Capture of Ribonucleotides in Yeast Genomic DNA Using Ribose-Seq.

Abstract

Experiments conducted in yeast cells have recently shown abundant presence of ribonucleotides (rNMPs) embedded both in nuclear and mitochondrial DNA. Indeed, rNMPs are the most frequent, nonstandard nucleotides found in cellular DNA. rNMPs have a highly reactive 2'-hydroxyl group in the ribose sugar that gives rise to genome instability by altering the structure, function, and properties of DNA. In order to profile rNMPs embedded in yeast genomic DNA, as well as any other genomic DNA of interest, we developed "ribose-seq." Ribose-seq utilizes Arabidopsis thaliana tRNA ligase (AtRNL), which enables ligation of 2'-phosphate termini of DNA molecules terminating with an rNMP to the 5'-phosphate end of the same DNA molecules. Thus, a unique feature of ribose-seq is its capacity to specifically and directly capture the rNMPs present in DNA. Here we describe how ribose-seq is applied to yeast Saccharomyces cerevisiae DNA to capture rNMPs that are incorporated in the yeast genome and build libraries of rNMP incorporation for high-throughput sequencing. We also provide the advancements over our original ribose-seq protocol at the end of Subheading 1, and the specific details are provided in the methods part of this chapter.