RESUMO
Although many bacterial chromosomes require only one replication initiator protein, e.g., DnaA, most plasmid replicons depend on dual initiators: host-encoded DnaA and plasmid-encoded Rep initiator protein for replication initiation. Using the plasmid pSC101 as a model system, this work investigates the biological rationale for the requirement for dual initiators and shows that the plasmid-encoded RepA specifically interacts with the replicative helicase DnaB. Mutations in DnaB or RepA that disrupt RepA-DnaB interaction cause failure to load DnaB to the plasmid ori in vitro and to replicate the plasmid in vivo. Although, interaction of DnaA with DnaB could not substitute for RepA-DnaB interaction for helicase loading, DnaA along with integration host factor, DnaC, and RepA was essential for helicase loading. Therefore, DnaA is indirectly needed for helicase loading. Instead of a common surface of interaction with initiator proteins, interestingly, DnaB helicase appears to have at least a limited number of nonoverlapping surfaces, each of which interacts specifically with a different initiator protein.
Assuntos
Proteínas de Bactérias , DNA Helicases/metabolismo , Replicação do DNA , DNA Bacteriano/biossíntese , Proteínas de Ligação a DNA , Plasmídeos/biossíntese , Transativadores , Sítios de Ligação , DnaB Helicases , Modelos Genéticos , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas/genética , Proteínas/metabolismoRESUMO
Escherichia coli-DNA-T protein is a key component of a multiprotein complex called the primosome which is involved in the initiation of DNA replication. The thermal and urea induced unfolding transition of this protein in the presence and absence of Mg2+ was studied using circular dichroism (CD) and fluorescence spectroscopy as probes. Quenching of the intrinsic fluorescence of DNA-T was observed in the thermal unfolding while formation of a hyperfluorescent form of the protein was found in the urea induced unfolding process. The CD studies showed a monophasic transition curve for thermal unfolding in the presence and absence of Mg2+. Biphasic curves indicative of the formation of intermediates was observed in the urea induced unfolding. The results suggest that the pathways of unfolding of thermal- and urea-induced transitions are different. MgCl2, which affects the conformation of the protein and stabilises the secondary structure, also affects the unfolding pattern.
Assuntos
Proteínas de Bactérias/química , Replicação do DNA , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Escherichia coli/química , Magnésio/química , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Dicroísmo Circular , Fluorescência , Magnésio/farmacologia , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Temperatura , Ureia/químicaRESUMO
A recombinant vector for overproduction of the E. coli single stranded DNA binding protein (E. coli SSBP) has been constructed. An E. coli strain carrying this plasmid produces up to 150 mg pure SSBP per litre of bacterial culture in a laboratory shake flask. Electron microscopy of the single stranded DNA complexed with SSBP shows characteristic "beaded string"-like appearance. Strong clustering of protein molecules on ssDNA is indicative of a highly cooperative binding.
Assuntos
Bacteriófago T7/genética , Proteínas de Ligação a DNA/biossíntese , Escherichia coli/metabolismo , Vetores Genéticos , Regiões Promotoras Genéticas , Escherichia coli/virologiaRESUMO
We have developed an in vitro replication system in which purified replication termination protein (Ter) elicits specific and polarized termination of DNA replication at terminator sites (tau) in a cell extract of tus- Escherichia coli that does not encode Ter protein. Using this system and two-dimensional agarose gel electrophoresis, we have identified intermediates with stalled replication forks. The replication bubbles contain both arrested leading strands and lagging strands that were initiated at the ColE1 origin of replication and that had progressed unidirectionally until arrested at tau. To dissect the system further, we have analyzed the kinetics of the formation of the termination intermediates and have discovered that the earliest termination intermediate had properties consistent with an arrested D loop. The D loop contained an arrested leading strand. Thus, in this test system, there appears to be a transient uncoupling of leading- and lagging-strand synthesis during termination of replication at tau sites.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Reguladores , Regiões Terminadoras Genéticas , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Teste de Complementação Genética , Cinética , Plasmídeos , Mapeamento por Restrição , Moldes GenéticosRESUMO
We have cloned the tus gene that encodes the replication terminator protein of Escherichia coli and have efficiently expressed its gene product. The overproducer strain has been used to purify the terminator (ter) protein in high yield to near homogeneity. The protein is a single 36 kd polypeptide. Using the ter protein and highly purified dnaB helicase, we show that the terminator protein is a DNA sequence-specific contra-helicase, i.e., the protein when bound to its recognition sequence (tau) strongly impedes the ATP-dependent unwinding of double-stranded DNA. This contra-helicase activity is polar, i.e., the impedance to unwinding takes place in only one orientation of the tau sequence. The results illuminate the mechanism of replication termination specifically at tau.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Replicação do DNA , Escherichia coli/enzimologia , Expressão Gênica , Biblioteca Gênica , Genes Bacterianos , Cinética , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Plasmídeos , Mapeamento por Restrição , Regiões Terminadoras GenéticasRESUMO
We have purified approximately 6600-fold an approximately 40-kDa protein (Ter protein) encoded by Escherichia coli that specifically binds to two sites at the 216-base-pair replication terminus (tau) of the plasmid R6K. Chemical footprinting experiments have shown that the Ter protein binds to two 14- to 16-base-pair sequences that exist as inverted repeats in the tau fragment. Site-directed mutagenesis of one of the terminus sequences (tau R) resulted in a mutant tau R that failed to bind to the Ter protein. The same mutant terminus also failed to terminate DNA replication in vivo. These experiments strongly suggest that the interaction of the Ter protein with tau sequences plays an essential role in the termination of DNA replication, specifically at tau.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Plasmídeos , Sequência de Bases , Bromodesoxiuridina , Clonagem Molecular , Reagentes de Ligações Cruzadas , Sondas de DNA , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease EcoRI , Desoxirribonuclease HindIII , Desoxirribonucleases de Sítio Específico do Tipo II , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , MutaçãoRESUMO
A preparative procedure for purifying bacteriophage lambda from large volumes of phage lysates by recirculating tangential flow ultrafiltration is described. Lambda DNA, isolated by deproteinization of the phage, is suitable for use in molecular biology.
