RESUMEN

yabA encodes a negative regulator of replication initiation in Bacillus subtilis and homologues are found in many other gram-positive species. YabA interacts with the beta-processivity clamp (DnaN) of DNA polymerase and with the replication initiator and transcription factor DnaA. Because of these interactions, YabA has been proposed to modulate the activity of DnaA. We investigated the role of YabA in regulating replication initiation and the activity of DnaA as a transcription factor. We found that YabA function is mainly limited to replication initiation at oriC. Loss of YabA did not significantly alter expression of genes controlled by DnaA during exponential growth or after replication stress, indicating that YabA is not required for modulating DnaA transcriptional activity. We also found that DnaN activates replication initiation apparently through effects on YabA. Furthermore, association of GFP-YabA with the replisome correlated with the presence of DnaN at replication forks, but was independent of DnaA. Our results are consistent with models in which YabA inhibits replication initiation at oriC, and perhaps DnaA function at oriC, but not with models in which YabA generally modulates the activity of DnaA in response to replication stress.

Asunto(s)

Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , ADN Bacteriano/biosíntesis , ADN Polimerasa Dirigida por ADN/genética , Regulación Bacteriana de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo de Reconocimiento del Origen , ARN Mensajero/metabolismo , Transcripción Genética

RESUMEN

DnaA functions as both a transcription factor and the replication initiator in bacteria. We characterized the DNA binding dynamics of DnaA on a genomic level. Based on cross-linking and chromatin immunoprecipitation data, DnaA binds at least 17 loci, 15 of which are regulated transcriptionally in response to inhibition of replication (replication stress). Six loci, each of which has a cluster of at least nine potential DnaA binding sites, had significant increases in binding by DnaA when replication was inhibited, indicating that the association of DnaA with at least some of its target sites is altered after replication stress. When replication resumed from oriC after inhibition of replication initiation, these high levels of binding decreased rapidly at origin-proximal and origin-distal regions, well before a replication fork could pass through each of the regulated regions. These findings indicate that there is rapid signaling to decrease activation of DnaA during replication and that interaction between DnaA bound at each site and the replication machinery is not required for regulation of DnaA activity in response to replication stress.

Asunto(s)

Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/genética , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Origen de Réplica , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Cromosomas Bacterianos/metabolismo , Proteínas de Unión al ADN/genética

RESUMEN

We investigated the genome-wide DNA binding of the chromosome partitioning and sporulation protein and ParB family member Spo0J in Bacillus subtilis using chromatin immunoprecipitation and DNA microarrays. We identified 10 parS loci to which Spo0J binds, two of which were unexpectedly distant (> 1 Mb) from the origin of replication. We used all 10 sites to refine the consensus sequence for parS. We found that Spo0J spreads along the DNA around each site. Binding was near maximal levels up to 1.6 kb away from parS, and significantly above background as far away as 18 kb. Deletion of soj (parA) had little or no effect on spreading. In contrast, the spo0J93 allele appeared to cause a significant decrease in spreading in vivo, without significantly affecting the DNA binding affinity in vitro. spo0J93 causes a phenotype similar to that of a spo0J null mutant and alters the region thought to be involved in interaction between Spo0J dimers. Our findings indicate that spreading is important for in vivo function of Spo0J. Gene expression in areas near parS sites was similar in wild type and a spo0J null mutant, indicating that binding and spreading of Spo0J on DNA does not normally silence transcription of nearby genes.

Asunto(s)

Bacillus subtilis/genética , Proteínas Bacterianas/genética , Cromosomas Bacterianos/genética , Genoma Bacteriano , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Inmunoprecipitación de Cromatina , Cromosomas Bacterianos/fisiología , Replicación del ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Microscopía Fluorescente , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Transcripción Genética

RESUMEN

Organisms respond to perturbations in DNA replication. We characterized the global transcriptional response to inhibition of DNA replication in Bacillus subtilis. We focused on changes that were independent of the known recA-dependent global DNA damage (SOS) response. We found that overlapping sets of genes are affected by perturbations in replication elongation or initiation and that this transcriptional response serves to inhibit cell division and maintain cell viability. Approximately 20 of the operons (>50 genes) affected have potential DnaA-binding sites and are probably regulated directly by DnaA, the highly conserved replication initiation protein and transcription factor. Many of these genes have homologues and recognizable DnaA-binding sites in other bacteria, indicating that a DnaA-mediated response, elicited by changes in DNA replication status, may be conserved.

