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J Biol Chem. 1993 Dec 25;268(36):27198-207.

The nucleotide binding site of the helicase/primase of bacteriophage T7. Interaction of mutant and wild-type proteins.

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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.


The helicase and primase activities of bacteriophage T7 are distributed between the 56- and 63-kDa gene 4 proteins. The 63-kDa protein catalyzes both helicase and primase activities. The 56-kDa gene 4 protein lacks the 63 amino acids at the N terminus of the colinear 63-kDa protein and catalyzes only helicase activity. Helicase activity is dependent on the hydrolysis of a nucleoside 5'-triphosphate. Sequence analysis reveals a single "A-type" nucleoside 5'-triphosphate binding site near the center of each gene 4 protein. We have examined the essential role of nucleoside triphosphate hydrolysis both in vivo and in vitro by using site-directed mutagenesis to alter the conserved, adjacent Gly and Lys residues within this nucleotide binding site. The mutant gene 4 proteins, expressed from plasmids carrying the cloned genes, do not complement a T7 phage lacking gene 4. Moreover, the mutations are dominant-lethal: they block productive infection by wild-type T7 phage. A nucleotide binding site mutant 56-kDa gene 4 protein, purified to homogeneity from cells over-expressing the gene, binds but lacks the ability to hydrolyze nucleotides and cannot bind to single-stranded DNA. Consequently, this mutant gene 4 protein also lacks helicase activity. The mutant gene 4 proteins inhibit the nucleotide hydrolysis activity of wild-type gene 4 proteins in a stoichiometric manner. The apparent inhibition constant (Ki = 22 +/- 4.5 nM) of this interaction may reflect the gene 4 oligomer dissociation constant in the presence of nucleotide and single-stranded DNA. Analysis of the inhibition reaction indicates that this is a linear mixed-type inhibition, indicating that the mutant protein binds the wild-type protein to form an inactive complex on single-stranded DNA. Furthermore, the mutant 56-kDa gene 4 protein has the same affinity for both the wild-type 63- and 56-kDa gene 4 proteins, suggesting that there is no preference for the formation of homo-oligomeric complexes. The ability of the mutant proteins to inhibit the activity of the wild-type gene 4 proteins indicates that nucleotide hydrolysis is coordinated and cooperative among the members of the gene 4 protein complex as it binds and translocates on single-stranded DNA.

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