Bacteria infected with bacteriophage T5 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 [H]-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 mol. 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 T5. One of these, T5. Bl, could not form the T5 phage-induced 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 T5 induced exonuclease, but the role of this enzyme was unclear.


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