Forrow, Stephen Michael; (1995) Comparative effects of A.T and G.C sequence selective DNA minor groove binding agents. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The DNA sequence preference of a series of DNA minor groove binding analogues of distamycin, containing from one to four imidazole moieties is described. Only the di- and the triimidazole analogues produced strong footprints and a detailed densitometric analysis revealed that the preferred binding site of the diimidazole compound ARI 114 was at 5'-(G.C)3(A.T), whereas the strongest footprinting sites for the triimidazole compound, ARI 144, was within two occurrences of the sequence 5'-TCGGGCT-3'. The analysis also showed that these two compounds bind to different sequences on the same DNA fragment, even though they differ by only one imidazole unit. An attempt was made to develop an assay for the binding sites of minor groove binders in a cellular environment. Although not entirely successful, the results from experiments involving a taq polymerase stop assay indicate that the DNA binding sites of some minor groove binders may be determined at doses lower than those required with existing techniques. The G.0 selective di and triimidazole compounds were compared to two A.T selective minor groove binders, distamycin and netropsin, for their ability to alter the distribution of guanine N7 monoalkylation products produced by three representative nitrogen mustards. All four of the ligands were capable of producing quantitative and/or qualitative changes in the distribution of the alkylation adducts. Pretreatment of the DNA with netropsin, distamycin or ARI 114 resulted in significantly altered patterns of guanine N7 monoalkylation, with some adducts being suppressed whilst others were enhanced. In contrast, ARI 144 produced only a strong inhibition of monoalkylation, but was unable to produce any qualitative alteration in the pattern of adducts. A comparison of the binding sites of the ligands (deduced by DNA footprinting) with patterns of enhancement or suppression of adducts yielded no simple correlation. In the third part of the study, netropsin and distamycin were compared to ARI 114 and ARI 144 for their ability to inhibit DNA cleavage by a panel of restriction endonucleases chosen to have either a high A.T or a high G.C content at the core of their recognition sequences. As predicted, netropsin and distamycin but not ARI 114 or ARI 144 were able to inhibit DNA cleavage by Eco RI and Eco RV. In contrast, neither ARI 114 or ARI 144 were very effective at inhibiting DNA cleavage by the G.C recognising enzymes Nru I and Bal I. Footprinting revealed that neither ARI 114 and ARI 144 bound at these enzyme recognition sequences under the restriction enzyme buffer conditions. DNA footprinting experiments showed that ARI 114 bound to two of three recognition sequences for the enzyme Fnu 4H1 in the fragment studied, and enzyme cleavage was shown to be inhibited only at these sites. ARI 144 bound strongly to two 5'-GGGCTC-3 sequences which are recognition sites for the enzyme Ban II. In this case a clear stimulation of DNA cleavage by Ban II was observed over a wide dose range of ARI 144. The experiments described in this thesis demonstrate that minor groove binding ligands can often have profound effects on DNA structure and that differing effects are seen between the A.T and the G.C selective binders.

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