DISSOCIATION OF THE AT-SPECIFIC BIFUNCTIONAL INTERCALATOR [N-MECYS(3),N-MECYS(7)]TANDEM FROM TPA SITES IN DNA

We have examined the dissociation of [N-MeCys(3),N-MeCys(7)]TANDEM, an AT-selective bifunctional intercalator, from TpA sites in mixed-sequence DNAs by a modification of the footprinting technique. Dissociation of complexes between the ligand and radiolabelled DNA fragments was initiated by adding a vast excess of unlabelled calf thymus DNA. Portions of this mixture were subjected to DNAse I footprinting at various times after adding the competitor DNA. Dissociation of the ligand from each site was seen by the time-dependent disappearance of the footprinting pattern. Within a natural DNA fragment (tyrT) the ligand dissociates from TTAT faster than from ATAT. We found that the stability of complexes with isolated TpA steps decreases in the order ATAT > TTAA > TATA. Dissociation from each of these sites is much faster than from longer regions of (AT)(n). These results confirm the requirement for A and T base-pairs surrounding the TpA step and suggest that the interaction is strongest with regions of alternating AT possibly as a result of its unusual structure. The ligand dissociates more slowly from the centre of (AT)(n) tracts than from the edges, suggesting that variations in dissociation rate arise from sequence-dependent variations in local DNA structure.