The kinetic properties of cruciform extrusion are determined by DNA base-sequence

Abstract

The extrusion kinetics of two cruciforms derived from unrelated DNA sequences differ markedly. Kinetic barriers exist for both reactions, necessitating elevated temperatures before extrusion proceeds at measureable speeds, but the dependence upon temperature and ionic strength is quite different for the two sequences. One, the ColE1 inverted repeat, exhibits a remarkably great temperature dependence of reaction rate and is suppressed by moderate amounts of NaCl or MgCl2. In contrast, the other, a synthetic inverted repeat present in pIRbke8, shows more modest temperature dependence and has a requirement for the presence of salt, with optimal concentrations being 50 mM NaCl or 100 microM MgCl2. Under optimal conditions, cruciform extrusion rates are fast (t1/2 less than 60m) at 37 degrees C for both sequences at native superhelix densities. In 50 mM NaCl the pIRbke8 inverted repeat is characterised by an Arrhenius activation energy of 42.4 +/- 3.2 kcal mole -1. The differences in kinetic properties between the two sequences indicate that DNA base sequence is itself an important factor in determining cruciform kinetics, and possibly even in the selection of the mechanistic pathway.