Structural basis of human PCNA sliding on DNA

Abstract

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 δ (pol δ). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol δ holoenzyme.

DNA Replication: How Does a Sliding Clamp Slide?

“…NMR studies and molecular dynamics simulations revealed that the lysines walk along successive backbone phosphates of one DNA strand during sliding [8], and predict a sliding rate far faster than the DNA polymerase, consistent with single-molecule measurements of rapid clamp diffusion on DNA [10]. The fast rate of clamp diffusion on DNA ensures that it will not hinder the DNA polymerase rate.…”

“…A recent study [8] has illuminated the clamp sliding process along DNA and the findings are fascinating. A crystal structure of human PCNA encircling a 10 base pair duplex DNA has been solved to high resolution, and the architecture of the clamp–DNA interface is now clear (Figure 1).…”

“…The fast rate of clamp diffusion on DNA ensures that it will not hinder the DNA polymerase rate. The molecular dynamics simulations also concluded that clamps mainly spiral when they diffuse along DNA [8,11]. The simulations show that the 30° tilt of DNA to the central axis of the clamp is maintained during sliding, such that when viewed from the side, a tilted clamp that runs along one DNA ‘rail’ propagates the tilt in a helical fashion and gives the appearance of alternating between forward and backward tilts every one-half turn of DNA (Figure 2A).…”

“…This is the natural consequence of sliding on a helical substrate, and De March et al . [8] refer to this sliding process as ‘cogwheeling’ along the DNA. They point out that certain biochemical studies of mutant proteins suggest that polymerases and clamp loaders are specific for the particular tilt that PCNA has adopted at the point that it is positioned at a primer terminus [12,13].…”

“…Studies of PCNA diffusion show that this type of switching does occur, but not as frequently as cogwheel diffusion [11]. It is speculated that this switch may be useful for proofreading by DNA polymerase δ, which has a 3′–5′ exonuclease site in a separate location from the polymerase site that removes nucleotides that are incorrectly placed into DNA by the polymerase [8]. Therefore, if the polymerase accidentally incorporates a wrong nucleotide into DNA, the polymerase–PCNA may switch between different DNA tracking sites and reposition the primer terminus from the polymerase site to the exonuclease site.…”