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FURTHER PARTICULARS
THE RESEARCH COMPLEX AT HARWELL
Structural Basis of Holliday Junction Resolution
Background to the post
The posts are supported by an MRC programme grant to Prof. Simon Phillips and Dr.
Stephen Carr at RCaH, as part of long-term study of the structure and function of
Holliday junction resolvases. The programme involves collaborations with the
groups of Prof. David Lilley (Dundee), Dr. Stephen West (CRUK Clare Hall), Dr. Ian
Hickson (Oxford) and Prof. Matthew Whitby (Oxford). The posts are based at RCaH
and will be concerned chiefly with the structural biology work, with functional
studies largely based in collaborators’ laboratories.
Summary of the research programme
Resolution of four-way DNA Holliday junctions in homologous recombination and
DNA repair is a ubiquitous process in living organisms, and DNA junctionresolvases are widespread in prokaryotes, eukaryotes and their viruses.
Junction-resolving enzymes bind to DNA junctions in a highly structure-specific
manner and bring about the coordinated cleavage of strands of the junction to
give two separate duplex products. Correct cleavage is critical to the biological
outcome, and errors in the system can lead to disease. The overall aim of the
programme is to determine three-dimensional structures for resolution complexes
and relate these to functional studies in collaborating laboratories to provide a
fuller mechanistic understanding of this fundamental biological process. In
particular we aim to:
1- Determine crystal structure of the recently discovered human Holliday junction
resolvase, GEN1, and of its complexes with DNA junctions.
2- Determine crystal structures for the Bloom Syndrome complex, its
subcomponents and their complexes with DNA.
3- Use complementary structural methods, such as Electron Microscopy (EM),
Small Angle X-ray Scattering (SAXS), Small Angle Neutron Scattering (SANS),
Atomic Force Microscopy (AFM) to study higher order complexes in solution and
relate these to the crystal structures and functional studies.
4- Extend our studies of EndoI-junction complex catalytic intermediates formed
during DNA cleavage to elucidate the reaction pathway in this model system and
the basis of bilateral cleavage.
5- Determine crystal structure of yeast resolvase CCE1, and of its complexes with
DNA junctions, to use it as a second model system to study catalysis in detail.
Research environment
The Research Complex at Harwell (www.rc-harwell.ac.uk) is a joint initiative
of the Research Councils (BBSRC, EPSRC, MRC, NERC and STFC) and the
Diamond Light Source, to provide new, state-of-the-art multidisciplinary research
laboratories on the Rutherford Appleton Laboratory (RAL) site adjacent to the
new Diamond third generation synchrotron source. The Complex is currently
nearing completion and will provide new, flexible laboratory space, amounting to
6500 m2 gross floor area. It is managed by MRC on behalf of the other partners,
and will have a core staff of about 10 to run the facility. It will accommodate up
to 150 physical and life scientists who will be funded by external grants to carry
out cutting edge multidisciplinary research. The research will concentrate on
areas requiring the use of the RAL major facilities: Diamond synchrotron, ISIS
neutron source and the Central Laser Facility (CLF).
The Research Complex's terms of reference are:
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To provide operational generic laboratory space for life and physical
sciences research that will attract world-class scientists;
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To provide common spaces and shared facilities to encourage interaction
between life and physical scientists, and users of Diamond, ISIS and CLF;
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To be sufficiently adaptable in design in order to respond to changes in
research requirements and opportunities in the future.
The core objective of the project is to deliver a multidisciplinary centre of
international research excellence to maximize the research capability and
scientific opportunities afforded by Diamond and the other facilities at the RAL
site. Many major advances in science take place at the boundaries between the
traditional disciplines, and increasingly utilise advanced technologies available at
centralised facilities. The Research Complex will house a number of long-,
medium- and short-term, research groups in life and physical sciences who will
benefit from the proximity of the major facilities and the synergy generated by
the multidisciplinary environment. In the initial phase there will be three core
groups:
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The Oxford Protein Production Facility UK (OPPF-UK) specialising in highthroughput methodologies for expressing, crystallising and determining
three-dimensional crystal structures of proteins, especially human proteins
of biomedical interest.
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A grouping from the RAL Lasers for Science Facility (LSF) specialising in
the development and use of advanced laser technologies for physical and
life sciences applications.
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The Collaborative Computing Project No.4 (CCP4) group specialising in the
generation and maintenance of the most widely used software for protein
crystallography.
All these groups provide a service element to the UK research community. They
will be joined by further grant funded research groups, selected on a competitive
basis, in areas such as:
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structural studies on membrane proteins and multi-protein complexes;
high throughput functional and structural genomics related to disease and
molecular mechanism in biology;
biological imaging, including living cells;
application of novel physical techniques in life sciences;
catalysis;
drug development and delivery;
matter under extreme conditions;
chemical processing;
surface science;
nanoscience and nanotechnology;
energy research.
The aim is to build an international reputation in multidisciplinary research,
provide a stimulating environment for training scientists at all levels, and
generate collaborations with academic institutes in the UK and abroad.
Structural Biology Facilities
RCaH provides outstanding facilities for structural biology, with the advantages of
the proximity of Diamond and ISIS. There will excellent facilities for large-scale
protein expression, purification and analysis, including those in the OPPF-UK, which
will be available for the programme. These include growth facilities for bacteria,
yeast, insect and mammalian cells, new chromatography systems and large-scale
robotic crystallization facilities. RCaH will also have its own new 200kV transmission
electron microscope, 400MHz NMR machine, and numerous facilities for biophysical
characterization. Computing facilities will very powerful, with a new Linux cluster
and numerous PC workstations. The CCP4 core team will also be in the building.