DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction.

Abstract:

We have used DNA footprinting techniques to analyze the interactions ...
We have used DNA footprinting techniques to analyze the interactions of five DNA replication proteins at a primer-template junction: the bacteriophage T4 DNA polymerase (the gene 43 protein), its three accessory proteins (the gene 44/62 and 45 proteins), and the gene 32 protein, which is the T4 helix-destabilizing (or single-stranded DNA-binding) protein. The 177-nucleotide-long DNA substrate consisted of a perfect 52-base pair hairpin helix with a protruding single-stranded 5' tail. As expected, the DNA polymerase binds near the 3' end of this molecule (at the primer-template junction) and protects the adjacent double-stranded region from cleavage. When the gene 32 protein binds to the single-stranded tail, it reduces the concentration of the DNA polymerase required to observe the polymerase footprint by 10-30-fold. Periodic ATP hydrolysis by the 44/62 protein is required to maintain the activity of the DNA polymerase holoenzyme (a complex of the 43, 44/62, and 45 proteins). Footprinting experiments demonstrate the formation of a weak complex between the DNA polymerase and the gene 45 protein, but there is no effect of the 44/62 protein or ATP on this enlarged footprint. We propose a model for holoenzyme function in which the complex of the three accessory proteins uses ATP hydrolysis to keep a moving polymerase tightly bound to the growing 3' end, providing a "clock" to measure polymerase stalling.

Polymerases:

T4

Topics:

Biotech Applications, Structure and Structure/Function, Accessory Proteins/Complexes, Nucleotide Incorporation, Enzyme Substrate Interactions

One line summary:

Analysis of T4 DNA polymerase (gp 43) and gp 44/62, gp 45 (T4 DNA replication accessory proteins), and gp 32 (T4 helix destabilizing protein) by DNA footprinting.

Status:

new topics/pols set partial results complete validated

Results:

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