The DNA-binding domain of the Chd1 chromatin-remodelling enzyme contains SANT and SLIDE domains

The ATP-dependent chromatin-remodelling enzyme Chd1 is a 168-kDa protein consisting of a double chromodomain, Snf2-related ATPase domain, and a C-terminal DNA-binding domain. Here, we show the DNA-binding domain is required for Saccharomyces cerevisiae Chd1 to bind and remodel nucleosomes. The crystal structure of this domain reveals the presence of structural homology to SANT and SLIDE domains previously identified in ISWI remodelling enzymes. The presence of these domains in ISWI and Chd1 chromatin-remodelling enzymes may provide a means of efficiently harnessing the action of the Snf2-related ATPase domain for the purpose of nucleosome spacing and provide an explanation for partial redundancy between these proteins. Site directed mutagenesis was used to identify residues important for DNA binding and generate a model describing the interaction of this domain with DNA. Through inclusion of Chd1 sequences in homology searches SLIDE domains were identified in CHD6-9 proteins. Point mutations to conserved amino acids within the human CHD7 SLIDE domain have been identified in patients with CHARGE syndrome.     

Snf2-family proteins: chromatin remodellers for any occasion

Chromatin facilitates the housing of eukaryotic DNA within the nucleus and restricts access to the underlying sequences. Thus, the regulation of chromatin structure provides an excellent platform for regulating processes that require information stored within genomic DNA. Snf2 proteins are a family of helicase-like proteins that direct energy derived from ATP hydrolysis into the mechanical remodelling of chromatin structure. Here, we highlight some of the recent discoveries regarding this family of proteins and show Snf2 proteins have roles in many aspects of genetic metabolism. Recent developments include new insights into the mechanism for nucleosome spacing and histone dimer exchange; together with growing evidence for the involvement of Snf2 proteins in DNA repair.     

Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin

In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecA-like recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p. Here we find that yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor. In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor. We also show that Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF. Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, our results indicate that Rad54p is a chromatin-remodeling enzyme that promotes homologous DNA pairing events within the context of chromatin.     

dMi-2 and ISWI chromatin remodelling factors have distinct nucleosome binding and mobilization properties

Mi-2 and ISWI, two members of the Snf2 superfamily of ATPases, reside in separate ATP-dependent chromatin remodelling complexes. These complexes differ in their biochemical properties and are believed to perform distinct functions in the cell. We have compared the remodelling activity of recombinant Drosophila Mi-2 (dMi-2) with that of recombinant ISWI. Both proteins are nucleosome-stimulated ATPases and promote nucleosome mobilization. However, dMi-2 and ISWI differ in their interaction with nucleosome core particles, in their substrate requirements and in the direction of nucleosome mobilization. We have used antibodies to immobilize a complex containing dMi-2 and the dRPD3 histone deacetylase from Drosophila embryo extracts. This complex shares the nucleosome-stimulated ATPase and nucleosome mobilization properties of recombinant dMi-2, demonstrating that these activities are maintained in a physiological context. Its functional properties distinguish dMi-2 from both SWI2/SNF2 and ISWI, defining a new family of ATP-dependent remodelling machines.     

Multiple Aspects of ATP-Dependent Nucleosome Translocation by RSC and Mi-2 Are Directed by the Underlying DNA Sequence

Background

Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted.

Methodology/Principal Findings

We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type) and native RSC from yeast (SWI/SNF-type) repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps.

Conclusions/Significance

Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase-catalyzed repositioning. Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA. This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo.     

Snf2 family ATPases and DExx box helicases: differences and unifying concepts from high-resolution crystal structures

Proteins with sequence similarity to the yeast Snf2 protein form a large family of ATPases that act to alter the structure of a diverse range of DNA–protein structures including chromatin. Snf2 family enzymes are related in sequence to DExx box helicases, yet they do not possess helicase activity. Recent biochemical and structural studies suggest that the mechanism by which these enzymes act involves ATP-dependent translocation on DNA. Crystal structures suggest that these enzymes travel along the minor groove, a process that can generate the torque or energy in remodelling processes. We review the recent structural and biochemical findings which suggest a common mechanistic basis underlies the action of many of both Snf2 family and DExx box helicases.     

