Effect of phosphorothioate substitutions on DNA cleavage by Escherichia coli DNA topoisomerase I.
To evaluate the structural influence of the DNA phosphate backbone on the activity of Escherichia coli DNA topoisomerase I, modified forms of oligonucleotide dA(7) were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone. A deoxy-iodo-uracil replaced the 5'-base to crosslink the oligonucleotides by ultraviolet (UV) and assess binding affinity. At the scissile phosphate there was little effect on the cleavage rate. At the +1 phosphate, the rectus phosphorus (Rp)-thio-substitution reduced the rate of cleavage by a factor of 10. At the +3 and -2 positions from the scissile bond, the Rp-isomer was cleaved at a faster rate than the sinister phosphorus (Sp)-isomer. The results demonstrate the importance of backbone contacts between DNA substrate and E. coli topoisomerase I.
Roche
C J
CJ
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
Tse-Dinh
Y C
YC
D001665:Binding Sites; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D008956:Models, Chemical; D009841:Oligonucleotides / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D013997:Time Factors
D009841:Oligonucleotides; D010710:Phosphates; D004247:DNA; D004264:DNA Topoisomerases, Type I; D010100:Oxygen