Single-Molecule Observation of the Rotational and Translational Movement of the PCNA Sliding Clamp Along DNA

Abstract

Using single-molecule TIR (total internal reflection) fluorescence microscopy we study the dynamics of the eukaryotic sliding clamp PCNA (proliferating cell nuclear antigen). PCNA is a homotrimeric ring that plays multiple roles at the replication fork as a processivity factor for polymerases and as a molecular tool belt tethering a variety of nucleic-acid enzymes to the DNA. First, we studied the 1-dimensional diffusion of PCNA loaded around a well-stretched and doubly-tethered λ-DNA molecule. We found that the diffusion coefficient of PCNA does not vary with ionic strength suggesting that PCNA maintains electrostatic contact with DNA as it slides. Further, we found that the diffusion coefficient of PCNA is relatively insensitive to changes in viscosity when high molecular viscogens are used. This observation suggests that PCNA tracks the DNA double helix as it slides. However, increasing the hydrodynamic radius of PCNA by coupling the protein to QDot, resulted in a diffusion coefficient that was over an order of magnitude higher than expected for a helically tracking protein. We therefore propose that PCNA uses both helically tracking and non-helically tracking modes of diffusion and speculate why this may be advantageous for the many roles played by PCNA. Finally, we seek to extend our dynamic studies of PCNA from naked DNA to the context of a replication fork. Towards that end, we are using Xenopus laevis egg extracts in combination with single-molecule fluorescence imaging to visualize individual PCNA trimers during polymerase-mediated DNA synthesis and eventually during DNA replication.