Genetic requirements for repair of lesions caused by single genomic ribonucleotides in S phase
Single ribonucleoside monophosphates (rNMPs) are transiently present in eukaryotic genomes. The RNase H2-dependent ribonucleotide excision repair (RER) pathway ensures error-free genomic rNMP removal. In pathological conditions, genomic rNMP levels can rise and persist. If these rNMPs hydrolyse in, or prior to, S phase, toxic single-ended double-strand breaks (seDSBs) can occur upon an encounter with replication forks. How such rNMP-derived seDSB lesions are repaired is unclear. We employed a cell cycle phase restricted allele of RNase H2 as a genetic tool to induce nicks at rNMPs specifically in S phase to generate such lesions and study their repair. Here, we introduce a network of genes that maintain DNA integrity when rNMP-derived nick lesions arise during DNA replication. We use genetic methods to characterise the molecular requirements of a Top1-independent, rNMP-derived nick lesion repair (NLR). In NLR, the RAD52 epistasis group becomes essential for homology-directed repair (HDR). Moreover, the previously described Rtt101Mms1-Mms22 dependent ubiquitylation of histone H3 is essential for NLR in cells with high rNMP load, and loss of Rtt101Mms1-Mms22 combined with RNase H2 dysfunction leads to compromised cellular fitness. We discuss the genetic NLR network in the context of human disease, where cancer therapies may be able to exploit these synthetic lethalities.