Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid I. Tertiary Structure of Early Replicative and Recombining Deoxyribonucleic Acid

Abstract

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.

Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid II. Single-Strand Breaks and Exposure of Uncomplemented Areas as a Prerequisite for Recombination

“…As with the wild-type phage (10), the integrity of the strands of DNA from DNA-deficient amber mutants is affected after infection of a nonpermissive host. This breakage of polynucleotide chains can be inhibited by CM, which implies that a new enzyme is required for this breakage.…”

“…Procedures for analysis in alkaline sucrose gradients have also been described (10), as have the details of using CM (11). Extraction of native intracellular DNA was by the sodium dodecyl sulfate-Pronase-1193 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from KOZINSKI AND FELGENHAUER phenol (SPP) method (12), which is similar to the method of Berns and Thomas (3).…”

“…The injected phage DNA is recovered as either an unreplicated or as a "hybrid" molecule. [The term "hybrid," as well as "heavy," "light," "phage equivalent length (PEL)," "round of replication," "replicative hybrid," and "recombinant molecule," were defined in the first paper of this series (10).] Addition of CM 7 min after infection did not interfere with molecular exchanges.…”

The Mechanism of Genetic Recombination

“…Since chloramphenicol is known to inhibit protein synthesis, it was concluded that the T 4 DNA coded for an enzyme necessary for recombination which cut the DNA, and that the production of this enzyme was being inhibited. Kozinski, Kozinski & James (1967) applied chloramphenicol at different times after infection, and found there was a gradient of inhibition of (u) DNA synthesis, (b) cutting of single chains and (c) repairing activities, in that order. They concluded that the enzyme responsible for cutting single chains in duplex DNA was coded by T 4 and was required for recombination.…”

Late events in T4 bacteriophage DNA replication. III. Specificity of DNA reinitiation as revealed by hybridization to cloned genetic fragments

“…In the experiments which follow, we will attempt to demonstrate the genetic nature of the pulse-labeled progeny DNA synthesized at late times after the onset of DNA replication under two conditions: when the covalent addition of progeny DNA to recombinant intersections is inhibited, and when there is covalent joining. It should be recalled here that in the past we have demonstrated that timely addition of CM serves as a fine dissecting tool, separating various replicative events (16). When CM is added at 5 to 6 min past infection there is no parent-toprogeny recombination, and the newly synthesized progeny DNA displays a genetic bias, being fixed in the amplifying mode (18).…”