J. gen. Virol. (I973), x8, 91-IO9 9I Printed in Great Britain Studies on the Replication of Bacteriophage T5

Bacteria infected with bacteriophage T 5 were disrupted with lysozyme and mild detergents and the intracellular phage-specific components resolved by sedimenta-tion through neutral sucrose gradients. In pulse-chase experiments with [aH]-thymi-dine 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 o 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 sedimenta-tion profile on alkaline-sucrose gradients. The pulse-labelled replicating DNA was very heterogeneous in tool. wt. and appeared to c ntain many single-strand nicks which were extensively repaired while the DNA was still in the fast-sedimenting form. The conversion sequence f s f--+ 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 phage-induced 5 ' exonuclease and in these infections there was no conversion of repli-cating 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