Advertisement

Measuring Ribonucleotide Incorporation into DNA In Vitro and In Vivo

  • Anders R. Clausen
  • Jessica S. Williams
  • Thomas A. KunkelEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1300)

Abstract

Ribonucleotides are incorporated into genomes by DNA polymerases, they can be removed, and if not removed, they can have deleterious and beneficial consequences. Here, we describe an assay to quantify stable ribonucleotide incorporation by DNA polymerases in vitro, and an assay to probe for ribonucleotides in each of the two DNA strands of the yeast nuclear genome.

Key words

Ribonucleotide incorporation DNA replication DNA polymerase Ribonucleotide excision repair Alkali-sensitive sites 

Notes

Acknowledgments

We thank Mercedes Arana and Katarzyna Bebenek for thoughtful comments on the manuscript and Kunkel lab members for technical expertise and discussions. This work was supported by Project Z01 ES065070 to T.A.K. from the Division of Intramural Research of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS).

References

  1. 1.
    Li Y, Breaker RR (1999) Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2′-hydroxyl group. J Am Chem Soc 121:5326–5372Google Scholar
  2. 2.
    Williams JS, Kunkel TA (2014) Ribonucleotides in DNA: origins, repair and consequences. DNA Repair 19:27–37CrossRefPubMedGoogle Scholar
  3. 3.
    Brown JA, Suo Z (2011) Unlocking the sugar "steric gate" of DNA polymerases. Biochemistry 50:1135–1142CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    McElhinny SAN, Watts BE, Kumar D, Watt DL, Lundstrom EB, Burgers PMJ, Johansson E, Chabes A, Kunkel TA (2010) Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proc Natl Acad Sci U S A 107:4949–4954CrossRefGoogle Scholar
  5. 5.
    Asturias FJ, Cheung IK, Sabouri N, Chilkova O, Wepplo D, Johansson E (2006) Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy. Nat Struct Mol Biol 13:35–43CrossRefPubMedGoogle Scholar
  6. 6.
    Clausen AR, Zhang S, Burgers PM, Lee MY, Kunkel TA (2013) Ribonucleotide incorporation, proofreading and bypass by human DNA polymerase delta. DNA Repair (Amst) 12:121–127CrossRefPubMedCentralGoogle Scholar
  7. 7.
    Lujan SA, Williams JS, Clausen AR, Clark AB, Kunkel TA (2013) Ribonucleotides are signals for mismatch repair of leading strand replication errors. Mol Cell 50:437–443CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    McElhinny SAN, Kumar D, Clark AB, Watt DL, Watts BE, Lundstrom EB, Johansson E, Chabes A, Kunkel TA (2010) Genome instability due to ribonucleotide incorporation into DNA. Nat Chem Biol 6:774–781CrossRefGoogle Scholar
  9. 9.
    Williams JS, Clausen AR, Nick McElhinny SA, Watts BE, Johansson E, Kunkel TA (2012) Proofreading of ribonucleotides inserted into DNA by yeast DNA polymerase epsilon. DNA Repair (Amst) 11:649–656CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Sambrook J, Russell DW (2001) Molecular cloning, a laboratory manuel, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  11. 11.
    Miyabe I, Kunkel TA, Carr AM (2011) The major roles of DNA polymerases epsilon and delta at the eukaryotic replication fork are evolutionarily conserved. PLoS Genet 7:e1002407CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Lujan SA, Williams JS, Pursell ZF, Abdulovic-Cui AA, Clark AB, Nick McElhinny SA, Kunkel TA (2012) Mismatch repair balances leading and lagging strand DNA replication fidelity. PLoS Genet 8:e1003016CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Williams JS, Smith DJ, Marjavaara L, Lujan SA, Chabes A, Kunkel TA (2013) Topoisomerase 1-mediated removal of ribonucleotides from nascent leading-strand DNA. Mol Cell 49:1010–1015CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Anders R. Clausen
    • 1
  • Jessica S. Williams
    • 1
  • Thomas A. Kunkel
    • 1
    Email author
  1. 1.Genome Integrity and Structural Biology LaboratoryNational Institute of Environmental Health Sciences, NIHResearch Triangle ParkUSA

Personalised recommendations