Sequence-specific recognition of DNA in the nucleosome by pyrrole-imidazole polyamides

The ability of DNA-binding proteins to recognize their cognate sites in chromatin is restricted by the structure and dynamics of nucleosomal DNA, and by the translational and rotational positioning of the histone octamer. Here, we use six different pyrrole-imidazole polyamides as sequence-specific molecular probes for DNA accessibility in nucleosomes. We show that sites on nucleosomal DNA facing away from the histone octamer, or even partially facing the histone octamer, are fully accessible and that nucleosomes remain fully folded upon ligand binding. Polyamides only failed to bind where sites are completely blocked by interactions with the histone octamer. Removal of the amino-terminal tails of either histone H3 or histone H4 allowed these polyamides to bind. These results demonstrate that much of the DNA in the nucleosome is freely accessible for molecular recognition in the minor groove, and also support a role for the amino-terminal tails of H3 and H4 in modulating accessibility of nucleosomal DNA.     

Unwinding of chromatin by the SV40 large T antigen DNA helicase.

We have analysed the unwinding of nucleosomally organized DNA by simian virus 40 large tumour (T) antigen. Isolated T antigen can bind to existing nucleosome cores containing the viral replication origin sequence, which results in displacement of the histone octamer and unwinding of the DNA. However, specific binding to nucleosome cores is salt sensitive and nearly completely blocked under ionic conditions that otherwise support DNA replication. Once started, the progressing T antigen helicase, like an elongating RNA polymerase, is not further repressed by histone octamers, irrespective of the presence or absence of linker histone H1. Disruption of the nucleosomal structure in the process of unwinding may be assisted by the demonstrated interaction of the hexameric T antigen complex with histone proteins H1 and H3. Finally, our studies reveal the inability of topoisomerase I and/or II to continually relieve the superhelical tension of covalently closed circular minichromosomes as generated during their unwinding by T antigen. This may indicate that chromatin relaxation during the process of DNA replication can only be efficiently performed by a topoisomerase that is (trans)activated by other factors.     

Crystal structures of nucleosome core particles in complex with minor groove DNA-binding ligands

We determined the crystal structures of three nucleosome core particles in complex with site-specific DNA-binding ligands, the pyrrole-imidazole polyamides. While the structure of the histone octamer and its interaction with the DNA remain unaffected by ligand binding, nucleosomal DNA undergoes significant structural changes at the ligand-binding sites and in adjacent regions to accommodate the ligands. Our findings suggest that twist diffusion occurs over long distances through tightly bound nucleosomal DNA. This may be relevant to the mechanism of ATP-dependent and spontaneous nucleosome translocation, and to the effect of bound factors on nucleosome dynamics.     

Rapid spontaneous accessibility of nucleosomal DNA

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for proteins that must bind it. How proteins gain access to DNA buried inside nucleosomes is not known. Here we report measurements of the rates of spontaneous nucleosome conformational changes in which a stretch of DNA transiently unwraps off the histone surface, starting from one end of the nucleosome, and then rewraps. The rates are rapid. Nucleosomal DNA remains fully wrapped for only approximately 250 ms before spontaneously unwrapping; unwrapped DNA rewraps within approximately 10-50 ms. Spontaneous unwrapping of nucleosomal DNA allows any protein rapid access even to buried stretches of the DNA. Our results explain how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale, and they imply that the major impediment to entry of RNA polymerase into a nucleosome is rewrapping of nucleosomal DNA, not unwrapping.     

The DNA chaperone HMGB1 facilitates ACF/CHRAC-dependent nucleosome sliding

Nucleosome remodelling complexes CHRAC and ACF contribute to chromatin dynamics by converting chemical energy into sliding of histone octamers on DNA. Their shared ATPase subunit ISWI binds DNA at the sites of entry into the nucleosome. A prevalent model assumes that DNA distortions catalysed by ISWI are converted into relocation of DNA relative to a histone octamer. HMGB1, one of the most abundant nuclear non-histone proteins, binds with preference to distorted DNA. We have now found that transient interaction of HMGB1 with nucleosomal linker DNA overlapping ISWI-binding sites enhances the ability of ACF to bind nucleosomal DNA and accelerates the sliding activity of limiting concentrations of remodelling factor. By contrast, an HMGB1 mutant with increased binding affinity was inhibitory. These observations are consistent with a role for HMGB1 as a DNA chaperone facilitating the rate-limiting DNA distortion during nucleosome remodelling.     

Structure-function relationships in nucleosomal arrays containing linker histone H5

To study the structural and functional changes accompanying the integration of histone H5 into the nucleosome structure, linear DNA species have been employed with a terminal promoter for bacteriophage T7 RNA polymerase followed by tandem repeats of a 207-bp nucleosome positioning sequence. The oligonucleosomes assembled from 12-repeat DNA and saturating amounts of core histone octamer plus histone H5 are compacted, in the presence of 1 mM free magnesium ions, to the level of the 30-nm fiber. Under these ionic conditions the efficiency in RNA synthesis and the size distribution of RNA chains obtained with this template are the same as those corresponding to the template without H5, indicating that the 30-nm fiber stabilized by H5 does not impair RNA elongation. Therefore, under our experimental conditions, incorporation of one molecule of histone H5 per nucleosome does not affect elongation of RNA even when a folded structure is produced. However, elongation is inhibited by binding of an excess of H5.     

A 'loop recapture' mechanism for ACF-dependent nucleosome remodeling

The ATPase ISWI is the molecular motor of several nucleosome remodeling complexes including ACF. We analyzed the ACF-nucleosome interactions and determined the characteristics of ACF-dependent nucleosome remodeling. In contrast to ISWI, ACF interacts symmetrically with DNA entry sites of the nucleosome. Two-color fluorescence cross-correlation spectroscopy measurements show that ACF can bind four DNA duplexes simultaneously in a complex that contains two Acf1 and ISWI molecules. Using bead-bound nucleosomal substrates, nucleosome movement by mechanisms involving DNA twisting was excluded. Furthermore, an ACF-dependent local detachment of DNA from the nucleosome was demonstrated in a novel assay based on the preferred intercalation of ethidium bromide to free DNA. The findings suggest a loop recapture mechanism in which ACF introduces a DNA loop at the nucleosomal entry site that propagates over the histone octamer surface and leads to nucleosome repositioning.