Processive chromosomal replication relies on sliding DNA clamps, which are loaded onto DNA by pentameric clamp loader complexes belonging to the AAA+ family of ATPases. We present structures for the ATP-bound state of the clamp loader complex from bacteriophage T4, bound to an open clamp and primer-template DNA. The clamp loader traps a spiral conformation of the open clamp so that both the loader and the clamp match the helical symmetry of DNA. One structure reveals that ATP has been hydrolyzed in one subunit, and suggests that clamp closure and ejection of the loader involves disruption of the ATP-dependent match in symmetry. The structures explain how synergy between the loader, the clamp and DNA can trigger ATP hydrolysis and release of the closed clamp on DNA.

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A–ssDNA complex defines the 5′–3′ directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics.

Summary Clamp loaders load sliding clamps onto primer-template DNA. The structure of the E. coli clamp loader bound to DNA reveals the formation of an ATP-dependent spiral of ATPase domains that tracks only the template strand, allowing recognition of both RNA and DNA primers. Unlike hexameric helicases, in which DNA translocation requires distinct conformations of the ATPase domains, the clamp loader spiral is symmetric and is set up to trigger release upon DNA recognition. Specificity for primed DNA arises from blockage of the end of the primer and accommodation of the emerging template along a surface groove. A related structure reveals how the ψ protein, essential for coupling the clamp loader to single-stranded DNA binding protein (SSB), binds to the clamp loader. By stabilizing a conformation of the clamp loader that is consistent with the ATPase spiral observed upon DNA binding, ψ binding promotes the clamp loading activity of the complex.