Asunto(s)

Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Transcripción Genética/genética , Sitios de Unión , División Celular , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/genética , Genes Bacterianos/genética , Operón/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rec A Recombinasas/genética

RESUMEN

Initiation of DNA replication from the Escherichia coli chromosomal origin is highly regulated, assuring that replication occurs precisely once per cell cycle. Three mechanisms for regulation of replication initiation have been proposed: titration of free DnaA initiator protein by the datA locus, sequestration of newly replicated origins by SeqA protein and regulatory inactivation of DnaA (RIDA), in which active ATP-DnaA is converted to the inactive ADP-bound form. DNA microarray analyses showed that the level of initiation in rapidly growing cells that lack datA was indistinguishable from that in wild-type cells, and that the absence of SeqA protein caused only a modest increase in initiation, in agreement with flow-cytometry data. In contrast, cells lacking Hda overinitiated replication twofold, implicating RIDA as the predominant mechanism preventing extra initiation events in a cell cycle.

Asunto(s)

Adenosina Trifosfatasas/fisiología , Proteínas Bacterianas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/fisiología , Escherichia coli/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/química , Alelos , Fenómenos Fisiológicos Bacterianos , Ciclo Celular , Proliferación Celular , ADN/química , Proteínas de Unión al ADN/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Citometría de Flujo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genotipo , Immunoblotting , Luz , Modelos Genéticos , Complejos Multiproteicos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Origen de Réplica , Dispersión de Radiación

RESUMEN

Target specificity for bacteriophage Mu was studied using a new phage derivative that enabled cloning of Mu-host junctions from phage DNA. Insertions distributed throughout the chromosome showed no orientation bias with respect to transcription or replication polarity. Genes with a high frequency of the triplet CGG were preferred targets.

Asunto(s)

Bacteriófago mu/genética , Escherichia coli/genética , Escherichia coli/virología , Repeticiones de Trinucleótidos , Secuencia de Aminoácidos , Cromosomas Bacterianos , Secuencia de Consenso , ADN Viral/genética , Datos de Secuencia Molecular , Origen de Réplica/genética

RESUMEN

In Escherichia coli DNA replication is carried out by the coordinated action of the proteins within a replisome. After replication initiation, the two bidirectionally oriented replisomes from a single origin are colocalized into higher-order structures termed replication factories. The factory model postulated that the two replisomes are also functionally coupled. We tested this hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA surrounding oriC in single, combed chromosomes showed instead that one replisome, usually the leftward one, was significantly ahead of the other 70% of the time. We next used microarrays to follow replication throughout the genome by measuring DNA copy number. We found in multiple E. coli strains that the replisomes are independent, with the leftward replisome ahead of the rightward one. The size of the bias was strain-specific, varying from 50 to 130 kb in the array results. When we artificially blocked one replisome, the other continued unabated, again demonstrating independence. We suggest an improved version of the factory model that retains the advantages of threading DNA through colocalized replisomes at about equal rates, but allows the cell flexibility to overcome obstacles encountered during elongation.

Asunto(s)

Cromosomas/fisiología , Replicación del ADN/fisiología , ADN/biosíntesis , Escherichia coli/genética , Escherichia coli/fisiología , Genoma Bacteriano , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos

RESUMEN

BACKGROUND: The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure. RESULTS: We measured the transcriptional response to a loss of supercoiling caused either by genetic impairment of a topoisomerase or addition of specific topoisomerase inhibitors during log-phase growth and identified genes whose changes are statistically significant. Transcription of 7% of the genome (306 genes) was rapidly and reproducibly affected by changes in the level of supercoiling; the expression of 106 genes increased upon chromosome relaxation and the expression of 200 decreased. These changes are most likely to be direct effects, as the kinetics of their induction or repression closely follow the kinetics of DNA relaxation in the cells. Unexpectedly, the genes induced by relaxation have a significantly enriched AT content in both upstream and coding regions. CONCLUSIONS: The 306 supercoiling-sensitive genes are functionally diverse and widely dispersed throughout the chromosome. We propose that supercoiling acts as a second messenger that transmits information about the environment to many regulatory networks in the cell.

