Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 408, Issue 2, Pages 187-204Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2011.01.003
Keywords
nucleosome; histone posttranslational modification; sequential native chemical ligation; fully synthetic histone; histone H3 lysine 56 acetylation
Categories
Funding
- American Heart Association [0815460D]
- National Institutes of Health [GM083055]
- National Science Foundation [MCB0845696]
- Burroughs Wellcome Fund
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [0845695] Funding Source: National Science Foundation
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Posttranslational modification (PTM) of histones plays a central role in genome regulation. Engineering histones with defined PTMs on one residue or on multiple residues is crucial for understanding their function within nucleosomes and chromatin. We introduce a sequential native chemical ligation strategy that is suitable for the preparation of fully synthetic histone proteins, allowing for site-specific incorporation of varied PTMs throughout the sequence. We demonstrate this method with the generation of histone H3 acetylated at lysine 56 [1-13(K56ac)]. H3(K56ac) is essential for transcription, replication, and repair. We examined the influence of H3 (K56ac) on the targeting of a model DNA binding factor (LexA) to a site 30 bp within the nucleosome. We find that H3(K56ac) increases LexA binding to its DNA target site by 3-fold at physiological ionic strength. We then demonstrate that H3(K56ac) facilitates LexA binding by increasing DNA unwrapping, not by nucleosome repositioning. Furthermore, we find that H3(K56Q) quantitatively imitates H3(K56ac) function. Together, these studies introduce powerful tools for the analysis of histone PTM functions. (C) 2011 Elsevier Ltd. All rights reserved.
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