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 concatermic 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-N509K, and A-R504C, have been isolated that restore the yield of lambda 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. lambda A-E(515)G, A-N509K, and A-R504C 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 lambda 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 lambda cosN G(2)C C(11)G. In a lambda 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 lambda terminase.