Douc-Rasy S, Kolb A, Prunell A
Centre National de la Recherche Scientifique, Université Paris VII, Institut Jacques Monod, France.
Nucleic Acids Res. 1989 Jul 11;17(13):5173-89. doi: 10.1093/nar/17.13.5173.
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 al. (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 frictional 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 degrees, and CAP, about 29 degrees, were in good agreement with previous measurements using topoisomerase I, the value found for lac repressor, 13 to 16 degrees, was significantly smaller.
本文描述了一种用于测量序列特异性蛋白质诱导的螺旋解旋的电泳方法。该方法应用于EcoR I、CAP和乳糖阻遏物，涉及携带单个蛋白质结合位点的正负超螺旋DNA小环与复合物的迁移。复合物相对于裸露DNA的迁移率变化似乎是由于：i）解旋；ii）分子摩擦系数增加导致迁移率降低；iii）弯曲，在CAP的特定情况下会导致迁移率增加；iv）成环，在乳糖阻遏物的情况下也会导致迁移率增加。在裸露拓扑异构体的迁移呈V形的条件下（拓扑异构体迁移率随正负超螺旋的增加呈相同的线性增加；Zivanovic等人，（1986年）《分子生物学杂志》，192卷，645 - 660页），假设蛋白质解旋对迁移率的贡献与热解旋情况下实验观察到的相同：所有负超螺旋拓扑异构体迁移率降低，所有正超螺旋拓扑异构体迁移率增加到相同程度。相比之下，摩擦项以及弯曲和成环对迁移率的贡献似乎随超螺旋程度变化很大，但随其极性变化很小。因此，在测量两个共迁移的正负超螺旋拓扑异构体观察到的迁移率变化差异时，后一种贡献可能大致抵消，从而可以计算解旋情况。虽然EcoR I的估计值为23±3°，CAP约为29°，与先前使用拓扑异构酶I的测量结果高度一致，但乳糖阻遏物的值为13至16°，明显较小。
