A novel 5′ DNA binding site in RFC facilitates PCNA loading for gap DNA repair

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases delta and epsilon;. The RFC pentamer forms a central chamber that binds a 3 primed DNA junction to load PCNA onto DNA during replication. We show here five structures revealing a novel second DNA binding site in RFC that binds a 5 prime junction. This 5 prime DNA site is located in the N-terminal BRCT domain and AAA module of the large Rfc1 subunit. It appears that 5 prime DNA binds after 3 prime DNA binding to RFC. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nucleotide gaps, consistent with the structural results. We propose that the 5 prime DNA site facilitates RFC PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5 prime DNA binding domain of Rfc1. We further observe that a 5 prime site facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5 prime DNA site at longer gaps including during lagging strand DNA synthesis. ### Competing Interest Statement The authors have declared no competing interest.