cDNA cloning, expression and functional characterization of an Arabidopsis thaliana homologue of the Escherichia coli DNA repair enzyme endonuclease III.
Reactive oxygen species (ROS) are ubiquitous DNA-damaging agents, and the repair of oxidative DNA lesions is essential to prevent mutations and cell death. Escherichia coli endonuclease III is the prototype repair enzyme for removal of oxidized pyrimidines from DNA. A database homology search identified a genomic sequence in Arabidopsis thaliana encoding a predicted protein with sequence similarity to E. coli endonuclease III. We cloned, sequenced and expressed the corresponding cDNA, which encodes a 39.1 kDa protein containing several sequence motifs conserved in endonuclease III homologues, including an iron-sulfur cluster domain and critical residues at the active site. The protein, designated AtNTH1, was over-expressed in E. coli and purified to apparent homogeneity. AtNTH1 exhibits DNA-glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polydeoxyribonucleotides. The enzyme also possesses an apurinic/apyrimidinic lyase activity on UV- and gamma-irradiated DNA substrates. The AtNTH1 gene contains 10 introns and 11 exons and is widely expressed in different plant tissues. Our results suggest that AtNTH1 is a structural and functional homologue of endonuclease III and probably plays a major role in plant defence against oxidative DNA damage.
Roldán-Arjona
T
T
Departamento de Genética, Facultad de Ciencias, Universidad de Córdoba, Spain.
García-Ortiz
M V
MV
Ruiz-Rubio
M
M
Ariza
R R
RR
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D015152:Blotting, Northern; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004260:DNA Repair; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010084:Oxidation-Reduction; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D018749:RNA, Plant; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase