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Structure paper

TitleMechanisms of loading and release of the 9-1-1 checkpoint clamp.
Journal, issue, pagesNat Struct Mol Biol, Vol. 29, Issue 4, Page 369-375, Year 2022
Publish dateMar 21, 2022
AuthorsJuan C Castaneda / Marina Schrecker / Dirk Remus / Richard K Hite /
PubMed AbstractSingle-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR. However, ...Single-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR. However, the basis for 9-1-1's recruitment to 5' junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-replication factor C (RFC), in complex with 9-1-1 and a 5' junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1's recruitment to 5' junctions and reveal new principles of sliding clamp loading.
External linksNat Struct Mol Biol / PubMed:35314831 / PubMed Central
MethodsEM (single particle)
Resolution2.2 - 3.9 Å
Structure data

25422

7st9

EMDB-25422, PDB-7st9:
Open state of Rad24-RFC:9-1-1 bound to a 5' ss/dsDNA junction
Method: EM (single particle) / Resolution: 2.2 Å

25423

7stb

EMDB-25423, PDB-7stb:
Closed state of Rad24-RFC:9-1-1 bound to a 5' ss/dsDNA junction
Method: EM (single particle) / Resolution: 2.72 Å

EMDB-25424: Mec3 focus refinement of Rad24-RFC:9-1-1:DNA in the open state
Method: EM (single particle) / Resolution: 2.43 Å

EMDB-25425: Rad24-Rfc4 focus refinement of Rad24-RFC-ADP
Method: EM (single particle) / Resolution: 3.9 Å

25426

7ste

EMDB-25426, PDB-7ste:
Rad24-RFC ADP state
Method: EM (single particle) / Resolution: 2.73 Å

Chemicals

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

ChemComp-MG:
Unknown entry

ChemComp-GLU:
GLUTAMIC ACID / Glutamic acid

ChemComp-THR:
THREONINE / Threonine

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

ChemComp-HOH:
WATER / Water

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

Source
  • saccharomyces cerevisiae (strain atcc 204508 / s288c) (yeast)
  • saccharomyces cerevisiae (brewer's yeast)
KeywordsREPLICATION/DNA / DNA damage / DNA replication / DNA sliding clamp / REPLICATION / REPLICATION-DNA complex

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