RESUMO
The chromosomal replication origin oriC and the gene encoding the replication initiator protein DnaA from Thermus thermophilus have been identified and cloned into an Escherichia coli vector system. The replication origin is composed of 13 characteristically arranged DnaA boxes, binding sites for the DnaA protein, and an AT-rich stretch, followed by the dnaN gene. The dnaA gene is located upstream of the origin and expresses a typical DnaA protein that follows the division into four domains, as with other members of the DnaA protein family. Here, we report the purification of Thermus-DnaA (Tth-DnaA) and characterize the interaction of the purified protein with the replication origin, with regard to the binding kinetics and stoichiometry of this interaction. Using gel retardation assays, surface plasmon resonance (SPR) and electron microscopy, we show that, unlike the E. coli DnaA, Tth-DnaA does not recognize a single DnaA box, instead a cluster of three tandemly repeated DnaA boxes is the minimal requirement for specific binding. The highest binding affinities are observed with full-length oriC or six clustered, tandemly repeated DnaA boxes. Furthermore, high-affinity DNA-binding of Tth-DnaA is dependent on the presence of ATP. The Thermus DnaA/oriC interaction will be compared with oriC complex formation generated by other DnaA proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Origem de Replicação/genética , Thermus thermophilus/enzimologia , Thermus thermophilus/genética , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Sequência de Bases , Sítios de Ligação , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/ultraestrutura , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/ultraestrutura , Genes Bacterianos/genética , Hidrólise , Cinética , Microscopia Eletrônica , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Sequências de Repetição em Tandem/genética , TermodinâmicaRESUMO
The gene order in the dnaA region of Thermus thermophilus was determined. Previously, we showed that the putative oriC of T. thermophilus is located in the dnaA-dnaN intergenic region. In the 4 kb region upstream of the dnaA gene four ORFs were found, all orientated in the same direction which is opposite to that of dnaA. The ORFs were identified as T. thermophilus homologs of gidA, gidB, soj and spo0J of Bacillus subtilis. The gene order spo0J-soj-gidB-gidA-dnaA-dnaN resembles that of B. subtilis, Pseudomonas putida, Coxiella burnetii, Streptomyces coelicolor, Mycobacterium leprae, and Mycobacterium tuberculosis. We identified the transcriptional start point of the dnaA gene. The -10 region shows significant homology to the Escherichia coli -10 consensus sequence. The putative -35 region shows homology neither to the E. coli -35 consensus sequence nor to known -35 sequences of T. thermophilus. There are no DnaA boxes in the promoter region, and consequently dnaA transcription is not repressed by DnaA protein in vitro, i.e. the dnaA gene of T. thermophilus is not autoregulated.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Thermus thermophilus/genética , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/química , Ordem dos Genes , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genéticaRESUMO
We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.
Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Origem de Replicação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , DnaB Helicases , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Streptomyces/genética , Streptomyces/metabolismo , Thermus thermophilus/genética , Thermus thermophilus/metabolismoRESUMO
The dnaA gene of Streptomyces lividans was cloned using the Escherichia coli medium-copy-number vector pSU18 and E. coli strain TC1963, which can by-pass the requirement for the DnaA protein. Its regulatory region was subcloned in the Streptomyces probe vector pIJ4083. Primer extension and S1 mapping studies allowed the identification of a class I Streptomyces promotor (P2). An additional, previously unknown promoter type (P1) was found by S1 mapping. The presence of two DnaA box motifs between P1 and P2 suggests that the transcriptions of the S. lividans dnaA gene is autoregulated by its gene product. It was shown that the transcription of the dnaA gene is significantly induced by mitomycin C, an agent known to inhibit DNA replication. The data suggest that, as in E. coli, one of the regulatory mechanisms governing the transcription of the dnaA gene in S. lividans is probably related to the SOS response network.
Assuntos
Replicação do DNA/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Streptomyces/genética , Transcrição Gênica , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Vetores Genéticos , Mitomicina/farmacologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
The tricho-rhino-phalangeal syndromes type I (TRPS I) and type II (TRPS II) result from the deletion of overlapping sets of genes within the Langer-Giedion syndrome chromosomal region (LGCR) on chromosome 8. In contrast to TRPS I patients, most TRPS II patients have cytogenetically visible deletions and are often mentally retarded. Using Southern blot and fluorescence in situ hybridization analysis, we searched for submicroscopic deletions in 12 patients with TRPS I and an apparently normal karyotype. One patient of normal intelligence was found to have a deletion of approximately 5 Mb. This suggests that mental retardation in TRPS is caused by genes outside the 5-Mb region. Using three LGCR microsatellite markers, we determined the parental origin of this TRPS I deletion and of eight TRPS II deletions. In six patients, the deletion was of paternal origin and in three patients it was of maternal origin.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 8 , Síndrome de Langer-Giedion/genética , Southern Blotting , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Feminino , Marcadores Genéticos , Impressão Genômica , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Repetições de Microssatélites , Mapeamento por RestriçãoRESUMO
The tricho-rhino-phalangeal syndrome type II (TRPS II, or Langer-Giedion syndrome) is an example of contiguous gene syndromes, as it comprises the clinical features of two autosomal dominant diseases, TRPS I and a form of multiple cartilaginous exostoses caused by mutations in the EXT1 gene. We have constructed a contig of cosmid, lambda-phage, PAC, and YAC clones, which covers the entire TRPS I critical region. Using these clones we identified a novel submicroscopic deletion in a TRPS I patient and refined the proximal border of the minimal TRPS1 gene region by precisely mapping the inversion breakpoint of another patient. As a first step towards a complete inventory of genes in the Langer-Giedion syndrome chromosome region (LGCR) with the ultimate aim to identify the TRPS1 gene, we analyzed 23 human expressed sequence tags (ESTs) and four genes (EIF3S3, RAD21, OPG, CXIV) which had been assigned to human 8q24.1. Our analyses indicate that the LGCR is gene-poor, because none of the ESTs and genes map to the minimal TRPS1 gene region and only two of these genes, RAD21 and EIF3S3, are located within the shortest region of deletion overlap of TRPS II patients. Two genes, OPG and CXIV, which are deleted only in some patients with TRPS II may contribute to the clinical variability of this syndrome.
Assuntos
Cromossomos Humanos Par 8/genética , Síndrome de Langer-Giedion/genética , Criança , Quebra Cromossômica/genética , Mapeamento de Sequências Contíguas , Cosmídeos , Eletroforese em Gel de Campo Pulsado , Etiquetas de Sequências Expressas , Feminino , Deleção de Genes , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Reação em Cadeia da PolimeraseRESUMO
The Langer-Giedion syndrome (tricho-rhino-phalangeal syndrome type II, TRPS II) is characterized by craniofacial dysmorphism and skeletal abnormalities. It combines the clinical features of TRPS I and multiple cartilaginous exostoses (EXT). We have used YAC cloning, Southern blotting, PCR analysis, and fluorescence in situ hybridization to study chromosome 8 deletions, translocations, an inversion, and an insertion in patients with TRPS I, TRPS II or EXT. Our results indicate that the TRPS gene maps more than 1,000 kb proximal to the EXT1 gene and that both genes are affected in TRPS II. We conclude that TRPS II is not due to pleiotropic effects of mutations in a single gene, but that it is a true contiguous gene syndrome.