Dissociation kinetics of echinomycin from CpG binding sites in different sequence environments

Fletcher, Michael C. and Fox, Keith R. (1996) Dissociation kinetics of echinomycin from CpG binding sites in different sequence environments. Biochemistry, 35 (3), 1064-1075. (doi:10.1021/bi9523623).

Record type: Article

Abstract

We have examined the kinetics of dissociation of echinomycin from CpG sites in several DNA fragments containing synthetic DNA inserts by a variation of the footprinting technique. Complexes of the ligand with radiolabeled DNA fragments were dissociated by adding an excess of unlabeled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the various CpG sites. At 20 °C, echinomycin dissociates more slowly from CpG sites flanked by (AT)(n) (t( 1/2 ) ~ .40 min) and (CA)n) · (TG)(n) (t( 1/2 ) ~ 11 min) than by A(n) · T(n) (t( 1/2 ) < 3 min). In each sequence context the dissociation from ACGT is slower than that from TCGA. (TAA)₄CG(TTA)₄ also represents a very good binding site (t 1/2 ) ~ 35 min), which is less sensitive to changes in temperature than most other sites. Within sequences (AT)₁₀(G/C)₄(AT)₁₀, the dissociation from CGGC is slower than that from CCCG or CCGC.

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