Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly.

Abstract:

The bacteriophage T4 DNA polymerase holoenzyme, consisting of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62), is loaded onto DNA in an ATP-dependent, multistep reaction. The trimeric, ring-shaped gp45 is loaded onto DNA such that the DNA passes through the center of the ring. gp43 binds to this complex, thereby forming a topological link with the DNA and increasing its processivity. Using stopped-flow fluorescence-resonance energy transfer, we have investigated opening and closing of the gp45 ring during the holoenzyme assembly process. Two amino acids that lie on opposite sides of the gp45 subunit interface, W91 and V162C labeled with coumarin, were used as the fluorescence donor and acceptor, respectively. Free in solution, gp45 has two closed subunit interfaces with W91 to V162-coumarin distances of 19 A and one open subunit interface with a W91 to V162C-coumarin distance of 40 A. Making the assumption that the distance across the two closed subunit interfaces is unchanged during the holoenzyme assembly process, we have found that the distance across the open subunit interface is first increased to greater than 45 A and is then decreased to 30 A during a 10-step assembly mechanism. The gp45 ring is not completely closed in the holoenzyme complex, consistent with previous evidence suggesting that the C-terminus of gp43 is inserted into the gp45 subunit interface. Unexpectedly, ATP-hydrolysis events are coupled to only a fraction of the total distance change, with conformational changes linked to binding DNA and gp43 coupled to the majority of the total distance change. Using the nonhydrolyzable ATP analogue ATP-gamma-S results in formation of a nonproductive gp45 x gp44/62 complex; however, adding an excess of ATP to this nonproductive complex results in rapid ATP/ATP-gamma-S exchange to yield a productive gp45 x gp44/62 complex within seconds.

Polymerases:

T4

Topics:

Status:

new topics/pols set partial results complete validated

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