THE STRUCTURAL CHARACTERISATION OF TWO HOLLIDAY JUNCTIONS

1
THE STRUCTURAL CHARACTERISATION OF TWO HOLLIDAY
JUNCTIONS Benjamin C. Gale , James H. Thorpe,
Susana C. M. Teixeira and Christine J. Cardin The
University of Reading Chemistry Department
Introduction The four-way junction, or the
Holliday junction plays an important role in the
physiology of cells, acting as a central
recombination intermediate for many recombination
enzymes1. These enzymes promote or catalyse
branch migration allowing the exchange of genetic
information between DNA duplexes. The Holliday
junction is formed through strand exchange of two
homologous DNA molecules to give a four-way
junction at the cross-over2. As such the Holliday
junction represents a structurally distinctive
motif that must be recognised at the molecular
level by the enzymes through a currently
unidentified mechanism. Here we present the
single crystal structures for two DNA Holliday
junctions formed by d(CCGGTACCGG)23 1and
d(TCGGTACCGA)2 2 at 2.35Å and 1.8Å resolutions
respectively in the monoclinic space group C2 and
discuss the essential features in the
stabilisation of the Holliday junction.
Data Collection and Processing d(CCGGTACCGG) Tempe
rature 100K Cryobuffer Perfluoropolyether (RS
3000) oil Instrument MAR345 image plate X-ray
Source Synchrotron radiation 1.073Å, X31
beamline DESY, Hamburg Germany Space group
C2 Cell a 64.9Å b 25.4Å c 37.4Å ?
110.6o No. of Observed Reflections 4466 No. of
Unique Reflections 2390 R-factor 22.64 Rmerge
0.056 (0.230) Completeness 96.41 Average
B-factor 39.94 Å2 Resolution Limits 30.429 -
2.350 Software DENZO and SCALEPACK
Data Collection and Processing d(TCGGTACCGA) 100K
Temperature Perfluoropolyether (RS 3000) oil
Cryobuffer CCD detector MAR-Research
Instrument Synchrotron radiation 0.8068Å, X11
beamline X-ray Source DESY, Hamburg
Germany C2 Space group a 66.1Å b 23.8Å c
73.8Å ? 110.58 Cell 9220 No. of Observed
Reflections 8780 No. of Unique Reflections 22.82
R-factor 0.0703 (0.2025) Rmerge 97.35
Completeness 23.5Å2 Average B-factor 69.007 -
1.850 Resolution Limits MOSFLM and CCP4 Software
Crystallisation Conditions d(CCGGTACCGG)2 d(TC
GGTACCGA)2 Temperature 290K 290K Crystallisin
g Solution 40mM sodium cacodylate (pH 7.0) 75mM
sodium cacodylate (pH 7.0) 12mM
spermine 35mM calcium chloride 80mM
potassium chloride 2.5 MPD 10 MPD
Equilibrated against 30 MPD Equilibrated
against 35 MPD DNA 1mM 1mM
Method Sitting Drop Vapour Diffusion Sitting
Drop Vapour Diffusion
Table 1 A summary of related native decamer
sequences
In this work the asymmetric unit is either a full
(Fig. 1) or half stacked-X (Fig. 2) with a two
fold symmetry at its centre. In the case of the
full quadruplex the c-axial length is halved and
the approximate two-fold symmetry is lost. In
either case however, the packing is remarkably
similar with helices crossing at approximately
40o in each case (Table 1). The strand exchange
at the centre of the structures leads four
phosphates to be closely packed (Figs. 3 4),
thus producing a region of highly negative
electrostatic potential. Here the better
resolution, when compared with 1 and other
Holliday sequences in Table 1 allows two
partially hydrated sodium ions near to the
phosphate junction in the minor and major groove
(Fig. 5) to be defined. Four distorted pentagonal
bipyramidal calcium atoms (Fig. 6) anchored in
the minor grooves to the G/C base pairs prior to
the terminal A/T sites can also be observed. It
is apparent that these sites aid in stabilising
the solvent structure throughout the minor
groove. In addition to these stabilisation
forces, well determined solvent bridging sites
between the phosphate oxygens at the cross-over
on the major groove face and the purine bases
either side are present in 2 (Fig 7). For both
structures, the minor groove face at the
cross-over shows direct interactions between the
cytosine N4 sites and the oxygens of the closely
packed phosphates (Figs. 8 9). Previously4 the
ACC step has been shown vital for the formation
of the Holliday junction within a crystalline
lattice. This again has been demonstrated but
further to this we have shown the presence of a
purine base either side of the cytosines to be
essential in the water mediated stabilisation at
the cross-over. Finally the ellusive ion
structure of the Holliday sequence has been
determined with a clarity highlighting the
importance of these sites in the stabilisation of
the solvent structure throughout the Holliday
structure.
References 1. D. M. Lilley M. F. White (2001).
Nature Mol. Cell Biol. 2, 433-44 2. R.
Holliday (1964). Genet. Res. 5, 283-304 3.
J. H. Thorpe, S. C. M. Teixeira, B. C. Gale C.
J. Cardin (2002). Acta Cryst. D58, 567-569. 4. B.
F. Eichman, B. H. M. Mooers, J. M. Vargason P.
Shing-Ho (2000). Proc. Natl Acad. Sci. USA. 97,
3971-3976 5. M. Oritz-Lombardia, A. Gonzalez, R.
Eritja, J. Aymani, F. Azorin M. Coll (1999).
Nature Struct Biol. 6, 913-917 6. A. A. Wood, C.
M. Nunn, O. J. Trent S. Niedle (1997). J. Mol.
Biol. 269, 827-841
Acknowledgements BCG would like to thank the
Association of International Cancer Research for
their essential funding and to JHT, SCMT and JHT
for their guidance. SCMT is grateful to the
Chemistry Department of the University of Reading
and the Portuguese Foundation for Science and
Technology