Genome-wide mutagenesis resulting from topoisomerase 1-processing of unrepaired ribonucleotides in DNA

Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2-5 base pair deletions (Δ2-5bp) in repetitive DNA are a signature of unrepaired ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol ε, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.