The Lord of the Rings: structural mechanism of a DNA polymerase sliding clamp loader

Seminar | November 4 | 4-5 p.m. | 106 Stanley Hall

 Brian Kelch, University of Massachusetts Medical School

 College of Chemistry

The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) is a central regulator of genomic integrity and cell proliferation pathways in all eukaryotes. PCNA is a ring-shaped complex that encircles and slides along DNA, serving as an essential cofactor of DNA polymerases and scores of other proteins to coordinate DNA replication with numerous cellular processes. PCNA is installed on DNA by a pentameric ATPase complex known as the clamp loader. Clamp loaders bind and open the PCNA ring, allowing primer-template DNA to bind inside the complex. Despite decades of study, the structural mechanism of clamp opening was still unknown. We visualized PCNA loading in high temporal and structural resolution using time-resolved small angle X-ray scattering (trSAXS) and single particle cryo-electron microscopy reconstruction (cryoEM). Our structures show that the clamp loader pries PCNA open in an unanticipated, multistep ‘crab claw’ motion. Furthermore, we find that PCNA is opened much wider than anticipated, such that dsDNA can directly enter the complex, in opposition to ‘filtering’ models that had been previously proposed. We also find that the clamp loader can unwind the first few base pairs of DNA, which allows PCNA loading at nicked DNA and sites of DNA repair. Finally, we identify a suite of interactions that inhibit DNA binding until the clamp is opened, which prevents the clamp loader from adopting a dead-end complex. Our work provides a high-resolution understanding of clamp loading, a reaction that occurs in all cellular life.