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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 δ and γ. The RFC pentamer forms a central chamber that binds 3′ ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures revealing a novel 2nd DNA binding site in RFC that binds a 5′ duplex. This 5′ DNA site is located in the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. It appears 5′ DNA binds after 3′ 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 nt gaps, consistent with the structural results. We propose that the 5′ site facilitates RFC’s 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′ DNA binding domain of Rfc1. We further observe that a 5′ end facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5′ DNA site in longer gaps including during lagging strand DNA synthesis.