RESUMEN
The initiation of chromosomal replication occurs only once during the cell cycle in both prokaryotes and eukaryotes. Initiation of chromosome replication is the first and tightly controlled step of a DNA synthesis. Bacterial chromosome replication is initiated at a single origin, oriC, by the initiator protein DnaA, which specifically interacts with 9-bp non-palindromic sequences (DnaA boxes) at oriC. In Escherichia coli, a model organism used to study the mechanism of DNA replication and its regulation, the control of initiation relies on a reduction of the availability and/or activity of the two key elements, DnaA and the oriC region. This review summarizes recent research into the regulatory mechanisms of the initiation of chromosomal replication in bacteria, with emphasis on organisms other than E. coli.
Asunto(s)
Bacterias/genética , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/fisiología , Replicación del ADN , Regulación Bacteriana de la Expresión Génica , Bacterias/metabolismo , Proteínas Bacterianas/genética , ADN Bacteriano/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Transducción de SeñalRESUMEN
We have developed a novel type of a positive screen for the discovery of antibacterial compounds that target the Escherichia coli replication initiator protein DnaA. DnaA is an essential replication protein, conserved in (almost) all bacteria--including all human pathogens--and no existing antibiotics target the main components of the DNA replication machinery. This makes DnaA an attractive target and compounds discovered by this screen will constitute a new group of antibiotics. The conditional mutant, dnaA219, has a cold sensitive phenotype due to overreplication. In the screen, a DnaA inhibitor will reduce DnaA overactivity and thus restore growth at the nonpermissive temperature. This positive type of selection utilizes the rare phenomenon of lethal overactivity. In addition, the mutant strain has been made independent of DnaA activity by introduction of an alternative initiation pathway that allows growth under conditions of complete knockdown of DnaA. The resulting dnaA219rnhA strain is the basis of a robust, cell-based assay amenable to high-throughput screening. The screening assay has been validated against (1) a library of microbial fermentation extracts and (2) a known intracellular DnaA inhibitor.
Asunto(s)
Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/antagonistas & inhibidores , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Evaluación Preclínica de Medicamentos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , TemperaturaRESUMEN
Bacterial chromosome replication is mediated by single initiator protein, DnaA, that interacts specifically with multiple DnaA boxes located within the origin (oriC). We compared the architecture of the DnaA-origin complexes of evolutionarily distantly related eubacteria: two Gram-negative organisms, Escherichia coli and Helicobacter pylori, and two Gram-positive organisms, Mycobacterium tuberculosis and Streptomyces coelicolor. Their origins vary in size (from approx. 200 to 1000 bp) and number of DnaA boxes (from 5 to 19). The results indicate that: (i) different DnaA proteins exhibit various affinities toward single DnaA boxes, (ii) spatial arrangement of two DnaA boxes is crucial for the H. pylori and S. coelicolor DnaA proteins, but not for E. coli and M. tuberculosis proteins, and (iii) the oriC regions are optimally adjusted to their cognate DnaA proteins. The primary functions of multiple DnaA boxes are to determine the positioning and order of assembly of the DnaA molecules. Gradual transition from the sequence-specific binding of the DnaA protein to binding through co-operative protein-protein interactions seems to be a common conserved strategy to generate oligomeric initiator complexes bound to multiple sites within the chromosomal, plasmid and virial origins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Helicobacter pylori/genética , Mycobacterium tuberculosis/genética , Origen de Réplica/genética , Streptomyces coelicolor/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , División Celular , Replicación del ADN/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/química , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Helicobacter pylori/metabolismo , Datos de Secuencia Molecular , Mycobacterium tuberculosis/metabolismo , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Streptomyces coelicolor/metabolismoRESUMEN
Huntington's disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant inheritance. The disease is caused by a CAG trinucleotide repeat expansion located in the first exon of the HD gene. The CAG repeat is highly polymorphic and varies from 6 to 37 repeats on chromosomes of unaffected individuals and from more than 30 to 180 repeats on chromosomes of HD patients. In this study, we show that the number of CAG repeats in the HD gene can be determined by restriction of the DNA with the endonuclease EcoP15I and subsequent analysis of the restriction fragment pattern by electrophoresis through non-denaturing polyacrylamide gels using the ALFexpress DNA Analysis System. CAG repeat numbers in the normal (30 and 35 repeats) as well as in the pathological range (81 repeats) could be accurately counted using this assay. Our results suggest that this high-resolution method can be used for the exact length determination of CAG repeats in HD genes as well as in genes affected in related CAG repeat disorders.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo III/metabolismo , Enfermedad de Huntington/genética , Proteínas/genética , Expansión de Repetición de Trinucleótido/genética , Análisis Mutacional de ADN/métodos , Exones/genética , Humanos , Proteína Huntingtina , Enfermedad de Huntington/patología , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Proteínas Nucleares , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The initiation of replication is the central event in the bacterial cell cycle. Cells control the rate of DNA synthesis by modulating the frequency with which new chains are initiated, like all macromolecular synthesis. The end of the replication cycle provides a checkpoint that must be executed for cell division to occur. This review summarizes recent insight into the biochemistry, genetics and control of the initiation of replication in bacteria, and the central role of the initiator protein DnaA.
