Stimulation of T4 bacteriophage DNA polymerase by the protein product of T4 gene 32

Abstract

The protein product of T4 bacteriophage gene 32 (32-protein) is required for replication of T4 DNA in vivo. Alberts, Amodio, Jenkins, Butmann & Ferris (1968) have shown that this protein binds tightly and co-operatively to single-stranded DNA under physiological ionic conditions in vitro, and in so doing it holds the otherwise highly folded DNA chain in a much more extended conformation (Alberts & Frey, 1970). We have now tested the effect of 32-protein on the activity of T4 DNA polymerase, an enzyme which requires a single-stranded DNA template, and find that it stimulates the in vitro rate of polymerization on such templates 5- to 10-fold. The rate of stimulated synthesis is about 10% of that measured for movements in vivo of growing points. The 32-protein from a temperature-sensitive mutant in gene 32 ( tsP7) is also temperature-sensitive in the stimulation of T4 polymerase in vitro, suggesting that this stimulation is related to the intracellular function of 32-protein in DNA replication. The stimulation of T4 DNA polymerase by 32-protein is greatest at low temperature, high ionic strength and at a level sufficient to bind most or all of the template DNA present. These results suggest that 32-protein stimulates this polymerase by removing inhibitory secondary structure from the template DNA strand. The activity of DNA polymerase from Escherichia coli on single-stranded DNA templates is not significantly affected by 32-protein. In addition, in the absence of DNA, a weak complex is formed between 32-protein and T4 DNA polymerase, whereas no such complex is formed with E. coli DNA polymerase. Thus, some specific interaction between polymerase and 32-protein may also be essential for the stimulation of DNA synthesis observed.