Abstract
Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET). The single-particle FRET (spFRET) approach for analysis of DNA uncoiling from the histone octamer during transcription through chromatin is described in detail.
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References
Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389:251–260
Gaykalova DA, Kulaeva OI, Pestov NA, Hsieh FK, Studitsky VM (2012) Experimental analysis of the mechanism of chromatin remodeling by RNA polymerase II. Methods Enzymol 512:293–314
Gaykalova DA, Kulaeva OI, Bondarenko VA, Studitsky VM (2009) Preparation and analysis of uniquely positioned mononucleosomes. Methods Mol Biol 523:109–123
Walter W, Studitsky VM (2004) Construction, analysis, and transcription of model nucleosomal templates. Methods 33:18–24
Walter W, Kashlev M, Studitsky VM (2004) Transcription through the nucleosome by mRNA-producing RNA polymerases. Methods Enzymol 377:445–460
Beard BC, Smerdon MJ (2004) Analysis of DNA repair on nucleosome templates. Methods Enzymol 377:499–507
Teng Y, Yu S, Reed SH, Waters R (2009) Lux ex tenebris: nucleotide resolution DNA repair and nucleosome mapping. Methods 48:23–34
Rowe CE, Narlikar GJ (2010) The ATP-dependent remodeler RSC transfers histone dimers and octamers through the rapid formation of an unstable encounter intermediate. Biochemistry 49:9882–9890
Hota SK, Bartholomew B (2012) Approaches for studying nucleosome movement by ATP-dependent chromatin remodeling complexes. Methods Mol Biol 809:367–380
Andrews AJ, Luger K (2011) A coupled equilibrium approach to study nucleosome thermodynamics. Methods Enzymol 488:265–285
Dyer PN, Edayathumangalam RS, White CL, Bao Y, Chakravarthy S, Muthurajan UM, Luger K (2004) Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol 375:23–44
Luger K, Rechsteiner TJ, Richmond TJ (1999) Preparation of nucleosome core particle from recombinant histones. Methods Enzymol 304:3–19
Vasudevan D, Chua EY, Davey CA (2010) Crystal structures of nucleosome core particles containing the ‘601’ strong positioning sequence. J Mol Biol 403:1–10
Mizuguchi G, Wu WH, Alami S, Luk E (2012) Biochemical assay for histone H2A.Z replacement by the yeast SWR1 chromatin remodeling complex. Methods Enzymol 512:275–291
Cirillo LA, Zaret KS (2004) Preparation of defined mononucleosomes, dinucleosomes, and nucleosome arrays in vitro and analysis of transcription factor binding. Methods Enzymol 375:131–158
Pennings S, Meersseman G, Bradbury EM (1991) Mobility of positioned nucleosomes on 5S rDNA. J Mol Biol 220:101–110
Meersseman G, Pennings S, Bradbury EM (1992) Mobile nucleosomes–a general behavior. EMBO J 11:2951–2959
Studitsky VM, Clark DJ, Felsenfeld G (1994) Mechanism of nucleosome displacement by a transcribing polymerase. In: Structural biology: the state of art. Adenine Press, New York, pp 125–131
Kireeva ML, Walter W, Tchernajenko V, Bondarenko V, Kashlev M, Studitsky VM (2002) Nucleosome remodeling induced by RNA polymerase II. Loss of the H2A/H2B dimer during transcription. Mol Cell 9:541–552
Thastrom A, Lowary PT, Widlund HR, Cao H, Kubista M, Widom J (1999) Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences. J Mol Biol 288:213–229
Lowary PT, Widom J (1998) New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J Mol Biol 276:19–42
Simon RH, Felsenfeld G (1979) A new procedure for purifying histone pairs H2A + H2B and H3 + H4 from chromatin using hydroxylapatite. Nucleic Acids Res 6:689–696
Owen-Hughes T, Utley RT, Steger DJ, West JM, John S, Cote J, Havas KM, Workman JL (1999) Analysis of nucleosome disruption by ATP-driven chromatin remodeling complexes. Methods Mol Biol 119:319–331
Buning R, van Noort J (2010) Single-pair FRET experiments on nucleosome conformational dynamics. Biochimie 92:1729–1740
Choy JS, Lee TH (2012) Structural dynamics of nucleosomes at single-molecule resolution. Trends Biochem Sci 37:425–435
Lee JY, Lee TH (2012) Effects of histone acetylation and CpG methylation on the structure of nucleosomes. Biochim Biophys Acta 1824:974–982
Lee JY, Lee TH (2012) Effects of DNA methylation on the structure of nucleosomes. J Am Chem Soc 134:173–175
Simon M, North JA, Shimko JC, Forties RA, Ferdinand MB, Manohar M, Zhang M, Fishel R, Ottesen JJ, Poirier MG (2011) Histone fold modifications control nucleosome unwrapping and disassembly. Proc Natl Acad Sci U S A 108:12711–12716
Murphy MC, Rasnik I, Cheng W, Lohman TM, Ha T (2004) Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy. Biophys J 86:2530–2537
Bondarenko VA, Steele LM, Ujvari A, Gaykalova DA, Kulaeva OI, Polikanov YS, Luse DS, Studitsky VM (2006) Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase II. Mol Cell 24:469–479
Kulaeva OI, Gaykalova DA, Pestov NA, Golovastov VV, Vassylyev DG, Artsimovitch I, Studitsky VM (2009) Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II. Nat Struct Mol Biol 16:1272–1278
Morozov AV, Fortney K, Gaykalova DA, Studitsky VM, Widom J, Siggia ED (2009) Using DNA mechanics to predict in vitro nucleosome positions and formation energies. Nucleic Acids Res 37:4707–4722
Hsieh FK, Fisher M, Ujvari A, Studitsky VM, Luse DS (2010) Histone Sin mutations promote nucleosome traversal and histone displacement by RNA polymerase II. EMBO Rep 11:705–710
Lee S, Lee J, Hohng S (2010) Single-molecule three-color FRET with both negligible spectral overlap and long observation time. PLoS One 5:e12270
Widengren J, Kudryavtsev V, Antonik M, Berger S, Gerken M, Seidel CA (2006) Single-molecule detection and identification of multiple species by multiparameter fluorescence detection. Anal Chem 78:2039–2050
Gansen A, Valeri A, Hauger F, Felekyan S, Kalinin S, Toth K, Langowski J, Seidel CA (2009) Nucleosome disassembly intermediates characterized by single-molecule FRET. Proc Natl Acad Sci U S A 106:15308–15313
Kireeva ML, Walter W, Tchernajenko V, Bondarenko V, Kashlev M, Studitsky VM (2002) Nucleosome remodeling induced by RNA polymerase II: loss of the H2A/H2B dimer during transcription. Mol Cell 9:541–552
Acknowledgements
Studies using radioactively labeled materials were supported by NIH grant (GM58650). Single-particle spectroscopy studies were supported by the Russian Science Foundation (RSF grant No 14-24-00031).
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Kudryashova, K.S. et al. (2015). Preparation of Mononucleosomal Templates for Analysis of Transcription with RNA Polymerase Using spFRET. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 1288. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2474-5_23
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DOI: https://doi.org/10.1007/978-1-4939-2474-5_23
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