This more common pathway is typified by the behaviour of plasmid pIRbke8. Following the formation of
a relatively small unpaired region, a proto-cruciform intermediate is produced, which then grows to equi-
librium size by branch migration through the four-way junction (Figure 2.26). The slower mechanism,
the C-pathway, involves the formation of a large bubble followed by its condensation to give the fully
developed cruciform. This behaviour explains the data for the pColl315 plasmid whose cruciform kinetics
show G‡about 180 kJ mol^1 with a large entropy of activation.
Such extrusion of cruciforms provides the most complete example of the characterisation of unusual
DNA structures by combined chemical, enzymatic, kinetic and spectroscopic techniques. However, it is
not clear whether cruciforms have any role in vivo. One reason may simply be that intracellular superhe-
lical densities may be too low to cause extrusion of inverted repeat sequences. Equally, the kinetics of the
process may also be too slow to be of physiological significance. However, cruciforms are formally equiva-
lent to Holliday junctions and these four-way junctions involve two DNA duplexes that are formed during
homologous recombination (Section 6.8).
Several X-ray crystallographic studies have provided a detailed picture of the 3D structure of the
Holliday junction.^28 Interestingly, DNA decamers with sequences CCGGGACCGG, CCGGTACCGG and
TCGGTACCGA fold into four-way junctions instead of adopting the expected B-form double helical geom-
etry (Figure 2.27). The tri-nucleotide ACC (underlined) forms the core of the junction and its 3-CG base
pairs helps to stabilise the arrangement by engaging in direct and water-mediated hydrogen bonds to phos-
phate groups at the strand crossover. The four strands exhibit a stacked-X conformation whereby the two
inter-connected duplexes form coaxially stacked arms that cross at an angle of ca.40°. Stable Holliday
junctions were also observed with DNA decamers that featured an AC(Me^5 C) tri-nucleotide core and the
tri-nucleotide AGC when covalently intercalated by psoralen.
2.3.3.3 Role of Metal Ions. NMR in solution, X-ray crystallography and computational simulations
(molecular dynamics, MD) have all shed light on the locations of metal cations surrounding nucleic acid
42 Chapter 2
Figure 2.26 Structures of a cruciform and alternative pathways for its formation (base-paired sections are helical
throughout)