The E. coli DnaX clamp loader sharply bends DNA to load β-clamp at nicks and small gaps.

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 Escherichia coli 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.