De novo reconstitution of chromatin using wheat germ cell‐free protein synthesis
| Autor: | Kei-Ichi Okimune, Nobuaki Takemori, Rohinton T. Kamakaka, Yaeta Endo, Hitoshi Onouchi, Taichi E. Takasuka, Szilvia K. Nagy |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
post‐translational chromatin assembly QH301-705.5 Method Context (language use) General Biochemistry Genetics and Molecular Biology 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Protein biosynthesis Methods Nucleosome Animals Drosophila Proteins Biology (General) Cell-free protein synthesis biology histone modifications DNA ATP‐dependent nucleosome assembly Chromatin Cell biology unmodified and soluble histone products 030104 developmental biology Histone Drosophila melanogaster chemistry 030220 oncology & carcinogenesis wheat germ cell‐free protein synthesis biology.protein DNA supercoil Mg2+ chromatin isolation |
| Zdroj: | FEBS Open Bio FEBS Open Bio, Vol 11, Iss 6, Pp 1552-1564 (2021) |
| ISSN: | 2211-5463 |
| Popis: | DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro. Here, Drosophila four core histones were produced in a native and unmodified form using wheat germ cell‐free protein synthesis. In the assembly reaction, four unpurified core histones and three chromatin assembly factors (dNAP‐1, dAcf1 and dISWI) were incubated with template DNA. We then assessed stoichiometry with the histones, nucleosome arrays, supercoiling and the ability of the chromatin to serve as a substrate for histone‐modifying enzymes. Overall, our method provides a new avenue to produce chromatin that can be useful in a wide range of chromatin research. We provide a new method to reconstitute chromatin using wheat germ cell‐free protein synthesis. The Drosophila four core histones were produced in a native and unmodified form. In the assembly reaction, four unpurified core histones and three assembly factors (dNAP‐1, dAcf1 and dISWI) were incubated with DNA, followed by Mg2+ isolation. The chromatin was evaluated and then used as substrates for histone‐modifying enzymes. |
| Databáze: | OpenAIRE |
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