Characterization of the DNA-dependent GTPase activity of T4 gene 41 protein, an essential component of the T4 bacteriophage DNA replication apparatus.

Abstract:

The product specified by T4 bacteriophage gene 41 is known from genetic analyses to be essential for phage DNA replication in vivo. Correspondingly, the purified gene 41 protein is an essential component of an efficient in vitro DNA replication system reconstructed from seven purified T4 replication proteins; it is required both for the synthesis of short RNA primers (in conjunction with the T4 gene 61 protein) and for the rapid unwinding of the double-helical DNA template at a replication fork. The purified gene 41 protein exhibits a DNA-dependent GTPase (and ATPase) activity. In this report, we have used this associated GTPase activity as a biochemical probe for the analysis of the interactions between DNA and the 41 protein. Our results suggest that, upon binding GTP, the 41 protein monomer is induced to form a dimer, which can them form a tight complex with single-stranded DNA. Driven by the repeated hydrolysis of GTP molecules, the 41 protein dimer appears to run rapidly along the bound DNA chain. Studies with the synthetic GTP analogue, GTP gamma S, suggest that GTP hydrolysis is required for this 41 protein movement, but that it is not essential for the function of the 41 protein in RNA primer synthesis. In sum, our observations suggest that a 41 protein dimer runs along the lagging strand template at a DNA replication fork; from this position, it functions as a DNA helicase and simultaneously interacts with the T4 gene 61 protein to make the pentaribonucleotide primers which initiate Okazaki pieces at specific primer initiation sites.

Polymerases:

T4

Topics:

Accessory Proteins/Complexes

Status:

new topics/pols set partial results complete validated

Results:

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