The structure of a ring-opened proliferating cell nuclear antigen–replication factor C complex revealed by fluorescence energy transfer

Abstract

We also follow the topological transitions of PCNA in the various steps of the clamp-loading pathway through both steady-state and stopped-flow fluorescence studies. We find that ATP effectively drives the clamp-loading process to completion with the formation of the closed PCNA bound to DNA, whereas ATP␥S cannot. The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm.clamp loader ͉ DNA replication T he replisome is a dynamic molecular complex responsible for the duplication of a genomic DNA template at the replication fork. The central components of the replisome are the replicative DNA polymerases that copy both leading-and lagging-strand DNA (reviewed in ref. 1). Processivity of DNA replication is accomplished through a clamp protein that forms a holoenzyme complex with the replicative DNA polymerase (2). The clamp proteins identified in different organisms show a common toroidal architecture with a central channel large enough to encircle duplex DNA (3). Once loaded onto duplex DNA, clamp proteins can freely slide in a 2D space. The eukaryotic clamp proliferating cell nuclear antigen (PCNA) forms a holoenzyme complex with DNA polymerases ␦ and through specific interaction(s) between the polymerase and the PCNA ring (4-6).The task of loading PCNA onto DNA at the primer-template junction is fulfilled by replication factor C (RFC) in an ATP-driven process. Eukaryotic RFC is a heteropentameric protein complex consisting of Rfc1, -2, -3, -4, and -5 subunits. Each Rfc subunit belongs to the AAA ϩ protein family that contains characteristic ATP-binding͞hydrolysis motifs and converts the chemical energy derived from ATP binding and hydrolysis to mechanic force that is required for clamp loading (7). The RFC complex shares partial similarities to its counterpart in lower organisms like bacteriophage T4 and Escherichia coli. However, the small subunits of eukaryotic RFC are more diverged compared with those in prokaryote and archaeon, which hints for a higher complexity in both structural and functional aspects. Indeed, the yeast RFC showed an interesting departure from its prokaryotic counterparts in terms of a step-wise binding of ATP molecules modulated by its interaction with either DNA or PCNA (8). Therefore, a substantial difference is expected in the mechanistic details for clamp loading as compared with its prokaryotic counterpart. Furthermore, additional RFC-like complexes have been identified that share the Rfc2-5 subunits with RFC but carry out specialized functions in the DNA damage checkpoint and in sister chromatid cohesion (reviewed in ref. 9).Although the clamp loaders from the three model systems, including yeast, E. coli, and bacteriophage T4, all use ATP for loading the clamp onto DNA, the timing and amount of ATP binding and of its hydrolysis differ. For the E. coli system, ATP binding is sufficient for opening the ␤ clamp (10), and subseque...

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

“…1C). Both RFC and ATP are required for the PCNA loading, which is consistent with earlier reports (12,19,20). Labeled PCNA in the presence of RFC without DNA showed no FRET signal upon addition of ATP ruling out mere acceptor fluorescence variation that was caused by environmental changes upon RFC binding.…”

“…A mutant PCNA with all four native cysteines mutated to serines, and lysine 107 and serine 179 changed to cysteine and tryptophan, respectively, was labeled with Cy5-maleimide and purified by size-exclusion chromatography (Fig. 1A) (19). The labeled mutant PCNA was as active as the wild type in terms of stimulating RFC mediated ATP hydrolysis and its loading onto DNA (Fig.…”

“…S2A). Both ATP and ATPγS promote opening of the PCNA ring in the presence of RFC from 13 to 34 and 27 Å, respectively (19).…”

“…The structure of the yeast RFC · PCNA complex (13) provided the coordinates for introducing a fluorescent donor-acceptor pair within the PCNA to probe the FRET efficiencies distance information on the conformation of the PCNA subunit interface (19). In the presence of ATP, the PCNA ring was opened by RFC; upon addition of DNA, PCNA closed around the DNA through a series of steps involving out-of-plane followed by in-plane closing of the ring.…”

“…In the presence of ATP, the PCNA ring was opened by RFC; upon addition of DNA, PCNA closed around the DNA through a series of steps involving out-of-plane followed by in-plane closing of the ring. In the presence of ATPγS the RFC · PCNA complex opened the clamp and bound to DNA but ring closure was not completed (19,20). Pre-steady-state global analysis of the accompanying ATP hydrolysis showed that the ATPase reaction proceeded through multiple steps within two distinct phases; first, assembly of the RFC · PCNA · DNA complex was coupled to ATP binding and second disassembly was coupled to ATP hydrolysis (21).…”