Abstract
R-loops are three-stranded nucleic acid structures composed of a DNA-RNA heteroduplex and a displaced single-stranded DNA. Although R-loops serve important roles in transcription and chromatin structure, they are also a major threat to genome stability. Cells prevent accumulation of genomic R-loops by mechanisms that remove these structures, such as ribonucleases which digest DNA-RNA hybrids and helicases which unwind R-loops. Here we describe methods to monitor resolvement of R-loops by the helicase DDX21 focussing on the impact of acetylation on helicase activity.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sanz LA, Hartono SR, Lim YW, Steyaert S, Rajpurkar A, Ginno PA et al (2016) Prevalent, dynamic, and conserved R-Loop structures associate with specific epigenomic signatures in mammals. Mol Cell 63(1):167–178
Santos-Pereira JM, Aguilera A (2015) R loops: new modulators of genome dynamics and function. Nat Rev Genet 16(10):583–597
Skourti-Stathaki K, Proudfoot NJ (2014) A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression. Genes Dev 28(13):1384–1396
Cerritelli SM, Crouch RJ (2009) Ribonuclease H: the enzymes in eukaryotes. FEBS J 276(6):1494–1505
Tran PLT, Pohl TJ, Chen CF, Chan A, Pott S, Zakian VA (2017) PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes. Nat Commun 8:15025
Hatchi E, Skourti-Stathaki K, Ventz S, Pinello L, Yen A, Kamieniarz-Gdula K et al (2015) BRCA1 recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair. Mol Cell 57(4):636–647
Chakraborty P, Grosse F (2011) Human DHX9 helicase preferentially unwinds RNA-containing displacement loops (R-loops) and G-quadruplexes. DNA Repair 10(6):654–665
Song C, Hotz-Wagenblatt A, Voit R, Grummt I (2017) SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability. Genes Dev 31:1370–1381
Sridhara SC, Carvalho S, Grosso AR, Gallego-Paez LM, Carmo-Fonseca M, de Almeida SF (2017) Transcription dynamics prevent RNA-mediated genomic instability through SRPK2-dependent DDX23 phosphorylation. Cell Rep 18(2):334–343
Popuri V, Bachrati CZ, Muzzolini L, Mosedale G, Costantini S, Giacomini E et al (2008) The human RecQ helicases, BLM and RECQ1, display distinct DNA substrate specificities. J Biol Chem 283(26):17766–17776
Wang X, Li J, Diaz J, You J (2014) Helicase assays. Bio-protocol 4(6):e1079
Kim JH, Seo YS (2009) In vitro assays for studying helicase activities. Methods Mol Biol 521:361–379
Valdez BC, Henning D, Perumal K, Busch H (1997) RNA-unwinding and RNA-folding activities of RNA helicase II/Gu—two activities in separate domains of the same protein. Eur J Biochem 250(3):800–807
Chen C, Okayama H (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7(8):2745–2752
Acknowledgment
Work in the Grummt lab is supported by the Deutsche Forschungsgemeinschaft (GR475/22-2 and SFB1036), CellNetworks (EcTop Survey 2014), and the Baden-Württemberg Stiftung.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Song, C., Grummt, I. (2019). Experimental Approaches to Investigate the Role of Helicase Acetylation in Regulating R-Loop Stability. In: Brosh, Jr., R. (eds) Protein Acetylation. Methods in Molecular Biology, vol 1983. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9434-2_14
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9434-2_14
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9433-5
Online ISBN: 978-1-4939-9434-2
eBook Packages: Springer Protocols