ABSTRACT
The yeast SWR1 complex catalyzes the exchange of histone H2A/H2B dimers in nucleosomes with Htz1/H2B dimers. We use cryoelectron microscopy to determine the structure of an enzyme-bound hexasome intermediate in the reaction pathway of histone exchange, in which an H2A/H2B dimer has been extracted from a nucleosome prior to the insertion of a dimer comprising Htz1/H2B. The structure reveals a key role for the Swc5 subunit in stabilizing the unwrapping of DNA from the histone core of the hexasome. By engineering a crosslink between an Htz1/H2B dimer and its chaperone protein Chz1, we show that this blocks histone exchange by SWR1 but allows the incoming chaperone-dimer complex to insert into the hexasome. We use this reagent to trap an SWR1/hexasome complex with an incoming Htz1/H2B dimer that shows how the reaction progresses to the next step. Taken together the structures reveal insights into the mechanism of histone exchange by SWR1 complex.
ABSTRACT
In Bacillus subtilis, a ParB-like nucleoid occlusion protein (Noc) binds specifically to Noc-binding sites (NBSs) on the chromosome to help coordinate chromosome segregation and cell division. Noc does so by binding to CTP to form large membrane-associated nucleoprotein complexes to physically inhibit the assembly of the cell division machinery. The site-specific binding of Noc to NBS DNA is a prerequisite for CTP-binding and the subsequent formation of a membrane-active DNA-entrapped protein complex. Here, we solve the structure of a C-terminally truncated B. subtilis Noc bound to NBS DNA to reveal the conformation of Noc at this crucial step. Our structure reveals the disengagement between the N-terminal CTP-binding domain and the NBS-binding domain of each DNA-bound Noc subunit; this is driven, in part, by the swapping of helices 4 and 5 at the interface of the two domains. Site-specific crosslinking data suggest that this conformation of Noc-NBS exists in solution. Overall, our results lend support to the recent proposal that parS/NBS binding catalyzes CTP binding and DNA entrapment by preventing the reengagement of the CTP-binding domain and the DNA-binding domain from the same ParB/Noc subunit.
Subject(s)
Bacillus subtilis , Bacterial Proteins , Chromosome Segregation , DNA, Bacterial , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Cell Division , DNA, Bacterial/chemistry , Protein Domains , Crystallography, X-RayABSTRACT
ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.
Subject(s)
Bacillus subtilis/cytology , Cytidine Triphosphate/metabolism , Pyrophosphatases/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Cell Division/genetics , Cell Division/physiology , Cell Membrane/metabolism , Chromosomes, Bacterial/genetics , Cytidine Triphosphate/physiology , Cytoskeletal Proteins/genetics , Pyrophosphatases/physiologyABSTRACT
Specific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins.
Subject(s)
Bacterial Proteins/genetics , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , Mutation/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , Conserved Sequence , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli/metabolism , Models, Biological , Protein Binding , Protein DomainsABSTRACT
Proper chromosome segregation during cell division is essential in all domains of life. In the majority of bacterial species, faithful chromosome segregation is mediated by the tripartite ParABS system, consisting of an ATPase protein ParA, a CTPase and DNA-binding protein ParB, and a centromere-like parS site. The parS site is most often located near the origin of replication and is segregated first after chromosome replication. ParB nucleates on parS before binding to adjacent non-specific DNA to form a multimeric nucleoprotein complex. ParA interacts with ParB to drive the higher-order ParB-DNA complex, and hence the replicating chromosomes, to each daughter cell. Here, we review the various models for the formation of the ParABS complex and describe its role in segregating the origin-proximal region of the chromosome. Additionally, we discuss outstanding questions and challenges in understanding bacterial chromosome segregation.
Subject(s)
Bacteria/genetics , Bacterial Proteins/metabolism , Chromosomes, Bacterial/genetics , Bacterial Proteins/chemistry , Chromosome SegregationABSTRACT
Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA-ParB-parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster â¼8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with â¼10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a â¼500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.