MOLECULAR-CLONING AND FUNCTIONAL-ANALYSIS OF A SCHIZOSACCHAROMYCES-POMBE HOMOLOG 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 a nd apurinic/apyrimidinic lyase activities. A database homology search identified an open reading frame in genomic sequences of Schizosacchar omyces pombe which encodes a protein highly similar to Nth-Eco. The ge ne 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 activi ty on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded poly mers. The eukaryotic protein removes urea more efficiently than the pr okaryotic 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. Thes e findings show that Nth protein is structurally and functionally cons erved from bacteria to fission yeast.