Mc. Fletcher et al., DISSOCIATION OF THE AT-SPECIFIC BIFUNCTIONAL INTERCALATOR [N-MECYS(3),N-MECYS(7)]TANDEM FROM TPA SITES IN DNA, Biochemical journal, 306, 1995, pp. 15-19
We have examined the dissociation of [N-MeCys(3),N-MeCys(7)]TANDEM, an
AT-selective bifunctional intercalator, from TpA sites in mixed-seque
nce DNAs by a modification of the footprinting technique. Dissociation
of complexes between the ligand and radiolabelled DNA fragments was i
nitiated by adding a vast excess of unlabelled calf thymus DNA. Portio
ns of this mixture were subjected to DNAse I footprinting at various t
imes after adding the competitor DNA. Dissociation of the ligand from
each site was seen by the time-dependent disappearance of the footprin
ting pattern. Within a natural DNA fragment (tyrT) the ligand dissocia
tes from TTAT faster than from ATAT. We found that the stability of co
mplexes with isolated TpA steps decreases in the order ATAT > TTAA > T
ATA. Dissociation from each of these sites is much faster than from lo
nger regions of (AT)(n). These results confirm the requirement for A a
nd T base-pairs surrounding the TpA step and suggest that the interact
ion is strongest with regions of alternating AT possibly as a result o
f its unusual structure. The ligand dissociates more slowly from the c
entre of (AT)(n) tracts than from the edges, suggesting that variation
s in dissociation rate arise from sequence-dependent variations in loc
al DNA structure.