Diversification of DNA-binding specificity via permissive and specificity-switching mutations in the ParB/Noc protein family
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ABSTRACT:
PROVIDER: PRJNA530746 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8842550.fastq.gz | Fastqsanger.gz | |||
| SRR8842560.fastq.gz | Fastqsanger.gz | |||
| SRR8842570.fastq.gz | Fastqsanger.gz | |||
| SRR8842551.fastq.gz | Fastqsanger.gz | |||
| SRR8842561.fastq.gz | Fastqsanger.gz |
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Project description: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 what were the mutational paths that led to changes in specificity. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB and Noc: both having 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 a 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 in understanding the evolution of other classes of DNA-binding proteins.
2019-08-05 | GSE129285 | GEO
Project description: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.
| S-EPMC7383237 | biostudies-literature
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:Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome
| PRJNA391136 | ENA
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: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: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: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:To identify ParB binding sites in vivo, we performed ChIP experiments followed by high throughput sequencing using a 3xflag tagged version of ParB, in different genetic backgrounds. This allowed us to show that ParB binds in vivo mostly to 4 parS sites, located within 15 kb of oriC. We also identify 9 secondary ParB binding sites.
2016-10-18 | GSE87409 | GEO
Project description:In Pseudomonas aeruginosa, partitioning protein ParB facilitates segregation of newly replicated chromosomes but is not essential for cell survival. Unlike in other bacteria, inactivation of parB leads to major changes of the transcriptome, suggesting that, directly or indirectly, ParB plays a role in regulation of gene expression in this organism. To identify primary targets of ParB, we analysed the impact of a slight increase in ParB amount on the transcriptome using microarrays. A several-fold increased ParB level does not cause recognizable phenotypic changes but leads to significant changes in the expression of 211 loci, including transcriptional regulators of operons involved in SOS response, virulence and adaptation. Most notably, the mRNA level of genes adjacent to high affinity ParB binding sites parS1-4 close to oriC is reduced. Our data support the role of partitioning protein ParB as a transcriptional regulator in Pseudomonas aeruginosa.
2017-07-25 | GSE95647 | GEO