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.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
We thank A. V. Bryksin, A. L. Gombolay, S. Biliya, and F. O. Vannberg for technical advises, and all of the Storici lab members for discussions and suggestions during the course of this project. This work was supported by the National Institutes of Health (R01ES026243-01 to F.S.), the Parker H. Petit Institute for Bioengineering and Bioscience at the Georgia Institute of Technology (12456H2 to F.S.), and the Howard Hughes Medical Institute Faculty Scholar grant (55108574 to F.S.).
Jinks-Robertson S, Klein HL (2015) Ribonucleotides in DNA: hidden in plain sight. Nat Struct Mol Biol 22:176–178CrossRefGoogle Scholar
Koh KD, Balachander S, Hesselberth JR, Storici F (2015) Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA. Nat Methods 12(3):251–257CrossRefGoogle Scholar
Clausen AR et al (2015) Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation. Nat Struct Mol Biol 22:185–191CrossRefGoogle Scholar
Keszthelyi A, Daigaku Y, Ptasinska K, Miyabe I, Carr AM (2015) Mapping ribonucleotides in genomic DNA and exploring replication dynamics by polymerase usage sequencing (Pu-seq). Nat Protoc 10:1786–1801CrossRefGoogle Scholar
Reijns MAM et al (2015) Lagging-strand replication shapes the mutational landscape of the genome. Nature 518:502–506CrossRefGoogle Scholar
Williams JS, Kunkel TA (2014) Ribonucleotides in DNA: origins, repair and consequences. DNA Repair 19:27–37CrossRefGoogle Scholar
Williams JS, Lujan SA, Kunkel TA (2016) Processing ribonucleotides incorporated during eu-karyotic DNA replication. Nat Rev Mol Cell Biol 17:350–363CrossRefGoogle Scholar
Vengrova S, Dalgaard JZ (2006) The wild-type Schizosaccharomyces pombe mat1 imprint consists of two ribonucleotides. EMBO Rep 7:59–65CrossRefGoogle Scholar