We recently showed that abasic sites, uracil mismatches, nicks, and gaps can trap DNA topoisomerase I (top1) when these lesions are introduced in the vicinity of a top1 cleavage site (Pourquier, P., Ueng, L. . In this study, we investigated the effects on top1 of an abundant base damage generated by various oxidative stresses: 7,8-dihydro-8-oxoguanine (8-oxoG). Using purified eukaryotic top1 and oligonucleotides containing the 8-oxoG modification, we found a 3-7-fold increase in top1-mediated DNA cleavage when 8-oxoG was present at the ؉1 or ؉2 position relative to the cleavage site. Another oxidative lesion, 5-hydroxycytosine, also enhanced top1 cleavage by 2-fold when incorporated at the ؉1 position of the scissile strand. 8-oxoG at the ؉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 was 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 site. Altogether, these results demonstrate that oxidative base lesions can increase top1 binding to DNA and induce top1 cleavage complexes.

Abasic sites and deamination of cytosine to uracil are probably the most common types of endogenous DNA damage. The effects of such lesions on DNA topoisomerase I (top1) activity were examined in oligonucleotides containing a unique top1 cleavage site. The presence of uracils and abasic sites within the first 4 bases immediately 5 to the cleavage site suppressed normal top1 cleavage and induced new top1 cleavage sites. Uracils immediately 3 to the cleavage site increased cleavage and produced a camptothecin mimicking effect. A mismatch with a bulge or abasic sites immediately 3 to the top1 cleavage site irreversibly trapped top1 cleavable complexes in the absence of camptothecin and produced a suicide cleavage complex. These results demonstrate that top1 activity is sensitive to physiological, environmental, and pharmacological DNA modifications and that top1 can act as a specific mismatch-and abasic site-nicking enzyme.

DNA topoisomerases I and II (top1 and top2, respectively) are ubiquitous enzymes that play an essential role in transcription, replication, chromosome segregation, and DNA repair. The basic enzymatic reaction of topoisomerases, namely reversible DNA nicking, is a transesterification reaction where a DNA phosphodiester bond is transferred to a specific enzyme tyrosine residue. Eukaryotic top1 and top2 exhibit major differences concerning their mechanism of action. Top1 acts as a monomer and forms a covalent bond with the 3'-terminus of a DNA single-strand break (1-3) whereas top2 acts as an homodimer and forms a covalent bond with the 5'-terminus of the DNA double-strand break with a four base-pairs overhang (Fig. 1) (1-4). No energy cofactor is required for top1 activity, whereas top2 hydrolyzes adenosine triphosphate (ATP) during its catalytic cycle. Fig. 1. Top1- and top2-cleavage complexes. (A) Top1 acts as a monomer, makes a single-strand break and covalently binds to the 3'-end of the break, leaving a 5'-hydroxyl end. (B) Top2 acts as a dimer, and generally makes a double-strand break. Each strand is cleaved by one monomer, with a 4-base overhang. Each monomer covalently binds to the 5'-end of the break and leaves a 3'-hydroxyl end.