7-aminoactinomycin binding to DNA sequences lacking GpC sites: A thermodynamic and kinetic study
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 (sto...
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2009
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00062960_v48_n1_p173_Biver http://hdl.handle.net/20.500.12110/paper_00062960_v48_n1_p173_Biver |
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| Sumario: | 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°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. © 2009 American Chemical Society. |
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