Relief of ParB autoinhibition by parS DNA catalysis and recycling of ParB by CTP hydrolysis promote bacterial centromere assembly.
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ABSTRACT:
SUBMITTER: Antar H
PROVIDER: S-EPMC8494293 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ParABS systems facilitate chromosome segregation and plasmid partitioning in bacteria and archaea. ParB protein binds centromeric parS DNA sequences and spreads to flanking DNA. We show that ParB is an enzyme that hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP). parS DNA stimulates cooperative CTP binding by ParB and CTP hydrolysis. A nucleotide cocrystal structure elucidates the catalytic center of the dimerization-dependent ParB CTPase. Single-molecule imaging and biochemical assays recapitulate features of ParB spreading from parS in the presence but not absence of CTP. These findings suggest that centromeres assemble by self-loading of ParB DNA sliding clamps at parS ParB CTPase is not related to known nucleotide hydrolases and might be a promising target for developing new classes of antibiotics.
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Project description:Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single-molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one-dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum dot labelled ParB. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations.
| S-EPMC8299390 | biostudies-literature
Project description:ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase, and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF-ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF-ParBF complex assembly. Together, parSF and CTP accelerate ParAF-ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF-ParBF and ParAF-ParBF interactions promoting efficient partitioning.
| S-EPMC8357417 | biostudies-literature
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: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:Spo0J (stage 0 sporulation protein J, a member of the ParB superfamily) is an essential component of the ParABS (partition system of ParA, ParB, and parS)-related bacterial chromosome segregation system. ParB (partition protein B) and its regulatory protein, ParA, act cooperatively through parS (partition S) DNA to facilitate chromosome segregation. ParB binds to chromosomal DNA at specific parS sites as well as the neighboring nonspecific DNA sites. Various ParB molecules can associate together and spread along the chromosomal DNA. ParB oligomer and parS DNA interact together to form a high-order nucleoprotein that is required for the loading of the structural maintenance of chromosomes proteins onto the chromosome for chromosomal DNA condensation. In this report, we characterized the binding of parS and Spo0J from Helicobacter pylori (HpSpo0J) and solved the crystal structure of the C-terminal domain truncated protein (Ct-HpSpo0J)-parS complex. Ct-HpSpo0J folds into an elongated structure that includes a flexible N-terminal domain for protein-protein interaction and a conserved DNA-binding domain for parS binding. Two Ct-HpSpo0J molecules bind with one parS. Ct-HpSpo0J interacts vertically and horizontally with its neighbors through the N-terminal domain to form an oligomer. These adjacent and transverse interactions are accomplished via a highly conserved arginine patch: RRLR. These interactions might be needed for molecular assembly of a high-order nucleoprotein complex and for ParB spreading. A structural model for ParB spreading and chromosomal DNA condensation that lead to chromosome segregation is proposed.
| S-EPMC4450421 | biostudies-literature
Project description:UnlabelledParB proteins bind centromere-like DNA sequences called parS sites and are involved in plasmid and chromosome segregation in bacteria. We previously showed that the opportunistic human pathogen Streptococcus pneumoniae contains four parS sequences located close to the origin of replication which are bound by ParB. Using chromatin immunoprecipitation (ChIP), we found here that ParB spreads out from one of these parS sites, parS(-1.6°), for more than 5 kb and occupies the nearby comCDE operon, which drives competence development. Competence allows S. pneumoniae to take up DNA from its environment, thereby mediating horizontal gene transfer, and is also employed as a general stress response. Mutating parS(-1.6°) or deleting parB resulted in transcriptional up-regulation of comCDE and ssbB (a gene belonging to the competence regulon), demonstrating that ParB acts as a repressor of competence. However, genome-wide transcription analysis showed that ParB is not a global transcriptional regulator. Different factors, such as the composition of the growth medium and antibiotic-induced stress, can trigger the sensitive switch driving competence. This work shows that the ParB-parS chromosome segregation machinery also influences this developmental process.ImportanceStreptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for more than a million deaths each year. Like all other organisms, S. pneumoniae must be able to segregate its chromosomes properly. Not only is understanding the molecular mechanisms underlying chromosome segregation in S. pneumoniae therefore of fundamental importance, but also, this knowledge might offer new leads for ways to target this pathogen. Here, we identified a link between the pneumococcal chromosome segregation system and the competence-developmental system. Competence allows S. pneumoniae to take up and integrate exogenous DNA in its chromosome. This process plays a crucial role in successful adaptation to--and escape from--host defenses, antibiotic treatments, and vaccination strategies. We show that the chromosome segregation protein ParB acts as a repressor of competence. To the best of our knowledge, this is the first example of a ParB protein controlling bacterial competence.
