EVIDENCE FOR DISTINCT PRIMASE AND HELICASE DOMAINS IN THE 63-KDA GENE-4 PROTEIN OF BACTERIOPHAGE-T7 - CHARACTERIZATION OF A NUCLEOTIDE-BINDING SITE MUTANT

Gene 4 of bacteriophage T7 encodes two co-linear proteins, the 56- and 63-kDa gene 4 proteins. The 56-kDa protein translocates 5' to 3' on s ingle-stranded DNA using nucleotide hydrolysis for energy and is a hel icase. The 63-kDa gene 4 protein catalyzes all of the activities of th e 56-kDa gene 4 protein and, in addition, catalyzes the synthesis of o ligoribonucleotides on single-stranded DNA. Two conserved residues in a putative nucleotide binding site of the 63-kDa gene 4 protein were m utated by substituting Val and Met for wild-type residues Gly and Lys, at positions 317 and 318, respectively. The mutant 63-kDa gene 4 prot ein lacks the ability to catalyze the hydrolysis of a nucleoside 5'-tr iphosphate in a single-stranded DNA-dependent reaction and inhibits nu cleotide hydrolysis by wild-type gene 4 proteins. The mutant primase c ontains 0.4% of the primase activity of the 63-kDa gene 4 protein on M 13 single-stranded DNA and 12% of the wild-type primase activity on an oligonucleotide with a single primase recognition site. Addition of w ild-type 56-kDa gene 4 protein stimulates the mutant primase activity over 50-fold on M13 single-stranded DNA and 8-fold on oligonucleotides . This increase in primase activity correlates with an increase in the affinity of the mutant primase-wild-type helicase complex for single- stranded DNA template.