Histone post-translational modifications are essential for regulating and facilitating biological processes such as RNA transcription and DNA repair. Fifteen modifications are located in the DNA-histone dyad interface and include the acetylation of H3-K115 (H3-K115Ac) and H3-K122 (H3-K122Ac), but the functional consequences of these modifications are unknown. We have prepared semisynthetic histone H3 acetylated at Lys-115 and/or Lys-122 by expressed protein ligation and incorporated them into single nucleosomes. Competitive reconstitution analysis demonstrated that the acetylation of H3-K115 and H3-K122 reduces the free energy of histone octamer binding. Restriction enzyme kinetic analysis suggests that these histone modifications do not alter DNA accessibility near the sites of modification. However, acetylation of H3-K122 increases the rate of thermal repositioning. Remarkably, Lys 3 Gln substitution mutations, which are used to mimic Lys acetylation, do not fully duplicate the effects of the H3-K115Ac or H3-K122Ac modifications. Our results are consistent with the conclusion that acetylation in the dyad interface reduces DNA-histone interaction(s), which may facilitate nucleosome repositioning and/or assembly/disassembly.All eukaryotic genomes are organized into strings of nucleosomes, where 147 bp of DNA are tightly wrapped around a histone protein octamer (1). Many biological processes are dependent on DNA-protein interactions. However, access to DNA-binding sites is often restricted by the nucleosome structure. Alterations in nucleosome structure, dynamics, and positioning have been hypothesized to play a gatekeeper role in regulating biological processes such as DNA replication, repair, and transcription (2).The post-translational modification (PTM) 3 of core histones (3) plays a central role in regulating the biological processing of eukaryotic genomes. Until recently, known histone PTMs were almost exclusively located on the unstructured histone tail regions, which extend from the structured core of the nucleosomes. PTMs in the histone tails can function to directly alter nucleosome (4 -6) and/or chromatin structure and stability (7,8) and function as protein-binding sites (9) in the "histone code" model (10).During the past 5 years over 30 additional histone PTMs were identified within structured regions of the nucleosome (11-13). Many of these modifications are buried within the nucleosome core and thus are not readily accessible for protein binding. Fifteen of these histone PTMs are located in the DNAhistone interface, where the histone octamer contacts the phosphate backbone of the wrapped DNA (14). Studies have suggested that only mild structural perturbations occur in coremodified nucleosomes, which implies that modifications buried beneath the DNA are unlikely to provide a protein-binding site (15). This has led to two additional models for the function of nucleosome core PTMs.Modifications such as Lys acetylation that reduce the positive charge of the histone octamer surface may reduce the bindi...

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Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. We prepared H3(T118ph) by expressed protein ligation and determined its influence on nucleosome dynamics. We find H3(T118ph) reduces DNA–histone binding by 2 kcal/mol, increases nucleosome mobility by 28-fold and increases DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increases the rate of hMSH2–hMSH6 nucleosome disassembly and enables nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions.