The BRCT domain of PARP1 binds intact DNA and mediates intrastrand transfer.

PARP1 is a key player in the response to DNA damage and is the target of clinical inhibitors for the treatment of cancers. Binding of PARP1 to damaged DNA leads to activation wherein PARP1 uses NAD+ to add chains of poly(ADP-ribose) onto itself and other nuclear proteins. PARP1 also binds abundantly to intact DNA and chromatin, where it remains enzymatically inactive. We show that intact DNA makes contacts with the PARP1 BRCT domain, which was not previously recognized as a DNA-binding domain. This binding mode does not result in the concomitant reorganization and activation of the catalytic domain. We visualize the BRCT domain bound to nucleosomal DNA by cryogenic electron microscopy and identify a key motif conserved from ancestral BRCT domains for binding phosphates on DNA and phospho-peptides. Finally, we demonstrate that the DNA-binding properties of the BRCT domain contribute to the "monkey-bar mechanism" that mediates DNA transfer of PARP1.

Investigating the Dynamics of Destabilized Nucleosomes Using Methyl-TROSY NMR.

The nucleosome core particle (NCP), comprised of histone proteins wrapped with ∼146 base pairs of DNA, provides both protection and controlled access to DNA so as to regulate vital cellular processes. High-resolution structures of nucleosomes and nucleosome complexes have afforded a clear understanding of the structural role of NCPs, but a detailed description of the dynamical properties that facilitate DNA-templated processes is only beginning to emerge. Using methyl-TROSY NMR approaches we evaluate the effect of point mutations designed to perturb key histone interfaces that become destabilized during nucleosome remodeling in an effort to probe NCP plasticity. Notably the NCP retains its overall structural integrity, yet relaxation experiments of mutant nucleosomes reveal significant dynamics within a central histone interface associated with alternative NCP conformations populated to as much as 15% under low salt conditions. This work highlights the inherent plasticity of NCPs and establishes methyl-TROSY NMR as a valuable compliment to current single molecule methods in quantifying NCP dynamic properties.

Structure of histone-based chromatin in Archaea.

Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps around an extended polymer, formed by archaeal histone homodimers, in a quasi-continuous superhelix with the same geometry as DNA in the eukaryotic nucleosome. Substitutions of a conserved glycine at the interface of adjacent protein layers destabilize archaeal chromatin, reduce growth rate, and impair transcription regulation, confirming the biological importance of the polymeric structure. Our data establish that the histone-based mechanism of DNA compaction predates the nucleosome, illuminating the origin of the nucleosome.

Histone chaperone FACT action during transcription through chromatin by RNA polymerase II.

FACT (facilitates chromatin transcription) is a histone chaperone that promotes chromatin recovery during transcription, with additional roles in cell differentiation. Although several models of the action of FACT during transcription have been proposed, they remain to be experimentally evaluated. Here we show that human FACT (hFACT) facilitates transcription through chromatin and promotes nucleosome recovery in vitro. FACT action depends on the presence of histone H2A/H2B dimers in the nucleosome. Kinetic analysis suggests that hFACT decreases the lifetime of nonproductive RNA polymerase II (Pol II)-nucleosome complexes and facilitates the formation of productive complexes containing nucleosomal DNA partially uncoiled from the octamer. Taken together, our data suggest that hFACT interacts with DNA-binding surfaces of H2A/H2B dimers, facilitating uncoiling of DNA from the histone octamer. Thus, hFACT-H2A/H2B interactions play a key role in overcoming the nucleosomal barrier by Pol II and promoting nucleosome survival during transcription.

Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA.

H2A.Bbd is an unusual histone variant whose sequence is only 48% conserved compared to major H2A. The major sequence differences are in the docking domain that tethers the H2A-H2B dimer to the (H3-H4)(2) tetramer; in addition, the C-terminal tail is absent in H2A.Bbd. We assembled nucleosomes in which H2A is replaced by H2A.Bbd (Bbd-NCP), and found that Bbd-NCP had a more relaxed structure in which only 118+/-2 bp of DNA is protected against digestion with micrococcal nuclease. The absence of fluorescence resonance energy transfer between the ends of the DNA in Bbd-NCP indicates that the distance between the DNA ends is increased significantly. The Bbd docking domain is largely responsible for this behavior, as shown by domain-swap experiments. Bbd-containing nucleosomal arrays repress transcription from a natural promoter, and this repression can be alleviated by transcriptional activators Tax and CREB. The structural properties of Bbd-NCP described here have important implications for the in vivo function of this histone variant and are consistent with its proposed role in transcriptionally active chromatin.

A new fluorescence resonance energy transfer approach demonstrates that the histone variant H2AZ stabilizes the histone octamer within the nucleosome.

Nucleosomes are highly dynamic macromolecular complexes that are assembled and disassembled in a modular fashion. One important way in which this dynamic process can be modulated is by the replacement of major histones with their variants, thereby affecting nucleosome structure and function. Here we use fluorescence resonance energy transfer between fluorophores attached to various defined locations within the nucleosome to dissect and compare the structural transitions of a H2A.Z containing and a canonical nucleosome in response to increasing ionic strength. We show that the peripheral regions of the DNA dissociate from the surface of the histone octamer at relatively low ionic strength, under conditions where the dimer-tetramer interaction remains unaffected. At around 550 mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)(2) tetramer-DNA complex. Significantly, this latter transition is stabilized in nucleosomes that have been reconstituted with the essential histone variant H2A.Z. Our studies firmly establish fluorescence resonance energy transfer as a valid method to study nucleosome stability, and shed new light on the biological function of H2A.Z.

Crystal structures of histone Sin mutant nucleosomes reveal altered protein-DNA interactions.

Here we describe 11 crystal structures of nucleosome core particles containing individual point mutations in the structured regions of histones H3 and H4. The mutated residues are located at the two protein-DNA interfaces flanking the nucleosomal dyad. Five of the mutations partially restore the in vivo effects of SWI/SNF inactivation in yeast. We find that even nonconservative mutations of these residues (which exhibit a distinct phenotype in vivo) have only moderate effects on global nucleosome structure. Rather, local protein-DNA interactions are disrupted and weakened in a subtle and complex manner. The number of lost protein-DNA interactions correlates directly with an increased propensity of the histone octamer to reposition with respect to the DNA, and with an overall destabilization of the nucleosome. Thus, the disruption of only two to six of the approximately 120 direct histone-DNA interactions within the nucleosome has a pronounced effect on nucleosome mobility and stability. This has implications for our understanding of how these structures are made accessible to the transcription and replication machinery in vivo.

Structure and dynamics of nucleosomal DNA.

The last five years have seen exciting advances in our understanding of the structure of the nucleosome core particle, the basic repeating unit in all eukaryotic chromatin. A picture emerges in which nucleosomal DNA, while distorted and compacted fivefold by tight interactions with the histone octamer core, is at the same time highly dynamic and adaptable. Here, we summarize the salient features from recent structural studies of nucleosome core particles (both published and unpublished) that concern the structure and dynamics of nucleosomal DNA, and the nature of protein-DNA interactions. Current mechanisms for chromatin remodeling and nucleosome sliding are discussed in light of new structural evidence. Finally, techniques to study nucleosome stability and ultimately dynamics are introduced.