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. 2010 Jun 2;132(21):7372-8.
doi: 10.1021/ja100365x.

Recognition of the ring-opened state of proliferating cell nuclear antigen by replication factor C promotes eukaryotic clamp-loading

John A Tainer  1 , J Andrew McCammon, Ivaylo Ivanov
Affiliations
Free PMC article

Recognition of the ring-opened state of proliferating cell nuclear antigen by replication factor C promotes eukaryotic clamp-loading

John A Tainer et al. J Am Chem Soc. .
Free PMC article

Abstract

Proliferating cell nuclear antigen (PCNA, sliding clamp) is a toroidal-shaped protein that encircles DNA and plays a pivotal role in DNA replication, modification and repair. To perform its vital functions, the clamp has to be opened and resealed at primer-template junctions by a clamp loader molecular machine, replication factor C (RFC). The mechanism of this process constitutes a significant piece in the puzzle of processive DNA replication. We show that upon clamp opening the RFC/PCNA complex undergoes a large conformational rearrangement, leading to the formation of an extended interface between the clamp and RFC. Binding of ring-open PCNA to all five RFC subunits transforms the free-energy landscape underlying the closed- to open state transition, trapping PCNA in an open conformation. Careful comparison of free-energy profiles for clamp opening in the presence and absence of RFC allowed us to substantiate the role of RFC in the initial stage of the clamp-loading cycle. RFC does not appreciably destabilize the closed state of PCNA. Instead, the function of the clamp loader is dependent on the selective stabilization of the open conformation of the clamp.

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Figures

Figure 1
Figure 1
(a) Structure of the trimeric sliding clamp protein PCNA with the three equivalent subunits displayed in red, blue, and green, respectively; (b) schematic representation of the clamp loading cycle involving the action of the multisubunit clamp loader RFC. (c) structure of the yeast RFC/PCNA complex. We denote the five RFC subunits A−E according to their sequential order in the pentamer. Herein, we also provide an alternative nomenclature for the RFC subunits as follows: subunit Rfc1 (A), Rfc4 (B), Rfc3 (C), Rfc2 (D), and Rfc5 (E). The subunits A−E are colored in blue, purple, green, yellow, and red, respectively. PCNA is shown in gray. (d) Steered molecular dynamics applied to PCNA; the Cα atoms involved are shown explicitly (yellow spheres) and the in-plane pulling direction is indicated by an arrow.
Figure 2
Figure 2
Computationally derived model for the complex of the clamp loader RFC with ring-open PCNA. (a) van der Waals surface representation of the model illustrates the out-of-plane twisting of the ring-open sliding clamp. Contacts with subunits D and E are highlighted by purple and blue rectangles. (b) Specific mode of interaction of the clamp with RFC subunits D and E.
Figure 3
Figure 3
Time evolution of characteristic structural parameters. (a) The blue line corresponds to the distance (dCZ-NZ) between the side chain nitrogen atom (NZ) of PCNA residue K107 and a side chain carbon atom (CZ) of PCNA residue Phe185 corresponding to the FRET donor−acceptor pair in the Zhuang et al. experiments. The red line represents the gap distance dgap defined as the closest distance between heavy atoms from the two PCNA subunits across the open subunit interface. (b) The blue line displays the displacement between the centers of mass of all backbone atoms from the two interfacial β-strands projected into the plane of the PCNA ring (in-plane strand displacement). The red line displays the displacement between the centers of mass of all backbone atoms from the two interfacial β-strands projected along the normal direction to the plane of the PCNA ring (out-of-plane strand displacement).
Figure 4
Figure 4
Electrostatic potential (ESP) mapped onto the molecular surface is shown for: (a) S. cerevisiae RFC and PCNA (anterior, posterior and lateral views are included for both proteins); (b) the internal cavity formed by the subunits of the clamp loader (labeled A−E) and the surfaces of the AAA+ modules responsible for binding to the sliding clamp. Regions of negative potential are shown in red; regions of positive potential are in blue.
Figure 5
Figure 5
Potentials of mean force (PMFs) for PCNA opening. (a) PMF for in-plane opening of an isolated sliding clamp (in the absence of RFC); (b) PMF for opening a clamp bound to RFC.
Figure 6
Figure 6
A proposed mechanism for the role of the clamp loader during the initial stage of the clamp loading cycle. (a) Schematic representation of the out-of-plane opening of PCNA upon binding of RFC and ATP. (b) Schematic representation of the change in the free energy landscape for clamp opening due to the complementary binding of RFC.
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