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

By Víctor M. Castillo-Acosta, Luis M. Ruiz-Pérez, Wei Yang, Dolores González-Pacanowska and Antonio E. Vidal


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 (H2O2). 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 H2O2. 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

Topics: Nucleic Acid Enzymes
Publisher: Oxford University Press
OAI identifier:
Provided by: PubMed Central

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  1. (1995). 3rd and Demple,B.
  2. (1997). 3rd and Povirk,L.F.
  3. (2007). A ‘moving metal mechanism’ for substrate cleavage by the DNA repair endonuclease APE-1.
  4. (1964). A deoxyribonucleic acid phosphatase-exonuclease from Escherichia coli.
  5. (1992). A mutant endonuclease IV of Escherichia coli loses the ability to repair lethal DNA damage induced by hydrogen peroxide but not that induced by methyl methanesulfonate.
  6. (1998). Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals.
  7. (2002). An exonucleolytic activity of human apurinic/apyrimidinic endonuclease on 30 mispaired DNA.
  8. (2004). AP endonuclease-independent DNA base excision repair in human cells.
  9. (2004). APE1 is the major 30-phosphoglycolate activity in human cell extracts.
  10. (2005). APE1-dependent repair of DNA single-strand breaks containing 30-end 8-oxoguanine.
  11. Aprataxin, causative gene product for EAOH/AOA1, repairs DNA single-strand breaks with damaged 30-phosphate and 30-phosphoglycolate ends.
  12. (1999). Apurinic/apyrimidinic endonuclease genes from the trypanosomatidae leishmania major and Trypanosoma cruzi confer resistance to oxidizing agents in DNA repair-deficient Escherichia coli.
  13. (1996). Asparagine 212 is essential for abasic site recognition by the human DNA repair endonuclease HAP1.
  14. (2006). Catalytic promiscuity and the divergent evolution of DNA repair enzymes.
  15. (1992). cDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Escherichia coli exonuclease III.
  16. (1985). Chemical changes induced in DNA by ionizing radiation.
  17. (1997). Cloning and characterization of a functional human homolog of Escherichia coli endonuclease III.
  18. (1991). Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes.
  19. (1996). Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a baseexcision DNA-repair protein superfamily.
  20. (2002). Conversion of phosphoglycolate to phosphate termini on 30 overhangs of DNA double strand breaks by the human tyrosyl-DNA phosphodiesterase hTdp1.
  21. (2007). Crystal structure and DNA repair activities of the AP endonuclease from Leishmania major.
  22. (2002). Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III.
  23. (1999). Determination of human DNA polymerase utilization for the repair of a model ionizing radiationinduced DNA strand break lesion in a defined vector substrate.
  24. (1996). DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes.
  25. (2006). DNA Repair and Mutagenesis. 2nd edn.,
  26. (2000). DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination [corrected].
  27. (1998). Dynamics of the interaction of human apurinic endonuclease (Ape1) with its substrate and product.
  28. (2004). Effects of backbone contacts 30 to the abasic site on the cleavage and the product binding by human apurinic/apyrimidinic endonuclease (APE1).
  29. (2000). endonuclease activity but stabilises substrate binding.
  30. (1976). Endonuclease II of Escherichia coli is exonuclease III.
  31. (1998). Excision of C-40-oxidized deoxyribose lesions from double-stranded DNA by human apurinic/ apyrimidinic endonuclease (Ape1 protein) and DNA polymerase beta.
  32. (1986). Exonuclease III and endonuclease IV remove 30 blocks from DNA synthesis primers in H2O2-damaged Escherichia coli.
  33. (2002). Free radical-induced damage to DNA: mechanisms and measurement. Free Radic.
  34. (1983). Gamma ray induced deoxyribonucleic acid strand breaks. 30 Glycolate termini.
  35. (1988). Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA.
  36. (2006). Human Ape2 protein has a 30-50 exonuclease activity that acts preferentially on mismatched base pairs.
  37. (1992). Human HeLa cell enzymes that remove phosphoglycolate 30-end groups from DNA.
  38. (2002). Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA.
  39. (2006). Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells.
  40. (1995). Identification of critical active-site residues in the multifunctional human DNA repair enzyme HAP1.
  41. (1999). Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease.
  42. (1991). Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E. coli xth (exonuclease III) mutants.
  43. (2000). Mapping the protein-DNA interface and the metal-binding site of the major human apurinic/apyrimidinic endonuclease.
  44. (1986). Mutagenesis by apurinic/apyrimidinic sites.
  45. (2003). Mutations in the alpha8 loop of human APE1 alter binding and cleavage of DNA containing an abasic site.
  46. (2005). Overexpression of AP endonuclease protects Leishmania major cells against methotrexate induced DNA fragmentation and hydrogen peroxide.
  47. (1999). Overexpression of the human HAP1 protein sensitizes cells to the lethal effect of bioreductive drugs.
  48. (1986). Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase.
  49. (1998). Rapid dissociation of human apurinic endonuclease (Ape1) from incised DNA induced by magnesium.
  50. (2002). Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins.
  51. (1994). Removal of 30-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1.
  52. (1989). Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase.
  53. (2006). Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism.
  54. (1999). Single-turnover analysis of mutant human apurinic/apyrimidinic endonuclease.
  55. (1990). Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression.
  56. (2003). Substrate specificity of human endonuclease III (hNTH1). Effect of human APE1 on hNTH1 activity.
  57. (1983). Superoxide radical: an endogenous toxicant.
  58. (1997). The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites.
  59. (1990). The enzymology of apurinic/apyrimidinic endonucleases.
  60. (2003). The exonuclease activity of human apurinic/apyrimidinic endonuclease (APE1). Biochemical properties and inhibition by the natural dinucleotide Gp4G.
  61. (2000). The human OGG1 gene: structure, functions, and its implication in the process of carcinogenesis.
  62. (2004). The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway.
  63. (1988). The relative importance of Escherichia coli exonuclease III and endonuclease IV for the hydrolysis of 30-phosphoglycolate ends in polydeoxynucleotides.
  64. (1999). The role of Mg 2+ and specific amino acid residues in the catalytic reaction of the major human abasic endonuclease: new insights from EDTA-resistant incision of acyclic abasic site analogs and site-directed mutagenesis.
  65. (1991). Two distinct human DNA diesterases that hydrolyze 30-blocking deoxyribose fragments from oxidized DNA.
  66. (2001). Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: implications for the catalytic mechanism.
  67. (1997). Use of a postlabelling assay to examine the removal of radiation-induced DNA lesions by purified enzymes and human cell extracts.
  68. (2001). XRCC1 coordinates the initial and late stages of DNA abasic site repair through protein-protein interactions.

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