Studies on the nature of replicating DNA in T4-infected Escherichia coli

Abstract

The replicating DNA of T4-infected Escherichia coli has been isolated as a rapidly sedimenting structure of about 200 s at 15 minutes after infection. This sedimentation, rate is approached from smaller values at earlier times after infection. Several properties suggest that it differs from ordinary molecules of phage DNA or nonspecific aggregates of phage DNA. The very fragile structure breaks into products that sediment like phage DNA but are less homogeneous and appear to be more sensitive to further breakage than phage DNA. Treatment with nucleases that act on denatured DNA results in the simultaneous formation of intermediate rapidly sedimenting material and heterogeneous fragments ranging from 30 s to 100 s. An early hydrolysis product sediments at 73 s. The latter resembles a DNA fraction recovered from chloramphenicol-treated phage infected cells ( Frankel, 1963 ). The buoyant density in CsCl of the isolated replicating DNA is almost identical with that of phage DNA. After recovery from the same density band, the two DNA's retain, in part, their difference in sedimentation rate. The replicating DNA is more sensitive to thermal denaturation than phage DNA, but after complete denaturation the densities of the two are identical. Preliminary experiments suggest that the polynucleotide strands of replicating DNA are not longer than those of phage DNA. It is concluded that T4 phage DNA in the replicating state has a structure that is different from mature phage DNA. The former may be longer and could contain all of the newly synthesized DNA in a single, bizarre structure. This structure would possess regions in which the usual base pairing of native DNA is interrupted. Other structures, however, are not excluded.