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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

View ORCID ProfileFengwei Zheng, Roxana E. Georgescu, Nina Y. Yao, View ORCID ProfileHuilin Li, Michael E. O’Donnell
doi: https://doi.org/10.1101/2022.02.04.479194
Fengwei Zheng
1Department of Structural Biology, Van Andel Institute, Grand Rapids, Michigan, USA
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Roxana E. Georgescu
2DNA Replication Laboratory, The Rockefeller University, New York, New York, USA
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Nina Y. Yao
2DNA Replication Laboratory, The Rockefeller University, New York, New York, USA
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Huilin Li
1Department of Structural Biology, Van Andel Institute, Grand Rapids, Michigan, USA
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  • For correspondence: huilin.li@vai.org odonnel@rockefeller.edu
Michael E. O’Donnell
2DNA Replication Laboratory, The Rockefeller University, New York, New York, USA
3Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA
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  • For correspondence: huilin.li@vai.org odonnel@rockefeller.edu
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Abstract

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 synthesis

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted May 11, 2022.
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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
Fengwei Zheng, Roxana E. Georgescu, Nina Y. Yao, Huilin Li, Michael E. O’Donnell
bioRxiv 2022.02.04.479194; doi: https://doi.org/10.1101/2022.02.04.479194
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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
Fengwei Zheng, Roxana E. Georgescu, Nina Y. Yao, Huilin Li, Michael E. O’Donnell
bioRxiv 2022.02.04.479194; doi: https://doi.org/10.1101/2022.02.04.479194

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