The interactions of yeast SWI/SNF and RSC with the nucleosome before and after chromatin remodeling

Interactions of the yeast chromatin-remodeling complexes SWI/SNF and RSC with nucleosomes were probed using site-specific DNA photoaffinity labeling. 5 S rDNA was engineered with photoreactive nucleotides incorporated at different sites in DNA to scan for the subunits of SWI/SNF in close proximity to DNA when SWI/SNF is bound to the 5 S nucleosome or to the free 5 S rDNA. The Swi2/Snf2 and Snf6 subunits of SWI/SNF were efficiently cross-linked at several positions in the nucleosome, whereas only Snf6 was efficiently cross-linked when SWI/SNF was bound to free DNA. DNA photoaffinity labeling of RSC showed that the Rsc4 subunit is in close proximity to nucleosomal DNA and not when RSC is bound to free DNA. After remodeling, the Swi2/Snf2 and Rsc4 subunits are no longer detected near the nucleosomal DNA and are evidently displaced from the surface of the nucleosome, indicating significant changes in SWI/SNF and RSC contacts with DNA after remodeling.     

Mutations to the histone H3 αN region selectively alter the outcome of ATP-dependent nucleosome-remodelling reactions

Mutational analysis of the histone H3 N-terminal region has shown it to play an important role both in chromatin function in vivo and nucleosome dynamics in vitro. Here we use a library of mutations in the H3 N-terminal region to investigate the contribution of this region to the action of the ATP-dependent remodelling enzymes Chd1, RSC and SWI/SNF. All of the enzymes were affected differently by the mutations with Chd1 being affected the least and RSC being most sensitive. In addition to affecting the rate of remodelling by RSC, some mutations prevented RSC from moving nucleosomes to locations in which DNA was unravelled. These observations illustrate that the mechanisms by which different ATP-dependent remodelling enzymes act are sensitive to different features of nucleosome structure. They also show how alterations to histones can affect the products generated as a result of ATP-dependent remodelling reactions.     

A conserved Swi2/Snf2 ATPase motif couples ATP hydrolysis to chromatin remodeling

Yeast (Saccharomyces cerevisiae) SWI/SNF is a prototype for a large family of ATP-dependent chromatin-remodeling enzymes that facilitate numerous DNA-mediated processes. Swi2/Snf2 is the catalytic subunit of SWI/SNF, and it is the founding member of a novel subfamily of the SF2 superfamily of DNA helicase/ATPases. Here we present a functional analysis of the diagnostic set of helicase/ATPase sequence motifs found within all Swi2p/Snf2p family members. Whereas many of these motifs play key roles in ATP binding and/or hydrolysis, we identify residues within conserved motif V that are specifically required to couple ATP hydrolysis to chromatin-remodeling activity. Interestingly, motif V of the human Swi2p/Snf2p homolog, Brg1p, has been shown to be a possible hot spot for mutational alterations associated with cancers.     

Open, repair and close again: chromatin dynamics and the response to UV-induced DNA damage

Due to its link with human pathologies, including cancer, the mechanism of Nucleotide Excision Repair (NER) has been extensively studied. Most of the pathway and players have been defined using in vitro reconstitution experiments. However, in vivo, the NER machinery must deal with the presence of organized chromatin, which in some regions, such as heterochromatin, is highly condensed but still susceptible to DNA damage. A series of events involving different chromatin-remodeling factors and histone-modifying enzymes target chromatin regions that contain DNA lesions. CPDs change the structure of the nucleosome, allowing access to factors that can recognize the lesion. Next, DDB1-DDB2 protein complexes, which mono-ubiquitinate histones H2A, H3, and H4, recognize nucleosomes containing DNA lesions. The ubiquitinated nucleosome facilitates the recruitment of ATP-dependent chromatin-remodeling factors and the XPC-HR23B-Centrin 2 complex to the target region. Different ATP-dependent chromatin-remodeling factors, such as SWI/SNF and INO80, have been identified as having roles in the UV irradiation response prior to the action of the NER machinery. Subsequently, remodeling of the nucleosome allows enzymatic reactions by histone-modifying factors that may acetylate, methylate or demethylate specific histone residues. Intriguingly, some of these histone modifications are dependent on p53. These histone modifications and the remodeling of the nucleosome allow the entrance of TFIIH, XPC and other NER factors that remove the damaged strand; then, gap-filling DNA synthesis and ligation reactions are carried out after excision of the oligonucleotide with the lesion. Finally, after DNA repair, the initial chromatin structure has to be reestablished. Therefore, factors that modulate chromatin dynamics contribute to the NER mechanism, and they are significant in the future design of treatments for human pathologies related to genome instability and the appearance of drug-resistant tumors.     

