Ontology highlight
ABSTRACT:
PROVIDER: PRJNA727457 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14425934.fastq.gz | Fastqsanger.gz | |||
| SRR14425935.fastq.gz | Fastqsanger.gz | |||
| SRR14425936.fastq.gz | Fastqsanger.gz | |||
| SRR14425937.fastq.gz | Fastqsanger.gz | |||
| SRR14425938.fastq.gz | Fastqsanger.gz |
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Similar Datasets
Project description:DNA partitioning CTPases of the ParB family mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centro-meric region of target DNA molecules and then spread laterally to form large nucleoprotein complexes that serve as docking points for the DNA segregation machinery. Here, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. Moreover, we solve crystal structures of ParB in the pre- and post-hydrolysis state and provide insight into the catalytic mechanism underlying nucleotide hydrolysis. The characterization of CTPase-deficient ParB variants reveals that CTP hydrolysis serves as a timing mechanism to control the sliding time of ParB. Hyperstable clamps are trapped on the DNA, leading to excessing spreading and severe chromosome segregation defects in vivo. These findings clarify the role of the ParB CTPase cycle in partition complex dynamics and function and thus complete our understanding of this prototypic CTP-dependent molecular switch.
2021-09-24 | GSE174037 | GEO
Project description:Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS to spread by sliding to the neighboring DNA. Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products may re-open the gates. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation and spreading.
2021-05-01 | GSE168968 | GEO
Project description:ParA and ParB homologs are involved in accurate chromosome segregation in bacteria. ParBs participate in proper folding and initial separation of ori domains by binding to specific parS sites (palindromic centromere-like sequences), mainly localized close to oriC. Bioinformatic analyses identified 10 parS sequences in the Pseudomonas aeruginosa PAO1 genome. One parS from the parS1-parS4 cluster is required for ParB mediated chromosome segregation. To verify the binding of ParB to all known parSs in vivo as well as to identify additional ParB binding sites we performed chromation immunoprecipitation (ChIP) using polyclonal anti-ParB antibodies followed by high throughput sequencing. ChIP was performed with P. aeruginosa PAO1161 (WT) cells, PAO1161 pKB9 (ParB+++) cells with a slight, non-toxic ParB overproduction as well as with 3 strains containing parS modifications impairing ParB binding to these sites. The data confirmed ParB binding to all known parS sequences with the exception of parS5. Moreover, we identified more than a 1000 of new ParB-bound regions, majority of which contained a DNA motif corresponding to inner 7 nt from one arm of the parS palindrome. ParB interactions with these numerous sites could affect chromosome topology, compaction and gene expression classifying P. aeruginosa ParB as a Nucleoid Associated Protein (NAP).
2018-03-27 | GSE110158 | GEO
Project description:Proper chromosome segregation is essential in all living organisms if daughter cells are each to inherit a full copy of genetic information. 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 recognized and bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. In this study, 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 ~8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites, then associates non-specifically with flanking DNA to form a high-order nucleoprotein complex that occupies an extensive ~10 kb DNA segment on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ~500 kb region surrounding the origin of replication and a ~100 kb region surrounding the terminus of the Caulobacter chromosome that are tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of the bacterial centromere-like parS is highly restricted and is crucial for chromosome segregation in Caulobacter.
2017-08-09 | GSE100233 | GEO
Project description:The tripartite ParA-ParB-parS complex ensures faithful chromosome segregation in the majority of bacterial species. ParB nucleates on the centromere-like parS site and spreads to neighboring DNA to form a network of protein-DNA complexes. This nucleoprotein network in turn interacts with ParA to partition the parS locus, hence the chromosome to each daughter cell. Here, we determine the co-crystal structure of the C-terminal domain truncated ParB-parS complex from Caulobacter crescentus, and show that its N-terminal domain is inherently flexible and adopts multiple different conformations. We propose that the flexibility of the N-terminal domain might facilitate the spreading of ParB on the chromosome. Next, using ChIP-seq we show that ParBs from different bacterial species exhibit variation in their intrinsic capability for spreading, and that the N-terminal domain rather than the C-terminal domain is the main determinant for the variation in spreading. Finally, we show that the C-terminal domain of Caulobacter ParB does not possess non-specific DNA-binding activity in vitro. Engineered ParB variants with enhanced non-specific DNA-binding activity condense DNA in vitro but do not spread further than a wild-type protein in vivo. Taken together, our results emphasize the central role of the N-terminal domain in ParB spreading and faithful chromosome segregation.
