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. • The bacterial DnaX clamp loader lacks a stable shoulder DNA-binding site • Unlike eukaryotic RFC, DnaX does not unwind DNA at nicks and small gaps • The E. coli β-clamp contains a conserved external DNA-binding site absent in PCNA • Sharp DNA bending enables β-clamp loading at nicks and small ssDNA gaps Zheng et al. show that the bacterial clamp-loader DnaX-complex uses a DNA-bending mechanism—rather than DNA unwinding—to load the β-clamp at nicks and small gaps, which reveals a clamp-loading strategy that is distinct from eukaryotic RFC and relevant to DNA damage repair.