![]() ![]() Recent biochemical and genetic studies show that DNA polymerases (Pols), such as Pols α, δ, and ε, readily incorporate ribonucleotides into DNA (Nick McElhinny et al, 2010a, b Williams et al, 2013). Taken together, these results define Top1 as a source of DSBs and genome instability when ribonucleotides incorporated by the replicative polymerases are not removed by RNase H2. ![]() Analysis of Top1-linked DNA from pull-down experiments revealed that Top1 is covalently linked to the end of DNA in RNase H2-deficient yeast cells, supporting this model. We demonstrate biochemically that irreversible DSBs are generated by subsequent Top1 cleavage on the opposite strand from the Top1-induced DNA nicks at ribonucleotide sites. Importantly, these phenotypes were rescued upon additional deletion of TOP1, implicating homologous recombination for the repair of Top1-induced damage at ribonuclelotide sites. We show that RNase H2-deficient yeast cells displayed elevated frequency of Rad52 foci, inactivation of RNase H2 and RAD52 led to synthetic lethality, and combined loss of RNase H2 and RAD51 induced slow growth and replication stress. Here, we provide genetic and biochemical evidence that DNA double-strand breaks (DSBs) can be directly generated by Top1 at sites of genomic ribonucleotides. ![]() Ribonuclease activity of topoisomerase I (Top1) causes DNA nicks bearing 2′,3′-cyclic phosphates at ribonucleotide sites.
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