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Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitro.

Martino, Fabrizio and Kueng, Stephanie and Robinson, Philip and Tsai-Pflugfelder, Monika and van Leeuwen, Fred and Ziegler, Mathias and Cubizolles, Fabien and Cockell, Moira M and Rhodes, Daniela and Gasser, Susan (2009) Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitro. Molecular Cell, 33 (3). pp. 323-334. ISSN 1097-4164

Abstract

At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.

Item Type: Article
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Additional Information: author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used
Keywords: DNA
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Date Deposited: 14 Dec 2009 13:50
Last Modified: 31 Jan 2013 01:02
URI: https://oak.novartis.com/id/eprint/969

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