SMC translocation is unaffected by an excess of nucleoid associated proteins in vivo
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ABSTRACT:
PROVIDER: PRJNA1123659 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29397400_1.fastq.gz | Fastqsanger.gz | |||
| SRR29397400_2.fastq.gz | Fastqsanger.gz | |||
| SRR29397401_1.fastq.gz | Fastqsanger.gz | |||
| SRR29397401_2.fastq.gz | Fastqsanger.gz | |||
| SRR29397402_1.fastq.gz | Fastqsanger.gz |
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Project description:Genome organization is important for DNA replication, gene expression, and chromosome segregation. In bacteria, two large family of proteins, nucleoid-associated proteins (NAPs) and the SMC complexes, play important roles organizing the genome. NAPs are abundant DNA-binding proteins that can bend, wrap, bridge and compact DNA, while SMC complexes load on the chromosome, translocate on the DNA, and extrude DNA loops. How SMC loop extrusion is influenced by various NAPs remains unknown. In this study, we expressed a collection of representative prokaryotic chromosome-associated proteins in Bacillus subtilis, which introduce distinct DNA structures and pose different challenges for SMC loop extrusion. By fluorescence microscopy and chromatin immunoprecipitation experiments, we observed that these proteins bound to the genome in characteristic manners. Through genome-wide chromosome conformation capture assays, we found that the SMC complex can traverse these DNA-binding proteins without slowing down. Our findings reveal that the DNA loop extrusion activity of the SMC complex is unaffected by the chromosome-associated proteins and highlight the robustness of SMC motors on the busy chromatin.
2025-01-17 | GSE269767 | GEO
Project description:Genome organization is important for DNA replication, gene expression, and chromosome segregation. In bacteria, two large families of proteins, nucleoid-associated proteins (NAPs) and SMC complexes, play important roles in organizing the genome. NAPs are highly abundant DNA-binding proteins that can bend, wrap, bridge, and compact DNA, while SMC complexes load onto the chromosome, translocate on the DNA, and extrude DNA loops. Although SMC complexes are capable of traversing the entire chromosome bound by various NAPs in vivo, it is unclear whether SMC translocation is influenced by NAPs. In this study, using Bacillus subtilis as a model system, we expressed a collection of representative bacterial and archaeal DNA-binding proteins that introduce distinct DNA structures and potentially pose different challenges for SMC movement. By fluorescence microscopy and chromatin immunoprecipitation, we observed that these proteins bound to the genome in characteristic manners. Using genome-wide chromosome conformation capture (Hi-C) assays, we found that the SMC complex traversed these DNA-binding proteins without slowing down. Our findings revealed that the DNA-loop-extruding activity of the SMC complex is unaffected by exogenously expressed DNA-binding proteins, which highlights the robustness of SMC motors on the busy chromatin.
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Project description:Structural maintenance of chromosomes (SMC) complexes organize genomes by extruding DNA loops. How collisions between SMC complexes and DNA polymerases are resolved in vivo remains poorly understood. Taking advantage of the ability to load SMC complexes at defined sites in the Bacillus subtilis genome, we investigated this problem. We engineered head-on and head-to-tail collisions between SMC complexes and the replisome, and monitored SMC translocation by time-resolved ChIP-seq and Hi-C and replisome progression by marker frequency analysis. We report that SMC complexes do not affect replication progression. By contrast, the replisome blocks SMC translocation regardless of collision orientation. Combining experimental data with simulations, we determined that SMC is first blocked by the replisome and then released from the chromosome. However, occasionally SMC can bypass the replisome and continue translocating. Our findings establish that the replisome is a barrier to SMC-mediated DNA-loop extrusion, with implications for chromosome organization in all organisms.
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Project description:Replisomes restrict SMC translocation in vivo
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Project description:Nucleoid remodeling facilitated by DNA supercoiling results in changes in nucleoid configuration and involves nucleoid-associated proteins (NAPs) and structural maintenance of chromosomes (SMC) proteins among others. Changes in nucleoid configurations regulated by NAPs are synchronized with cellular adaptation and influence the simultaneous expression of several genes. HU, a ubiquitous bacterial histone-like protein, is among the most conserved and abundant NAPs in eubacteria. In Escherichia coli, HU forms dimers by HUα self-association (HUαα) or by HUα-HUβ interactions (HUαβ). HUα is mostly expressed during lag and early exponential growth phase and HUβ is expressed only during the later exponential and stationary phase, pointing to distinct HUαα/DNA and HUαβ/DNA packaging of the nucleoid in regulating expression patterns during growth and stasis. Mutations or the deletion of HU transform the E. coli nucleoid to a different form and alter overall transcription program; thus, HU interactions with DNA directly affect global gene regulation. Recently, analysis of high-resolution contact maps of the E. coli nucleoid revealed an important role of HU to promote long-range DNA-DNA contacts within the nucleoid. Yet, the molecular connections between HU-DNA interactions and changes in nucleoid architecture that regulate gene expression globally remain unknown. Here, we explored the higher-order E. coli nucleoid organization by soft x-ray tomography (SXT) and revealed an effect of HU surface charges in overall nucleoid organization and rearrangements. We also studied global transcription by next-generation RNA sequencing (RNA-Seq) and found a link between nucleoid rearrangement and changes in global transcription. To determine the functional relationships of observed nucleoid rearrangement and HU-DNA interactions, we also characterized the overall organization of HU nucleoprotein complexes in solution by small angle x-ray scattering (SAXS). We found that HUαα-mediated DNA networks are different at different ionic strengths and pHs. By means of macromolecular crystallography, we additionally elucidate HUαα dependent molecular switches that modulate DNA networking. This integrative structural study explains how HUαα regulates dynamic transformations of the nucleoid by DNA bridging to control nucleoid rearrangement and global gene regulation.
