Transcript X-ray Anomalous Diffraction Studies Quadruplex

DNA studies within the School of Chemistry
J.H. Thorpe, S.C.M. Teixeira, B.C. Gale, Y. Gan, M.I.A.A. Moraes and C.J. Cardin
The DNA Holliday Junction
The role of metal ions in determining the solution
conformation of the Holliday junction is well
established, but to date the picture of metal ion
binding from structural studies of the four-way
DNA Holliday junction is very incomplete. Here
we present two refined
structures of the Holliday
junction
formed
by
d(TCGGTACCGA) in the
presence of Na+ and Ca2+
ions and separately Sr2+
ions, to resolutions of
1.85Å and 1.65Å. This
sequence includes the
ACC core found to
promote spontaneous junction formation. The Na+
sites, the most convincing observations in junctions
to date, are on either face of the junction crossover
region, and stabilise the highly ordered hydration
within the arms. The four Ca2+ sites in the same
structure are at the CG/CG steps in the
minor groove. The Sr2+ ions occupy the TC/AG,
GG/CC and TA/TA sites in the minor groove, giving
ten positions forming two spines of ions spiralling
through each arm of the X-stacked structure. Both
structures show an opening of the minor groove face
of the junction of 8.4° in
the Ca2+ and Na+
containing structure and
13.4°
in
the
Sr2+
containing structure. The
crossover angles at the
junction are 39.3° and
43.3° respectively, and a
relative shift in the base
pair stack alignment of
the arms of 2.3Å is observed for the strontium
structure. Overall these results provide an insight
into the so-far elusive stabilising ion structure of the
DNA Holliday junction.
Ref:
JMB, (2003), 327, p97-109
Many thanks go to the AICR for support in this project
Drug Binding Studies – Crystallography and Competitive Dialysis
The binding modes of intercalating experimental
anti-tumour agents to DNA has proved far more
complex than the simple model of intercalation
represented in most texts. Works carried out here
have revealed the disordered nature of intercalating
agents and the multiple
modes
of
binding
observed both for the
chromophore and more
importantly the anchoring
sidechain. Atomic and
high resolution studies
have revealed the messy
multiplicity now known
to affect the properties of
these chemotherapeutic agents. Further to the
model of intercalation into duplex DNA, our
studies have focussed on the modes of binding to
higher order DNA structure as a potential target for
many highly active clinical agents. These studies
have yielded a previously unobserved mode of
binding for a bis-intercalator, where we have shown
it to specifically target a larger intercalation site
formed at the junction of four helices. The drug is
therefore observed to thread through the enlarged
binding pocket, rather than the more commonly
accepted sandwich style
binary modes of binding.
The identification of
sequence specific binding
sites for experimental
anti-tumour agents is now
undertaken
prior
to
crystallisation
studies
through the technique of
competitive dialysis. The
methodology involves the soaking of several
selected DNA sequences in a low concentration of
drug solution, resulting in the drug binding to those
preferred sequence sites, and is studied through UV
spectrometry.
Ref:
JMB, (2002), 323, p167-171.
Many thanks go to Dr. Peter Charlton of
Quadruplex DNA
Accumulating evidence within the literature to date
regarding the biological significance of quadruplex
DNA, provides compelling support for its
existence and functions
within
biological
systems, making it an
enticing
therapeutic
target. The synthetic
DNA
heptamer
d(GCATGCT)
under
certain ion stabilisation conditions,
forms a hair-pin looped quadruplex,
composed of four G/C base pairs, as
opposed to the more orthodox G
tetrads. Thus far the stabilisation of
this unusual motif has been observed in the
presence of magnesium, cobalt hexamine and
barium where each hydrated ion is observed to
bind within solvent cavities to phosphate oxygens.
X-ray measurements of this structure have been
carried out to atomic resolution, revealing the
highly stable nature of this quadruplex hairpin.
Studies into the addition of more toxic metal species
to quadruplex DNA have revealed results thus far
for nickel, where it is observed to distort the hairpin
structure
through
the
covalent binding of the
two available guanine N7
positions and with four
bound solvent oxygens,
the ion attains a complete
octahedral coordination
shell. A C/G quadruplex is still
maintained through the binding of a
central sodium ion which tethers the
bases in a similar fashion to that
observed for many G tetrads. The
aim of this study is therefore to
catalogue the interactions of a series
of metals with DNA and hence relate these
crystallographic studies to the toxic nature of the
cations. Ref: Nucleic Acids Research, (2003), 31 (3), p844-849.
X-ray Anomalous Diffraction Studies
Strontium is known to mimic calcium within
natural systems, making it a good candidate for
derivatising calcium binding macromolecules. It is
ideally suited for use with synchrotron radiation
sources as an anomalous scatterer, having
wavelengths achievable at most
modern facilities utilising the Sr
K-edge at ~0.77Å. We therefore
present the crystal structure of the
four-way
Holliday
junction
formed by the DNA sequence
d(TCGGTACCGA)
solved
through the multi-wavelength
anomalous dispersive (MAD)
phasing of the hydrated group II
metal ion, strontium, to a resolution of 1.6Å, and
final refinement to 1.5Å. Within the PDB to date
there are numerous examples of macromolecular
calcium binding derivatives, whereas there are
surprisingly few examples illustrating strontium
binding. Although strontium has no direct
biological function of its own, it follows the
metabolic pathways of calcium and behaves
similarly, although it is not homeostatically
controlled. Structurally, the strontium ion is larger
than the calcium ion, thereby requiring a slightly
larger binding site within a
macromolecule. However their
hydration numbers are the same,
with a value of 29, illustrating
further similarities between the
chemistries of both cations and a
feature that provides each with a
similar
charge
stabilising
potential. In conclusion we
propose strontium as a good
candidate for derivatising calcium binding
macromolecules for use in anomalous diffraction
measurements. The figure illustrates the MAD phases
achieved from strontium using the tuneable facilities
at DESY Hamburg (beam line BW7A).
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Ref:
To be published.