Yes / Ribonucleoside monophosphate (rNMP) incorporation in genomic DNA poses a significant threat to genomic integrity. In addition to repair, DNA damage tolerance mechanisms ensure replication progression upon encountering unrepaired lesions. One player in the tolerance mechanism is Rad5, which is an E3 ubiquitin ligase and helicase. Here, we report a new role for yeast Rad5 in tolerating rNMP incorporation, in the absence of the bona fide ribonucleotide excision repair pathway via RNase H2. This role of Rad5 is further highlighted after replication stress induced by hydroxyurea or by increasing rNMP genomic burden using a mutant DNA polymerase (Pol ε - Pol2-M644G). We further demonstrate the importance of the ATPase and ubiquitin ligase domains of Rad5 in rNMP tolerance. These findings suggest a similar role for the human Rad5 homologues helicase-like transcription factor (HLTF) and SNF2 Histone Linker PHD RING Helicase (SHPRH) in rNMP tolerance, which may impact the response of cancer cells to replication stress-inducing therapeutics.

Our previous studies have demonstrated that RNA can serve as a template for double-strand break (DSB) repair in the yeast Saccharomyces cerevisiae using synthetic RNA-containing oligonucleotides (oligos). Following this initial work, we show that the RNA tract of RNA-containing oligos can be copied into DNA to transfer a genetic change at the chromosomal level also in the bacterium Escherichia coli and in human cells. Exploiting the use of oligos containing ribonucleoside monophosphates (rNMPs), we developed a molecular approach to generate RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in both yeast and E. coli cells. Such technique allows us to study how rNMPs present in the DNA genome of cells are tolerated by cells, what factors recognize and target rNMPs in DNA and to what extent the embedded rNMPs may alter genome integrity. Here we proved that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis during chromosomal replication and produce a genetic change. We discovered that mispaired rNMPs that are embedded in genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in yeast and in E. coli. Our data reveal novel substrates for the MMR system, and also uncover an unpredicted competition between RNase H and MMR for the RNA/DNA mispairs.

<p>Topoisomerase 1 (Top1) resolves transcription-associated supercoils by generating transient single-strand breaks in DNA and is a major source of transcription-associated mutagenesis in Saccharomyces cerevisiae. Top1 generates a distinctive mutation signature characterized by deletions in short, tandem repeats, and a similar signature is associated with ribonucleoside monophosphates (rNMPs) in DNA. DNA polymerases incorporate rNMPs into genomic DNA, and such rNMPs are efficiently removed in an error-free manner by ribonuclease (RNase) H2. In the absence of RNase H2, persistent rNMPs give rise to short deletions via a mutagenic process initiated by a Top1 incision at an rNMP. There is only partial overlap, however, between Top1-dependent deletion hotspots identified in highly transcribed DNA and those associated with rNMPs, suggesting the existence of both rNMP-dependent and rNMP-independent events. Here I present evidence that rNMP-independent hotspots reflect processing of a trapped Top1 cleavage complex (Top1cc), and that rNMP-dependent hotspots reflect sequential Top1 reactions. A sequential-cleavage model for rNMP-dependent deletions is tested in vivo and in vitro, employing Top1 cleavage and ligation assays. In addition, I report that rNMP-dependent hotspot activity is significantly enhanced when Top1 incises the non-transcribed strand of an actively transcribing reporter gene. Finally, I describe a novel type of mutagenesis that reflects repair of multiple Top1ccs. Specifically, expression of a mutant Top1 with reduced ligation activity (Top1-T722A) caused large deletion mutations that are distinct from Top1-dependent short deletions. Genetic data indicates that Top1-T722A-dependent large deletions are non-homologous end joining events.</p> / Dissertation