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

Sensitivity of Leishmania cells to H2O2 and MMS-induced DNA damages. (A) Western blot showing LMAP protein levels in whole cell extracts from 2.5 × 106 parasites harboring vector pX63-Neo (cell line pX), expressing wild-type LMAP (cell line pX-LMAP) or LMAPA138D (cell line pX-138). As control of charge, level of mevalonate kinase protein was measured in the three cell lines. (B) Immunofluorescence analysis was performed to establish the intracellular localization of LMAP in L. major cell lines pX, pX-LMAP and pX138. Nuclear and kinetoplast DNA were stained with DAPI and LMAP and LMAPA138D were detected with anti-LmAP and FITC conjugated anti-rabbit secondary antibody. Images were collected with a Zeiss Axiophot microscope. (C) The 3′-phosphodiesterase and (D) AP endonuclease activities of whole cell extracts from cell lines pX, pX-LMAP and pX-138 were tested on 21-mer DNA duplex containing a nick with a 3′-PG residue or a THF residue, respectively, and labeled at the 5′ of the lesion-carrying strand. Increasing amounts of protein (0–100 ng) were incubated for 30 min at 37°C with the oligonucleotide substrate (2000 nM) under standard reaction conditions. The products of the reactions were separated by denaturing 20% PAGE. (E) A total of 107 cells/ml from mid-log phase Leishmania cultures were exposed to increasing concentrations of H2O2 (left) or MMS (right) concentrations for 1 h at 28°C in M199 medium supplemented with 10% fetal bovine serum. After exposure to the genotoxic agent, cells were centrifuged, washed, resuspended in the same volume of PBS buffer and incubated with resazurin for 1 h at 28°C. Fluorescence was measured at 570 nm excitation wavelength and 590 nm emission wavelength. Experiments for each compound were performed three times, in duplicate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Sensitivity of Leishmania cells to H2O2 and MMS-induced DNA damages. (A) Western blot showing LMAP protein levels in whole cell extracts from 2.5 × 106 parasites harboring vector pX63-Neo (cell line pX), expressing wild-type LMAP (cell line pX-LMAP) or LMAPA138D (cell line pX-138). As control of charge, level of mevalonate kinase protein was measured in the three cell lines. (B) Immunofluorescence analysis was performed to establish the intracellular localization of LMAP in L. major cell lines pX, pX-LMAP and pX138. Nuclear and kinetoplast DNA were stained with DAPI and LMAP and LMAPA138D were detected with anti-LmAP and FITC conjugated anti-rabbit secondary antibody. Images were collected with a Zeiss Axiophot microscope. (C) The 3′-phosphodiesterase and (D) AP endonuclease activities of whole cell extracts from cell lines pX, pX-LMAP and pX-138 were tested on 21-mer DNA duplex containing a nick with a 3′-PG residue or a THF residue, respectively, and labeled at the 5′ of the lesion-carrying strand. Increasing amounts of protein (0–100 ng) were incubated for 30 min at 37°C with the oligonucleotide substrate (2000 nM) under standard reaction conditions. The products of the reactions were separated by denaturing 20% PAGE. (E) A total of 107 cells/ml from mid-log phase Leishmania cultures were exposed to increasing concentrations of H2O2 (left) or MMS (right) concentrations for 1 h at 28°C in M199 medium supplemented with 10% fetal bovine serum. After exposure to the genotoxic agent, cells were centrifuged, washed, resuspended in the same volume of PBS buffer and incubated with resazurin for 1 h at 28°C. Fluorescence was measured at 570 nm excitation wavelength and 590 nm emission wavelength. Experiments for each compound were performed three times, in duplicate.

Mentions: The biochemical analysis of the mutants described in this work evidences subtle but significant differences between the parasite and human AP endonucleases that could affect the catalytic process during oxidative DNA-damage repair. To further examine the role of residue A138 of the LMAP enzyme in parasite survival under genotoxic stress, the mutant enzyme LMAPA138D was over-expressed in L. major cells. A cell line over-expressing LMAPA138D (pX-138) was generated by transfection with the expression vector pX63Neo-LMAPA138D and selection of clones resistant to G418 at 32 µg/ml. Cell lines over-expressing LMAP wild-type (pX-LMAP) and a control cell line (pX) harboring the empty expression vector have been obtained and described elsewhere (47). The level of expression of the selected clones was determined by western blot using a purified antibody directed against pure recombinant LMAP (Figure 7A). Protein quantity standardization was carried out using an antibody directed against L. major mevalonate kinase (MVK). Cell lines pX-LMAP and pX-138 expressed 9- and 16-fold higher levels of LMAP, respectively, than cell line pX. It was also checked that growth rate was not affected by the protein over-expression.Figure 7.


