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Mapping Ribonucleotides Incorporated into DNA by Hydrolytic End-Sequencing

  • Clinton D. Orebaugh
  • Scott A. Lujan
  • Adam B. Burkholder
  • Anders R. Clausen
  • Thomas A. Kunkel
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1672)

Abstract

Ribonucleotides embedded within DNA render the DNA sensitive to the formation of single-stranded breaks under alkali conditions. Here, we describe a next-generation sequencing method called hydrolytic end sequencing (HydEn-seq) to map ribonucleotides inserted into the genome of Saccharomyce cerevisiae strains deficient in ribonucleotide excision repair. We use this method to map several genomic features in wild-type and replicase variant yeast strains.

Key words

HydEn-seq DNA replication DNA repair DNA polymerase Ribonucleotide excision repair RNase H2 Next-generation sequencing Bioinformatics Genomics 

Notes

Acknowledgment

We thank Dr. Jessica Williams and Dr. Kin Chan for helpful comments on the manuscript. This work was supported by the Division of Intramural Research of the US National Institutes of Health (NIH), National Institute of Environmental Health Sciences (project Z01 ES065070 to T.A.K.).

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Clinton D. Orebaugh
    • 1
  • Scott A. Lujan
    • 1
  • Adam B. Burkholder
    • 2
  • Anders R. Clausen
    • 3
  • Thomas A. Kunkel
    • 1
  1. 1.Genome Integrity and Structural Biology LaboratoryNational Institute for Environmental Health Sciences, National Institute of Health (NIH)Research Triangle ParkUSA
  2. 2.Integrative BioinformaticsNational Institute for Environmental Health Sciences, National Institute of Health (NIH)Research Triangle ParkUSA
  3. 3.Department of Medical Biochemistry and Cell BiologyUniversity of GothenburgGothenburgSweden

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