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Capture of Ribonucleotides in Yeast Genomic DNA Using Ribose-Seq

  • Sathya Balachander
  • Taehwan Yang
  • Gary Newnam
  • Waleed M. M. El-Sayed
  • Kyung Duk Koh
  • Francesca StoriciEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2049)

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.

Key words

Ribonucleotides Yeast Saccharomyces cerevisiae Ribose-seq tRNA ligase AtRNL Alkali 

Notes

Acknowledgments

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.).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sathya Balachander
    • 1
  • Taehwan Yang
    • 1
  • Gary Newnam
    • 1
  • Waleed M. M. El-Sayed
    • 1
    • 2
  • Kyung Duk Koh
    • 3
  • Francesca Storici
    • 1
    Email author
  1. 1.School of Biological SciencesGeorgia Institute of TechnologyAtlantaUSA
  2. 2.Marine Microbiology DepartmentNational Institute of Oceanography and FisheriesRed SeaEgypt
  3. 3.Department of MedicineUniversity of California, San FranciscoSan FranciscoUSA

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