Dynamics of Loading theEscherichia coliDNA Polymerase Processivity Clamp

Abstract

Sliding clamps and clamp loaders are processivity factors required for efficient DNA replication. Sliding clamps are ring-shaped complexes that tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders assemble these ring-shaped clamps onto DNA in an ATP-dependent reaction. The overall process of clamp loading is dynamic in that protein-protein and protein-DNA interactions must actively change in a coordinated fashion to complete the mechanical clamp-loading reaction cycle. The clamp loader must initially have a high affinity for both the clamp and DNA to bring these macromolecules together, but then must release the clamp on DNA for synthesis to begin. Evidence is presented for a mechanism in which the clamp-loading reaction comprises a series of binding reactions to ATP, the clamp, DNA, and ADP, each of which promotes some change in the conformation of the clamp loader that alters interactions with the next component of the pathway. These changes in interactions must be rapid enough to allow the clamp loader to keep pace with replication fork movement. This review focuses on the measurement of dynamic and transient interactions required to assemble the Escherichia coli sliding clamp on DNA.

Mechanism of ATP-Driven PCNA Clamp Loading by S. cerevisiae RFC

“…Clamps are formed by association of two or more identical or homologous subunits into strikingly similar ring-shaped oligomers, with large enough diameters to encompass duplex DNA. They are loaded onto DNA by multi-protein complexes known as clamp loaders, in a reaction driven by ATP binding and hydrolysis 4,5. In eukaryotes, including humans, the PCNA clamp (Proliferating Cell Nuclear Antigen) is a trimer of identical subunits arranged in head-to-tail fashion 6,7.…”

A single hydrophobic cleft in the Escherichia coli processivity clamp is sufficient to support cell viability and DNA damage-induced mutagenesis in vivo

“…Like their eukaryotic counterparts, these clamps are loaded onto DNA in an ATP-dependent manner by a multi-subunit clamp loader complex [7]. Once loaded, they recruit the replicative Pol (Pol III), as well as other partner proteins involved in various aspects of DNA replication, repair, and damage tolerance [5].…”

“…Our current appreciation of clamp functionality is based largely on results of in vitro biochemical assays (reviewed in [5,7,23]). For example, recent in vitro experiments exploiting heterodimeric clamps comprised of two distinct β protomers revealed that a single cleft of the clamp was both necessary and sufficient for supporting a switch between Pol IV and a stalled Pol III [5,12,13].…”

Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA

“…The clamp is loaded onto DNA by the clamp loader, a five-subunit complex of AAAϩ family proteins that couple ATP binding and hydrolysis to mechanical work (8 -13). Structural and mechanistic analyses of clamp loaders such as E. coli ␥ complex (11,14), bacteriophage T4 gp44/62 (8,15,16), and Saccharomyces cerevisiae RFC (10,(17)(18)(19) among others, have shown that ATP binding enables the clamp loader to bind and open the clamp and bind ptDNA, and ATP hydrolysis leads to the release of the clamp-ptDNA product (see Fig. 1A), which can then be used by DNA polymerase and other proteins.…”