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...

Eukaryotic genomes are repetitively wrapped into nucleosomes that then regulate access of transcription and DNA repair complexes to DNA. The mechanisms that regulate extrinsic protein interactions within nucleosomes are unresolved. We demonstrate that modulation of the nucleosome unwrapping rate regulates protein binding within nucleosomes. Histone H3 acetyl-lysine 56 [H3(K56ac)] and DNA sequence within the nucleosome entry-exit region additively influence nucleosomal DNA accessibility by increasing the unwrapping rate without impacting rewrapping. These combined epigenetic and genetic factors influence transcription factor (TF) occupancy within the nucleosome by at least one order of magnitude and enhance nucleosome disassembly by the DNA mismatch repair complex, hMSH2–hMSH6. Our results combined with the observation that ∼30% of Saccharomyces cerevisiae TF-binding sites reside in the nucleosome entry–exit region suggest that modulation of nucleosome unwrapping is a mechanism for regulating transcription and DNA repair.

SUMMARY DNA nucleotide mismatches and lesion arise on chromosomes that are a complex assortment of protein and DNA (chromatin). The fundamental unit of chromatin is a nucleosome that contains ~146 bp DNA wrapped around an H2A, H2B, H3, and H4 histone octamer. We demonstrate that the mismatch recognition heterodimer hMSH2-hMSH6 disassembles a nucleosome. Disassembly requires a mismatch that provokes the formation of hMSH2-hMSH6 hydrolysis-independent sliding clamps, which translocate along the DNA to the nucleosome. The rate of disassembly is enhanced by actual or mimicked acetylation of histone H3 within the nucleosome entry-exit and dyad axis that occurs during replication and repair in vivo and reduces DNA-octamer affinity in vitro. Our results support a passive mechanism for chromatin remodeling where hMSH2-hMSH6 sliding clamps trap localized fluctuations in nucleosome positioning and/or wrapping that ultimately leads to disassembly, and highlights unanticipated strengths of the Molecular Switch Model for mismatch repair (MMR).

Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA–histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure.

The SWI/SNF chromatin remodeling complex changes the positions where nucleosomes are bound to DNA, exchanges out histone dimers, and disassembles nucleosomes. All of these activities depend on ATP hydrolysis by the catalytic subunit Snf2, containing a DNA-dependent ATPase domain. Here we examine the role of another domain in Snf2 called SnAC (Snf2 ATP coupling) that was shown previously to regulate the ATPase activity of SWI/SNF. We have found that SnAC has another function besides regulation of ATPase activity that is even more critical for nucleosome remodeling by SWI/SNF. We have found that deletion of the SnAC domain strongly uncouples ATP hydrolysis from nucleosome movement. Deletion of SnAC does not adversely affect the rate, processivity, or pulling force of SWI/SNF to translocate along free DNA in an ATP-dependent manner. The uncoupling of ATP hydrolysis from nucleosome movement is shown to be due to loss of SnAC binding to the histone surface of nucleosomes. While the SnAC domain targets both the ATPase domain and histones, the SnAC domain as a histone anchor plays a more critical role in remodeling because it is required to convert DNA translocation into nucleosome movement. The packing of DNA into chromatin involves the wrapping of 147 bp of DNA around a histone octamer to form a nucleosome and then assembly of nucleosomes into higher-order structures. Chromatin makes genomic DNA less accessible to protein factors important for transcription, replication, repair, and recombination. Chromatin structure is dynamic due to remodeling factors, some of which are ATP dependent, that facilitate making discrete regions accessible to other factors. ATP-dependent chromatin remodelers are single or large multisubunit assemblies composed of 1 to 17 subunits ranging from several kilodaltons to over a megadalton in molecular mass (1, 2). Each has a catalytic subunit with a conserved ATPase domain related to that of ATPdependent DNA helicases. In helicases, this domain couples ATP hydrolysis to DNA translocation and subsequent unwinding of double-stranded nucleic acid substrates by means of a translocase domain and a duplex destabilizing wedge domain (3-5). Unlike helicases, chromatin remodelers do not have nucleic acid unwinding activity, but have retained the translocase activity, which in turn repositions or disassembles nucleosomes (5, 6).Nucleosome movement by SWI/SNF-and ISWI-type complexes requires the ATPase domain to translocate along nucleosomal DNA near the dyad axis (7-10). DNA gaps near superhelical location 2 (SHL2) of the nucleosome block movement without interfering with binding of the remodeler. DNA translocation this far inside nucleosomes is challenging because there is no easy path for the ATPase domain to initially move. As the ATPase domain begins to translocate, it encounters histone-DNA interactions in both directions and has to overcome multiple histone-DNA interactions while trying to pull DNA into nucleosomes. The force required to disrupt histone-DNA interactions, as shown by mechanically ...

of RNA polymerase through the nucleosome. We develop a statistical-mechanics model of a nucleosome as a wormlike chain bound to a spool, incorporating fluctuations in the number of bases bound, the spool orientation, and the conformations of the unbound polymer segments. With the resulting free-energy surface, we perform dynamic simulations that permit a direct comparison with single-molecule experiments on a single nucleosome. This simple approach demonstrates that the experimentally observed structural states at nonzero tension are a consequence of the tension. Therefore, our model plays an important role in extrapolating the behavior to zero tension. This mechanism would arise in any system where the tether molecule is deformed in the transition state under the influence of tension. Using our statistical-mechanics model, we also study the translocation of RNA polymerase through a nucleosome. We consider RNA polymerase as a Brownian ratchet and model the translocation process using dynamic Monte Carlo simulation. We incorporate the effect of DNA elasticity on protein movement by considering the probability of RNA polymerase going into the pause state due to the force being applied by the bent DNA. Our theory suggests that RNA polymerase translocation is in pseudo-equilibrium with local DNA fluctuations and is not rate-limiting. Our theory predicts a very small change in translocation velocity of RNA polymerase in presence of TFIIS, suggesting that RNA polymerase generates sufficient forces to unravel the nucleosome in the absence of TFIIS.