1. De novo reconstitution of chromatin using wheat germ cell-free protein synthesis.
- Author
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Endo Y, Takemori N, Nagy SK, Okimune KI, Kamakaka R, Onouchi H, and Takasuka TE
- Subjects
- Animals, Chromatin chemistry, DNA chemistry, Drosophila Proteins chemistry, Drosophila melanogaster, Chromatin metabolism, DNA metabolism, Drosophila Proteins metabolism
- Abstract
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., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Published
- 2021
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