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 single-stranded DNA (ssDNA) gaps-common DNA repair intermediates-remains unclear. Here, we show that the bacterial E. coli DnaX clamp loader uses a mechanism distinct from its eukaryotic counterpart. Whereas eukaryotic replication factor C (RFC) unwinds DNA at the recessed 3' end and stabilizes the 5'-dsDNA (double-stranded DNA) at a 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 contains a conserved external DNA-binding site that bends gapped DNA by ∼150°, promoting insertion of 3'-dsDNA into the clamp. This DNA-bending mechanism enables efficient β-clamp loading at nicks and small gaps and reveals a distinct bacterial strategy likely important for DNA repair.