PURIFICATION OF PROTEIN-DNA COMPLEXES BY NATIVE GEL ELECTROPHORESIS FOR ELECTRON MICROSCOPY STUDY
Abstract
Electrophoretic separation under native conditions may be used for purification of protein molecules and their complexes with DNA and other ligands. Here, we employed this approach to separate protein-DNA complexes with a molecular weight of about 200 kDa: mono- and dinucleosomes. The purified mononucleosomes were subjected to single particle electron microscopy study using negative stain contrasting, and the two-dimensional projections of the nucleosomes were obtained. A comparison of the nucleo some projections before and after separation in the native PAGE revealed different orientation of particles on the carbon film.
About the Authors
M. E. Valieva
Lomonosov Moscow State University
Russian Federation
Russian Federation
Department of Bioengineering, School of Biology,
Leninskiye Gory 1–12, Moscow, 119234
N. I. Derkacheva
Evdokimov University of Medicine and Dentistry
Russian Federation
Russian Federation
Department of Biochemistry,
Delegatskaya ul. 20–1, Moscow, 127473
O. S. Sokolova
Lomonosov Moscow State University
Russian Federation
Russian Federation
Department of Bioengineering, School of Biology,
Leninskiye Gory 1–12, Moscow, 119234
References
1. Clapier C., Cairns B. The biology of chromatin remodeling complexes // Annu. Rev. Biochem. 2009. Vol. 78. P. 273–304.
2. Vignali M., Hassan A., Neely K., Workman J. ATPdependent chromatin-remodeling complexes// Mol. Cell. Biol. 2000. Vol. 20. N 6. P. 1899–1910.
3. Kornberg R.D., Thomas J.O. Chromatin structure; oligomers of the histones // Science. 1974. Vol. 184. N 4139. P. 865–868.
4. Luger K., Mäder A.W., Richmond R.K., Sargent D.F., Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 Å res-olution // Nature. 1997. Vol. 389. N 6648. P. 251–260.
5. Arents G., Moudrianakis E.N. The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerization // Proc. Natl. Acad. Sci. U.S.A. 1995. Vol. 92. N 24. P. 11170–11174.
6. Dechassa M.L., Zhang B., Horowitz-Scherer R., Persinger J., Woodcock C.L., Peterson C.L., Bartholomew B. Architecture of the SWI/SNF-nucleosome complex // Mol. Cell. Biol. 2008. Vol. 28. N 19. P. 6010–6021.
7. Feser J., D.T., Das C., Carson J.J., Kieft J., Harkness T., Tyler J.K. Elevated histone expression promotes life span extension // Mol. Cell. 2010. Vol. 39. N 5. P. 724–735.
8. Ljungman M., Hanawalt P. Efficient protection against oxidative DNA damage in chromatin // Mol. Carcinog. 1992. Vol. 5. N 4. P. 264–269.
9. Enright H.U., Miller W.J., Hebbel R.P. Nucleosomal histone protein protects DNA from iron mediated damage // Nucleic. Acids Res. 1992. Vol. 20. N 13. P. 3341–3346.
10. van Heel M., Gowen B., Matadeen R., Orlova E.V., Finn R., Pape T., Cohen D., Stark H., Schmidt R., Schatz M., Patwardhan A. Single-particle electron cryo-microscopy: towards atomic resolution // Q. Rev. Biophys. 2000. Vol. 33. N 4. P. 307–369.
11. Knispel R.W., Kofler C., Boicu M., Nickel W.B.S. Blotting protein complexes from native gels to electron microscopy grids // Nat. Methods. 2012. Vol. 9. N 2. P. 182–184.
12. 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.
13. 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.
14. van Heel M., Harauz G., Orlova E.V., Schmidt R., Schatz M. A new generation of the IMAGIC image processing system // J. Struct. Biol. 1996. Vol. 116. N 1. P. 17–24.
15. Frank J., Wagenknecht T., McEwen B.F., Marko M., Hsieh C.E., Mannella C.A. Three-dimensional imaging of biological complexity // 2002. J. Struct. Biol. Vol. 138. N 1–2. P. 85–91.
16. Henderson R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules // Q. Rev. Biophys. 1995. Vol. 28. N 2. P. 171–193.
17. Glaeser R.M. How good can cryo-EM become? // Nat. Methods. 2012. Vol. 13. N 1. P. 28–32.
18. Asturias F.J., Chung W.H., Kornberg R.D., Lorch Y. Structural analysis of the RSC chromatin remodeling complex // Proc. Natl. Acad. Sci. U. S. A. 2002. Vol. 99. N 21. P. 13477–13480.
19. Davey C.A., Sargent D.F., Luger K., Maeder A.W., Richmond T.J. Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution // J. Mol. Biol. 2002. Vol. 319. N 5. P. 1097–1113.
Review
For citations:
Valieva M.E., Derkacheva N.I., Sokolova O.S. PURIFICATION OF PROTEIN-DNA COMPLEXES BY NATIVE GEL ELECTROPHORESIS FOR ELECTRON MICROSCOPY STUDY. Vestnik Moskovskogo universiteta. Seriya 16. Biologiya. 2017;72(1):3-8. (In Russ.)
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