Structural Basis of Human PCNA Sliding on DNA
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ARTICLE Received 14 Jul 2016 | Accepted 14 Nov 2016 | Published 10 Jan 2017 DOI: 10.1038/ncomms13935 OPEN Structural basis of human PCNA sliding on DNA Matteo De March1, Nekane Merino2, Susana Barrera-Vilarmau3, Ramon Crehuet3, Silvia Onesti1, Francisco J. Blanco2,4 & Alfredo De Biasio1 Sliding clamps encircle DNA and tether polymerases and other factors to the genomic template. However, the molecular mechanism of clamp sliding on DNA is unknown. Using crystallography, NMR and molecular dynamics simulations, here we show that the human clamp PCNA recognizes DNA through a double patch of basic residues within the ring channel, arranged in a right-hand spiral that matches the pitch of B-DNA. We propose that PCNA slides by tracking the DNA backbone via a ‘cogwheel’ mechanism based on short-lived polar interactions, which keep the orientation of the clamp invariant relative to DNA. Mutation of residues at the PCNA–DNA interface has been shown to impair the initiation of DNA synthesis by polymerase d (pol d). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol d holoenzyme. 1 Structural Biology Laboratory, Elettra-Sincrotrone Trieste S.C.p.A., 34149 Trieste, Italy. 2 CIC bioGUNE, Parque Tecnologico de Bizkaia Edificio 800, 48160 Derio, Spain. 3 Institute of Advanced Chemistry of Catalonia (IQAC), CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain. 4 IKERBASQUE, Basque Foundation for Science, Bilbao 48013, Spain. Correspondence and requests for materials should be addressed to S.O. (email: [email protected]) or to F.J.B. (email: [email protected]) or to A.D.B. (email: [email protected]). NATURE COMMUNICATIONS | 8:13935 | DOI: 10.1038/ncomms13935 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13935 rocessive chromosomal replication requires ring-shaped residues lining the clamp channel are conserved in eukaryotes sliding clamp factors that encircle DNA and anchor and a subset of these residues function in DNA synthesis by Ppolymerases and other proteins of the replisome. Prolifer- pol d and clamp loading by replication factor C (RFC)7–9, ating cell nuclear antigen (PCNA)—the eukaryotic sliding pointing to the existence of direct interactions between clamp—is a homotrimeric ring of 86 kDa featuring a central PCNA and the DNA phosphodiester backbone. In a channel lined with lysine and arginine-rich a-helices through crystallographically derived model of yeast PCNA bound to a which DNA is threaded1–3. The central channel of PCNA 10 bp primed DNA, the DNA in the clamp channel protrudes is a conserved structural feature among sliding clamps and is from the back face at a B40° tilt angle, is held in place B35 Å in diameter, significantly larger than the diameter of the by a crystal contact, and would collide with PCNA if it B-form DNA double helix (B24 Å)4. Since the first was lengthened towards the front face. The PCNA–DNA determination of the structure of a sliding clamp, the bacterial interface only shows some charge complementarity and the b-clamp5, this feature has raised questions regarding the protein residues involved largely do not overlap with those interaction with DNA and the sliding mechanism. The crystal found to have an effect on clamp loading or pol d function7–9, structure of b-clamp bound to primed DNA6 showed the making it difficult to ascribe this model to a functional state of the DNA duplex threaded through the clamp at a 22° tilt angle, complex. Because of the inherent lability of the PCNA–DNA making contacts with residues in the channel and on protruding interaction, structural and biophysical characterization of loops on the back face of the clamp. However, extensive this interface has been challenging. As a consequence, how interactions are also established between the single-strand eukaryotic clamps recognize DNA has remained controversial portion of DNA and the protein binding pocket of a and a molecular mechanism of PCNA sliding on DNA has crystallographically related b-clamp molecule. The b-clamp- not been proposed. Here we have determined the crystal structure ssDNA inter-molecular interaction in the crystal is intra- of human PCNA bound to a 10 bp long double-stranded molecular in solution, and DNA competes with DNA DNA (dsDNA). We analysed the DNA-induced perturbations polymerase binding6. in the solution NMR spectrum of PCNA, and computationally Despite structural conservation, the absence of patterns characterized the interactions in multinanosecond molecular of sequence similarity in bacterial and eukaryotic clamps1 does dynamics (MD) simulations. We show that PCNA recognizes not allow a simple correlation among the DNA-binding sites the DNA structure through a set of basic residues within the ring in the two systems. On the other hand, the positively charged channel organized to match