Two types of replication proteins increase the rate at which T4 DNA polymerase traverses the helical regions in a single-stranded DNA template.


We have recently developed an in vitro DNA synthesis system in which a ...
We have recently developed an in vitro DNA synthesis system in which a synthetic heptaribonucleotide pairs with a unique site on a single-stranded fd DNA molecule and thereby primes the growth of new DNA strands from this single point (Huang, C.-C., and Hearst, J. E. (1980) Anal. Biochem. 103, 127-139). In this report, we use this system to investigate the mechanism by which various bacteriophage T4 DNA replication proteins stimulate the T4 DNA polymerase. We find that with the "polymerase accessory proteins" present (the T4 gene 44/62 and 45 proteins), the DNA polymerase proceeds rather rapidly through the occasional hairpin helices which otherwise interrupt the progress of this enzyme along single-stranded DNA templates. By using a potent inhibitor of the 44/62 ATPase, ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)), we have obtained data which suggest that ATP hydrolysis is required for the formation of a polymerase accessory protein-DNA template complex, and that this complex then persists, serving as a sliding clamp which greatly increases the strength of binding between a T4 DNA polymerase molecule and its 3'OH primer template end. The progress of the T4 DNA polymerase though hairpin helices in the DNA template is also stimulated by addition of the T4 helix-destabilizing protein (gene 32 protein). The effect of the 44/62 and 45 proteins is independent of the effect of the 32 protein in this assay, and the rate of polymerase travel over the strongest hairpin helices is increased more than 40-fold in the presence of these four additional proteins.




Kinetic Parameters, Nucleotide Incorporation


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