Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting

Fletcher, Michael C. and Fox, Keith R. (1993) Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting. Nucleic Acids Research, 21 (6), 1339-1344. (doi:10.1093/nar/21.6.1339).

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Abstract

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.

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Published date: 25 March 1993

Additional Information: Funding Information: This work was supported by grants from the Science and Engineering Research Council and the Cancer Research Campaign. We thank Gabrielle Turner for technical assistance. KRF is a Lister Institute Research Fellow.