Cloning and characterization of a functional human homolog of Escherichia coli endonuclease III.
Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death. Endonuclease III is the major DNA glycosylase activity in Escherichia coli that catalyzes the excision of pyrimidines damaged by ring opening or ring saturation, and it also possesses an associated lyase activity that incises the DNA backbone adjacent to apurinic/apyrimidinic sites. During analysis of the area adjacent to the human tuberous sclerosis gene (TSC2) in chromosome region 16p13.3, we identified a gene, OCTS3, that encodes a 1-kb transcript. Analysis of OCTS3 cDNA clones revealed an open reading frame encoding a predicted protein of 34.3 kDa that shares extensive sequence similarity with E. coli endonuclease III and a related enzyme from Schizosaccharomyces pombe, including a conserved active site region and an iron/sulfur domain. The product of the OCTS3 gene was therefore designated hNTH1 (human endonuclease III homolog 1). The hNTH1 protein was overexpressed in E. coli and purified to apparent homogeneity. The recombinant protein had spectral properties indicative of the presence of an iron/sulfur cluster, and exhibited DNA glycosylase activity on double-stranded polydeoxyribonucleotides containing urea and thymine glycol residues, as well as an apurinic/apyrimidinic lyase activity. Our data indicate that hNTH1 is a structural and functional homolog of E. coli endonuclease III, and that this class of enzymes, for repair of oxidatively damaged pyrimidines in DNA, is highly conserved in evolution from microorganisms to human cells.
Aspinwall
R
R
Medical Research Council Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom.
Rothwell
D G
DG
Roldan-Arjona
T
T
Anselmino
C
C
Ward
C J
CJ
Cheadle
J P
JP
Sampson
J R
JR
Lindahl
T
T
Harris
P C
PC
Hickson
I D
ID
D000230:Adenocarcinoma / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D002885:Chromosomes, Human, Pair 16; D003001:Cloning, Molecular; D003106:Colon / Q000201:enzymology; D004260:DNA Repair; D018076:DNA, Complementary / Q000235:genetics; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D006801:Humans; D008190:Lyases / Q000378:metabolism; D008969:Molecular Sequence Data; D011740:Pyrimidine Dimers / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D011740:Pyrimidine Dimers; 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; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase