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Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair
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 second DNA binding site in RFC that binds a 5′ du...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9293004/ https://www.ncbi.nlm.nih.gov/pubmed/35829698 http://dx.doi.org/10.7554/eLife.77469 |
Sumario: | 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 second 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 synthesis. |
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