Abstract
Ribonucleotides (rNTPs) are incorporated into genomic DNA at a relatively high frequency during replication. They have beneficial effects but, if not removed from the chromosomes, increase genomic instability. Here, we describe a fast method to easily estimate the amounts of embedded ribonucleotides into the genome. The protocol described is performed in Saccharomyces cerevisiae and allows us to quantify altered levels of rNMPs due to different mutations in the replicative polymerase ε. However, this protocol can be easily applied to cells derived from any organism.
M. Muzi-Falconi and F. Lazzaro are co-last authors.
This is a preview of subscription content, log in via an institution.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Joyce CM (1997) Choosing the right sugar: how polymerases select a nucleotide substrate. Proc Natl Acad Sci U S A 94:1619–1622
McElhinny SAN, Watts BE, Kumar D et al (2010) Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proc Natl Acad Sci U S A 107:4949–4954
Ghodgaonkar MM, Lazzaro F, Olivera-Pimentel M et al (2013) Ribonucleotides misincorporated into DNA act as strand-discrimination signals in eukaryotic mismatch repair. Mol Cell 50:323–332
Lujan SA, Williams JS, Clausen AR et al (2013) Ribonucleotides are signals for mismatch repair of leading-strand replication errors. Mol Cell 50:437–443
Dalgaard JZ (2012) Causes and consequences of ribonucleotide incorporation into nuclear DNA. Trends Genet 28:592–597
Sparks JL, Chon H, Cerritelli SM et al (2012) RNase H2-initiated ribonucleotide excision repair. Mol Cell 47:980–986
Lazzaro F, Novarina D, Amara F et al (2012) RNase H and postreplication repair protect cells from ribonucleotides incorporated in DNA. Mol Cell 45:99–110
Pizzi S, Sertic S, Orcesi S et al (2015) Reduction of hRNase H2 activity in Aicardi-Goutières syndrome cells leads to replication stress and genome instability. Hum Mol Genet 24:649–658
Nick McElhinny SA, Kumar D, Clark AB et al (2010) Genome instability due to ribonucleotide incorporation into DNA. Nat Chem Biol 6:774–781
Günther C, Kind B, Reijns MAM et al (2014) Defective removal of ribonucleotides from DNA promotes systemic autoimmunity. J Clin Invest 125(1):413–424
Reijns MAM, Rabe B, Rigby RE et al (2012) Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development. Cell 149:1008–1022
Hiller B, Achleitner M, Glage S et al (2012) Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity. J Exp Med 209:1419–1426
Crow YJ, Manel N (2015) Aicardi-Goutières syndrome and the type I interferonopathies. Nat Rev Immunol 15(7):429–440
Acknowledgments
This work was supported by grants from AIRC (n.15631) and Telethon (GGP15227) to M.M.-F., from AIRC and Fondazione Cariplo (TRIDEO 2014 Id. 15724) to F.L., and from Fondazione Cariplo (grant number 2013-0798) to P.P.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Meroni, A., Nava, G.M., Sertic, S., Plevani, P., Muzi-Falconi, M., Lazzaro, F. (2018). Measuring the Levels of Ribonucleotides Embedded in Genomic DNA. In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7306-4_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7306-4_22
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7305-7
Online ISBN: 978-1-4939-7306-4
eBook Packages: Springer Protocols