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 5-bromodeoxyuridine) bacteria with light (not substituted with 5-bromodeoxyuridine) radioactive bacteriophage was isolated from a CsCl 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.