Evidence for distinct primase and helicase domains in the 63-kDa gene 4 protein of bacteriophage T7. Characterization of 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 single-stranded DNA using nucleotide hydrolysis for energy and is a helicase. The 63-kDa gene 4 protein catalyzes all of the activities of the 56-kDa gene 4 protein and, in addition, catalyzes the synthesis of oligoribonucleotides on single-stranded DNA. Two conserved residues in a putative nucleotide binding site of the 63-kDa gene 4 protein were mutated by substituting Val and Met for wild-type residues Gly and Lys, at positions 317 and 318, respectively. The mutant 63-kDa gene 4 protein lacks the ability to catalyze the hydrolysis of a nucleoside 5'-triphosphate in a single-stranded DNA-dependent reaction and inhibits nucleotide hydrolysis by wild-type gene 4 proteins. The mutant primase contains 0.4% of the primase activity of the 63-kDa gene 4 protein on M13 single-stranded DNA and 12% of the wild-type primase activity on an oligonucleotide with a single primase recognition site. Addition of wild-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.
Mendelman
L V
LV
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Notarnicola
S M
SM
Richardson
C C
CC
D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D017354:Point Mutation; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D013942:Thymine Nucleotides; D014764:Viral Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases; C024157:thymidine 5'-triphosphate