RNA primer handoff in bacteriophage T4 DNA replication: the role of single-stranded DNA-binding protein and polymerase accessory proteins.

Abstract:

In T4 phage, coordinated leading and lagging strand DNA synthesis is carried out by an eight-protein complex termed the replisome. The control of lagging strand DNA synthesis depends on a highly dynamic replisome with several proteins entering and leaving during DNA replication. Here we examine the role of single-stranded binding protein (gp32) in the repetitive cycles of lagging strand synthesis. Removal of the protein-interacting domain of gp32 results in a reduction in the number of primers synthesized and in the efficiency of primer transfer to the polymerase. We find that the primase protein is moderately processive, and this processivity depends on the presence of full-length gp32 at the replication fork. Surprisingly, we find that an increase in the efficiency of primer transfer to the clamp protein correlates with a decrease in the dissociation rate of the primase from the replisome. These findings result in a revised model of lagging strand DNA synthesis where the primase remains as part of the replisome after each successful cycle of Okazaki fragment synthesis. A delay in primer transfer results in an increased probability of the primase dissociating from the replication fork. The interplay between gp32, primase, clamp, and clamp loader dictates the rate and efficiency of primer synthesis, polymerase recycling, and primer transfer to the polymerase.

Polymerases:

T4

Topics:

Accessory Proteins/Complexes

One line summary:

Gp32 (single stranded binding protein was examined during lagging synthesis. Gp32's protein interaction affects the quantity of primer produced and its efficiency of transfer to polymerases. It was found that the more efficient primer transfer was, the less primase dissociation occured from the replisome.

Status:

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Results:

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