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: To provide operational generic laboratory space for life and physical sciences research that will attract world-class scientists; To provide common spaces and shared facilities to encourage interaction between life and physical scientists, and users of Diamond, ISIS and CLF; 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: 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. 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. 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: 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.