title: 7-Aminoactinomycin binding to DNA sequences lacking GpC sites: a thermodynamic and kinetic study.
abstract: The interaction of 7-aminoactinomycin (7AAMD) with selected DNA sequences (TAGTTA, R5, HP5, and HP1) of different lengths and secondary structures, all containing a 5'-TAGT-3' block, was studied at an ionic strength of 0.02 M and pH 7.7 by means of fluorescence equilibrium and kinetic (stopped-flow) measurements. Both approaches indicated that the antibiotic binds strongly to both the single-stranded and hairpin (HP1) structures, although the sequences lacked the canonical GpC sites favored by actinomycin. Binding isotherms and initial rate analyses revealed that the binding stoichiometry was 1:1 in all cases. While the single-stranded sequences displayed a simple monoexponential kinetic behavior, the binding of 7AAMD to HP1 at <30 degrees C was biphasic and could be rationalized in terms of a sequential formation of two isomeric bound forms or alternatively in terms of an ssHP1-hpHP1 equilibrium, with both HP1 forms reacting with 7AAMD. The rates of complex dissociation induced by the detergent SDS were also measured. After correction of the kinetic traces for spurious effects that can be attributed to the SDS, monoexponential traces were obtained, with relaxation times in agreement with the kinetics of complex formation.
authors.affiliation: Dipartimento di Chimica e Chimica Industriale, Universita di Pisa, Via Risorgimento 35, 56126 Pisa, Italy.
authors.forename: Tarita
authors.initials: T
authors.lastname: Biver
authors.forename: Marcella
authors.initials: M
authors.lastname: Venturini
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Jares-Erijman
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Jovin
authors.forename: Fernando
authors.initials: F
authors.lastname: Secco
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D003609:Dactinomycin / Q000031:analogs & derivatives* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D013816:Thermodynamics
chemical_list: D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D004247:DNA