DNA-protein interaction at the replication termini of plasmid R6K.
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.
Sista
P R
PR
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Hutchinson
C A
CA
Bastia
D
D
D001345:Autoradiography; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004247:DNA / Q000648:ultrastructure; D016172:DNA Fingerprinting; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008745:Methylation; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004247:DNA