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.
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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.
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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
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DOI: https://doi.org/10.1007/978-1-4939-9434-2_14
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