At an early stage of replication, parental T4 DNA shows a loop structure often displaying two 3'-ended, single-stranded "whiskers", located in trans configuration at the branching-points. Several Replicative loops were first demonstrated by Cairns in the DNA of Escherichia coli (1). Schnos and Inman showed that the replication of lambda DNA always starts at the same place on the DNA molecule, and usually proceeds from that site in both directions (2).We showed that partially replicated molecules of T4 DNA that have not yet completed one round of replication cannot be resolved by shearing into pure parental and hybrid moieties, which suggests that multiple initiation occurs (3). In this paper we describe the results of an electron microscopic study of the partially replicated from of replicating T4 DNA.
A replicative hybrid resulting from the infection of heavy (substituted with 5bromodeoxyuridine) bacteria with light (not substituted with 5-bromodeoxyuridine) radioactive bacteriophage was isolated from a CsCI density gradient. Sedimentation studies indicate that 60% of the deoxyribonucleic acid (DNA) behaves as if it were in units more than four times as large as an intact reference molecule. Under the electron microscope, hybrid molecules appeared tangled, showed puffs and loops, occupied a small area, and often had a total length twice that of mature phage. This indicates that sucrose gradient sedimentation is not applicable as a method for estimating the relative molecular size of replicative forms of DNA. After denaturation, the separated strands of hybrid were of the same size as those of reference DNA. CsCl density gradient analysis revealed no terminal covalent addition of new material to the old parental strand. The possibility of a continuous growth of the DNA molecule, either on a single-stranded level or as a double helical structure, is disproved. When chloramphenicol (CM) was added at critical times after infection, DNA synthesis continued at a constant rate. The parental label soon assumed and retained a hybrid density, despite concomitant synthesis of DNA, throughout the rest of the period of incubation in CM. The hybrid moiety, however, actively participated in replication and exchanged its partner strand for a new one; this was demonstrated by changing the density label during incubation in CM. A new enzyme synthesized shortly after infection introduced single-stranded "nicks" into the parental DNA. Since nicking can be inhibited by chloramphenicol, the responsible enzyme is not of host origin.The time of the appearance of this enzyme coincided with the onset of molecular recombination. Another enzyme, which mediates the repair of the continuity of the polynucleotide chain after recombination, appeared after recombination. If selectively inhibited by chloramphenicol, recombinant molecules remained unrepaired, and, upon denaturation, the parental fragment was liberated in pure form.
Soon after infection parental deoxyribonucleic acid (DNA) enters a structure sedimenting fast to the bottom of a sucrose gradient. The addition of chloramphenicol (CM) prevents formation of this structure, whereas treatment with Pronase releases DNA which sediments thereafter with the speed characteristic of phenolextracted replicative DNA. It is assumed therefore that the structure responsible for fast sedimentation of replicative DNA is a newly synthesized protein. Those fast-sedimenting complexes contain preferentially the replicative form of parental DNA; this was proven by density labeling experiments. Progeny DNA labeled with 3H-thymidine added after infection can also be detected preferentially in this fastsedimenting moiety. The association of the DNA with the complexing protein is of a colinear or quasi-colinear type. This was proven by introducing double-strand scissions into DNA embedded in the replicative complex; double-strand scissions do not liberate DNA from the fast-sedimenting complex. Despite the apparent intimate relation between protein and DNA, DNA residing in complexes is fully sensitive to the action of nucleases. Shortly prior to the appearance of the fastsedimenting complex, parental DNA displays still another characteristic: at about 3 min after infection, it sediments faster than reference, but sizeably slower than the complex which appears at roughly 4 to 5 min after infection. The transition between these two fast-sedimenting types of moieties is not continuous. This fastsedimenting intermediate, which appears at 3 min after infection, cannot be inhibited by the addition of CM either at the moment or prior to infection. Fastsedimenting intermediate can be destroyed by sodium dodecyl sulfate, Pronase, or phenol extraction. The progeny DNA labeled with 3H-thymidine between 3 and 3.5 min after infection can be recovered in fast-sedimenting intermediate. The contribution of newly synthesized progeny DNA is so small that it cannot be detected as a shift of the parental density in a density labeling experiment. Small fragments of progeny DNA recovered in fast-sedimenting intermediate are not covalentlv attached to parental molecules and represent both strands of T4 DNA.In 1965 Kozinski and Lin (11) reported the formation of a complex between a protein moiety and replicative deoxyribonucleic acid (DNA) during reproduction of bacteriophage T4 in Escherichia coli B. Formation of the complex was postulated because replicative, intracellular, 32P-labeled, parental phage DNA was not recovered in the aqueous phase during phenol extraction unless the cell lysates were first incubated with Pronase. Kozinski reported results (Bio-I Submitted by Robert C. Miller, Jr., in partial fulfillment of the requirements for the Ph.D. degree from the
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