Understanding the DNA Holliday Junction

1
Understanding the DNA Holliday Junction Benjamin
C. Gale, James H. Thorpe, Nick H. Hopcroft and
Christine J. Cardin. The School of Chemistry,
University of Reading.
Crystallographic Studies
Competitive Dialysis
Homologous recombination (HR) is key for life,
acting to create genetic diversity and to repair
double strand breaks in DNA. However the key role
now appears to be in the repair and resetting of
DNA replication forks that have stalled or
collapsed at sites of DNA damage1. Either way HR
This work has investigated two sequences,
d(TCGGTACCGA) and d(CCGGTACCGG) in the presence
of Ca2 and Sr2. In the presence of Sr2,
d(TCGGTACCGA) forms the half-X structure with a
spine of five Sr2 sites that spiral down each
B-DNA arm. By contrast in the

presence
Competitive dialysis or competition dialysis was
initially described by Ren and Chaires1 and has
since then been adapted in this laboratory. The
aim of such an experiment
intercalate into some of the oligonucleotide
sequences and further displace the osmosis
equilibrium so that more drug will
pass from the beaker to the
is to determine which sequence a drug
preferentially binds to in solution and hence
provides great insight into the drugs that should
be crystallised with specific sequences. The
method works by equilibrium dialysis. A
macromolecule is paced inside a semi-permeable
membrane, called a dispodialyser, that have pore
sizes to prevent escape of the macromolecule but
allow the surrounding drug solution to enter. The
dialysers are then left to equilibrate in a
beaker containing a stirred solution of drug,
which can cross the membrane of the
dispodialysers and bind to the most preferred
DNA sequence. More technically
dispodialysers to compensate for the binding to
DNA. Osmotic pressure and intercalation will
therefore start to compete up to a point when
equilibrium is reached. The amount of drug up
taken by each sequence is compared by UV-visible
spectroscopy after equilibrating overnight. This
work has initially focussed on adopting the
protocol described by Ren and Chaires to suit our
requirements. Though there has been only limited
success to date it is expected that this
technique will provide the rationalisation for
crystallisation studies within this research
area. A range of drugs are
is characterised by the formation of
branched DNA molecules called Holliday Junctions
(HJs)2. The structures of the DNA HJs solved
in this group have provided strong evidence of
features now associated with this motif. In
particular the requirement of the central d(ACC)
core and quite specific stabilising contacts have
been shown to be key3,4,5,6. Until recently
however7,8 the role of metal ions in the crystal
structure of the HJ has been limited despite
their dramatic effect in solution9.
of Ca2, only two ion sites have
been refined in the short arms, removed from the
terminal bases. For the second sequence, four
Ca2 ion sites can be located. Two are in the
long arms, found removed from the terminal bases
whereby in the short arms the two sites are found
at the terminus of the sequence causing the
junction to be closed up. Preliminary results for
Sr2 indicate two ion sites, one in the short arm
at the terminus and one in the long arm removed
from the terminal bases.
available for use with this study including
XR5944, which has been shown to have
sub-nanomolar IC50 values in tumour cell lines
and entered Phase 1 clinical trials in the UK in
July 2003.
the drug in the beaker will
first enter the dispodialysers by simple osmosis,
to equilibrate the pressure difference between
both sides of the membranes of the
dialysers. The drug will then
1 McGlynn, P. et al, PNAS, 2001. 98 p.
8227-8234. 2 Holliday, R., Genet. Res., 1964. 5
p. 282-304. 3 Eichman, B. F. et al, PNAS, 2000.
97 p. 3971-3976. 4 Ho, P.S. et al, Curr. Opin.
Struct. Biol. 2001. 11 p. 302-308. 5
Ortiz-Lombardia, M. et al, Nat. Struct. Biol.
1999 6 p. 913-917. 6 Thorpe, J.H. et al Acta
Cryst, 2002. D58 p. 567-569. 7 Thorpe, J.H. et
al, J. Mol. Biol, 2003. 327 p. 97-109. 8
Vargason, J.M. et al, J. Biol. Chem, 2002. 277
p. 21041-21049. 9 Lilley, D.M., DNA-Protein
Structural Interactions, OUP
1 Ren, J. and Chaires, J.B. Methods in
Enzymology, 2001. 340 p. 99 - 108.
p53 Consensus Sequence
Fundamental Points

• Crystallographic Studies
• Homologous recombination is characterised by the
  formation of branched DNA molecules called
  Holliday Junctions.
• The role of metal ions in solution is key to the
  HJ.
• The crystal structures reveal the binding of
  metal ions to the HJ.
• Small differences gives rise to quite dramatic
  effects which could be important to junction
  recognition.
• p53 Consensus Sequence
• The tumour suppressor protein p53 is mutated in
  over 50 of all human cancers.
• The p53 consensus sequence defines the DNA
  sequence elements with which p53 interacts.
• The half site of this sequence has a striking
  similarity to the HJ forming sequences.
• The sequence d(GGGCTAGCCC) will be characterised
  to see if it adopts the HJ conformation.

The pivotal role of the tumour suppressor protein
p53 in cancer is well documented with over 50 of
all cancers having associated with them,
mutations in p531,2. Previous work has identified
the p53 consensus sequence, which defines the DNA
sequence elements with which p53 interacts3.
565 base pair sequence, this represents a high
level of specificity. This study will look to
characterise the half site 5'-PuPuPuC(A/T)(T/A)GPy
PyPy-3 of the p53 consensus

• Competitive Dialysis
• This experiment will seek to determine which
  sequence a drug preferentially binds to in
  solution.
• This will allow crystallographic studies to be
  focussed on the most relevant sequences and
  drugs.
• The method is based on simple equilibrium
  dialysis.
• A range of drugs are available for study
  including those that have sub-nanomolar IC50
  values in tumour cell lines and have entered
  Phase 1 clinical trials.
• Acknowledgements
• For funding I thank the EPSRC, the BBSRC and the
  Association for International Cancer Research.
• Thanks also to Xenova, and in particular to
  Peter Charlton for supplying many of the drugs
  being used.
• For useful correspondence on the competition
  dialysis method I would like to thank Jonathan
  Chaires.
• Finally I would like to thank the many
  colleagues who have helped me including staff at
  DESY in Hamburg.

sequence, which has a striking similarity to the
HJ forming sequences to see if it is pre-disposed
to being a HJ or if the binding of p53 or a range
of anti-cancer drugs effects the conformation
adopted.
X-ray crystallography has revealed how
one core domain, the central half of p53 binds to
a quarter site4 and further a model has been
proposed in which four identical domains can
occupy all four quarter isites
in the full consensus sequence though this has
never been seen in an X-ray crystallography
experiment4. Further to this it has been
demonstrated through NMR that human p53 can bind
to the Holliday junction5. In this work it was
demonstrated that 80-96 of p53 were specifically
located at the junction with only 4 at the
ends. For an
Crystallisation studies are currently underway
for the sequence d(GGGCTAGCCC), which has been
initially screened with the Hampton Research
Nucleic Acid screen to yield small microcrystals.
Optimised conditions are now being utilised and
it is hoped that suitable crystals will be
available for data collection in the later half
of this year.
1 Levine, A.J. et al, Nature, 1991. 351. 2
Hollstein, M. et al, Science, 1991. 253 p.
49-53. 3 el-Deiry, W. S. et al, Nat Genet, 1992.
1 p. 45-49. 4 Cho, Y. et al, Science, 1994, 15
p. 346-355. 5 Suman, L. et al, J Biol Chem,
1997. 272 p. 7532 - 7539.