Structural basis for inhibition of DNA replication by aphidicolin

Abstract

Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.

Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome

“…4B). However, electron density for these residues was missing in the p180core structures (31), indicating that they could become a part of the 180core-p180 C linker in the case of p180core dissociation from the rest of primosome. The p58 Np58 C linker has a well defined density (Fig.…”

“…This includes the crystal structures of the intact human primase (21) and its truncated variant without p58 C (22), as well as the high resolution structures of p180core (31), p180 C -p70 (14), p58 C (25,30), p49 (33), and their yeast orthologs (15,29,32,33). The interaction of eukaryotic primases with substrates has never been structurally characterized except for the binding site for an incoming nucleotide triphosphate (NTP) (22,33).…”

DNA replication stress underlies renal phenotypes in CEP290-associated Joubert syndrome

“…Together, our results provide a link between CEP290 and DNA replication stress and suggest CDK inhibition as a potential treatment strategy for a wide range of ciliopathy syndromes. (20) (400 nM, 18 hours; n = 3; P < 0.05; Figure 2, B and C).…”

7-(2-Thienyl)-7-Deazaadenosine (AB61), a New Potent Nucleoside Cytostatic with a Complex Mode of Action

“…The next day, cells were pretreated with medium or inhibitor of replicative DNA polymerases aphidicolin (ref. 22; 10 mmol/L) for 1 hour. Afterwards, cells were pulsed with 10 mmol/L of PNH404 (11) or medium for 3 hours.…”