The replication terminus of the plasmid R6K has been cloned into the single-stranded DNA phage vector M13mpS and also into the plasmid vectors pBR313 and pBR322. By using single-stranded DNA templates prepared from the recombinant DNA clones, the sequence of 215 base pairs of DNA containing the replication terminus has been determined. The DNA sequence of the region ofthe terminus does not contain any 2-fold rotational symmetry. Therefore, folding of the DNA at the region of the terminus is unlikely to be a cause -for replication termination. Interaction of a host-specified protein(s) with the sequence of the replication terminus is probably the basis of the mechanism of replication termination.In spite of extensive work, the molecular mechanism of termination of DNA replication still remains unclear. In efforts to elucidate the mechanism of termination of replication, analysis of the DNA sequences involved in replication termination and identification of proteins that interact with the replication terminus are of utmost importance.The drug-resistance factor R6K which carries genes for ampicillin and streptomycin resistance (1) is a model system for this study because its replication is asymmetrically bidirectional. The asymmetry in the bidirectional replication is manifested in the confluence of the two moving forks of the replicating chromosome at a point that is not located at 1800 from the replication origins (2, 3). The asymmetrically bidirectional replication strongly suggested the existence of a genetically determined terminus of replication.In this paper we report the cloning ofthe replication terminus into the vector M13mp5, its precise localization in a 215-basepair DNA fragment, and analysis of its nucleotide sequence. The nucleotide sequence of the replication terminus suggests a mechanism of termination that probably involves interaction of the terminus sequence of the plasmid with one or more proteins specified by the host genome.
MATERIAL AND METHODSBacterial, Plasmid, and phage Strains. The bacterial strain JC411 thy-was obtained from D. Helinski and the strain 71-18, from J. Messing (4). The plasmid vectors pBR313 and pBR322 (5) were obtained from H. Boyer through R. Curtiss III. The single-stranded DNA phage vector M13mp5, which contains a single HindIII site was a gift from J. Messing.Chemicals, Enzymes, and Labeled Precursors. dNTPs (Sigma), dideoxynucleotide triphosphates (P-L Biochemicals), [a-32P]dATP (100-300 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels; ICN), 5-bromo-4-chloroindolyl /3-galactoside (Sigma) and isopropyl 13-thiogalactoside; (Sigma) were obtained as indicated.The restriction enzymes HindIII (6), Hpa II (7), Alu 1 (8), Hinf (9), Hae III and Hae 11 (10), and Hha (11) were purified according to published procedures. DNA polymerase I holoenzyme and the Klenow fragment of Escherichia coli were purchased from Boehringer Mannheim. Exonuclease III and BamHI were purchased from New England BioLabs. T4 ligase was purified as described by Weiss (12).Molecular Cloning. These experime...