Cleavage of dT8 and dT8 phosphorothioyl analogues by Escherichia coli DNA topoisomerase I: product and rate analysis.
Escherichia coli DNA topoisomerase I catalyzes the cleavage of short, single-stranded oligodeoxynucleotides with dT8 as the shortest cleavable oligo(thymidylic acid). The 5'-32P-labeled products formed from the cleavage of [5'-32P]dT8 are dT5, dT4, and dT3 with over 70% of the substrate cleaved to dT4. Mg(II) ions affect this product distribution by increasing the percentage of dT4 formed. The substitution of a sulfur atom for a nonbridging oxygen atom in a phosphodiester linkage yields oligodeoxynucleotide phosphorothioyl (PS) analogues. The epimers of the analogues were separated, and the position and stereochemistry of the phosphorothiodiester bond were determined. Topoisomerase I is stereospecific in its reactivity toward these analogues. With the oligodeoxynucleotide PS analogue substrates, the rate of cleavage, the stereospecificity, and the product distribution depend upon the position and the stereochemistry of the phosphorothiodiester linkage.
Domanico
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Central Research and Development Department, E. I. du Pont de Nemours and Co., Wilmington, Delaware 19898.
Tse-Dinh
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D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D009946:Organothiophosphorus Compounds / Q000378:metabolism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D009946:Organothiophosphorus Compounds; C027903:oligo (dT); D004264:DNA Topoisomerases, Type I; D008274:Magnesium