ANALYSIS OF NUCLEOSOME STRUCTURE IN POLYACRYLAMIDE GEL USING FŐRSTER RESONANCE ENERGY TRANSFER METHOD
Abstract
On the basis of Förster resonance energy transfer (FRET) effect a method to analyze the structure of (Cy3, Cy5)-labeled nucleosomes in polyacrylamide gel after electrophoresis in native conditions was developed. It is shown that the correct application of the method requires monitoring of nonspecific intermolecular FRET and fluorescence re-absorption. A comparative analysis of the results of the FRET measurements of two types of nucleosomes and their complexes with yeast protein FACT was performed, which confirmed the similarity of the structural features of nucleosomes detected in the gel and in aqueous solution. An application of FRET analysis in combination with electrophoresis allows one to separate and visualize components of a complex mixture, evaluate their relative content, and, in addition, to characterize the structural differences between these complexes in situ.
About the Authors
O. V. Chertkov
Lomonosov Moscow State University
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
Russian Federation
Russian Federation
Bioengineering Department, School of Biology
Leninskiye Gory 1–12, Moscow, 119234, Russia
ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia
M. E. Valieva
Lomonosov Moscow State University
Russian Federation
Russian Federation
Bioengineering Department, School of Biology
Leninskiye Gory 1–12, Moscow, 119234, Russia
N. V. Malyuchenko
Lomonosov Moscow State University
Russian Federation
Russian Federation
Bioengineering Department, School of Biology
Leninskiye Gory 1–12, Moscow, 119234, Russia
A. V. Feofanov
Lomonosov Moscow State University
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
Russian Federation
Russian Federation
Bioengineering Department, School of Biology
Leninskiye Gory 1–12, Moscow, 119234, Russia
ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia
References
1. Valieva M.E., Armeev G.A., Kudryashova K.S., Gerasimova N.S., Shaytan A.K., Kulaeva O.I., McCullough L.L., Formosa T., Georgiev P.G., Kirpichnikov M.P., Studitsky V.M., Feofanov A.V. Large-scale ATP-independent nucleosome unfolding by a histone chaperone // Nat. Struct. Mol. Biol. 2016. Vol. 23. N 12. P. 1111–1116.
2. Dechassa M.L., Sabri A., Pondugula S., Kassabov S.R., Chatterjee N., Kladde M.P., Bartholomew B. SWI/SNF has intrinsic nucleosome disassembly activity that is dependent on adjacent nucleosomes // Mol. Cell. 2010. Vol. 38. N 4. P. 590–602.
3. Kulaeva O.I., Hsieh F.K., Studitsky V.M. RNA polymerase complexes cooperate to relieve the nucleosomal barrier and evict histones // Proc. Natl. Acad. Sci. U.S.A. 2010. Vol. 107. N 25. P. 11325–11330.
4. Chang H.W., Pandey M., Kulaeva O.I., Patel S.S., Studitsky V.M. Overcoming a nucleosomal barrier to replication // Sci. Adv. 2016. Vol. 2. N 11. e1601865.
5. Pestov N.A., Gerasimova N.S., Kulaeva O.I., Studitsky V.M. Structure of transcribed chromatin is a sensor of DNA damage // Sci. Adv. 2015. Vol. 1. N 6. e1500021.
6. Luger K., Mäder A.W., Richmond R.K., Sargent D.F., Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 A resolution // Nature. 1997. Vol. 389. N 6648. P. 251–260.
7. Gaykalova D.A., Kulaeva O.I., Volokh O., Shaytan A.K., Hsieh F.K., Kirpichnikov M.P., Sokolova O.S., Studitsky V.M. Structural analysis of nucleosomal barrier to transcription // Proc. Natl. Acad. Sci. U. S. A. 2015. Vol. 112. N 43. P. 5787– 5795.
8. Kudryashova K.S., Chertkov O.V., Nikitin D.V., Pestov N.A., Kulaeva O.I., Efremenko A.V., Solonin A.S., Kirpichnikov M.P., Studitsky V.M., Feofanov A.V. Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET // Methods Mol. Biol. 2015. Vol. 1288. P. 395–412.
9. Buning R., Kropff W., Martens K., van Noort J. spFRET reveals changes in nucleosome breathing by neighboring nucleosomes // J. Phys. Condens. Matter. 2015. Vol. 27. N 6. 064103.
10. Gansen A., Tóth K., Schwarz N., Langowski J. Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure––a FRET study // Nucleic Acids Res. 2015. Vol. 43. N 3. P. 1433–1443.
11. Kudryashova K.S., Nikitin D.V., Chertkov O.V., Gerasimova N.S., Valieva M.E. Studitsky V.M., Feofanov A.V. Development of fluorescently labeled mononucleosomes for the investigation of transcription mechanisms by single complex microscopy // Moscow Univ. Biol. Sci. Bull. 2015. Vol. 70. N 4. P. 189–193.
12. Sultanov D., Gerasimova N., Kudryashova K., Maluchenko N., Kotova E., Langelier M.-F., Pascal J., Kirpichnikov M., Feofanov A., Studitsky V. Unfolding of core nucleosomes by PARP- 1 revealed by spFRET microscopy // AIMS Genetics. 2017. Vol. 4. N 1. P. 21–31.
13. Valieva M.E., Gerasimova N.S., Kudryashova K.S., Kozlova A.L., Kirpichnikov M.P., Hu Q., Botuyan M.V., Mer G., Feofanov A.V., Studitsky V.M. Stabilization of nucleosomes by histone tails and by FACT revealed by spFRET microscopy // Cancers (Basel). 2017. Vol. 9. N 1, 3. DOI:10.3390/cancers9010003.
14. Shimahara H., Hirano T., Ohya K., Matsuta S., Seeram S.S., Tate S. Nucleosome structural changes induced by binding of non-histone chromosomal proteins HMGN1 and HMGN2 // FEBS Open Bio. 2013. Vol. 3. P. 184–191.
15. Kulaeva O.I., Gaykalova D.A., Pestov N.A., Golovastov V.V., Vassylyev D.G., Artsimovitch I., Studitsky V.M. Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II // Nat. Struct. Mol. Biol. 2009. Vol. 16. N 12. P. 1272–1278.
16. Gaykalova D.A., Kulaeva O.I., Bondarenko V.A., Studitsky V.M. Preparation and analysis of uniquely positioned mononucleosomes // Methods Mol. Biol. 2009. Vol. 523. P. 109–123.
17. Xin H., Takahata S., Blanksma M., McCullough L., Stillman D.J., Formosa T. yFACT induces global accessibility of nucleosomal DNA without H2AH2B displacement // Mol. Cell. 2009. Vol. 35. N 3. P. 365–376.
Review
For citations:
Chertkov O.V., Valieva M.E., Malyuchenko N.V., Feofanov A.V. ANALYSIS OF NUCLEOSOME STRUCTURE IN POLYACRYLAMIDE GEL USING FŐRSTER RESONANCE ENERGY TRANSFER METHOD. Vestnik Moskovskogo universiteta. Seriya 16. Biologiya. 2017;72(4):229-234. (In Russ.)
Views:
322
Email this article 