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Project description:The Structural Maintenance of Chromosomes (SMC) complex plays an important role in chromosome organization and segregation in most living organisms. In Caulobacter crescentus, SMC is required to align the left and the right arms of the chromosome that run in parallel down the long axis of the cell. However, the mechanism of SMC-mediated alignment of chromosomal arms remains elusive. Here, using genome-wide methods and microscopy of single cells, we show that Caulobacter SMC is recruited to the centromeric parS site and that SMC-mediated arm alignment depends on the chromosome partitioning protein ParB. We provide evidence that SMC likely tethers the parS-proximal regions of the chromosomal arms together, promoting arm alignment. Furthermore, we show that highly-transcribed genes near parS that are oriented against SMC translocation disrupt arm alignment, suggesting that head-on transcription interferes with SMC translocation. Our results demonstrate a tight interdependence of bacterial chromosome organization and global patterns of transcription.
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Project description:Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosomeâpresumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc. ChIP-Seq experiments were performed on wild type and mutant cells of Bacillus subtilis 1A700.
2016-02-18 | E-GEOD-76949 | biostudies-arrayexpress
Project description:Structural maintenance of chromosomes (SMC) complexes fold genomes by extruding DNA loops. In eukaryotes, loop-extruding SMC complexes form topologically associating domains (TADs) by being stalled by roadblock proteins. It remains unclear whether a similar mechanism of domain formation exists in prokaryotes. Using high-resolution chromosome conformation capture sequencing, we show that an archaeal homolog of the bacterial Smc-ScpAB complex organizes the genome of Thermococcus kodakarensis into TAD-like domains. We also find that TrmBL2, a nucleoid-associated protein that forms a stiff nucleoprotein filament, stalls the T. kodakarensis SMC complex and establishes a boundary at the site-specific recombination site dif. TrmBL2 stalls the SMC complex at tens of additional non-boundary loci with lower efficiency. Intriguingly, the stalling efficiency is correlated with shape properties of underlying DNA sequences. Our study illuminates not only a eukaryotic-like mechanism of domain formation in archaea, but also an unforeseen role of intrinsic DNA shape in large-scale genome organization.
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Project description:Structural maintenance of chromosomes (SMC) complexes fold genomes by extruding DNA loops. In eukaryotes, loop-extruding SMC complexes form topologically associating domains (TADs) by being stalled by roadblock proteins. It remains unclear whether a similar mechanism of domain formation exists in prokaryotes. Using high-resolution chromosome conformation capture sequencing, we show that an archaeal homolog of the bacterial Smc-ScpAB complex organizes the genome of Thermococcus kodakarensis into TAD-like domains. We also find that TrmBL2, a nucleoid-associated protein that forms a stiff nucleoprotein filament, stalls the T. kodakarensis SMC complex and establishes a boundary at the site-specific recombination site dif. TrmBL2 stalls the SMC complex at tens of additional non-boundary loci with lower efficiency. Intriguingly, the stalling efficiency is correlated with shape properties of underlying DNA sequences. Our study illuminates not only a eukaryotic-like mechanism of domain formation in archaea, but also an unforeseen role of intrinsic DNA shape in large-scale genome organization.
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Project description:Structural maintenance of chromosomes (SMC) complexes fold genomes by extruding DNA loops. In eukaryotes, loop-extruding SMC complexes form topologically associating domains (TADs) by being stalled by roadblock proteins. It remains unclear whether a similar mechanism of domain formation exists in prokaryotes. Using high-resolution chromosome conformation capture sequencing, we show that an archaeal homolog of the bacterial Smc-ScpAB complex organizes the genome of Thermococcus kodakarensis into TAD-like domains. We also find that TrmBL2, a nucleoid-associated protein that forms a stiff nucleoprotein filament, stalls the T. kodakarensis SMC complex and establishes a boundary at the site-specific recombination site dif. TrmBL2 stalls the SMC complex at tens of additional non-boundary loci with lower efficiency. Intriguingly, the stalling efficiency is correlated with shape properties of underlying DNA sequences. Our study illuminates not only a eukaryotic-like mechanism of domain formation in archaea, but also an unforeseen role of intrinsic DNA shape in large-scale genome organization.
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