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)

Sensitivity of Leishmania cells to H2O2 and MMS-induced DNA damages. (A) Western blot showing LMAP protein levels in whole cell extracts from 2.5 × 106 parasites harboring vector pX63-Neo (cell line pX), expressing wild-type LMAP (cell line pX-LMAP) or LMAPA138D (cell line pX-138). As control of charge, level of mevalonate kinase protein was measured in the three cell lines. (B) Immunofluorescence analysis was performed to establish the intracellular localization of LMAP in L. major cell lines pX, pX-LMAP and pX138. Nuclear and kinetoplast DNA were stained with DAPI and LMAP and LMAPA138D were detected with anti-LmAP and FITC conjugated anti-rabbit secondary antibody. Images were collected with a Zeiss Axiophot microscope. (C) The 3′-phosphodiesterase and (D) AP endonuclease activities of whole cell extracts from cell lines pX, pX-LMAP and pX-138 were tested on 21-mer DNA duplex containing a nick with a 3′-PG residue or a THF residue, respectively, and labeled at the 5′ of the lesion-carrying strand. Increasing amounts of protein (0–100 ng) were incubated for 30 min at 37°C with the oligonucleotide substrate (2000 nM) under standard reaction conditions. The products of the reactions were separated by denaturing 20% PAGE. (E) A total of 107 cells/ml from mid-log phase Leishmania cultures were exposed to increasing concentrations of H2O2 (left) or MMS (right) concentrations for 1 h at 28°C in M199 medium supplemented with 10% fetal bovine serum. After exposure to the genotoxic agent, cells were centrifuged, washed, resuspended in the same volume of PBS buffer and incubated with resazurin for 1 h at 28°C. Fluorescence was measured at 570 nm excitation wavelength and 590 nm emission wavelength. Experiments for each compound were performed three times, in duplicate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Sensitivity of Leishmania cells to H2O2 and MMS-induced DNA damages. (A) Western blot showing LMAP protein levels in whole cell extracts from 2.5 × 106 parasites harboring vector pX63-Neo (cell line pX), expressing wild-type LMAP (cell line pX-LMAP) or LMAPA138D (cell line pX-138). As control of charge, level of mevalonate kinase protein was measured in the three cell lines. (B) Immunofluorescence analysis was performed to establish the intracellular localization of LMAP in L. major cell lines pX, pX-LMAP and pX138. Nuclear and kinetoplast DNA were stained with DAPI and LMAP and LMAPA138D were detected with anti-LmAP and FITC conjugated anti-rabbit secondary antibody. Images were collected with a Zeiss Axiophot microscope. (C) The 3′-phosphodiesterase and (D) AP endonuclease activities of whole cell extracts from cell lines pX, pX-LMAP and pX-138 were tested on 21-mer DNA duplex containing a nick with a 3′-PG residue or a THF residue, respectively, and labeled at the 5′ of the lesion-carrying strand. Increasing amounts of protein (0–100 ng) were incubated for 30 min at 37°C with the oligonucleotide substrate (2000 nM) under standard reaction conditions. The products of the reactions were separated by denaturing 20% PAGE. (E) A total of 107 cells/ml from mid-log phase Leishmania cultures were exposed to increasing concentrations of H2O2 (left) or MMS (right) concentrations for 1 h at 28°C in M199 medium supplemented with 10% fetal bovine serum. After exposure to the genotoxic agent, cells were centrifuged, washed, resuspended in the same volume of PBS buffer and incubated with resazurin for 1 h at 28°C. Fluorescence was measured at 570 nm excitation wavelength and 590 nm emission wavelength. Experiments for each compound were performed three times, in duplicate.
Mentions: The biochemical analysis of the mutants described in this work evidences subtle but significant differences between the parasite and human AP endonucleases that could affect the catalytic process during oxidative DNA-damage repair. To further examine the role of residue A138 of the LMAP enzyme in parasite survival under genotoxic stress, the mutant enzyme LMAPA138D was over-expressed in L. major cells. A cell line over-expressing LMAPA138D (pX-138) was generated by transfection with the expression vector pX63Neo-LMAPA138D and selection of clones resistant to G418 at 32 µg/ml. Cell lines over-expressing LMAP wild-type (pX-LMAP) and a control cell line (pX) harboring the empty expression vector have been obtained and described elsewhere (47). The level of expression of the selected clones was determined by western blot using a purified antibody directed against pure recombinant LMAP (Figure 7A). Protein quantity standardization was carried out using an antibody directed against L. major mevalonate kinase (MVK). Cell lines pX-LMAP and pX-138 expressed 9- and 16-fold higher levels of LMAP, respectively, than cell line pX. It was also checked that growth rate was not affected by the protein over-expression.Figure 7.

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