Before dividing, cells need to accurately duplicate their DNA, to ensure that each daughter cell has an identical copy. DNA replication is therefore a fundamental cellular process and requires a complex multi-protein machinery called replisome. The replisome functions like a factory where molecular players separate the two DNA parental strands and use them as templates to create the new daughter strands. Central players of the replisome are the DNA sliding clamps, ring-shaped proteins that encircle DNA and recruit the polymerases, the enzymes that replicate DNA, preventing them from falling off the genomic template. Due to their critical role in cell proliferation, these proteins are an important hallmark of tumors, as well as potential drug targets for anti-cancer therapy.
A long-standing mystery surrounds DNA sliding clamps: How do they slide on DNA? Is their motion controlled by chemical interactions with DNA? Or do they rather “levitate” on DNA? How does this motion affect the catalytic activity of the polymerase bound to the clamp?
Our work allows for the first time to visualize, at an atomic level, the interactions between the human sliding clamp PCNA and DNA, and to follow how they evolve in time. This provides the molecular basis for explaining how PCNA slides on DNA – a helical motion based on short-lived polar interactions – and sheds new light onto previous biological observations on PCNA function in DNA replication.
PCNA is a “moving clamp”, and its ability to transit along the DNA duplex is critical for the function. Therefore, in order to allow motion, the interactions that the clamp may establish with DNA have to be weak and transient. Such labile interactions are inherently difficult to observe. In order to provide a full description of the PCNA–DNA interaction, we applied a hybrid approach that combined X-ray crystallography, Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD) simulations. In the Structural Biology Laboratory of Elettra, we crystallized a complex made of human PCNA bound to a 10 base pair DNA duplex and, partly making use of the Elettra XRD-1 beamline, we determined the X-ray structure of this complex at 2.8 Å resolution. In this structure, the DNA within the PCNA central channel is tilted and contacts a set of protein residues arranged in a right-hand spiral that matches the pitch of DNA, forming polar contacts with consecutive DNA phosphate groups.
Figure 1. (a) 2.8 Å crystal structure of the human PCNA homotrimer bound to a 10 bp DNA duplex: the surface of the trimeric PCNA molecule is shown in different shades of blue while DNA is in orange. A close-up shows the detailed interactions between the DNA phosphates (yellow spheres) and a set of positively charged residues within the PCNA central channel. (b) NMR analysis: PCNA residues whose amide chemical shifts are significantly perturbed by DNA are colored red. The crystallographic position of DNA is shown in orange (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 a 100 ns MD simulation of the complex (d) Proposed PCNA sliding mechanism: Interacting side chains are able to rapidly switch between adjacent phosphates in a non-coordinated manner (illustrated by the thin and thick lines). When this stochastic process generates a state in which a sufficient number of electrostatic contacts are simultaneously established with adjacent phosphates in one direction of the DNA helical axis, a net rotation of the protein occurs and results in the advancement of one base pair.
This mode of binding was validated by analyzing the DNA-induced perturbations in the solution NMR spectrum of PCNA, in collaboration with Francisco Blanco’s group at CIC bioGUNE in Spain. Our MD simulations, done in collaboration with Ramon Crehuet’s group at the Institute of Advanced Chemistry of Catalonia, show that many PCNA interfacial residues can randomly switch between adjacent DNA phosphates on a sub-nanosecond time scale. This stochastic process must eventually generate a state where a sufficient number of contacts with adjacent phosphates in one direction of the DNA helical axis are simultaneously established, resulting in a net rotation of the protein and the advancement of one DNA base pair. This “cogwheel” mechanism would allow DNA backbone tracking in both directions while retaining DNA-protein contacts that keep the clamp in a defined orientation relative to DNA.
Earlier evidence showed that a single mutation among PCNA residues composing the PCNA‒DNA interface defined in our work severely reduces the replicative polymerase ability to incorporate an incoming nucleotide at the initiation of DNA synthesis. These residues are critical for PCNA‒DNA recognition and for orienting the clamp on DNA. Thus, we propose that this orientation is necessary for the assembly of a functional PCNA‒polymerase complex, able to initiate DNA replication. In addition, our results show that, despite a pronounced structural similarity, prokaryotic and eukaryotic clamps show fundamental differences in interacting with DNA.
Matteo De March1, Silvia Onesti1, Alfredo De Biasio1, Nekane Merino2,Francisco J. Blanco2, Susana Barrera-Vilarmauand Ramon Crehuet3
1 Sincrotrone Trieste S.C.p.A., Trieste, Italy
2 CIC bioGUNE, Derio, Spain
3 Advanced Chemistry of Catalonia (IQAC), CSIC, Barcelona, Spain
Alfredo De Biasio, email: email@example.com
M. De March, N. Merino, S. Barrera-Vilarmau, R. Crehuet, S. Onesti, F. J. Blancoand A. De Biasio,“Structural basis of human PCNA sliding on DNA”, Nat. Commun. 7, 13935 (2016) doi: 10.1038/ncomms13935