Mutations That Extend the Specificity of the Endonuclease Activity of λ Terminase

Terminase, an enzyme encoded by the Nu1 and A genes of bacteriophage lambda, is crucial for packaging concatemeric DNA into virions. cosN, a 22-bp segment, is the site on the virus chromosome where terminase introduces staggered nicks to cut the concatemer to generate unit-length virion chromosomes. Although cosN is rotationally symmetric, mutations in cosN have asymmetric effects. The cosN G(2)C mutation (a G-to-C change at position 2) in the left half of cosN reduces the phage yield 10-fold, whereas the symmetric mutation cosN C(11)G, in the right half of cosN, does not affect the burst size. The reduction in phage yield caused by cosN G(2)C is correlated with a defect in cos cleavage. Three suppressors of the cosN G(2)C mutation, A-E(515)G, A-N(509)K, and A-R(504)C, have been isolated that restore the yield of λ cosN G(2)C to the wild-type level. The suppressors are missense mutations that alter amino acids located near an ATPase domain of gpA. λ A-E(515)G, A-N(509)K, and A-R(504)C phages, which are cosN(+), also had wild-type burst sizes. In vitro cos cleavage experiments on cosN G(2)C C(11)G DNA showed that the rate of cleavage for A-E(515)G terminase is three- to fourfold higher than for wild-type terminase. The A-E(515)G mutation changes residue 515 of gpA from glutamic acid to glycine. Uncharged polar and hydrophobic residues at position 515 suppressed the growth defect of λ cosN G(2)C C(11)G. In contrast, basic (K, R) and acidic (E, D) residues at position 515 failed to suppress the growth defect of λ cosN G(2)C C(11)G. In a λ cosN(+) background, all amino acids tested at position 515 were functional. These results suggest that A-E(515)G plays an indirect role in extending the specificity of the endonuclease activity of λ terminase.