Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA

Abstract

Abundant ribonucleotide incorporation in DNA during replication and repair has profound consequences for genome stability, but the global distribution of ribonucleotide incorporation is unknown. We developed Ribose-seq, a method to capture unique products generated by alkaline cleavage of DNA at embedded ribonucleotides. High-throughput sequencing of these fragments from yeast Saccharomyces cerevisiae DNA revealed widespread ribonucleotide distribution with a strong preference for cytidine and guanosine, and identified hotspots of ribonucleotide incorporation in nuclear and mitochondrial DNA. Ribonucleotides were primarily incorporated on the newly-synthesized leading strand of nuclear DNA and were present upstream of G+C-rich tracts in the mitochondrial genome. Ribose-seq is a powerful tool for the systematic profiling of ribonucleotide incorporation in genomic DNA.

Unveiling the non-repair face of the Base Excision Repair pathway in RNA processing: A missing link between DNA repair and gene expression?

“…It has been studied how the additional presence of a reactive 2′-hydroxyl group on the sugar ring may: i) alter the DNA physical properties, reducing its elasticity [102,103] and structure in a sequence dependent manner [104][105][106][107][108]; ii) induce DNA replication or transcription arrest making the DNA backbone prone to hydrolysis triggering a persistent genomic instability [103,108,109]. This incorporation occurs quite frequently into the cell [110,111], potentially during every DNA replication reaction [110]. It has been estimated that a few thousands of rNMPs are embedded in budding yeast genome [110] and over a million in mouse genome [112].…”

“…Moreover, oxidation can occur not only in DNA but also in the nucleotides pool and then rNMP may be incorporated into neo-replicated DNA. During last years, several approaches have been developed [119], including Ribose-seq, in order to map rNMPs sites into genomic DNA [111]. By using these methods, it has been discovered that the incorporation of rNMPs has a widespread but not random distribution in chromosomal DNA of budding and fission yeast [111] and their number, per nuclear chromosome, seems to be proportional to the chromosome size [111].…”

Elevated Genome-Wide Instability in Yeast Mutants Lacking RNase H Activity

“…Despite the efficiency with which replicative DNA polymerases discriminate between rNTP and dNTP precursors, there are 15,000 rNMPs inserted into yeast DNA during each round of replication, which translates into 1 rNMP per 1000 nucleotides (Nick McElhinny et al 2010b). rNMPs in budding yeast genomic DNA have recently been mapped to single-nucleotide resolution by several groups (Clausen et al 2015;Koh et al 2015;Reijns et al 2015). In contrast to R-loops, which are removed by RNase H1 (encoded by RNH1) or RNase H2 (catalytic subunit encoded by RNH201), only RNase H2 is capable of removing of single rNMPs embedded in DNA (Cerritelli and Crouch 2009).…”

Measuring the Elasticity of Ribonucleotide(s)-Containing DNA Molecules Using AFM

“…Not only do polymerases incorporate rNMPs into the genome, but also there are other sources of rNMPs in DNA [13][14][15]. More recently, we developed an approach, ribose-seq, to profile these rNMPs in yeast genomic DNA [16]. These misincorporated rNMPs may have a significant effect on genome instability, resulting from DNA-protein and DNA-DNA interactions, which are crucial for many cellular processes.…”