A Central Swivel Point in the RFC Clamp Loader Controls PCNA Opening and Loading on DNA

摘要

Replication Factor C (RFC) is a 5-subunit complex that loads PCNA clamps onto primer-template DNA (ptDNA) during replication. RFC subunits belong to the AAA+ superfamily, and their ATPase activity drives interactions between the clamp loader, clamp, and ptDNA, leading to topologically linked PCNA•ptDNA. We report the kinetics of transient events in S. cerevisiae RFC catalyzed PCNA loading, including ATP-induced RFC activation, PCNA opening, ptDNA binding, ATP hydrolysis, PCNA closing and PCNA•ptDNA release. This detailed perspective enables assessment of individual RFC-A, -B, -C, -D, -E subunit functions in the reaction mechanism. Functions have been ascribed to RFC subunits previously, based on steady state analysis of ‘arginine finger’ ATPase mutants; however, pre-steady state analysis provides a different view. The central subunit, RFC-C, serves as a critical swivel point in the clamp loader. ATP binding to this subunit initiates RFC activation and the clamp loader adopts a spiral conformation that stabilizes PCNA in a corresponding open spiral. The importance of RFC subunit response to ATP binding decreases as: RFC-C > RFC-D > RFC-B, with RFC-A being unnecessary. RFC-C-dependent activation of RFC also enables ptDNA binding, leading to formation of the RFC•ATP•PCNAopen•ptDNA complex. Subsequent ATP hydrolysis leads to complex dissociation, with RFC-D activity contributing the most to rapid ptDNA release. The pivotal role of the RFC-BCD subunit ATPase core in clamp loading is consistent with the similar central location of all three ATPase-active subunits of the E. coli clamp loader.