THE NUCLEOTIDE-BINDING SITE OF THE HELICASE-PRIMASE OF BACTERIOPHAGE-T7 - INTERACTION OF MUTANT AND WILD-TYPE PROTEINS

The helicase and primase activities of bacteriophage T7 are distribute d between the 56- and 63-kDa gene 4 proteins. The 63-kDa protein catal yzes both helicase and primase activities. The 56-kDa gene 4 protein l acks the 63 amino acids at the N terminus of the colinear 63-kDa prote in and catalyzes only helicase activity. Helicase activity is dependen t on the hydrolysis of a nucleoside 5'-triphosphate. Sequence analysis reveals a single ''A-type'' nucleoside 5'-triphosphate binding site n ear the center of each gene 4 protein. We have examined the essential role of nucleoside triphosphate hydrolysis both in vivo and in vitro b y 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 dom inant-lethal: they block productive infection by wild-type T7 phage. A nucleotide binding site mutant 56-kDa gene 4 protein, purified to hom ogeneity from cells over-expressing the gene, binds but lacks the abil ity to hydrolyze nucleotides and cannot bind to single-stranded DNA. C onsequently, 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 (K(i) = 22 +/- 4.5 nM) of this interaction may ref lect the gene 4 oligomer dissociation constant in the presence of nucl eotide and single-stranded DNA. Analysis of the inhibition reaction in dicates that this is a linear mixed-type inhibition, indicating that t he mutant protein binds the wild-type protein to form an inactive comp lex on single-stranded DNA. Furthermore, the mutant 56-kDa gene 4 prot ein 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 inhi bit the activity of the wild-type gene 4 proteins indicates that nucle otide hydrolysis is coordinated and cooperative among the members of t he gene 4 protein complex as it binds and translocates on single-stran ded DNA.