Cruciform formation in a negatively supercoiled DNA may be kinetically forbidden under physiological conditions.

Cruciform formation in a pBR322 derivative containing a 68 base pair perfect palindromic sequence has been studied. In relaxed DNA the cruciform is unstable by about 17 kcal mole-1 in free energy. In a supercoiled DNA with a negative specific linking difference (superhelical density) of 0.03 or higher, however, the cruciform becomes the stable species. Nevertheless, even at native superhelical densities (typically around -0.06), cruciform formation is extremely slow, unless the DNA is subjected to conditions that destabilize base pairing. When precautions are taken to avoid artificial triggering of cruciform formation, the 68 bp perfect palindrome-containing plasmid isolated from cells is devoid of the cruciform. The rate of disappearance of the cruciform in linear DNA and the deletion of the palindromic sequence in vivo have also been examined.