Abstract
Ribonucleotide monophosphates (rNMPs) are among the most frequent form of DNA aberration, as high ratios of ribonucleotide triphosphate:deoxyribonucleotide triphosphate pools result in approximately two misincorporated rNMPs/kb of DNA. The main pathway for the removal of rNMPs is by RNase H2. However, in a RNase H2 knock-out 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 and/or 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 led 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. A 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 for repair by the error-free RNase H2-dependent pathway in vivo. Our results provide direct evidence for the generation of short deletions by sequential Top1 cleavage events and for the promotion of nick religation at rNMP sites by Top1.
Highlights
DNA deletions at short repeat sites containing ribonucleotides are Top1-dependent
Top1 Binding at Ribonucleotide monophosphates (rNMPs) Sites Generates DNA Nicks with 2Ј,3ЈCyclic Phosphate Ends—To study the mechanism of Top1-dependent DNA deletions at short repeat sites on a high rNMP background, we focused on the (AT)2 hot spot from the CAN1 gene, which exhibits a strong Top1-dependent deletion signature (16 –18)
Genomic misincorporation of rNMPs occurs in many domains of living organisms, from bacteria to yeast to mammalian cells [5, 6, 15, 41]
Summary
DNA deletions at short repeat sites containing ribonucleotides are Top1-dependent. Results: Top generates DNA deletions at short repeat sites in vitro and reverses nicks at ribonucleotide sites. In a RNase H2 knock-out 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 and/or sequential Top binding, but experimental support has been lacking far. The catalytic mechanism of Top involves a reversible transesterification reaction, initiated by the nucleophilic attack by the active-site tyrosine (Tyr-723 for human Top1) on the scissile phosphate, generating a DNA nick and a covalent protein-DNA complex (termed the Top cleavage complex (Top1cc)). We show that Top promotes the religation of rNMP-induced DNA nicks, allowing the RNase H2-dependent repair pathway to carry out error-free removal of rNMPs
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