Bacterial DNA ligases

DNA ligases join breaks in the phosphodiester backbone of DNA molecules and are used in many essential reactions within the cell. All DNA ligases follow the same reaction mechanism, but they may use either ATP or NAD+ as a cofactor. All Bacteria (eubacteria) contain NAD+-dependent DNA ligases, and the uniqueness of these enzymes to Bacteria makes them an attractive target for novel antibiotics. In addition to their NAD+-dependent enzymes, some Bacteria contain genes for putative ATP-dependent DNA ligases. The requirement for these different isozymes in Bacteria is unknown, but may be related to their utilization in different aspects of DNA metabolism. The putative ATP-dependent DNA ligases found in Bacteria are most closely related to proteins from Archaea and viruses. Phylogenetic analysis suggests that all NAD+-dependent DNA ligases are closely related, but the ATP-dependent enzymes have been acquired by Bacterial genomes on a number of separate occasions.     

Structural studies of the human PBAF chromatin-remodeling complex

ATP-dependent chromatin remodeling is one of the central processes responsible for imparting fluidity to chromatin and thus regulating DNA transactions. Although knowledge on this process is accumulating rapidly, the basic mechanism (or mechanisms) by which the remodeling complexes alter the structure of a nucleosome is not yet understood. Structural information on these macromolecular machines should aid in interpreting the biochemical and genetic data; to this end, we have determined the structure of the human PBAF ATP-dependent chromatin-remodeling complex preserved in negative stain by electron microscopy and have mapped the nucleosome binding site using two-dimensional (2D) image analysis. PBAF has an overall C-shaped architecture--with a larger density to which two smaller knobs are attached--surrounding a central cavity; one of these knobs appears to be flexible and occupies different positions in each of the structures determined. The 2D analysis of PBAF:nucleosome complexes indicates that the nucleosome binds in the central cavity.     

Collaborative competition mechanism for gene activation in vivo

The mechanism by which gene regulatory proteins gain access to their DNA target sites is not known. In vitro, binding is inherently cooperative between arbitrary DNA binding proteins whose target sites are located within the same nucleosome. We refer to such competition-based cooperativity as collaborative competition. Here we show that arbitrarily chosen foreign DNA binding proteins, LexA and Tet repressor, cooperate with an adjacently binding endogenous activator protein, Gcn4, to coactivate expression of chromosomal reporter genes in Saccharomyces cerevisiae. Coactivation requires that the cooperating target sites be within a nucleosome-length distance; it leads to increased occupancy by Gcn4 at its binding site; and it requires both Gcn5 and Swi/Snf which, at an endogenous Gcn4-dependent promoter, act subsequent to Gcn4 binding. These results imply that collaborative competition contributes to gene regulation in vivo. They further imply that, even in the presence of the cell's full wild-type complement of chromatin remodeling factors, competition of regulatory proteins with histone octamer for access to regulatory target sites remains a quantitative determinant of gene expression levels. We speculate that initial target site recognition and binding may occur via spontaneous nucleosomal site exposure, with remodeling factor action required downstream to lock in higher levels of regulatory protein occupancy.     

Identification of multiple distinct Snf2 subfamilies with conserved structural motifs

The Snf2 family of helicase-related proteins includes the catalytic subunits of ATP-dependent chromatin remodelling complexes found in all eukaryotes. These act to regulate the structure and dynamic properties of chromatin and so influence a broad range of nuclear processes. We have exploited progress in genome sequencing to assemble a comprehensive catalogue of over 1300 Snf2 family members. Multiple sequence alignment of the helicase-related regions enables 24 distinct subfamilies to be identified, a considerable expansion over earlier surveys. Where information is known, there is a good correlation between biological or biochemical function and these assignments, suggesting Snf2 family motor domains are tuned for specific tasks. Scanning of complete genomes reveals all eukaryotes contain members of multiple subfamilies, whereas they are less common and not ubiquitous in eubacteria or archaea. The large sample of Snf2 proteins enables additional distinguishing conserved sequence blocks within the helicase-like motor to be identified. The establishment of a phylogeny for Snf2 proteins provides an opportunity to make informed assignments of function, and the identification of conserved motifs provides a framework for understanding the mechanisms by which these proteins function.