Effect of magnesium on cruciform extrusion in supercoiled DNA

Recently, it was reported that Mg2+ greatly facilitates cruciform extrusion in the short palindromes of supercoiled DNA, thereby allowing the formatio n of cruciform structures in vivo. Because of the potential biological impo rtance of this phenomenon, we undertook a broader study of the effect of Mg 2+ on a cruciform extrusion in supercoiled DNA. The method of two-dimension al gel electrophoresis was used to detect the cruciform extrusion both in t he absence and in the presence of these ions. Our results show that Mg2+ sh ifts the cruciform extrusion in the d(CCC(AT)(16)GGG) palindrome to a highe r, rather than to a lower level of supercoiling. in order to study possible sequence-specific properties of the short palindromes for which the unusua l cruciform extrusion in the presence Mg2+ was reported, we constructed a p lasmid with a longer palindromic region. This region bears the same sequenc es in the hairpin loops and four-arm junction as the short palindrome, exce pt that the short stems of the hairpins are extended. The extension allowed us to overcome the limitation of our experimental approach which cannot be used for very short palindromes. Our results show that Mg2+ also shifts th e cruciform extrusion in this palindrome to a higher level of supercoiling. These data suggest that cruciform extrusion in the short palindromes at lo w supercoiling is highly improbable. We performed a thermodynamic analysis of the effect of Mg2+ on cruciform extrusion. The treatment accounted for t he effect of Mg2+ on both free energy of supercoiling and the free energy o f cruciform structure per se. Our analysis showed that although the level o f supercoiling required for the cruciform extrusion is not reduced by Mg2+, the ions reduce the free energy of the cruciform structure. (C) 1999 Acade mic Press.