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Project description:Polo-like kinase 4 (Plk4) is a master regulator of centriole duplication, and its hyperactivity induces centriole amplification. Homodimeric Plk4 has been shown to be ubiquitinated as a result of autophosphorylation, thus promoting its own degradation and preventing centriole amplification. Unlike other Plks, Plk4 contains three rather than two Polo box domains, and the function of its third Polo box (PB3) is unclear. Here, we performed a functional analysis of Plk4's structural domains. Like other Plks, Plk4 possesses a previously unidentified autoinhibitory mechanism mediated by a linker (L1) near the kinase domain. Thus, autoinhibition is a conserved feature of Plks. In the case of Plk4, autoinhibition is relieved after homodimerization and is accomplished by PB3 and by autophosphorylation of L1. In contrast, autophosphorylation of the second linker promotes separation of the Plk4 homodimer. Therefore, autoinhibition delays the multiple consequences of activation until Plk4 dimerizes. These findings reveal a complex mechanism of Plk4 regulation and activation which govern the process of centriole duplication.
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Project description:Organization and segregation of replicated chromosomes are essential processes during cell division in all organisms. Similar to eukaryotes, bacteria possess centromere-like DNA sequences (parS) that cluster at the origin of replication and the structural maintenance of chromosomes (SMC) complexes for faithful chromosome segregation. In Bacillus subtilis, parS sites are bound by the partitioning protein Spo0J (ParB), and we show here that Spo0J recruits the SMC complex to the origin. We demonstrate that the SMC complex colocalizes with Spo0J at the origin and that insertion of parS sites near the replication terminus targets SMC to this position leading to defects in chromosome organization and segregation. Consistent with these findings, the subcellular localization of the SMC complex is disrupted in the absence of Spo0J or the parS sites. We propose a model in which recruitment of SMC to the origin by Spo0J-parS organizes the origin region and promotes efficient chromosome segregation.
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Project description:Plasmid pCXC100 from the Gram-positive bacterium Leifsonia xyli subsp. cynodontis uses a type Ib partition system that includes a centromere region, a Walker-type ATPase ParA and a centromere-binding protein ParB for stable segregation. However, ParB shows no detectable sequence homology to any DNA-binding motif. Here, we study the ParB centromere interaction by structural and biochemical approaches. The crystal structure of the C-terminal DNA-binding domain of ParB at 1.4?Å resolution reveals a dimeric ribbon-helix-helix (RHH) motif, supporting the prevalence of RHH motif in centromere binding. Using hydroxyl radical footprinting and quantitative binding assays, we show that the centromere core comprises nine uninterrupted 9-nt direct repeats that can be successively bound by ParB dimers in a cooperative manner. However, the interaction of ParB with a single subsite requires 18 base pairs covering one immediate repeat as well as two halves of flanking repeats. Through mutagenesis, sequence specificity was determined for each position of an 18-bp subsite. These data suggest an unique centromere recognition mechanism by which the repeat sequence is jointly specified by adjacent ParB dimers bound to an overlapped region.
| S-EPMC3074162 | biostudies-literature