Studies on the Replication of Bacteriophage T5

Abstract

SUMMARYBacteria infected with bacteriophage T 5 were disrupted with lysozyme and mild detergents and the intracellular phage-specific components resolved by sedimentation through neutral sucrose gradients. In pulse-chase experiments with [aH]-thymidine most of the radioactivity initially appeared in a fast-sedimenting form of DNA (fsf) which was very shear-sensitive and bound tightly to nitrocellulose. Label next appeared in a slower sedimenting form (ssf), then phage heads and finally virus particles. The ssf showed susceptibility to shear similar to that of DNA from intact virus, and sedimented with it on neutral gradients. The ssf DNA differed from virus DNA by binding to nitrocellulose and showing a different sedimentation profile on alkaline-sucrose gradients. The pulse-labelled replicating DNA was very heterogeneous in tool. wt. and appeared to contain many single-strand nicks which were extensively repaired while the DNA was still in the fast-sedimenting form. The conversion sequence fsf--+ ssf ---+ heads ---~ phage was supported by the accumulation of components in non-permissive host bacteria infected with certain amber mutants of T 5. One of these, T 5. B1, could not form the T 5 phageinduced 5' exonuclease and in these infections there was no conversion of replicating DNA to ssf, and net DNA synthesis stopped prematurely. It was concluded that maturation of T5 virus involved mature virus-size pieces of DNA of unusual structure as intermediates between replicating DNA and phage heads. The process appeared to require functional T 5 induced exonuclease, but the role of this enzyme was unclear.

DNA Replication of Bacteriophage T5. 2. Structure and Properties of the Slow Sedimenting Form of Intracellular T5 DNA

“…The ssf does not accumulate (Carrington & Lunt, 1973): presumably it could be lost by recombination, by degradation or some may appear in aberrant heads or particles. Examples of the latter could include the T 5 'del' particles described by Saigo (1976) in which the'right-hand'terminally redundant region of virion DNA is missing, or the defective particles described by Labedan & Legault-Demare (1974).…”

“…It was stated that one of the unusual properties of T 5, and hence its importance in this context, was the existence of a pool of intracellular T5 DNA of similar sedimentation rate to that of mature T5 virion DNA. This intracellular DNA, the slow sedimenting form (ssf), appeared to be a precursor of encapsulated DNA on the basis of pulse-chase experiments and studies using T5 strains defective in DNA replication (Carrington & Lunt, 1973). These observations implied that the excision from concatemers and packaging of T5 DNA may be fundamentally different from that of other similar phages.…”

DNA Replication of Bacteriophage T5. 3. Studies on the Structure of Concatemeric T5 DNA

“…A control incubation without enzyme showed that most of the original sample maintained its fast-sedimenting character throughout the manipulations (Fig. lb); the variable extent of breakdown of the control was probably due to the extreme shear-sensitivity of T5 concatemeric DNA (Carrington & Lunt, 1973). Therefore, it appears that single-stranded regions are abundant in T5 concatemeric DNA.…”

“…There was little material of greater than mature length in the preparation, although similar experiments with native DNA showed that more than 90% of the fast-sedimenting preparations was retained at the top of the gel (results not shown). Carrington & Lunt (1973) showed that the distribution of radioactive label among the single-strand fragments of pulse-labelled T5 concatemeric DNA was more heterogeneous than that of uniformally labelled DNA. They suggested that this result could be indicative of repair of single-stranded regions or nicks in vivo.…”

“…Later, it was shown that this intracellular pool of DNA was shear-sensitive, would bind to nitrocellulose (and therefore presumably contained single-stranded regions) and produced a distribution of single-stranded fragments upon alkali denaturation which bore some resemblance to that of mature T5 DNA (Carrington & Lunt, 1973). More recently, Bourguignon et al (1976) intermediates of T5 DNA replication and showed that a number of different origins of replication were used prior to the formation of larger, branched molecules.…”