Cryo-EM structures reveal that RFC recognizes both the 3'- and 5'-DNA ends to load PCNA onto gaps for DNA repair

Abstract

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases {delta} and {varepsilon}. 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 that identify a 2nd DNA binding site in RFC that binds a 5 duplex. This 5 DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. 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. Because both 3 and 5 ends are present at a ssDNA gap, we propose that the 5 site facilitates RFCs 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 30-nt gap, suggesting a potential role of the RFC 5-DNA site in lagging strand DNA synthesis