Abstract
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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Spelling of the first authors name has been corrected
