Induction of reversible complexes between eukaryotic DNA topoisomerase I and DNA-containing oxidative base damages - 7,8-dihydro-8-oxoguanine and 5-hydroxycytosine

We recently showed that abasic sites, uracil mismatches, nicks, and gaps ca n trap DNA topoisomerase I (top1) when these lesions are introduced in the vicinity of a top1 cleavage site (Pourquier, P., Ueng, L.-M., Kohlhagen, G. , Mazumder, A, Gupta, Ri., Kohn, K. W., and Pommier, Y, (1997) J. Biol. Che m. 272, 7792-7796; Pourquier, P., Pilon, A. A, Kohlhagen, G., Mazumder, A., Sharma, A, and Pommier, Y. (1997) J. Biol. Chem. 26441-26447). In this stu dy, we investigated the effects on top1 of an abundant base damage generate d by various oxidative stresses: 7,8-dihydro-8-oxoguanine (8-oxoG). Using p urified eukaryotic top1 and oligonucleotides containing the 8-oxoG modifica tion, we found a 3-7-fold increase in top1-mediated DNA cleavage when 8-oxo G was present at the +1 or +2 position relative to the cleavage site. Anoth er oxidative lesion, 8-hydroxycytosine, also enhanced top1 cleavage by a-fo ld when incorporated at the +1 position of the scissile strand. 8-oxoG at t he +1 position enhanced noncovalent top1 DNA binding and had no detectable effect on DNA religation or on the incision step. top1 trapping by 8-oxoG w as markedly enhanced when asparagine adjacent to the catalytic tyrosine was mutated to histidine, suggesting a direct interaction between this residue and the DNA major groove immediately downstream from the top1 cleavage sit e. Altogether, these results demonstrate that oxidative base lesions can in crease top1 binding to DNA and induce top1 cleavage complexes.