A CTP-dependent gating mechanism enables ParB spreading on DNA
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA714673 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13970453.fastq.gz | Fastqsanger.gz | |||
| SRR13970454.fastq.gz | Fastqsanger.gz | |||
| SRR13970455.fastq.gz | Fastqsanger.gz | |||
| SRR13970456.fastq.gz | Fastqsanger.gz | |||
| SRR15194706.fastq.gz | Fastqsanger.gz |
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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: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 and spread by sliding to the neighboring DNA (Jalal et al., 2020). 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 are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.
| S-EPMC8367383 | biostudies-literature
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:Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1-parS4), which is positioned within the cell by ParA. Remarkably, ParB of P. aeruginosa binds to multiple heptanucleotides (half-parSs) scattered in the genome. In this work we analysed the influence of culturing conditions on ParB binding to DNA. Using chromatin immunoprecipitation-sequencing (ChIP-seq), we analysed patterns of genome occupancy by ParB in cells, with either coupling or uncoupling between replication and cell division. Our data indicated no altered preference of ParB to bind to individual half-parS sites under varying growth conditions, however a shift from parSs to half-parSs was evident in response to extended cell division time. The ChIP-seq analysis of strains expressing ParB variants unable to dislocate from parSs showed that ParB spreading ability is not required for ParB binding to half-parSs. Finally, a P. aeruginosa strain with mutated 27 half-parSs forming the strongest ParB ChIP-seq peaks was constructed and analysed showing changes in the ParB coverage of oriC region. Overall this work suggests the role of half-parSs in retaining ParB on the nucleoid within P. aeruginosa cells.
2023-08-03 | GSE233378 | 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:The ATP- and CTP-dependent ParA-ParB-parS segrosome is a macromolecular complex that segregates chromosomes/plasmids in most bacterial species. CTP binding and hydrolysis enable ParB to slide along DNA and to bridge and condense DNA, thereby dictating the size and dynamics of the tripartite ParABS complex. Several other evolutionarily distinct systems can also segregate DNA, although the full diversity of bacterial DNA partition systems remains unknown. Here, we identify a CTP-independent ParABS system that maintains the conjugative plasmid SCP2 in the filamentous bacterium Streptomyces coelicolor. We demonstrate that an SCP2 ParB-like protein, ParT, loads onto DNA at an 18-bp parS site and diffuses away to the adjacent DNA despite lacking an apparent CTPase domain and detectable NTPase activity. We further show that parS DNA facilitates ParT transition from loading to a diffusing state, allowing ParT to accumulate on DNA, and that ParT activates the ATPase activity of its cognate partner protein, ParA. Additionally, we identify numerous structural homologs of ParT, suggesting that CTP-independent diffusion on DNA might be more common than previously recognized. Overall, our findings reveal a CTP-independent DNA translocation as an alternative and unexpected mechanism for assembling a bacterial DNA segregation complex and suggest that CTP binding and hydrolysis are not universal features of ParABS-like systems.
2025-06-22 | GSE263222 | 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:We report the genome-wide analysis from chromatin immunoprecipitated DNA (ChIP-sequencing) of the DNA binding pattern of ParBF (SopB) of plasmid F. This study, performed in E. coli, investigates the impact of mutations in the CTP binding motif BoxIII on ParBF DNA binding profiles in vivo. We found that ParBF-N153A allele binds to parS as WT but is impaired in clamp formation thus abolishing the long distance DNA binding on parS-proximal DNA. By contrast, ParBF-R156A DNA binding profile reveals that it binds to parS, converts to clamps that diffuse over several Kb and unbinds non-specific DNA in a similar way as WT. However, the reduction in ParB binding at proximity of parS revealed that ParBF-R156A is impaired in ParB-ParB interaction, thus preventing the assembly of ParB condensates.
2025-06-01 | GSE289093 | 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: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