Abstract
Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.
Highlights
Gene 4 protein and DNA polymerase of bacterio- and Richardson, 1985; Nakai and Richardson, 1986)
The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primedDNA synthesis in the pres
When functioning as a helicase, it catalyzes the unwinding of the helical structure of duplex DNA (Matson et al, 1983) and stimulates DNA synthesis catalyzed by T 7 DNA polymerase on such templates(Kolodner et al, 1978; LechnerandRichardson, 1983)
Summary
From the Departmentof Biological Chemistry, Haruard Medical School, Boston, Massachusetts 02115. Inthe single-stranded DNA, ATP, and CTP, a primer-tem- presence of T 7 DNA polymerase, the oligoribonucleotides are plate complex is formed; it is likely that gene 4 protein used as primersto initiate DNA synthesis(Scherzinger et al, mediates stable binding of the oligonucleotide to the 1977a, 1977b; Romano and Richardson1,979a). The complex, purified free of unbound pro- panying paper we used such an RNA-primedDNA synthesis teins and nucleotides, supports DNA synthesis upon assay to monitor thegene 4 protein. Both of these activities addition of DNA polymerase and deoxynucleoside 5’- of the gene 4 protein are dependent on the bindingof gene 4 triphosphates. In the accompanying paper (Nakai and Richardson, 1986) we described three basic physical interactions of the DNA
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