title: Monte-Carlo simulation of multisite echinomycin-DNA interactions detected by in vitro transcription analysis.
abstract: The interaction of echinomycin with DNA was analyzed at 37 degrees C by in vitro transcription analysis using a 497 bp fragment of DNA containing the lac UV5 promoter. Sixteen discrete drug binding sites were detected. The mole fraction of blocked transcript at each site was monitored over 4 h, and the kinetic profile was analyzed by Monte-Carlo simulation. The time course for all 16 sites was fully described by this process. For each drug site, three parameters were resolved with the following variation between sites: relative drug occupancy (1-26), dissociation rate constant (0.06-0.70 min-1), and probability of termination of transcription (0-48%). Eight low-occupancy binding sites were at 5'-CA sequences (relative occupancy of 1.0-2.9). The eight major sites were all at 5'-CG sequences (relative occupancy of 6.3-26) and exhibited an average occupancy some 13-fold greater than the CA sites, corresponding to an average additional stability of approximately 1.6 kcal. The dissociation rates from apparent high-affinity sites were only partially correlated with relative occupancy. Ten binding sites exhibited a 3-48% probability of termination of transcription immediately adjacent to the 5'-CG central sequence. Termination probably arises from distortion of the DNA helix in regions flanking the binding site and was most dramatic (48% probability) where two adjacent CG sites were separated by only 1 bp. This termination phenomenon may well account for the observed effects of echinomycin in vivo.
authors.affiliation: Biochemistry Department, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: D R
authors.initials: DR
authors.lastname: Phillips
authors.forename: R J
authors.initials: RJ
authors.lastname: White
authors.forename: D
authors.initials: D
authors.lastname: Dean
authors.forename: D M
authors.initials: DM
authors.lastname: Crothers
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004448:Echinomycin / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D011810:Quinoxalines / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D011810:Quinoxalines; D004448:Echinomycin; D004247:DNA