Nucleotide-dependent binding of the gene 4 protein of bacteriophage T7 to single-stranded DNA.
The gene 4 protein of bacteriophage T7 is a multifunctional enzyme that catalyzes (i) the hydrolysis of nucleoside 5'-triphosphates, (ii) the synthesis of tetraribonucleotide primers at specific recognition sequences on a DNA template, and (iii) the unwinding of duplex DNA. All three activities depend on binding of gene 4 protein to single-stranded DNA followed by unidirectional 5' to 3' translocation of the protein (Tabor, S., and Richardson, C. C. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 205-209). Binding of gene 4 protein to single-stranded DNA, assayed by retention of DNA-protein complexes on nitrocellulose filters, is random with regard to DNA sequence. Although gene 4 protein does not bind to duplex DNAs, the presence of a 240-nucleotide-long single-stranded tail on a 7200-base pair duplex DNA molecule is sufficient for gene 4 protein to cause retention of the DNA on a filter. The binding reaction requires, in addition to MgCl2, the presence of a nucleoside 5'-triphosphate, but binding is not dependent on hydrolysis; nucleoside 5'-diphosphate will substitute for nucleoside 5'-triphosphate. Of the eight common nucleoside triphosphates, dTTP promotes optimal binding. The half-life of the gene 4 protein-DNA complex depends on both the secondary structure of the DNA and on whether or not the nucleoside 5'-triphosphate cofactor can be hydrolyzed. Using the nonhydrolyzable nucleoside 5'-triphosphate analog, beta,gamma-methylene dTTP, the half-life of the gene 4 protein-DNA complex is greater than 80 min. In the presence of the hydrolyzable nucleoside 5'-triphosphate, dTTP, the half-life of the gene 4 protein-DNA complex using circular M13 DNA is at least 4 times longer than that observed using linear M13 DNA.
D001483:Base Sequence; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009711:Nucleotides / Q000378:metabolism / Q000494:pharmacology*; D013604:T-Phages / Q000235:genetics / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000378:metabolism / Q000494:pharmacology; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009711:Nucleotides; D013942:Thymine Nucleotides; D014764:Viral Proteins; D015636:Magnesium Chloride; C041083:beta, gamma-methylenedeoxythymidine 5'-triphosphate; D008274:Magnesium; C024157:thymidine 5'-triphosphate