Enzymic synthesis of deoxyribonucleic acid. XXII. Replication of a circular single-stranded DNA template by DNA polymerase of Escherichia coli

DNA from the bacteriophage M13, specific for male strains of Escherichia coli, was found to have a ring structure similar to that of φX174 DNA. E. coli DNA polymerase, free of endonuclease, uses this DNA as a template for a complete or even more extensive replication. The template-product complex is separable in alkali, yielding the original circular template and an open-chain product strand. The molecular weights of the template, the full replication product and the 1:1 hybrid of template and product were calculated from sedimentation coefficients to be 2 × 10⁶, 2 × 10⁶ and 4 × 10⁶ daltons, respectively. Electron microscopic studies of the hybrid reveal circular, helical molecules about 2 μ in contour length; with only partial replication, circular molecules are seen which appear to have one single-stranded region linking the ends of a helical segment; with extensive replication, helical branches are seen attached to 2-μ long helical circles. Analysis of the product reveals the initiating termini of the strands as the 5′-(mono)-phosphates of each of the four deoxynucleotides. Replication of circular M13 DNA thus appears to be initiated without covalent linkage to the template, without a specific nucleotide locus on the template and with the elimination of the terminal pyrophosphoryl group of the initial deoxynucleoside triphosphate.