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

Abstract

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 ~ 76μM) and repair of DNA containing an O6-methylguanine residue (IC50 ~ 63μM). 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.

Global Multi-Method Analysis of Affinities and Cooperativity in Complex Systems of Macromolecular Interactions

“…This may not be trivial considering the different susceptibility of methods to experimental imperfections. For example, protein modifications are required for surface binding in SPR, as well as for the attachment of fluorophores, both with the potential to profoundly alter the binding properties 30,31 . Preparative impurities can bias AUC, but, if consisting of unreactive species, may be irrelevant for ITC and SPR, and preparations subject to time-dependent degradation can behave differently in different techniques 32 .…”

Lesion-specific DNA-binding and repair activities of human O6-alkylguanine DNA alkyltransferase

“…Cleavage is restored when the methyl group is removed by AGT (25). To assay duplex DNA repair, oligonucleotides 4 and 5 containing the NarI sequence (Table 1) were annealed and transferred into reaction buffer (20 mM Tris-acetate (pH 7.9 at 25°C), 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol) by dialysis.…”

“…As a partial solution to this problem, alkyl-transfer-inactive forms of AGT have been studied, including active-site mutants, such as the C145S protein (24) and chemically modified forms such as the C145-methyl and C145-benzyl adducts (18,25). This strategy allowed DNase I mapping of a 6mG-specific complex, showing protection of 18 nt on the 6mG-strand and 14 nt on its complement (24), although the number of proteins and their distribution within the complex were not established.…”

Measuring Protein Interactions by Optical Biosensors

“…can introduce all the problems well known in chromophoric labeling and related covalent protein modifications, including artificial reduction or strengthening of binding affinities (Melikishvili, Rodgers, & Fried, 2011;. For all these reasons, the use of this technique requires careful planning and the execution of a number of different experiments, with time and effort more or less comparable to any of the other techniques available for the study of protein-protein interactions.…”