Multistep loading of a DNA sliding clamp onto DNA by replication factor C

• DOI: 10.7554/elife.78253
• Publication Date: 2022-08-08T16:00:25Z
• Authors: Marina Schrecker; Juan C Castaneda; Sujan Devbhandari; Charanya Kumar; Dirk Remus; Richard K Hite
• Keywords: DNA clamp; Replication factor C; DNA polymerase delta

The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic metabolic enzymes. PCNA loaded around by the ATP-dependent loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded (dsDNA) junctions harboring a recessed 3’ end (3’ ss/dsDNA junctions) and nicks. To illuminate loading mechanism we have investigated structure of RFC:PCNA bound to ATPγS or nicked using cryogenic electron microscopy. Unexpectedly, observe open closed conformations in RFC:PCNA:DNA complex, revealing that can adopt open, planar conformation allows direct insertion dsDNA, raising question whether ring closure mechanistically coupled ATP hydrolysis. By resolving multiple DNA-bound states partial melting facilitates lateral into central channel formed RFC:PCNA. We also resolve Rfc1 N-terminal domain demonstrate its single BRCT participates coordinating prior RFC channel, promotes on lagging strand forks vitro. Combined, our data suggest comprehensive fundamentally revised model RFC-catalyzed onto DNA.