Application Notes

The Decondensation factor 31 binds to mono-nucleosomes

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1 Characterization of a Protein - Nucleosome Interaction Application Note NT-MO-017 The Decondensation factor 31 binds to mono-nucleosomes Dr. Thomas Schubert Lab of Prof. Gernot Längst, University of Regensburg in cooperation with 2bind GmbH, Germany Abstract Chromatin, the packaging form of the genome possesses two fundamental functions. On the one hand it compacts the DNA to fit into the nucleus and on the other hand it allows access to the underlying sequences for essential DNA dependent processes. Nucleosomes represent the basic structural component of chromatin. Here we characterize the specific interaction of the Decondensation factor 31 (Df31) with mono-nucleosomes. Df31 was recently shown to form a complex with snoRNAs and chromatin, to generate accessible higher order structures of chromatin. Introduction Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a eukaryotic cell. Chromatin compacts the DNA, so it fits into the cell nucleus, helps to prevent DNA damage, and to control gene expression and other DNA dependent processes. The primary protein components of chromatin are histones that associate with DNA in 1.65 helical turns, forming an octamer of the histone proteins H2A, H2B, H3 and H4. The resulting structure, the nucleosome is the basic structural component of chromatin (van- Holde, 1989). Although the nucleosome is a very stable protein-DNA complex, it is not static and has been shown to undergo a number of different structural re-arrangements including nucleosome sliding and DNA site exposure (Langst and Becker, 2001). In addition the nucleosome represents a binding partner for various proteins and serves as scaffold to establish multi-protein complexes at chromatin. In a recent study the Drosophila Decondensation factor 31 (Df31) was shown to be involved in regulating chromatin compaction and to be tethered to chromatin (Schubert et al., 2012). To identify the interaction partner of Df31 within the nucleosome, MicroScale Thermophoresis analyses were performed. Results In this application note, the binding behavior of Df31 to salt assembled mono-nucleosomes was evaluated, employing MicroScale Thermophoresis. Fig. 1 Measurement of Df31 binding to Cy5-labeled mono- nucleosomes. MST values were normalized to fraction bound. 3 independent measurements where performed to obtain the depicted binding curve. The K d of the Df31-mono-nucleosome interaction was 3.72 ± 0.56 µM.

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