Molecular cloning and functional analysis of a Schizosaccharomyces pombe homologue of Escherichia coli endonuclease III.
The Escherichia coli endonuclease III (Nth-Eco) protein is involved in the removal of damaged pyrimidine residues from DNA by base excision repair. It is an iron-sulphur enzyme possessing both DNA glycosylase and apurinic/apyrimidinic lyase activities. A database homology search identified an open reading frame in genomic sequences of Schizosaccharomyces pombe which encodes a protein highly similar to Nth-Eco. The gene has been subcloned in an expression vector and the protein purified to apparent homogeneity. The S.pombe Nth homologue (Nth-Spo) is a 40.2 kDa protein of 355 amino acids. Nth-Spo possesses glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polymers. The eukaryotic protein removes urea more efficiently than the prokaryotic enzyme, whereas its efficiency in excising thymine glycol is lower. A nicking assay was used to show that the enzyme also exhibits an AP lyase activity on UV- and gamma-irradiated DNA substrates. These findings show that Nth protein is structurally and functionally conserved from bacteria to fission yeast.
Roldán-Arjona
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Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
Anselmino
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Lindahl
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005057:Eukaryotic Cells; D008190:Lyases / Q000032:analysis; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013941:Thymine / Q000031:analogs & derivatives / Q000378:metabolism; D014508:Urea / Q000378:metabolism
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C029389:thymine glycol; D014508:Urea; 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; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D013941:Thymine