Genome-wide binding of replication initiation proteins in Bacillus subtilis
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ABSTRACT:
ORGANISM(S): Escherichia coli Bacillus subtilis Bacillus subtilis subsp. subtilis str. 168
PROVIDER: GSE23686 | GEO | 2010/12/07
SECONDARY ACCESSION(S): PRJNA130913
REPOSITORIES: GEO
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Project description:Initiation of DNA replication requires binding of the initiator protein, DnaA, to specific binding sites in the chromosomal origin of replication, oriC. In low G+C Gram-positive bacteria, the primosomal proteins DnaD and DnaB, in conjunction with loader ATPase DnaI, load the replicative helicase at oriC, and this depends on DnaA. DnaD and DnaB are also required to load the replicative helicase outside of oriC during replication restart, in a DnaA-independent manner. DnaA also binds to many sites around the chromosome, outside of oriC, and acts as a transcription factor at several of these. Using chromatin immunoprecipitation, we found that DnaD and DnaB, but not the replicative helicase, are associated with many of the chromosomal regions bound by DnaA in vivo in Bacillus subtilis. This association was dependent on DnaA and the order of recruitment was the same as that at oriC, but was independent of a functional oriC. The presence of DnaD and DnaB at the secondary (non-oriC) targets of DnaA in the absence of helicase loading indicates a possible role for DnaD and DnaB in modulating the activity of DnaA. The genome-wide binding profiles of DnaA, DnaD, DnaB and DnaC were determined. Binding profiles were determined in exponentially growing cells with and without HPUra treatment. Three biological replicates were analyzed per protein/treatment (one per array). Enrichment in immunoprecipitated samples versus total genomic DNA were determined.
2010-12-07 | E-GEOD-23686 | biostudies-arrayexpress
Project description:To identify potential targets of co-regulation by DnaA and Rok, we compared the transcriptional profiles of dnaA null, rok null, and dnaA null rok null mutants. Because a dnaA null mutant requires an oriC- strain background, we used an oriC- oriN+ background for all strains, to allow direct comparisons.
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Project description:To identify potential targets of co-regulation by DnaA and Rok, we compared the transcriptional profiles of dnaA null, rok null, and dnaA null rok null mutants. Because a dnaA null mutant requires an oriC- strain background, we used an oriC- oriN+ background for all strains, to allow direct comparisons. We compared biological triplicates for each of the following strains: dnaA null (CAL2074), rok null (CAS196), dnaA null rok null (CAS192), and dnaA+ rok+ (CAL2083; control).
2015-06-13 | E-GEOD-69788 | biostudies-arrayexpress
Project description:The dpiA and dpiB genes of Escherichia coli, which are orthologs of genes that regulate citrate uptake and utilization in Klebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in the K. pneumoniae promoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response in E. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size-both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB-both of which act at A+T-rich replication origin sequences in the E. coli chromosome and DpiA-targeted plasmids-reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics.
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Project description:The stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response the cell produces large quantities of the nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which ppGpp inhibits DNA replication initiation in Escherichia coli remains unclear. Prior work suggested that ppGpp blocks new rounds of DNA replication by inhibiting transcription of the essential initiation factor dnaA, but we found that replication is still inhibited by ppGpp in cells ectopically producing DnaA. Instead, we provide evidence that a global reduction of transcription by ppGpp prevents replication initiation by modulating the supercoiling state of the origin of replication, oriC. Active transcription normally introduces negative supercoils into oriC to help promote replication initiation, so the accumulation of ppGpp reduces initiation potential at oriC. We find that maintaining transcription near oriC, either by expressing a ppGpp-blind RNA polymerase mutant or by inducing transcription from a ppGpp-insensitive promoter, can strongly bypass the inhibition of replication by ppGpp. Additionally, we show that increasing global negative supercoiling by inhibiting topoisomerase I or by deleting the nucleoid-associated protein seqA, also relieves inhibition. We propose a model, potentially conserved across proteobacteria, in which ppGpp indirectly creates an unfavorable energy landscape for initiation by preventing the introduction of negative supercoils into oriC.
