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Replication factor C (RFC) is a heteropentameric AAA+ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, gamma complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA+ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli gamma complex in which only one subunit makes strong contact with the beta clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric beta ring.

Replication factor C (RFC) catalyzes assembly of circular proliferating cell nuclear antigen clamps around primed DNA, enabling processive synthesis by DNA polymerase during DNA replication and repair. In order to perform this function efficiently, RFC must rapidly recognize primed DNA as the substrate for clamp assembly, particularly during lagging strand synthesis. Earlier reports as well as quantitative DNA binding experiments from this study indicate, however, that RFC interacts with primer-template as well as single- and double-stranded DNA (ssDNA and dsDNA, respectively) with similar high affinity (apparent K(d) approximately 10 nm). How then can RFC distinguish primed DNA sites from excess ssDNA and dsDNA at the replication fork? Further analysis reveals that despite its high affinity for various DNA structures, RFC selects primer-template DNA even in the presence of a 50-fold excess of ssDNA and dsDNA. The interaction between ssDNA or dsDNA and RFC is far less stable than between primed DNA and RFC (k(off) > 0.2 s(-1) versus 0.025 s(-1), respectively). We propose that the ability to rapidly bind and release single- and double-stranded DNA coupled with selective, stable binding to primer-template DNA allows RFC to scan DNA efficiently for primed sites where it can pause to initiate clamp assembly.