Error‐free and mutagenic processing of topoisomerase 1‐provoked damage at genomic ribonucleotides

Genomic ribonucleotides incorporated during DNA replication are commonly repaired by RNase H2‐dependent ribonucleotide excision repair (RER). When RNase H2 is compromised, such as in Aicardi‐Goutières patients, genomic ribonucleotides either persist or are processed by DNA topoisomerase 1 (Top1) by either error‐free or mutagenic repair. Here, we present a biochemical analysis of these pathways. Top1 cleavage at genomic ribonucleotides can produce ribonucleoside‐2′,3′‐cyclic phosphate‐terminated nicks. Remarkably, this nick is rapidly reverted by Top1, thereby providing another opportunity for repair by RER. However, the 2′,3′‐cyclic phosphate‐terminated nick is also processed by Top1 incision, generally 2 nucleotides upstream of the nick, which produces a covalent Top1–DNA complex with a 2‐nucleotide gap. We show that these covalent complexes can be processed by proteolysis, followed by removal of the phospho‐peptide by Tdp1 and the 3′‐phosphate by Tpp1 to mediate error‐free repair. However, when the 2‐nucleotide gap is associated with a dinucleotide repeat sequence, sequence slippage re‐alignment followed by Top1‐mediated religation can occur which results in 2‐nucleotide deletion. The efficiency of deletion formation shows strong sequence‐context dependence.