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TitleCryo-EM structures reveal that RFC recognizes both the 3'- and 5'-DNA ends to load PCNA onto gaps for DNA repair.
Journal, issue, pagesElife, Vol. 11, Year 2022
Publish dateJul 13, 2022
AuthorsFengwei Zheng / Roxana Georgescu / Nina Y Yao / Huilin Li / Michael E O'Donnell /
PubMed AbstractRFC 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 ...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.
External linksElife / PubMed:35829698 / PubMed Central
MethodsEM (single particle)
Resolution3.09 - 3.41 Å
Structure data

25872

7tfh

EMDB-25872: Cryo-EM 3D map of the S. cerevisiae clamp-clamp loader complex PCNA-RFC bound to two DNA molecules, one at the 5'-recessed end and the other at the 3'-recessed end
PDB-7tfh: Atomic model of the S. cerevisiae clamp-clamp loader complex PCNA-RFC bound to two DNA molecules, one at the 5'-recessed end and the other at the 3'-recessed end
Method: EM (single particle) / Resolution: 3.09 Å

25873

7tfi

EMDB-25873: Cryo-EM 3D map of the S. cerevisiae clamp-clamp loader complex PCNA-RFC bound to DNA with an open clamp
PDB-7tfi: Atomic model of the S. cerevisiae clamp-clamp loader complex PCNA-RFC bound to DNA with an open clamp
Method: EM (single particle) / Resolution: 3.41 Å

25874

7tfj

EMDB-25874: Cryo-EM 3D map of S. cerevisiae clamp-clamp loader complex PCNA-RFC bound to DNA with a closed clamp ring
PDB-7tfj: Atomic model of S. cerevisiae clamp-clamp loader complex PCNA-RFC bound to DNA with a closed clamp ring
Method: EM (single particle) / Resolution: 3.3 Å

25875

7tfk

EMDB-25875: Cryo-EM 3D map of S. cerevisiae clamp loader RFC bound to two DNA molecules, one at the 5'-recessed end and the other at the 3'-recessed end
PDB-7tfk: Atomic model of S. cerevisiae clamp loader RFC bound to two DNA molecules, one at the 5'-recessed end and the other at the 3'-recessed end
Method: EM (single particle) / Resolution: 3.25 Å

25876

7tfl

EMDB-25876: Cryo-EM 3D map of S. cerevisiae clamp loader RFC bound to DNA
PDB-7tfl: Atomic model of S. cerevisiae clamp loader RFC bound to DNA
Method: EM (single particle) / Resolution: 3.33 Å

Chemicals

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

ChemComp-MG:
Unknown entry

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

Source
  • synthetic construct (others)
  • saccharomyces cerevisiae (brewer's yeast)
KeywordsDNA BINDING PROTEIN/DNA / DNA replication / DNA damage repair / RFC loader / PCNA clamp / DNA polymerase processivity factor / DNA BINDING PROTEIN-DNA complex

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