Nucleic Acids Research, 1989, Vol. 17, No. 13 5173-5190
© 1989
MOLECULAR BIOLOGY |
Protein-induced unwinding of DNA: measurement by gel electrophoresis of complexes with DNA minicircles. Application to restriction endonuclease EcoRI, catabolite gene activator protein and lac repressor
Centre National de la Recherche Scientifique, Université Paris VII, Institut Jacques Monod 2 Place Jussieu, 75251 Paris, Cédex 05 1Département de Biologie Moléculaire, InstitutPasteur 75724 Paris, Cédex 15, France
Received May 9, 1989. Accepted June 7, 1989.
An electrophoretic procedure for the measurement of the helix unwinding induced by a sequence-specific protein is described. The method, which was applied here to EcoR I, CAP and lac repressor. involved the migration of the complexes with positively and negatively supercoiled DNA minicircles carrying a single protein binding site. Mobility shifts of complexes relative to naked DNAs appeared to be a result of i) the unwinding; of ii) an increase in the molecular frictional coefficient, which led to a retardation; of iii) bending, in the particular case of CAP, which induced an acceleration; and of iv) looping, in the case of lac repressor, which also resulted In an acceleration. Under conditions where the migration of the naked topoisomers was V-like (topoisomer mobility showed the same linear increase with both negative and positive supercoilings; Zivanovic et at. (1986) J. Mol. Biol., 192. 645–660), the protein unwinding contribution to mobility was assumed to be Identical to that experimentally observed In the case of a thermal unwinding: all negatively supercoiled topoisomers were retarded and all positively supercoiled topoisomers were accelerated to the same extent. In contrast, the mobility contribution of the frictlonal term, as well as those of bending and looping, appeared to vary strongly with the magnitude of the supercoiling, but only weakly with its polarity. As a consequence, these latter contributions may approximately cancel when one Is measuring the difference between the shifts observed for two comigrating, negatively and positively supercoiled, topoisomers, allowing the unwinding to be calculated. While estimates obtained for EcoR I, 23 ± 3°, and CAP, about 29°, were in good agreement with previous measurements using topoisomerase I, the value found for lac repressor, 13 to 16°, was significantly smaller.
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