Structural basis for aphidicolin inhibition of human processive DNA synthesis

Eukaryotic DNA replication requires RNA–DNA primer synthesis by Pol α and processive lagging- and leading-strand synthesis by Pol δ and Pol ε, respectively, tethered to the PCNA clamp. Aphidicolin, a fungal diterpenoid, is a selective inhibitor of these B-family polymerases and a tool for inducing controlled replication stress, yet its structural mechanism has been defined only for the catalytic core of Pol α. Here we report cryo-EM structures of human Pol δ and Pol ε holoenzymes bound to primer-template DNA, PCNA and aphidicolin at 3.0 Å and 3.1 Å, respectively. In both, aphidicolin occupies the incoming dNTP pocket and flips the templating base outward, locking the fingers open and arresting synthesis while preserving the holoenzyme architecture. Aphidicolin occupies an architecturally conserved pocket in Pol α, Pol δ and Pol ε, yet Pol δ is approximately 4-5-fold more sensitive than Pol ε in reconstituted PCNA-dependent assays. We attribute this to a grip beyond the templating base that anchors the template strand in Pol δ but engages it only loosely in Pol ε, allowing the strand to escape the aphidicolin trap. Selectivity therefore resides not in the inhibitor pocket but in template handling, defining a route to next-generation B-family polymerase inhibitors.