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Phage T4 terminase is an enzyme that binds to the portal protein of proheads and cuts and packages concatemeric DNA. The T4 terminase is composed of two subunits, gene products (gp) 16 and 17. The role of the small subunit, gp16, in T4 DNA packaging is not well characterized. We developed a new purification procedure to obtain large quantities of purified gp16 from an overexpression vector. The pure protein is found in two molecular weight forms, due to specific C-terminal truncation, displays in vitro packaging activity, and binds but does not hydrolyze ATP. gp16 forms specific oligomers, rings, and side-by-side double rings, as judged by native polyacrylamide gel electrophoresis and scanning transmission electron microscopy measurements. Packaging of DNA into most dsDNA 1 bacteriophage heads requires an enzyme, terminase, which interacts with the portal vertex of the prohead and concatemeric DNA to form a packaging machine. Terminases in most of the phages contain two subunits. The large subunit generally has DNA-dependent ATPase and endonuclease activities, whereas the DNA-binding specificity resides in the small subunit (cf. Ref. 1). terminase specifically binds and cuts at a unique cos site to leave 12-base 5Ј protruding complementary ends at both termini (2, 3). For phages P1 or P22, the terminases bind and cut specifically near an initiation packaging (pac) site and then generate nonspecific ends, which result from processive headful packaging (4, 5).Although it was first established in phage T4 that the mature DNA ends result from headful packaging, in the case of T4 the mechanism of DNA end formation in packaging is less well defined (6). In fact, the phage T4 terminase displays many of the features of other phage terminases, including the small (gp16) and large (gp17) subunit structure (1, 7). Our genetic studies implicate gp16, the small subunit of T4 terminase, in amplifying a gene 17-gene 19 fragment. This is achieved by recombination following alignment of two homologous 24-base pair segments within gene 16 and gene 19 in T4 Hp17 (amplification) mutants. A synapsis model was proposed to correlate this novel activity with the known role of gene 16 in initiation of DNA packaging and in controlling activity of gp17, the terminase large subunit. This amplification suggests DNA binding by gp16 whose sequence specificity remains to be assessed (8, 9).ATP hydrolysis is required for phage in vitro DNA packaging (1, 10). ATP not only provides an energy source for DNA translocation into the prohead, but also acts as an allosteric effector to control terminase holoenzyme specificity (11). terminase has two ATP binding sites, where the high affinity site is located in the large subunit, and the low affinity site exists in the small subunit in which a weak ATPase activity was detected (12-14). It is possible that ATP binding to the terminase small subunit may serve mainly as an allosteric effector rather than in energy transduction. Thus in the Bacillus subtilis phage SPP1, the small subunit binds but does not ...

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Phage T4 terminase, comprising the products of genes 16 and 17, packages headfuls of DNA from a concatemer but its mechanism of DNA recognition remains to be determined. Phage T4 terminase gene sequences were introduced into prophage lambda imm434 and plasmids in order to assess their effect on packaging as measured by transduction frequency and DNA content of T4-transducing particles. Multiple copy prophage lambda imm434 genes were transduced at 100-fold higher frequency, and high copy plasmids were transduced at 1000-fold higher frequency than single copy prophage or chromosomal genes T4 16 gene inserts enhanced both prophage and plasmid packaging; terminase gene-containing plasmid DNA in T4 transducing particles could exceed 10% of the total. Deletion or base change of the 24-bp gene 16 3' region which is required for sequence specific amplification of terminase gene 17 (Hp 17 mutations) depressed these elevated plasmid transduction frequencies, suggesting that this is a preferred T4 pac sequence. Moreover, a specific gene 16-containing pac fragment could be detected in mature, packaged phage T4 DNA following restriction endonuclease digestion. We conclude that both the copy number of homologous sequences and the DNA pac sequence(s) themselves are important for packaging, consistent with a synapsis model for regulation of terminase cutting and packaging in phage T4.