Research objectives and content
This project will make use of the atomic force microscope (AFM) to investigate DNA translocating enzymes using EcoKI type restriction endonuclease as a test system. The translocation properties of these enzymes make them ideal candidates for direct visualisation of protein-DNA interactions. They bind to a specific site, translocation produces large loops and the loops are maintained after cleavage. Furthermore, EcoKI is available in homogenous form in milligram amounts and mutations have been produced which affect different aspects of the cleavage process. Early studies of EcoKI by electron microscopy produced clear pictures of the DNA loops produced by the translocation but the presence of either 1 or 2 DNA loops which could be relaxed or supercoiled, coupled with low resolution and harsh. variable sample preparation methods, precluded the construction of a good model of the translocation process. The higher resolution of the atomic force microscope, improved quality of protein and DNA samples and the ease of simple preparation in conditions which maintain enzyme activity indicate that this is an opportune time to study this translocation process again. It is hoped to follow the translocation process and growth of DNA loops as a function of time and, possibly, visualise the orientation of the protein subunits on the DNA substrate before and during the translocation process. Translocation of DNA by proteins is of fundamental importance in the life of any cell but is with few exceptions not well characterised with respect to mechanism and structure.
Training content (objective, benefit and expected impact)
Training will be given in the molecular biological techniques required for purification of the proteins and DNA substrates and in the use of an atomic force microscope specifically designed for structural analysis of these biological macromolecules. The project will be carried out as a study for the degree of PhD.
Links with industry / industrial relevance (22)