have shown that APE1 mutants at residue D70 display lower AP endonuclease-specific activity than the wild-type enzyme (43,44). To determine the effect of the A138D mutation on the incision of AP sites by LMAP, initial reaction rates were measured by incubating the enzymes with increasing concentrations of 21-nt duplex DNA substrate containing a THF residue. In agreement with the above-mentioned studies, we found that wild-type APE1 cleaves AP sites more efficiently than D70A mutant with a kcat value for the incision of an AP site aproximately 10-fold higher (202 versus 17 min−1) (Figure 5A). No gain of incision activity was observed mutating LMAP to LMAPA138D as might have been expected (Figure 5B). Instead, the mutation produced a 3-fold decrease in the kcat value with apparently no effect on the Km, indicating that D138 in LMAP cannot exert the same function as D70 in APE1. Even though the human and parasite AP endonucleases have structurally similar active sites, subtle differences must exist that affect the processing of AP sites by the two enzymes.Figure 5.


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)

Michaelis–Menten plots for the calculation of the Km and Vmax values of the AP endonuclease activity. (A) APE1 (filled circle), APE1D70A (open circle); (B) LMAP (filled square), LMAPA138D (open square). The kcat and Km values represent the average from three independent determinations. Standard deviations did not exceed 20% for any of the values calculated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Michaelis–Menten plots for the calculation of the Km and Vmax values of the AP endonuclease activity. (A) APE1 (filled circle), APE1D70A (open circle); (B) LMAP (filled square), LMAPA138D (open square). The kcat and Km values represent the average from three independent determinations. Standard deviations did not exceed 20% for any of the values calculated.
Mentions: Mutagenesis studies have shown that APE1 mutants at residue D70 display lower AP endonuclease-specific activity than the wild-type enzyme (43,44). To determine the effect of the A138D mutation on the incision of AP sites by LMAP, initial reaction rates were measured by incubating the enzymes with increasing concentrations of 21-nt duplex DNA substrate containing a THF residue. In agreement with the above-mentioned studies, we found that wild-type APE1 cleaves AP sites more efficiently than D70A mutant with a kcat value for the incision of an AP site aproximately 10-fold higher (202 versus 17 min−1) (Figure 5A). No gain of incision activity was observed mutating LMAP to LMAPA138D as might have been expected (Figure 5B). Instead, the mutation produced a 3-fold decrease in the kcat value with apparently no effect on the Km, indicating that D138 in LMAP cannot exert the same function as D70 in APE1. Even though the human and parasite AP endonucleases have structurally similar active sites, subtle differences must exist that affect the processing of AP sites by the two enzymes.Figure 5.

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