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Structure paper

TitleCryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader.
Journal, issue, pagesElife, Vol. 11, Year 2022
Publish dateFeb 18, 2022
AuthorsChristl Gaubitz / Xingchen Liu / Joshua Pajak / Nicholas P Stone / Janelle A Hayes / Gabriel Demo / Brian A Kelch /
PubMed AbstractSliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. ...Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.
External linksElife / PubMed:35179493 / PubMed Central
MethodsEM (single particle)
Resolution3.3 - 4.0 Å
Structure data

25568

7thj

EMDB-25568, PDB-7thj:
Structure of the yeast clamp loader (Replication Factor C RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen PCNA) in an autoinhibited conformation
Method: EM (single particle) / Resolution: 3.9 Å

25569

7tic

EMDB-25569, PDB-7tic:
Structure of the yeast clamp loader (Replication Factor C RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen PCNA) in an autoinhibited conformation
Method: EM (single particle) / Resolution: 3.9 Å

25614

7thv

EMDB-25614, PDB-7thv:
Structure of the yeast clamp loader (Replication Factor C RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen PCNA) in an autoinhibited conformation
Method: EM (single particle) / Resolution: 4.0 Å

25615

7tku

EMDB-25615, PDB-7tku:
Structure of the yeast clamp loader (Replication Factor C RFC) bound to the open sliding clamp (Proliferating Cell Nuclear Antigen PCNA)
Method: EM (single particle) / Resolution: 4.0 Å

25616

7tib

EMDB-25616, PDB-7tib:
Structure of the yeast clamp loader (Replication Factor C RFC) bound to the open sliding clamp (Proliferating Cell Nuclear Antigen PCNA) and primer-template DNA
Method: EM (single particle) / Resolution: 3.4 Å

25617

7tid

EMDB-25617, PDB-7tid:
Structure of the yeast clamp loader (Replication Factor C RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen PCNA) and primer-template DNA
Method: EM (single particle) / Resolution: 3.3 Å

25753

7ti8

EMDB-25753, PDB-7ti8:
Structure of the yeast clamp loader (Replication Factor C RFC) bound to the open sliding clamp (Proliferating Cell Nuclear Antigen PCNA)
Method: EM (single particle) / Resolution: 3.5 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

Source
  • saccharomyces cerevisiae (brewer's yeast)
  • synthetic construct (others)
KeywordsREPLICATION / sliding clamp / DNA replication / AAA+ / clamp loader / REPLICATION/DNA / REPLICATION-DNA complex

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