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. 2006 Feb 21;103(8):2546-51.
doi: 10.1073/pnas.0511263103. Epub 2006 Feb 13.

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

Affiliations

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

Zhihao Zhuang et al. Proc Natl Acad Sci U S A. .

Abstract

Numerous proteins that function in DNA metabolic pathways are known to interact with the proliferating cell nuclear antigen (PCNA). The important function of PCNA in stimulating various cellular activities requires its topological linkage with DNA. Loading of the circular PCNA onto duplex DNA requires the activity of a clamp-loader [replication factor C (RFC)] complex and the energy derived from ATP hydrolysis. The mechanistic and structural details regarding PCNA loading by the RFC complex are still developing. In particular, the positive identification of a long-hypothesized structure of an open clamp-RFC complex as an intermediate in loading has remained elusive. In this study, we capture an open yeast PCNA clamp in a complex with RFC through fluorescence energy transfer experiments. 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 ATPgammaS 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.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
The subunit interface of PCNA. The residues located at the interfacial antiparallel β-strands are highlighted. The residues (Phe-185 and Lys-107) selected for introducing the FRET pair are shown as purple and gold, respectively.
Fig. 2.
Fig. 2.
Fluorescence spectra of PCNA-WC (magenta) and PCNA-WCad (green) (A) and PCNA-WCad (magenta) and PCNA-Cad (green) (B) with 290 nm excitation.
Fig. 3.
Fig. 3.
Steady-state fluorescence spectra of RFC catalyzed PCNA-WCad loading in the presence of ATP (A) and ATPγS (B).
Fig. 4.
Fig. 4.
Stopped-flow fluorescence traces of mixing RFC versus PCNA-WCad in the presence of ATP (A), RFC-PCNA-WCad complex formed in the presence of ATP versus DNA bound with streptavidin (B), RFC versus PCNA-WCad in the presence of ATPγS (C), and RFC-PCNA-WCad complex formed in the presence of ATPγS versus DNA–streptavidin (D). The solid line represents the best fit of the trace to the exponential equations, as described in Results.
Fig. 5.
Fig. 5.
Modeling of the opened PCNA–RFC structure. (A) The S. cerevisiae RFC–PCNA crystal structure. The RFC five subunits are rendered as ribbon and colored as red, gold, green, cyan, and blue, respectively. The PCNA exists as a closed trimer with three subunits colored as yellow (subunit I), blue (subunit II), and magenta (subunit III), respectively. (B) Modeled in-plane opening PCNA structure with 34-Å distance between Trp-185 and Cys-107-AEDANS. (C) Modeled out-of-plane opening PCNA structure with 34-Å distance between Trp-185 and Cys-107-AEDANS.
Fig. 6.
Fig. 6.
The proposed steps of RFC catalyzed PCNA loading. (I) In-plane opening PCNA complexed with RFC(ATP). (II) Out-of-plane closing of PCNA onto duplex DNA. (III) Final in-plane closing of PCNA onto DNA.

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