A mutation correcting the DNA interaction defects of a mutant phage lambda terminase, gpNu1 K35A terminase.
Terminase, the DNA packaging enzyme of bacteriophage lambda, is a heteromultimer composed of gpNu1 (181 aa) and gpA (641 aa) subunits, encoded by the lambda Nu1 and A genes, respectively. Similarity between the deduced amino acid sequences of gpNu1 and gpA and the nucleotide binding site consensus sequence suggests that each terminase subunit has an ATP reactive center. Terminase has been shown to have two distinct ATPase activities. The gpNu1 subunit has a low-affinity ATPase stimulated by nonspecific DNA and gpA has a high-affinity ATPase. In previous work, a mutant terminase, gpNu1 K35A holoterminase, had a mild defect in interactions with DNA, such that twofold increased DNA concentrations were required both for full stimulation of the low-affinity ATPase and for saturation of the cos cleavage reaction. In addition, the gpNu1 K35A terminase exhibited a post-cleavage defect in DNA packaging that accounted for the lethality of the Nu1 K35A mutation [Y. Hwang and M. Feiss (1997) Virology 231, 218-230]. In the work reported here, a mutation in the turn of the putative helix-turn-helix DNA binding domain has been isolated as a suppressor of the gpNu1 K35A change. This suppressor mutation causes the change A14V in gpNu1. A14V reverses the DNA-binding defects of gpNu1 K35A terminase, both for stimulation of the low-affinity ATPase and for saturation of the cos cleavage defect. A14V suppresses the post-cleavage DNA packaging defect caused by the gpNu1 K35A change.
Hwang
Y
Y
College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
Feiss
M
M
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000201:enzymology*; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013489:Suppression, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C050856:lambda phage protein Nu 1; D004706:Endodeoxyribonucleases; C030894:terminase; D000251:Adenosine Triphosphatases