PT  - JOURNAL ARTICLE
AU  - Fengwei Zheng
AU  - Roxana E. Georgescu
AU  - Nina Y. Yao
AU  - Huilin Li
AU  - Michael E. O’Donnell
TI  - Cryo-EM structures reveal that RFC recognizes both the 3’- and 5’-DNA ends to load PCNA onto gaps for DNA repair
AID  - 10.1101/2022.02.04.479194
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.02.04.479194
4099  - http://biorxiv.org/content/early/2022/05/11/2022.02.04.479194.short
4100  - http://biorxiv.org/content/early/2022/05/11/2022.02.04.479194.full
AB  - RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. 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 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 30-nt gap, suggesting a potential role of the RFC 5’-DNA site in lagging strand DNA synthesisCompeting Interest StatementThe authors have declared no competing interest.