Topoisomerase Poisons Bound at a DNA Junction-like Quadruplex

1
Topoisomerase Poisons Bound at a DNA
Junction-like Quadruplex James H. Thorpe,
Jeanette R. Hobbs, Susana C. M. Teixeira and
Christine J. Cardin The University of Reading
Chemistry Department
Introduction Topoisomerases are responsible for
the interconversion of the topological states of
DNA generating transient single or double strand
breaks in the DNA phosphodiester backbone to
allow passage of one (topo I) or two (topo II)
DNA strands during replication, resulting in an
associated relaxation of their supercoiled state.
For this reason the topoisomerases have long
been an attractive target in the fight against
cancer. A series of third generation anti-tumour
agents have been developed in New Zealand to
target malignant melanomas amongst other forms of
tumour, with some inducing DNA cleavage in the
presence of both topoisomerase I and II making
them part of only a small range of drugs to
exhibit such properties and a major step forward
in the fight against cancer.
Experimental Crystals of the duplex
dCG(5BrU)ACG2 with 9-bromo-phenazine-1-carboxami
de in the orthorhombic space group C222 were
grown by vapour diffusion over a period of 12
months. MAD data collection was carried out at
the DESY synchrotron facility, where several
wavelengths were measured to maximise the
anomalous and dispersive signals. Data processing
was carried out with mosflm and SCALA. Two heavy
atom positions were determined by SOLVE
and
We have previously show that the related 9-amino
- acridine carboxamides, which are known to
inhibit only topoisomerase II at the point of DNA
cleaving through catalytic inhibition, can
intercalate into duplex DNA with the carboxamide
side chain located in the major groove binding to
the N7/O6 of the adjacent guanine. Here we
present the first examples of a range of
cytotoxic agents bound at DNA junction-like
quadruplex.
phase calculations carried out with MLPHARE and
DM. The initial model was built ab initio into
the 2Å MAD map with the map fitting program XFIT
from the XTALVIEW program suite. Crystallographic
refinement was carried out using SHELX97 with
SHELXPRO used for map production.
Results The structure of the duplex dCGTACG2
bound to a range of topo poisons exhibits an
unusual intercalation site and helical fraying
(Figure 1) stabilised through disordered modes of
drug binding and an array of cations. The
intercalation cavity is formed by the strand
exchange of a cytosine base rotated to pair with
a guanine of a symmetry related helix at 90 and
a distance of 15Å generating a pseudo-Holliday
type junction (Figure 2). Of the cytotoxic
agents shown to bind at this junction (Figure 4)
the acridine based drugs have thus far only
stabilised this strained system with the aid of a
Mg2 ion, bound at the centre of the cross-over,
to the four cytosine phosphates, a feature which
is thus far absent for the phenazines. Two such
cavities are linked through a quadruplex formed
by the minor groove interactions of the N2/N3
guanine cavity sites (Figure 3) at an angle
of 40, effectively
(1) 9Br-phenazine (XN, YBr, Z2) (2) DACA
(XCH, YH, Z2) (3) 9-aminoDACA (XNH2, YH,
Z2) (4) DACA3 (XCH, YH, Z3) (5) Bis-DACA
(X9-aminooctylDACA, YH, Z2)
Three views of the 2Å MAD map used to build the
initial model. Green 1? and yellow 2?.
Figure 2. Two views of the four way helical
junction and drug cavity formed by the DNA
cross-over and quadruplex. For clarity the bound
drugs have been removed and cobalt positions are
shown in purple.
junctions as well as duplex DNA and and even
strand-nicked DNA (hemi-intercalated) as in the
cleavable complex, suggesting a structural basis
for the dual poisoning of topoisomerase I and II
by this family of drugs. It must be noted however
that we only obtained crystals in the presence of
Co2 ions, and although they do not directly
influence the helix cross-over and intercalation
site, they are essential for this junction
formation.
Figure 4 The structural formulae of the tricyclic
drug systems which have currently been shown to
help stabilise this unusual X-stacked DNA
junction.
Figure 1 Schematic view of the numbering and
labelling scheme for the X-stacked junction and
bound cobalt ions.
Figure 3. Two illustrations of the DNA
quadruplex forming the large intercalation
cavity. (a) A stereoview of the cavity with drugs
removed for clarity. (b) A 2Å sigma-A map showing
the floor of the cavity and the minor groove
interactions of the guanine N2/N3 positions.
Conclusions DNA junctions have been known for
some time to provide high-affinity binding sites
for intercalators and as this work suggests may
help stabilise the X-stacked forms through charge
neutralisation from their cationic side chains,
as does Mg2. Resolution of these junctions can
occur by resolvase enzymes but also some
eukaryotic topo I enzymes. Human topo II? has
also been shown to preferentially bind to such
sites. The stabilising effects exhibited by this
class of compound therefore illustrates their
potential to bind with DNA
doubling the size of the intercalation site and
allowing for a large degree of disordered binding
of the drug chromophore despite the carboxamide
side chain anchoring to the N7/O6 guanine
positions in the major groove. The other end of
the duplex exhibits a terminal base fraying in
the presence of Co2 ions linking symmetry
related guanine bases (see MAD pictures)
intertwined through the minor groove, yielding a
quasi-continuous stack. A second hydrated cobalt
ion is bound to the pre-ultimate guanine G8 and
linked through water sites to its symmetry
related partner at 7.5Å.
Further Work This work suggests a possible
explanation for the dual poisoning properties of
certain anti-tumour agents, and as an extension
of these studies, work looking at the formation
of the cleavable complex between this family of
cytotoxins and the topoisomerases has begun.
Reference Biochem., 49, 15055-15061, 2000
Acknowledgements W.A.Denny (University of
Auckland, NZ) P. Charlton (Xenova plc) DESY
synchrotron and staff A.K. Todd (Institute of
Cancer Research, London) A. Adams (Trinity
College, Dublin).