Interaction of minor groove binding ligands with long AT tracts

Abu-Daya, Anita and Fox, Keith R. (1997) Interaction of minor groove binding ligands with long AT tracts. Nucleic Acids Research, 25 (24), 4962-4969. (doi:10.1093/nar/25.24.4962).

Abstract

We have used quantitative DNase I footprinting to examine the ability of distamycin and Hoechst 33258 to discriminate between different arrangements of AT residues, using synthetic DNA fragments containing multiple blocks of (A/T)₆ or (A/T)₁₀ in identical sequence environments. Previous studies have shown that these ligands bind less well to (A/T)₄ sites containing TpA steps. We find that in (A/T)₆ tracts distamycin shows little discrimination between the various sites, binding ~2-fold stronger to TAATTA than (TA)₃, T₃A₃ and GAATTC. In contrast, Hoechst 33258 binds ~20-fold more tightly to GAATTC acid TAATTA than T₃A₃ and (TA)₃. Hydroxyl radical footprinting reveals that both ligands bind in similar locations at the centre of each AT tract. At (A/T)₁₀ sites distamycin binds with similar affinity to T₅A₅, (TA)₅ and AATT, though bands in the centre of (TA)₅ are protected at ~50-fold lower concentration than those towards the edges. Hoechst 33258 shows a similar pattern of preference, with strong binding to AATT, T₅A₅ and the centre of (TA)₅. Hydroxyl radical footprinting reveals that at low concentrations both ligands bind at the centre of (TA)₅ and A₅T₅, while at higher concentrations ligand molecules bind to each end of the (A/T)₁₀ tracts. At T₅A₅ two ligand molecules bind at either end of the site, even at the lowest ligand concentration, consistent with the suggestion that these compounds avoid the TpA step. Similar DNase I footprinting experiments with a DNA fragment containing T(n) (n = 3-6) tracts reveals that both ligands bind in the order T₃ < T₄ << T₅ = T₆.

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Contributors

Author: Keith R. Fox

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