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 {beta}-clamp itself contains a conserved external DNA-binding site that enables sharp bending of gapped DNA by [~]150{degrees}, promoting insertion of the 3'-dsDNA into the clamp. This DNA-bending mechanism allows efficient {beta}-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. In briefZheng et al. show that the bacterial clamp loader DnaX-complex uses a DNA-bending mechanism--rather than DNA unwinding--to load the {beta}-clamp at nicks and small gaps, revealing a clamp-loading strategy distinct from eukaryotic RFC and relevant to DNA damage repair. HighlightsO_LIThe bacterial DnaX clamp loader lacks a stable shoulder DNA-binding site C_LIO_LIUnlike eukaryotic RFC, DnaX does not unwind DNA at nicks and small gaps C_LIO_LIThe E. coli {beta}-clamp contains a conserved external DNA-binding site absent in PCNA C_LIO_LISharp DNA bending enables {beta}-clamp loading at nicks and small ssDNA gaps C_LI