The anomalous gel migration of a stable cruciform: temperature and salt dependence, and some comparisons with curved DNA
Diekmann, Stephan; Lilley, David M.; Max-Planck-Institut für Biophysikalische ChemieAm Fassberg, D-3400 Göttingen-Nikolausberg, FRG; Lilley David M.; Department of Biochemistiy, The University; Institut Pasteur (Biologie Moléculaire, chez H.Buc)
Журнал:
Nucleic Acids Research
Дата:
1987
Аннотация:
We have made an analysis of the gel electrophoretic properties of a pseudo-cruciform fragment, a linear DNA molecule containing a stable cruciform. The migration of this construct was analysed in polyacrylamide gels at various temperatures in the range 50° to 55°C, and in the presence of NaCl, MgCl<sub>2</sub> or ethidium bromide. The magnitude of the anomalous migration (retardation) was almost temperature independent up to 40°C, but decreased strongly beyond this point, extrapolating to normal migration at 70°C. Addition of salts reduced the anomaly. This took the form of a continuous reduction in anomalous migration with the addition of NaCl up to 60 mM, while with MgCl<sub>2</sub> there was a sharp reduction in the anomaly to a constant value which is reached by 10 mM. Under these conditions, moreover, the migration of the fragment became almost temperature-independent over the entire range. These results have been interpreted to reflect the influence of ion binding at the four-way junction on the relative disposition of the cruciform arms. The detailed electrophoretic properties of the pseudo-cruciform are in marked contrast to those of sequence-directed curved DNA fragments. In particular, the response to the addition of 1 μ/ml ethidium bromide offers a convenient method for distinguishing between anomalous retardation arising from curvature (greatly reduced anomaly) or a cruciform junction (enhanced anomaly).
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