RESUMO
Understanding the molecular mechanism of specific and polarized termination of DNA replication at a sequence-specific replication terminus requires detailed analyses of the interaction of terminator protein (ter) with specific DNA sequences (tau), constituting the replication terminus. Such analyses should provide the structural basis of the functional polarity of replication inhibition observed in vivo and in vitro at tau sites. With this objective in mind, we have purified the replication terminator protein of Escherichia coli to homogeneity and have analyzed the interaction of the protein with the replication termini of R6K, using chemical probes and by site-directed mutagenesis. The results show that one monomer of ter protein binds to a single tau site with an equilibrium dissociation constant of 5 x 10(-9) moles/liter. Furthermore, a combination of alkylation interference and protection, hydroxyradical footprinting, and site-directed mutagenesis has revealed the phosphate groups and base residues of the tau core sequence that make contacts with ter protein and those residues that are important for both DNA-protein interaction and for termination of replication in vivo. The overall picture that emerges from these analyses reveals that ter forms an asymmetric complex with a tau sequence. Thus, the asymmetric ter-tau complex provides a structural basis for the functional polarity of the arrest of a moving replication fork at a tau site.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Plasmídeos , Autorradiografia , Sequência de Bases , Reagentes de Ligações Cruzadas , DNA/ultraestrutura , Impressões Digitais de DNA , Eletroforese em Gel de Poliacrilamida , Metilação , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , FenótipoRESUMO
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.