Summary
A bioRxiv preprint reports cryo-EM structures showing aphidicolin blocking human DNA polymerases δ and ε at the site where they add DNA building blocks. Reconstituted assays and the structures suggest that differences in how the enzymes grip the DNA template help explain why Pol δ is more sensitive.
A bioRxiv preprint reports cryo-electron microscopy structures showing how aphidicolin blocks two human DNA-copying enzymes, DNA polymerases δ and ε. The structures place the inhibitor in the pocket where each enzyme normally receives an incoming DNA building block. Reconstituted assays found Pol δ to be about 4–5 times more sensitive to aphidicolin than Pol ε.
During eukaryotic DNA replication, Pol δ and Pol ε carry out much of the synthesis on the lagging and leading DNA strands, respectively. Both work with PCNA, a ring-shaped protein clamp that helps keep a polymerase attached to DNA as it copies. Aphidicolin is a fungal compound used to inhibit B-family DNA polymerases and induce controlled replication stress. Before this study, the authors say, its structural mechanism had been defined only for the catalytic core of Pol α, another DNA polymerase involved in replication.
How aphidicolin stops DNA synthesis
The researchers determined cryo-EM structures of human Pol δ and Pol ε holoenzymes bound to primer-template DNA, PCNA and aphidicolin. The structures had resolutions of 3.0 Å for Pol δ and 3.1 Å for Pol ε. A holoenzyme is the working assembly of an enzyme with its associated components; here, the structures capture the polymerases alongside the DNA and PCNA clamp.
In both polymerases, aphidicolin occupies the incoming nucleotide pocket—the site where the next DNA building block would bind. The inhibitor also pushes the DNA template’s next base out of position. According to the study, this leaves the polymerase’s “fingers” region open and prevents DNA synthesis from proceeding, while the wider enzyme assembly remains intact.
Template handling explains a difference in sensitivity
Although the inhibitor occupies a structurally similar pocket in Pol α, Pol δ and Pol ε, the reconstituted PCNA-dependent assays showed a sensitivity difference: Pol δ was approximately 4–5-fold more sensitive than Pol ε. The authors attribute that difference to how each enzyme holds the DNA template beyond the base that is being copied.
In their interpretation, Pol δ grips this part of the template strand firmly, helping hold it in the position where aphidicolin blocks synthesis. Pol ε engages the strand more loosely, so the template can escape the inhibitor’s trap more readily. The proposed explanation puts the difference in sensitivity in the enzymes’ handling of DNA, rather than in a substantially different inhibitor-binding pocket.
The findings offer a structural explanation for aphidicolin’s action on these two human replication polymerases and point to template handling as a possible consideration in designing inhibitors of B-family polymerases. The report is a bioRxiv preprint, and its evidence here comes from cryo-EM structures and reconstituted biochemical assays.