Nucleic Acids Research, 1993, Vol. 21, No. 6 1339-1344
© 1993
CHEMISTRY |
Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting
Department of Physiology and Pharmacology, University of Southampton Bassett Crescent East, Southampton SO9 3TU, UK
*To whom correspondence should be addressed
Received January 18, 1993. Accepted February 24, 1993.
We have Investigated the kinetics of dissociation of actlnomycin D from DNA by a variation of the foot-printing technique. Complexes of actlnomycin with a radiolabelied DNA fragment (tyrT) were dissociated by addition of a large excess of uniabelled calf thymus DNA and the mixture subjected to DNase I footprinting at subsequent intervals. The rates at which the footprints disappeared varied between the different binding sites. The dissociation was temperature dependent with average time constants of 30 s, 10 mins and 2 hours at temperatures of 37°C, 20°C and 4°C respectively. The dissociation from a DNA fragment containing the synthetic insert T9GCA9 was significantly faster, with a half-life of about 1 min at 20°C. In contrast, the dissociation of distamycln was too fast to measure (<5 s) even at 4°C.