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TitleA second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA.
Journal, issue, pagesElife, Vol. 11, Year 2022
Publish dateJun 22, 2022
AuthorsXingchen Liu / Christl Gaubitz / Joshua Pajak / Brian A Kelch /
PubMed AbstractClamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are ...Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader replication factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC's central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC's external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.
External linksElife / PubMed:35731107 / PubMed Central
MethodsEM (single particle)
Resolution3.0 - 3.7 Å
Structure data

EMDB-26280: RFC bound to PCNA and two primer/template DNA molecules
Method: EM (single particle) / Resolution: 3.4 Å

26297

7u19

EMDB-26297, PDB-7u19:
RFC:PCNA bound to nicked DNA
Method: EM (single particle) / Resolution: 3.7 Å

26298

7u1a

EMDB-26298, PDB-7u1a:
RFC:PCNA bound to dsDNA with a ssDNA gap of six nucleotides
Method: EM (single particle) / Resolution: 3.3 Å

26302

7u1p

EMDB-26302, PDB-7u1p:
RFC:PCNA bound to DNA with a ssDNA gap of five nucleotides
Method: EM (single particle) / Resolution: 3.0 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
  • saccharomyces cerevisiae (brewer's yeast)
  • synthetic construct (others)
KeywordsREPLICATION / sliding clamp / DNA replication&repair / AAA+ / clamp loader / BRCT domain / REPLICATION/DNA / REPLICATION-DNA complex

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