2020-07-22 | GSE134665 | GEO
Project description:Chromosomes readily unlink from one another and segregate to daughter cells during cell division highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes mediate chromosome folding by DNA loop extrusion. In most bacteria, SMC complexes start loop extrusion at the ParB/parS partition complex formed near the replication origin. Whether they are recruited by recognizing a specific DNA structure in the partition complex or a protein component is unknown. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate chromosomes when transplanted into another organism. Using chimeric proteins and chemical cross-linking, we find that ParB binds to the Smc subunit directly. We map a binding interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex to initiate DNA loop extrusion.
2022-03-01 | GSE190491 | GEO
Project description:Chromosomes readily unlink from one another and segregate to daughter cells during cell division highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes mediate chromosome folding by DNA loop extrusion. In most bacteria, SMC complexes start loop extrusion at the ParB/parS partition complex formed near the replication origin. Whether they are recruited by recognizing a specific DNA structure in the partition complex or a protein component is unknown. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate chromosomes when transplanted into another organism. Using chimeric proteins and chemical cross-linking, we find that ParB binds to the Smc subunit directly. We map a binding interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex to initiate DNA loop extrusion.
2022-06-23 | GSE206446 | GEO
Project description:Segregation of replicated chromosomes during cell division is an essential process in all organisms. Chromosome segregation is promoted by the action of the DNA-binding ParB protein in the rod-shaped model bacterium Bacillus subtilis. How oval shaped bacteria, such as the human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes is poorly understood. Here, we show that the pneumococcal homolog of ParB is enriched at four centromere-like DNA sequences (parS sites) that are present near the origin of replication.
2011-06-11 | GSE26839 | GEO
Project description:In the majority of bacterial species, the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s), assists in the chromosome partitioning. ParB forms large nucleoprotein complexes at parS(s), located in the vicinity of oriC, which after replication are subsequently relocated by ParA to polar positions. It was shown that ParB-parS complexes are loading platforms for structural maintenance of chromosome (Smc) proteins, which juxtapose the two arms of the circular chromosome. In this work, we characterized the Pseudomonas aeruginosa ParB interactions with DNA in the absence of Smc and interaction with the cognate ParA using chromatin immunoprecipitation-sequencing (ChIPseq). We show that in strains lacking Smc or strains with disturbed ParB-ParA interactions (ParA L84K or ParB G11A mutations) ParB is able to bind and spread around parS1-4 cluster and still binds to half-parS sites. Comparison of the ratio between the number of ChIP-seq reads mapping to region around parS1-4 with those mapping to ChIPseq peaks containing half-parSs indicate no major effect of the twod factors on the extent of ParB spreading, thus suggesting that the mechanisms controlling ParB association with the DNA do not involve interaction with cognate ParA partner and Smc.
2022-12-17 | GSE213881 | GEO
Project description:The transcriptional antisilencer VirB acts as a master regulator of virulence gene expression in the human pathogen Shigella flexneri. It binds defined sequences (virS) upstream of VirB-dependent promoters and counteracts their silencing by the nucleoid-organizing protein H-NS. However, its precise mode of action remains unclear. Notably, VirB is not a classical transcription factor but related to DNA partitioning pro- teins of the ParB family, which have recently been recognized as DNA-sliding clamps using CTP binding and hydrolysis to control their DNA entry gate. Here, we show that VirB binds CTP, embraces DNA in a clamp-like fashion upon its CTP-dependent loading at virS sites and slides laterally on DNA after clamp closure. Mutations that prevent CTP binding block the loading of VirB clamps in vitro and the formation of VirB nucleoprotein complexes in vivo. Thus, VirB represents a CTP-dependent molecular switch that uses a loading-and-sliding mechanism to control transcription during bacterial pathogenesis.
2023-12-06 | PXD046358 | Pride