Abstract
Despite the abundance of ribonucleoside monophosphates (rNMPs) in DNA, sites of rNMP incorporation remain poorly characterized. Here, by using ribose-seq and Ribose-Map techniques, we built and analyzed high-throughput sequencing libraries of rNMPs derived from mitochondrial and nuclear DNA of budding and fission yeast. We reveal both common and unique features of rNMP sites among yeast species and strains, and between wild type and different ribonuclease H-mutant genotypes. We demonstrate that the rNMPs are not randomly incorporated in DNA. We highlight signatures and patterns of rNMPs, including sites within trinucleotide-repeat tracts. Our results uncover that the deoxyribonucleotide immediately upstream of the rNMPs has a strong influence on rNMP distribution, suggesting a mechanism of rNMP accommodation by DNA polymerases as a driving force of rNMP incorporation. Consistently, we find deoxyadenosine upstream from the most abundant genomic rCMPs and rGMPs. This study establishes a framework to better understand mechanisms of rNMP incorporation in DNA.
Highlights
Despite the abundance of ribonucleoside monophosphates in DNA, sites of rNMP incorporation remain poorly characterized
The percentages of rA, rC, rG, and rU among these libraries were similar, regardless of the RE set used; we revealed some variation among the strains (Figs. 1a and 2a). rA, followed by rC and rG, is the most abundant rNMP found in almost all libraries examined
We examined whether rA, rC, rG, and rU were randomly incorporated in nuclear DNA (nDNA) of the wild type and mutant RNase H strains by determining the frequency of the dNMPs preceding or following each rNMP, and comparing this frequency with the given frequency of each dinucleotide in S. cerevisiae nDNA
Summary
Despite the abundance of ribonucleoside monophosphates (rNMPs) in DNA, sites of rNMP incorporation remain poorly characterized. By using ribose-seq and Ribose-Map techniques, we built and analyzed high-throughput sequencing libraries of rNMPs derived from mitochondrial and nuclear DNA of budding and fission yeast We reveal both common and unique features of rNMP sites among yeast species and strains, and between wild type and different ribonuclease H-mutant genotypes. The first studies reporting rNMP identity and distribution at the genome level used two yeast backgrounds for budding yeast Saccharomyces cerevisiae and one for fission yeast Schizosaccharomyces pombe, in which the gene coding for the catalytic subunit of RNase H2 (RNH201) was inactivated These reports revealed some initial features of rNMPs in DNA: widespread distribution along chromosomal and mitochondrial DNA (mtDNA), abundance of rC and rG, low level of rU in budding yeast, and biased presence of rNMPs on the leading vs lagging strand of DNA replication in association with specific low-fidelity mutants of DNA polymerases[19,20,21,22]. By analyzing 34 ribose-seq libraries, and comparatively examining five emRiboSeq libraries[22], we uncover conserved, new features, patterns, motifs, and hotspots of rNMPs in yeast DNA, all pointing towards a major role played by the upstream deoxyribonucleotide in rNMP incorporation
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