Topoisomerase I alone is sufficient to produce short DNA deletions and can also reverse nicks at ribonucleotide sites [Enzymology]

April 17th, 2015 by Huang, S.-y. N., Ghosh, S., Pommier, Y.

Ribonucleotides monophosphates (rNMPs) are among the most frequent form of DNA aberration, as high ratios of ribonucleotide triphosphate/deoxyribonucleotide triphosphate (rNTP/dNTP) pools result in an estimated two misincorporated rNMPs per kilobase of DNA. The main pathway for the removal of rNMPs is by RNase H2. However, in RNase H2 knockout yeast strain, a topoisomerase I (Top1)-dependent mutator effect develops with accumulation of short deletions within tandem repeats. Proposed models for these deletions implicated processing of Top1-generated nicks at rNMP sites or/and sequential Top1 binding, but experimental support has been lacking thus far. Here, we investigated the biochemical mechanism of the Top1-induced short deletions at the rNMP sites by generating nicked DNA substrates bearing 2′,3′-cyclic phosphates at the nick sites, mimicking the Top1-induced nicks. We demonstrate that a second Top1 cleavage complex adjacent to the nick and subsequent faulty Top1 religation leads to the short deletions. Moreover, when acting on the nicked DNA substrates containing 2′,3′-cyclic phosphates, Top1 generated not only the short deletion, but also a full-length religated DNA product. Catalytically inactive Top1 mutant (Top1-Y723F) also induced the full-length products, indicating that Top1 binding independent of its enzymatic activity promotes the sealing of DNA backbones via nucleophilic attacks by the 5′-hydroxyl on the 2′,3′-cyclic phosphate. The resealed DNA would allow renewed attempt by the error-free RNase H2-dependent repair in vivo. Our results provide direct evidence for the generation of short deletions by sequential Top1 cleavage events and for Top1 promoting the religation of nicks at rNMP sites.