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

Abstract

© 2015 The Authors. 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. Synopsis Genomic ribonucleotides misincorporated during DNA replication can be processed by RNase H2 or topoisomerase I (Top1). In vitro reconstitution shows that Top1-mediated ribonucleotide removal comprises two consecutive catalytic steps and formation of a covalent Top1-DNA complex, which is repaired in either an error-free or error-prone manner. Top1 reversibly cleaves genomic ribonucleotides with formation of a ribonucleotide 2′,3′-cyclic phosphate. Top1 cleaves two nucleotides upstream of the ribonucleotide 2′,3′-cyclic phosphate to yield a covalent Top1-DNA complex. The Top1-DNA complex can be repaired by proteolysis and degradation of the modified 3′-phosphate by Tdp1 and Tpp1. Religation after slippage realignment at a dinucleotide repeat sequence can result in 2-nt deletions. In vitro reconstitution reveals how covalent Top1-DNA complexes formed during removal of misincorporated ribonucleotides can be repaired in either an error-free or error-prone manner.