are sensitive to single-strand nucleases, such as S1 and P1, and especially to chemical reagents such as
bromoacetaldehyde, osmium tetroxide, bisulfite and glyoxal (Chapter 8). In addition, the junctions are
cleavage sites for yeast resolvase and for T4 endonuclease VII.
David Lilley has shown that the formation of two such loops requires the unpairing and unstacking of
three or more base pairs and so will be thermodynamically unstable compared to the corresponding single
helix.^27 While there can be some stacking of bases in the loops, the adverse energy of formation of a sin-
gle cruciform has been calculated to be some 75 kJ mol^1. In experiments on cruciforms using closed cir-
cular superhelical DNA, this energy can be provided by the release of strain energy in the form of negative
supercoiling (see the following section) and is directly related to the length of the arms of the cruciform:
the formation of an arm of 10.5 bp unwinds the supercoil by a single turn.
There is also a kinetic barrier to cruciform formation and Lilley has suggested two mechanisms that
have clearly distinct physical parameters and may be sequence-dependent. The faster process for cruci-
form formation, the S-pathway, has G‡of about 100 kJ mol^1 with a small positive entropy of activation.
DNA and RNA Structure 41
Figure 2.25 (a) The hairpin loop formed by d(CGCGCGTTTTCGCGCG). (b) Formation of a cruciform from an
inverted repeat sequence of the bacterial plasmid pBR322. The inverted palindromic regions, each of
11 bp, are shown in colour