Substrate specificity of Schizosaccharomyces pombe Nth protein for products of oxidative DNA damage.
A gene from Schizosaccharomyces pombe, which encodes a protein with a strong sequence similarity to the Nth protein of Escherichia coli, has recently been identified [Roldán-Arjona, T., Anselmino, C., and Lindahl, T. (1996) Nucleic Acids Res. 24, 3307-3312]. The functional analysis of this eukaryotic enzyme indicated that it is a homologue of E. coli Nth protein. The gene has been subcloned and the protein (Nth-Spo) purified to apparent homogeneity. We investigated the substrate specificity of this eukaryotic enzyme for modified bases in oxidatively damaged DNA, using the technique of gas chromatography/isotope-dilution mass spectrometry (GC/IDMS). DNA substrates containing up to 17 types of modified bases were prepared by gamma-irradiation or by treatment with H2O2 in the presence of Fe(III)-EDTA or Cu(II). The results revealed an efficient excision of five pyrimidine-derived lesions, 5-hydroxycytosine, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydroxycytosine, and 5-hydroxyuracil. None of the other pyrimidine or purine lesions was excised. Excision was measured as a function of enzyme concentration, time, substrate concentration, and temperature. Kinetic constants were determined. Although some DNA base lesions removed by Nth-Spo protein were similar to those previously described for E. coli Nth protein, differences between substrate specificities of these two enzymes were noted.
Karahalil
B
B
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.
Roldán-Arjona
T
T
Dizdaroglu
M
M
D004249:DNA Damage; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008401:Gas Chromatography-Mass Spectrometry; D010084:Oxidation-Reduction; D011743:Pyrimidines / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D011743:Pyrimidines; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli