The dynamic processivity of the T4 DNA polymerase during replication.

Abstract:

The polymerase (gp43) processivity during T4 replisome mediated DNA replication has been investigated. The size of the Okazaki fragments remains constant over a wide range of polymerase concentrations. A dissociation rate constant of approximately 0.0013 sec(-1) was measured for the polymerases from both strands, consistent with highly processive replication on both the leading and lagging strands. This processive replication, however, can be disrupted by a catalytically inactive mutant D408N gp43 that retains normal affinity for DNA and the clamp. The inhibition kinetics fit well to an active exchange model in which the mutant polymerase (the polymerase trap) displaces the replicating polymerase. This kinetic model was further strengthened by the observation that the sizes of both the Okazaki fragments and the extension products on a primed M13mp18 template were reduced in the presence of the mutant polymerase. The effects of the trap polymerase therefore suggest a dynamic processivity of the polymerase during replication, namely, a solution/replisome polymerase exchange takes place without affecting continued DNA synthesis. This process mimics the polymerase switching recently suggested during the translesion DNA synthesis, implies the multiple functions of the clamp in replication, and may play a potential role in overcoming the replication barriers by the T4 replisome.

Polymerases:

Topics:

Kinetic Parameters, Accessory Proteins/Complexes, Source / Purification

One line summary:

The use of mutant T4 D809N (an inactive polymerase) with wild-type polymerase shows a decrease in the rate of replication, whereby the mutant polymerase displaces the wild type polymerase. Suggests multiple polymerases may be integrated into a replisome complex during replication and may show a greater number roles in the clamp.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 Yang J2004 Full length or truncated Full length
T4 Yang J2004 Vmax 150 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
T4 Yang J2004 Kd 145nM Reaction: Nucleotide incorporation; Substrate: dNTPs

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