Mutations that extend the specificity of the endonuclease activity of lambda 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 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.
Arens
J S
JS
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA.jean-sippy@uiowa.edu
Hang
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Q
Hwang
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Tuma
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Max
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Feiss
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D019943:Amino Acid Substitution; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D002874:Chromosome Mapping; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D005814:Genes, Viral; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013489:Suppression, Genetic; D010948:Viral Plaque Assay
D004706:Endodeoxyribonucleases; C030894:terminase