Topoisomerase‐Induced DNA Cleavage at Ribonucleotide Misincorporation Sites

Abstract

An estimated two ribonucleotides (rNMPs) per kilobase are mis‐incorporated into DNA due to high ratios of rNTP/dNTP pools in vivo, placing rNMPs as the most frequent source of DNA damage to date (Williams and Kunkel, 2014). The main pathway for rNMPs removal is the RNaseH2‐dependent pathway. When RNaseH2 is deleted in yeast, a specific topoisomerase I (Top1)‐dependent mutator effect develops: accumulation of short deletions within tandem repeats at genomic ribonucleotide sites generated by Top1 (Kim et al., 2011). We will discuss how ribonucleotide misincorporations induce replication stress and hyper‐recombination phenotype, which are alleviated upon deletion of the Top1 gene. On the other hand, ribonucleotide misincorporation is aggravated by inactivation of homologous repair (HR) in yeast strains. These in vivo data suggest that HR is important in the repair of Top1‐induced DNA damage in the presence of high levels of rNMPs. Using oligonucleotides with rNMPs incorporated at different positions, we show that Top1 can generate frequent DNA nicks with 3′ blocking ends (2′,3′‐cyclophosphates). In addition, we will demonstrate how a single rNMP is sufficient to generate recombination products and double‐strand breaks, a potential source of replication stress in vivo. Finally, we will show that faulty religation by Top1 generated 2‐nucleotide deletion, and the deletion products no longer contained rNMPs. Together these findings demonstrate the potential danger of Top1 activity at ribonucleotide misincorporation sites· Kim, N., Huang, S. Y., Williams, J. S., Li, Y. C., Clark, A. B., Cho, J. E., Kunkel, T. A., Pommier, Y., and Jinks‐Robertson, S. (2011). Mutagenic processing of ribonucleotides in DNA by yeast topoisomerase I. Science 332, 1561‐1564· Williams, J. S., and Kunkel, T. A. (2014). Ribonucleotides in DNA: origins, repair and consequences. DNA Repair (Amst) 19, 27‐37.