6-Carboxyfluorescein and structurally similar molecules inhibit DNA binding and repair by O6-alkylguanine DNA alkyltransferase

Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. These activities protect normal cells and tumor cells against drugs that alkylate DNA; drugs that inactivate AGT are under test as chemotherapeutic enhancers. In studies using 6-carboxyfluorescein (FAM)-labeled DNAs, AGT reduced the fluorescence intensity by ∼40% at binding saturation, whether the FAM was located at the 5′ or the 3′ end of the DNA. AGT protected residual fluorescence from quenching, indicating a solute-inaccessible binding site for FAM. Sedimentation equilibrium analyses showed that saturating AGT-stoichiometries were higher with FAM-labeled DNAs than with unlabeled DNAs, suggesting that the FAM provides a protein binding site that is not present in unlabeled DNAs. Additional fluorescence and sedimentation measurements showed that AGT forms a 1:1 complex with free FAM. Active site benzylation experiments and docking calculations support models in which the primary binding site is located in or near the active site of the enzyme. Electrophoretic analyses show that FAM inhibits DNA binding (IC50 ∼ 76 μM) and repair of DNA containing an O6-methylguanine residue (IC50 ∼ 63 μM). Similar results were obtained with other polycyclic aromatic compounds. These observations demonstrate the existence of a new class of non-covalent AGT-inhibitors. After optimization for binding-affinity, members of this class might be useful in cancer chemotherapy.

► Binding and repair by O6-alkylguanine DNA alkyltransferase (AGT) were studied.
► Higher stoichiometries were found with FAM-labeled DNAs than with unlabeled DNAs.
► Fluorescence and activity assays suggest FAM occupies the AGT active site.
► Unconjugated FAM inhibits DNA binding and repair.
► FAM-like molecules belong to a new class of AGT-inhibitors.