the pitch of B-DNA, establishing a c X-ray structure MD model K77 P1 P2 K20 P3 R149 P4 P4 K217 P5 P3 P2 P1 R149 H153 K217 K80 K20 K77 K80 b NMR data 0.05 K77 0.04 T73 180° 0.03 D21 A218 0.02 CSP (p.p.m.) ± KD= 0.7 0.1 mM N213 0.01 ex << 38 ms A145 G83 S152 0.00 0.0 0.5 1.0 1.5 2.0 K14 T224 [DNA] (mM) Figure 1 | Structural basis of DNA recognition by human PCNA. (a) 2.8 Å crystal structure of PCNA bound to a 10 bp DNA duplex. PCNA and DNA are shown in surface and ribbon representation, respectively. PCNA subunits are coloured in different shades of blue and DNA in orange. The expansion shows the complex in ribbon representation. Interacting PCNA side chains and DNA phosphates (interatomic side chain nitrogen—DNA phosphorus distance o5 Å) are shown as sticks and yellow spheres, respectively, and interactions as dashed lines (b) NMR analysis. Left: front- and back-face views of PCNA surface. PCNA residues whose amide chemical shifts are significantly perturbed by DNA are coloured red. The crystallographic position of DNA is shown in orange. The interacting region in the clamp channel overlaps with that seen in the crystal structure, whereas in the crystal, the side chains can be discriminated, in solution the perturbations involve the backbone amides. Right: chemical shift perturbation of the amide signal of PCNA residue T73at different DNA concentrations. Fitting was performed using a single-site binding model. Extrapolated dissociation and exchange time constants are indicated. (c) Model interface from MD simulation. The crystallographic position of the DNA segment is shown in orange, whereas in black the DNA is shown in a position corresponding to the final state of the 100 ns MD simulation of the complex. 2 NATURE COMMUNICATIONS | 8:13935 | DOI: 10.1038/ncomms13935 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13935 ARTICLE short-lived polar interactions with consecutive DNA phosphates. another residue (K80) on the proximal a-helix of the adjacent The interacting side chains are able to switch between adjacent subunit. The side chains of the PCNA interfacial residues form phosphates in a non-coordinated manner, supporting a a right-hand spiral that closely matches the pitch of B-DNA, helical sliding mechanism in which the clamp rotates and and are involved in polar contacts with five consecutive phos- tilts by keeping a fixed orientation relative to the DNA backbone. phates of a single DNA strand (Fig. 1a). We discuss the implications of these findings for the function The dynamic PCNA–DNA interface observed in the crystal of the PCNA-pol d holoenzyme in DNA replication, and also is recapitulated by the NMR analysis of the binding in solution for clamp loading by RFC. (Fig. 1b, Supplementary Figs 2a and 2b and 3). Titration of PCNA with dsDNA shows weak binding (KDB0.7 mM) to the inner side of the ring. In this assay, time averaging of the NMR signal Results restores the symmetry of the PCNA ring that is broken in Structure of the PCNA–DNA complex. We obtained crystals the crystal. Backbone amide chemical shift perturbations of PCNA–dsDNA complex with one PCNA trimer per asym- are small (CSPo0.06 p.p.m.), and the exchange rate is fast metric unit and diffracting to 2.8 Å resolution (Supplementary (Fig. 1b, Supplementary Figs 2 and 3). These observations are Table 1). The dsDNA molecule threads through the PCNA ring consistent with the low affinity of the interaction, the contacts with its longitudinal axis and the C3 axis of the ring forming an involving long amino-acid side chains, and the high crystal- angle of 15° (Fig. 1a and Supplementary Fig. 1). No crystal lattice lographic temperature factors of DNA. contacts between DNA and symmetry-related molecules Using NMR, we tested the binding of PCNA to the primed- are present, and the crystal packing does not affect the DNA template DNA (pDNA) substrate (Supplementary Table 2) that position. The high temperature factors of dsDNA are compatible was co-crystallized with the bacterial b-clamp6. In the b-pDNA with a partial occupancy of dsDNA and/or with the existence structure, the ssDNA template strand is anchored to the main of a subpopulation of complexes with slightly different DNA protein-binding pocket of the clamp, thus competing with orientations. The complex interface involves the side chains of DNA polymerase binding6. Our NMR mapping, however, five basic residues (K20, K77, R149, H153 and K217), distributed shows that pDNA binds the inner wall of the human PCNA on four a-helices of one PCNA subunit, and the side chain of channel but not the protein binding (PIP-box) pocket a 30 20 10 Angle (°) PCNA rotation DNA tilt 0 0 50 100 150 200 250 Time (ns) b i+1 i i–1 R149 i+1 i i–1 K77 i+1 i DNA P index i–1 R210 050100 150 200 250 Time (ns) c K77* R210* K20 K20 180° N84 K80 R149* K217 K80 N84* S10 S223 K14* Figure 2 | MD simulation of human PCNA bound to a 30 bp DNA.