Nucleic Acids Research, Vol 25, Issue 24 4962-4969, Copyright © 1997 by Oxford University Press
A Abu-Daya and KR Fox
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)6or (A/T)10in identical sequence environments. Previous studies have
shown that these ligands bind less well to (A/T)4sites containing TpA
steps. We find that in (A/T)6tracts distamycin shows little discrimination
between the various sites, binding approximately 2-fold stronger to TAATTA
than (TA)3, T3A3and GAATTC. In contrast, Hoechst 33258 binds approximately
20-fold more tightly to GAATTC and TAATTA than T3A3and (TA)3. Hydroxyl
radical footprinting reveals that both ligands bind in similar locations at
the centre of each AT tract. At (A/T)10sites distamycin binds with similar
affinity to T5A5, (TA)5and AATT, though bands in the centre of (TA)5are
protected at approximately 50-fold lower concentration than those towards
the edges. Hoechst 33258 shows a similar pattern of preference, with strong
binding to AATT, T5A5and the centre of (TA)5. Hydroxyl radical footprinting
reveals that at low concentrations both ligands bind at the centre of
(TA)5and A5T5, while at higher concentrations ligand molecules bind to each
end of the (A/T)10tracts. At T5A5two 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.
ARTICLES
Interaction of minor groove binding ligands with long AT tracts
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.
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