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Structure paper

TitleThe DnaX clamp loader sharply bends DNA to load β-clamp at nicks and small gaps.
Journal, issue, pagesbioRxiv, Year 2026
Publish dateJan 20, 2026
AuthorsFengwei Zheng / Nina Y Yao / Roxana E Georgescu / Meinan Lyu / Michael E O'Donnell / Huilin Li /
PubMed AbstractDNA sliding clamps are essential for processive DNA synthesis in all domains of life and are loaded by ATP-dependent clamp loaders that recognize recessed 3' ends. How clamp loaders function at nicks ...DNA sliding clamps are essential for processive DNA synthesis in all domains of life and are loaded by ATP-dependent clamp loaders that recognize recessed 3' ends. How clamp loaders function at nicks and small ssDNA gaps-common intermediates during DNA repair-remains incompletely understood. Here, we show that the bacterial DnaX clamp loader employs a fundamentally different mechanism from its eukaryotic counterpart. Whereas eukaryotic RFC unwinds DNA at the recessed 3' end and stabilizes the 5'-dsDNA at a dedicated shoulder site, the bacterial DnaX-complex neither unwinds DNA nor stably binds the 5'-dsDNA in vitro. Instead, cryo-EM structures reveal that the β-clamp itself contains a conserved external DNA-binding site that enables sharp bending of gapped DNA by ~150°, promoting insertion of the 3'-dsDNA into the clamp. This DNA-bending mechanism allows efficient β-clamp loading at nicks and small gaps and reveals a distinct bacterial strategy for clamp loading. Because small DNA gaps are frequently associated with DNA damage, clamps loaded at these sites are likely important for DNA repair.
External linksbioRxiv / PubMed:41648351 / PubMed Central
MethodsEM (single particle)
Resolution2.53 - 3.14 Å
Structure data

EMDB-71013: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 1-nt gapped DNA in state 1 conformer 1 with flexibly bound DNA at the shoulder
PDB-9oyb: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 1-nt gapped DNA in state 1 conformer 1 with flexibly bound DNA at the shoulder
Method: EM (single particle) / Resolution: 3.14 Å

EMDB-71014: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 1-nt gapped DNA in state 1 conformer 2 with flexibly bound DNA at the shoulder
PDB-9oyc: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 1-nt gapped DNA in state 1 conformer 2 with flexibly bound DNA at the shoulder
Method: EM (single particle) / Resolution: 2.71 Å

EMDB-71015: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 1-nt gapped DNA in state 1 conformer 3 with disordered DNA at the shoulder
PDB-9oyd: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 1-nt gapped DNA in state 1 conformer 3 with disordered DNA at the shoulder
Method: EM (single particle) / Resolution: 2.73 Å

EMDB-71016: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 1-nt gapped DNA in state 2 conformer 1 with sharply bent DNA
PDB-9oye: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 1-nt gapped DNA in state 2 conformer 1 with sharply bent DNA
Method: EM (single particle) / Resolution: 2.72 Å

EMDB-71017: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 1-nt gapped DNA in state 2 conformer 2 with less bent DNA
PDB-9oyf: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 1-nt gapped DNA in state 2 conformer 2 with less bent DNA
Method: EM (single particle) / Resolution: 3.12 Å

EMDB-71018: Cryo-EM map of the E. coli clamp loader DnaX-complex alone
PDB-9oyg: Structure of the E. coli clamp loader DnaX-complex alone
Method: EM (single particle) / Resolution: 2.95 Å

EMDB-71021: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 1 the DNA recognition state
PDB-9oyh: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 1 the DNA recognition state
Method: EM (single particle) / Resolution: 2.54 Å

EMDB-71022: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 1 with fully open clamp and unsettled DNA
PDB-9oyi: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 1 with fully open clamp and unsettled DNA
Method: EM (single particle) / Resolution: 2.54 Å

EMDB-71023: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 2 with fully open clamp and settled DNA
PDB-9oyj: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 2 with fully open clamp and settled DNA
Method: EM (single particle) / Resolution: 2.53 Å

EMDB-71024: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 3 with partially closed clamp
PDB-9oyk: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 3 with partially closed clamp
Method: EM (single particle) / Resolution: 2.6 Å

EMDB-71025: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 4 with fully closed clamp
PDB-9oyl: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 4 with fully closed clamp
Method: EM (single particle) / Resolution: 2.88 Å

EMDB-71026: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 5 with fully closed clamp
PDB-9oym: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 5 with fully closed clamp
Method: EM (single particle) / Resolution: 2.6 Å

EMDB-71027: Cryo-EM map of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 6 with fully closed clamp
PDB-9oyn: Structure of the E. coli clamp loader DnaX-complex loading beta-clamp onto 10-nt gapped DNA in state 2 conformer 6 with fully closed clamp
Method: EM (single particle) / Resolution: 2.7 Å

Chemicals

ChemComp-ZN:
Unknown entry

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
  • escherichia coli (E. coli)
  • dna molecule (others)
KeywordsREPLICATION/DNA / E. coli / DNA replication / DNA damage repair / clamp loading complex / clamp beta / clamp loader DnaX-complex / REPLICATION / REPLICATION-DNA complex / TRANSFERASE

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