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Project description:The dpiA and dpiB genes of Escherichia coli, which are orthologs of genes that regulate citrate uptake and utilization in Klebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in the K. pneumoniae promoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response in E. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size-both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB-both of which act at A+T-rich replication origin sequences in the E. coli chromosome and DpiA-targeted plasmids-reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
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Project description:Initiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. The absence or failure of DNA replication can result in bacterial cell growth arrest or death. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. For this purpose, DNA replication was blocked using a CRISPRi approach specifically targeting DnaA boxes 6 and 7, which are essential for replication initiation. We characterized the phenotype of these cells and analyzed the overall changes in the proteome using quantitative mass spectrometry. Cells with arrested replication were elongating and not dividing but showed no evidence of DNA damage response (DDR). Moreover, these cells did not cease translation over time. This study sets the ground for future research on non-replicating but translationally active B. subtilis, which might be valuable for biotechnological applications.
2024-03-26 | PXD036876 | Pride
Project description:The dpiA and dpiB genes of Escherichia coli, which are orthologs of genes that regulate citrate uptake and utilization in Klebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in the K. pneumoniae promoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response in E. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size-both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB-both of which act at A+T-rich replication origin sequences in the E. coli chromosome and DpiA-targeted plasmids-reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
2006-01-31 | GSE4112 | GEO
Project description:The Origin Recognition Complex (ORC) is essential for the initiation of eukaryotic chromosome replication as it loads the replicative helicase, the minichromosome maintenance (MCM) complex, at replication origins. Origins display a stereotypic chromatin structure with nucleosome depletion at ORC binding sites and flanking arrays of regularly spaced nucleosomes. Although discovered long ago, it is unknown how this chromatin struture is established and if it matters for replication. By genome-scale biochemical reconstitution we screened ~300 origins and 17 purified budding yeast chromatin factors and found that ORC established nucleosome depletion over origins and flanking nucleosome arrays by orchestrating the chromatin remodelers INO80, ISW1a, ISW2 and Chd1. The functional importance of ORC’s chromatin organizing activity was directly demonstrated by ORC mutations that maintained classical MCM loader activity but lost array generation activity. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, besides its canonical role as MCM loader, has a second essential function as master regulator of the origin chromatin structure that is a crucial prerequisite for efficient chromosome replication.
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Project description:Recent advances in the study of archaeal DNA replication have uncovered defined replication origins (oriC) and demonstrated specific binding of the Cdc6/Orc1 protein and Mcm helicase to oriC in vivo and/or in vitro. The oriC of the hyperthermophilic archaeon Pyrococcus abyssi is characterized by 13 bp repeats, AT-rich regions and an inverted repeat whose precise roles remain unclear. We report here that the 13 bp repeats are widespread in the three Pyrococcus genomes. Nevertheless, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip), we found that binding of P. abyssi Cdc6/Orc1 to oriC in vivo was highly specific both in exponential and stationary phases, allowing oriC to be distinguished in the 1.8 M bp genome. ChIP-chip analysis also indicated that a single 13 bp repeat is not sufficient for stable binding of Cdc6/Orc1. Purified Cdc6/Orc1 binds a DNA fragment containing the inverted repeat of oriC with a relatively low affinity, suggesting that multiple clusters of the 13 bp repeat discovered in this study contribute to the stable binding of Cdc6/Orc1 to oriC. Our ChIP-chip analysis revealed that Mcm also binds oriC only in proliferating cells, consistent with its role as a licensing factor. Finally, we found that both Cdc6/Orc1 and Mcm have one additional target site. Notably, Mcm binds constitutively to a GC-rich region containing two rRNA genes and a tRNA gene, suggesting a role of archaeal Mcm in DNA replication and/or transcription of this peculiar region. Keywords: ChIP-chip
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