Arens J S, Hang Q, Hwang Y, Tuma B, Max S, Feiss M
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242,
J Bacteriol. 1999 Jan;181(1):218-24. doi: 10.1128/JB.181.1.218-224.1999.
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 G2C 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 C11G, in the right half of cosN, does not affect the burst size. The reduction in phage yield caused by cosN G2C is correlated with a defect in cos cleavage. Three suppressors of the cosN G2C mutation, A-E515G, A-N509K, and A-R504C, have been isolated that restore the yield of lambda cosN G2C to the wild-type level. The suppressors are missense mutations that alter amino acids located near an ATPase domain of gpA. lambda A-E515G, A-N509K, and A-R504C phages, which are cosN+, also had wild-type burst sizes. In vitro cos cleavage experiments on cosN G2C C11G DNA showed that the rate of cleavage for A-E515G terminase is three- to fourfold higher than for wild-type terminase. The A-E515G 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 G2C C11G. In contrast, basic (K, R) and acidic (E, D) residues at position 515 failed to suppress the growth defect of lambda cosN G2C C11G. In a lambda cosN+ background, all amino acids tested at position 515 were functional. These results suggest that A-E515G plays an indirect role in extending the specificity of the endonuclease activity of lambda terminase.
末端酶是由噬菌体λ的Nu1和A基因编码的一种酶，对于将串联DNA包装到病毒粒子中至关重要。cosN是一个22个碱基对的片段，是病毒染色体上末端酶引入交错切口以切割串联体从而产生单位长度病毒粒子染色体的位点。尽管cosN是旋转对称的，但cosN中的突变具有不对称效应。cosN左半部分的cosN G2C突变（第2位的G到C的变化）使噬菌体产量降低10倍，而cosN右半部分的对称突变cosN C11G则不影响裂解量。cosN G2C导致的噬菌体产量降低与cos切割缺陷相关。已经分离出cosN G2C突变的三个抑制子，即A-E515G、A-N509K和A-R504C，它们将λ cosN G2C的产量恢复到野生型水平。这些抑制子是错义突变，改变了位于gpA的ATP酶结构域附近的氨基酸。cosN+的λ A-E515G、A-N509K和A-R504C噬菌体也具有野生型的裂解量。对cosN G2C C11G DNA进行的体外cos切割实验表明，A-E515G末端酶的切割速率比野生型末端酶高3至4倍。A-E515G突变将gpA的第515位残基从谷氨酸变为甘氨酸。第515位的不带电荷的极性和疏水残基抑制了λ cosN G2C C11G的生长缺陷。相反，第515位的碱性（K、R）和酸性（E、D）残基未能抑制λ cosN G2C C11G的生长缺陷。在λ cosN+背景下，在第515位测试的所有氨基酸都是有功能的。这些结果表明，A-E515G在扩展λ末端酶内切核酸酶活性的特异性方面起间接作用。
