The effects of sequence selective antitumour antibiotics on DNA structure

Waterloh, Kerstin (1991) The effects of sequence selective antitumour antibiotics on DNA structure. University of Southampton, Doctoral Thesis.

Abstract

Actinomycin, echinomycin and [N-Me Cys³ N-Me Cys⁷] TANDEM bind to double stranded DNA by a sequence selective mechanism involving intercalation. Previous studies have identified the sequence selectivity of these ligands as GpC, CpG and regions of alternating AT respectively and shown that they can affect local DNA structure. In this thesis a variety of footprinting techniques have been used to determine the effects of these antibiotics on the structure of flanking DNA sequences and further to investigate their sequence selectivity. Structural effects. I have investigated the effects of these drugs on DNA structure by footprinting techniques using a variety of chemical and enzymatic probes sensitive to DNA structure. The novel approach described in this thesis used DNA fragments which have been prepared by cloning synthetic oligonucleotides, each containing a central drug binding site flanked by other repetitive regions, into longer DNA sequences. I have demonstrated that a common feature of selective drug binding is the ability to distort the structure of surrounding regions. These conformational changes can be propagated over several turns of the DNA helix, vary according to the length and the position of the particular sequence and are peculiar to each ligand under study. In general regions of polydA.polydT and poly(dA-dT) are more easily distorted than (dG-dT).(dA-dC). Sequence selectivity. By using a large number of synthetic fragments I have identified the sequence selectivity of [N-MeCys³ N-MeCys⁷] TANDEM as TpA. The nominal sequence selectivity of actinomycin (GpC) and echinomycin (CpG) is shown to be affected by surrounding regions. Not all canonical drug binding sites are bound by the ligands, whilst others reveal unusually good drug/interactions. The structure of DNA flanking each binding site is important in determining the precise sequence selectivity. Secondary binding sites for actinomycin are found at (dG-dT)(dA-dC); sites flanked by GT and AC rich sites for actinomycin are more strongly bound by the ligand. In addition to secondary binding of echinomycin to CC (GG) this drug shows a cooperative interaction with ApT affected by the binding of the ligand to adjacent canonical CpG sites. Preliminary studies are presented for the development of the footprinting technique as a mean to study drug dissociation kinetics.

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