Abstract
The Escherichia coli beta dimer is a ring-shaped protein that encircles DNA and acts as a sliding clamp to tether the replicase, DNA polymerase III holoenzyme, to DNA. The gamma complex (gammadeltadelta'chipsi) clamp loader couples ATP to the opening and closing of beta in assembly of the ring onto DNA. These proteins are functionally and structurally conserved in all cells. The eukaryotic equivalents are the replication factor C (RFC) clamp loader and the proliferating cell nuclear antigen (PCNA) clamp. The delta subunit of the E. coli gamma complex clamp loader is known to bind beta and open it by parting one of the dimer interfaces. This study demonstrates that other subunits of gamma complex also bind beta, although weaker than delta. The gamma subunit like delta, affects the opening of beta, but with a lower efficiency than delta. The delta' subunit regulates both gamma and delta ring opening activities in a fashion that is modulated by ATP interaction with gamma. The implications of these actions for the workings of the E. coli clamp loading machinery and for eukaryotic RFC and PCNA are discussed.
Highlights
Chromosomal replicases are highly processive machines owing to a sliding clamp subunit that encircles and slides on DNA, acting as a mobile tether for the replicase during synthesis [1,2,3,4]
The RFC1 clamp loader assembles the ring-shaped proliferating cell nuclear antigen (PCNA) clamp onto DNA for processive DNA polymerase action [12, 13]
What is the role of ATP if it is not required for clamp opening? Our studies on this subject reveal that the ␦ subunit is buried within ␥ complex such that its interaction with  is weak compared with the ␦1⁄7B  complex [26]
Summary
␥ or any other subunit of ␥ complex [26]. ␥ binds  weaker than ␦ and is ϳ20-fold less efficient in unloading  from DNA compared with ␦ (k␦unloading ϭ 0.42 minϪ1; k␥unloading ϭ 0.016 minϪ1). ␦Ј is essential for coupling ATP to the action of ␥ and ␦ with , even though ATP binds ␥ and not ␦Ј. These interactions between ␥ complex subunits among themselves and , and their regulation by ␦Ј and ATP, are discussed in terms of a molecular model of ␥ complex mechanism
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