A Deoxyribonucleic Acid-Binding Protein Induced by Bacteriophage T7

Abstract

Abstract A protein which binds strongly and preferentially to single-stranded DNA has been purified by DNA-cellulose chromatography from Escherichia coli infected with bacteriophage T7. The protein, synthesized between 5 and 18 min following infection, comprises more than 0.1% of soluble protein in T7-infected cells. In vitro studies show that this protein can specifically stimulate the rate of polymerization catalyzed by the T7-induced DNA polymerase. At low temperatures, where the T7-induced DNA polymerase utilizes single-stranded DNA poorly as template primer, the binding protein stimulates the rate of synthesis 10- to 15-fold. The stimulatory effect is also observed employing single-stranded DNA at 37°; however, the stimulation is not as great (2- to 3-fold) at the higher temperature. Similarly, when a double-stranded DNA containing internal single-stranded regions is used as template, stimulation is observed, but is not as pronounced (approximately 5-fold). T7 DNA polymerase activity can be stimulated 20- to 50-fold by salt when a gapped DNA is used as template but only 1.5-fold when a single-stranded DNA is used as template. On the latter template, stimulation by binding protein is relatively constant over a range of salt concentrations, whereas on the former template, stimulation occurs only at salt concentrations less than optimal for polymerase activity. The stimulatory reaction is relatively insensitive to pH. The protein has a molecular weight of 31,000 and forms aggregates at high protein concentrations in the presence of magnesium ions. It is capable of binding to single-stranded DNA in quantities which exceed the weight of the DNA by a factor of 20. The protein does not bind to native DNA. The T7 DNA-binding protein dissociates from single-stranded T7 DNA with a rate constant of about 1 x 10-4 s-1 and a half-life of dissociation between 97 and 120 min under the conditions studied.