Identification of a residue critical for the excision of 3'-blocking ends in apurinic/apyrimidinic endonucleases of the Xth family.
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.
Castillo-Acosta
Víctor M
VM
Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Armilla (Granada), Spain.
Ruiz-Pérez
Luis M
LM
Yang
Wei
W
González-Pacanowska
Dolores
D
Vidal
Antonio E
AE
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018320:Leishmania major / Q000201:enzymology*; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D016877:Oxidants; D015800:Protozoan Proteins; D006861:Hydrogen Peroxide; D005092:Exonucleases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium