Structural basis of bilateral cleavage in Holliday junction resolution

Homologous recombination is a natural mechanism, which can take place in any living cell. Using this mechanism it is possible for the cell to move segments of DNA, and therefore its genes, from one location on a chromosome to another. This is the mechanism that allows a person to inherit some characterstics 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 (e.g. one form each parent) are side by side, one strand of DNA on each chromosome is broken and then attached to the broken strand of DNA on the other chromosome. The crossover point, which is called the Holliday junction, is able to slide up and down between the two chromosomes, so that a little or a lot of DNA can ultimately be switched between them. We are interested in the mechanism cell uses to 'break' or 'cut' the Holliday junction once enough DNA has been exchanged between the chromosomes. Cells typically use proteins to cut Holliday junctions and we have previously studied the structure of one of these proteins (EndoI) in fine detail. Very recently, we have been able to make a complex showing how EndoI actually sticks to a Holliday junction. For this work we would like to study the structure of this complex. We would specifically like to: Study the structure of the EndoI / Holliday junction complex in fine detail. Try and trap intermediate states during the cutting process and study the structure of these. Relate these structures to ongoing functional studies to attempt to reconstruct the exact mechanism EndoI uses to cut Holliday junctions.

We have recently solved the structure, at medium resolution, of a complex between T7 Endonuclease I (EndoI) and a synthetic Holliday junction with data collected at the SRS, Daresbury. This provides an opportunity to determine the structural basis of the catalysis in this well-defined system, and, in particular, the mechanism that ensures bilateral cleavage. For the proposed work we wish to: 1) Determine the crystal structure of the complex between EndoI and a DNA junction to (at least) 3Å resolution to elucidate the basis of structure-specific recognition in this system. We will prepare complexes from recombinant protein and synthetic DNA junctions, grow crystals, and collect X-ray diffraction data at the ESRF to obtain the necessary high intensity beam (or Diamond Light Source from 2007) to increase the resolution of our current structure. 2) Determine crystal structures for intermediate forms where only one DNA strand has been cleaved to elucidate structural changes that might accelerate cleavage of the second strand. We will address this by attempting to determine structures of unilaterally-cleaved complexes. 3) Determine crystal structures for product complexes where both strands have been cleaved but before the product has dissociated. We will approach this in 2 ways: (i) we will incubate intact junctions with wt EndoI and Mn2+ ions, before setting up for crystallization trials. (ii) We will prepare bilaterally cleaved junctions, by annealing appropriate sets of four oligonucleotides. 4) Attempt to trap intermediate states by flash cooling crystals of the complex at different stages of the reaction. We will start from crystals of junction complexes grown in Ca2+ ions, soak these crystals in Mn2+ ions and flash-cool samples in liquid nitrogen at a series of time points.