Asunto(s)

Cromosomas Bacterianos/genética , ADN Superhelicoidal/genética , Escherichia coli K12/genética , Transcripción Genética/genética , Composición de Base/genética , Codón Iniciador/genética , Cumarinas/farmacología , ADN-Topoisomerasas/deficiencia , ADN-Topoisomerasas/genética , ADN-Topoisomerasas/fisiología , ADN Bacteriano/genética , Inhibidores Enzimáticos/farmacología , Escherichia coli K12/enzimología , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/estadística & datos numéricos , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Genes Bacterianos/genética , Genes Bacterianos/fisiología , Cinética , Mutación/genética , Mutación/fisiología , Norfloxacino/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Inhibidores de Topoisomerasa

RESUMEN

Transposable elements have influenced the genetic and physical composition of all modern organisms. Defining how different transposons select target sites is critical for understanding the biochemical mechanism of this type of recombination and the impact of mobile genes on chromosome structure and function. Phage Mu replicates in Gram-negative bacteria using an extremely efficient transposition reaction. Replicated copies are excised from the chromosome and packaged into virus particles. Each viral genome plus several hundred base pairs of host DNA covalently attached to the prophage right end is packed into a virion. To study Mu transposition preferences, we used DNA microarray technology to measure the abundance of >4,000 Escherichia coli genes in purified Mu phage DNA. Insertion hot- and cold-spot genes were found throughout the genome, reflecting >1,000-fold variation in utilization frequency. A moderate preference was observed for genes near the origin compared to terminus of replication. Large biases were found at hot and cold spots, which often include several consecutive genes. Efficient transcription of genes had a strong negative influence on transposition. Our results indicate that local chromosome structure is more important than DNA sequence in determining Mu target-site selection.

Asunto(s)

Bacteriófago mu/genética , Elementos Transponibles de ADN/genética , Escherichia coli/genética , Genes Bacterianos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción Genética/genética , Integración Viral/genética , ADN Viral/genética , Escherichia coli/virología , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Familia de Multigenes/genética , Mutagénesis/genética , Origen de Réplica/genética

Asunto(s)

Caulobacter crescentus/genética , Segregación Cromosómica/genética , Cromosomas Bacterianos/genética , ADN Bacteriano/genética

RESUMEN

BACKGROUND: Autonomously replicating sequences (ARSs) function as replication origins in Saccharomyces cerevisiae. ARSs contain the 17 bp ARS consensus sequence (ACS), which binds the origin recognition complex. The yeast genome contains more than 10,000 ACS matches, but there are only a few hundred origins, and little flanking sequence similarity has been found. Thus, identification of origins by sequence alone has not been possible. RESULTS: We developed an algorithm, Oriscan, to predict yeast origins using similarity to 26 characterized origins. Oriscan used 268 bp of sequence, including the T-rich ACS and a 3' A-rich region. The predictions identified the exact location of the ACS. A total of 84 of the top 100 Oriscan predictions, and 56% of the top 350, matched known ARSs or replication protein binding sites. The true accuracy was even higher because we tested 25 discrepancies, and 15 were in fact ARSs. Thus, 94% of the top 100 predictions and an estimated 70% of the top 350 were correct. We compared the predictions to corresponding sequences in related Saccharomyces species and found that the ACSs of experimentally supported predictions show significant conservation. CONCLUSIONS: The high accuracy of the predictions indicates that we have defined near-sufficient conditions for ARS activity, the A-rich region is a recognizable feature of ARS elements with a probable role in replication initiation, and nucleotide sequence is a reliable predictor of yeast origins. Oriscan detected most origins in the genome, demonstrating previously unrecognized generality in yeast replication origins and significant discriminatory power in the algorithm.

Asunto(s)

ADN de Hongos/genética , Origen de Réplica/genética , Saccharomyces cerevisiae/genética , Algoritmos , Composición de Base/genética , Sitios de Unión/genética , Mapeo Cromosómico/métodos , Cromosomas Fúngicos/genética , Secuencia Conservada/genética , Replicación del ADN/genética , ADN de Hongos/química , Proteínas de Unión al ADN/genética , Estudios de Evaluación como Asunto , Evolución Molecular , Complejo de Reconocimiento del Origen , Valor Predictivo de las Pruebas , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Diseño de Software , Termodinámica

RESUMEN

Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained. In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal. By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC. Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability. The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell.

Asunto(s)

Replicación del ADN/genética , Escherichia coli/genética , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Infecciones por Escherichia coli/inmunología , Cinética , Ratones , Mutagénesis Insercional , Fenotipo , Reacción en Cadena de la Polimerasa , Origen de Réplica , Eliminación de Secuencia , Linfocitos T/inmunología