Asunto(s)
Bacterias/genética , Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas Virales/fisiología , Bacterias/clasificación , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Cromosomas Bacterianos/fisiología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Complejo de Reconocimiento del Origen , Plásmidos/clasificación , Plásmidos/genética , Plásmidos/metabolismo , Estructura Terciaria de Proteína/fisiología , Homología de Secuencia de Aminoácido , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Virales/genéticaRESUMEN
Streptomyces coelicolor A3(2) and Escherichia coli are quite different in their life-style and the structures of their genomes. Streptomyces exhibit complex multicellular development including formation of multigenomic hyphae during growth. These organisms possess a large linear (8.7 Mb) and GC-rich (approximately 72%) chromosome. The genome sequence of S. coelicolor has just been completed. The difference between E. coli and Streptomyces making them an excellent model organisms for a comparison of their replication modes. In this review, we compare initiation of chromosome replication in both organisms. Their replication origins are different in size, but both have DnaA boxes--a binding motifs for initiator DnaA protein. The two DnaA proteins have practically the same biochemical properties. Many aspects of the control of initiation seem to be similar. A comparison of the two systems thus allows us to define those aspects of replication initiation that are universally used in the eubacterial kingdom.
Asunto(s)
Cromosomas Bacterianos/genética , Replicación del ADN , Escherichia coli/genética , Streptomyces/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Origen de RéplicaAsunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bioquímica/métodos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/enzimología , Transcripción Genética , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Huella de ADN/métodos , Proteínas de Unión al ADN/aislamiento & purificación , Desoxirribonucleasa I/metabolismo , Escherichia coli/genética , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Apart from its function as an initiator of DNA replication, the Escherichia coli DnaA protein is also a specific transcription factor. It activates and represses a number of promoters. However, mechanisms of transcription stimulation by DnaA remained unknown. Bacteriophage lambda pR promoter is one of the promoters activated by DnaA. It was reported previously that DnaA binds downstream of the pR promoter and perhaps interacts with the RNA polymerase beta subunit. Here we demonstrate that DnaA positively regulates transcription from pR by stimulation of two steps in transcription initiation: RNA polymerase binding to the promoter region and promoter escape. For this transcription activation, two weak DnaA boxes located downstream of pR are necessary and sufficient. Such a mechanism of transcription activation and location of the activator-binding sites relative to the transcription start point are unusual in prokaryotes. Changes in the distance between the transcription start point and the first DnaA box by 5 and 10 bp and alterations in the orientation of these boxes did not abolish the stimulation of transcription by DnaA, but the efficiency of the promoter activation was different for various mutations. It seems plausible that formation of higher order nucleoprotein structures, involving DNA looping, is necessary for effective stimulation of the pR promoter. At high concentrations, DnaA is a repressor of pR rather than an activator. This repression was found to be because of inhibition of RNA polymerase binding to the promoter region.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacteriófago lambda/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Regulación Viral de la Expresión Génica , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Secuencia de Consenso , Cartilla de ADN , Replicación del ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/virología , Proteínas de Escherichia coli/metabolismoRESUMEN
For plasmids derived from bacteriophage lambda, the initiation of bidirectional DNA replication from orilambda depends on the stimulation of transcription from the p(R) promoter by the host replication initiator protein DnaA. Certain Escherichia coli dnaA(ts) mutants cannot be transformed by wild-type lambda plasmids even at the temperature permissive to cell growth. This plasmid-host incompatibility appeared to be due to inefficient stimulation of transcription from the p(R) promoter by the mutant DnaA protein. This paper shows that there is a second mechanism for the incompatibility between lambda plasmids and dnaA(ts) hosts, exemplified in this study by the dnaA46 mutant. This is based on the competition between the lambda P protein and the host DnaA and DnaC proteins for DnaB helicase. Both mechanisms must be operative for the incompatibility.
Asunto(s)
Proteínas Bacterianas/genética , Bacteriófago lambda/genética , Replicación del ADN , Proteínas de Unión al ADN/genética , Escherichia coli/virología , Plásmidos/genética , ADN Helicasas/metabolismo , AdnB Helicasas , Mutación , Regiones Promotoras Genéticas/genética , Origen de Réplica/genética , Activación TranscripcionalRESUMEN
Streptomycetes differ from other prokaryotic organisms in their mycelial life cycle and in possessing a large, linear, GC-rich chromosome. To deduce structural features of the Streptomyces origin of chromosomal replication, the oriC sequences of three Streptomyces species (S. antibioticus, S. chrysomallus and S. lividans) were compared. In Streptomyces, the oriC region contains 19 DnaA boxes whose location, orientation and spacing are conserved. The consensus sequence of the DnaA box identified within Streptomyces oriC is (T/C)(T/C)(G/A/C)TCCACA (preferred bases underlined). The interactions of DnaA with DNA fragments containing single, two or three DnaA boxes were studied using surface plasmon resonance. The dissociation constant (KD) for specific binding of individual DnaA boxes varied between 12 and 78 nM. Streptomyces oriC does not contain the three AT-rich 13-mer direct repeats present in the 5' part of the Escherichia coli oriC region. However, short AT-rich sequences are distributed among the DnaA boxes of Streptomyces oriC. Repeated attempts to unwind Streptomyces oriC have been unsuccessful. It remains to be elucidated whether DnaA interacts with putative accessory proteins which help in unwinding Streptomyces oriC.