Structural studies of protein-DNA complexes in recombination and repair

Human cells have two copies of each chromosome (one inherited from each parent) which carry their genes encoded in DNA. There are therefore two copies of each gene, one from each parent, and the equivalent pairs of DNA molecules are called ?homologues?. Each DNA molecule is a double helix with two strands. Homologous recombination is a natural mechanism, by which it is possible for the cell to move segments of DNA, and therefore genes or parts of genes, from one DNA molecule to the equivalent place on another ?homologous? one. This is the mechanism that allows a person to inherit some characteristics from his/her mother, and others from his/her father, as it mixes and matches the genes. It is therefore a very important evolutionary process. When two chromosomes are side by side, one strand of DNA on each chromosome is broken and then attached to a broken strand of DNA on the other chromosome at the equivalent position. The crossover point, which is called the ?Holliday junction?, is able to slide up and down between the two chromosomes, and a little or a lot of DNA from one molecule can be switched over from one to the other. As well as being used to exchange genes between chromosomes, and generate the obvious mixture of inherited characteristics in children, it is also used in the important process of DNA repair. Our DNA is constantly being damaged, each of our cells suffering thousands of lesions per day, and such damage leads to diseases such as cancer. Homologous recombination allows the equivalent healthy chromosome DNA to be used as a template to repair the damaged DNA, and without this system we would not survive for long. We are interested in the mechanism the cell uses to cut the Holliday junction once enough DNA has been exchanged between the chromosomes, and allow the two DNA molecules to separate again. Cells typically use specialised proteins (enzymes) to cut Holliday junctions and we have previously studied the structure of a simple one from a virus. In this project we will study the structure of several human enzymes that carry out this job to understand how they work in detail. People who inherit defective versions of these enzymes often develop cancers, and a full understanding of the mechanism should help the design of specific treatments in the future.

Resolution of four-way DNA Holliday junctions in homologous recombination and DNA repair is a ubiquitous process in living organisms, and DNA junction-resolvases of various classes are widespread in prokaryotes, eukaryotes and their viruses. Junction-resolving enzymes bind to DNA junctions in a highly structure-specific manner and bring about 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: determine crystal structure of the recently discovered human Holliday junction resolvase, GEN1, and of its complexes with DNA junctions; determine crystal structures for the Bloom Syndrome complex, its subcomponents and their complexes with DNA junctions; 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; extend our studies of EndoI-junction complex catalytic intermediates, to elucidate the reaction pathway in this model system and the basis of bilateral cleavage; 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.