Structure and mechanism of DNA replication and repair enzymes

Previous and current research
The replication and repair of DNA requires a vast array of different proteins. Although the overall biological mechanisms involved in these processes are beginning to become clearer, the molecular mechanisms of most of the proteins are still very poorly understood. In order to learn more about these enzymes at the molecular level, we utilise a variety of techniques ranging from X-ray crystallography and site-specific mutagenesis to more standard enzymology and molecular biology methods. By combining these techniques in the same group we take a multidisciplinary approach which tackles the problem from several different angles and helps us to gain a greater insight into the mechanism of these enzymes than could be achieved by any single method in isolation. We continue to study a number of different systems including topoisomerases, ligases, primases, recombinases and helicases. Typically, projects are initiated by determining the crystal structure of the enzyme under study. Using the information provided by the structure, we then design biochemical experiments, such as site-specific mutants, to test out ideas about the mechanism and then utilise a variety of different methods (including DNA footprinting, fluorescence spectroscopy, pre-steady state kinetics, etc., as appropriate) to study the wildtype and mutant proteins. These continuing studies often aid our understanding sufficiently to allow us to determine further crystal structures to try and trap the proteins at different stages in the catalytic cycle and hence gain a fuller understanding of the complete mechanism. It is the synergy between these different aspects of the work which leads to a detailed understanding of the proteins.

Future projects
Much of our previous work has been focused on prokaryotic enzymes. On moving to Clare Hall, however, we are expanding our interests to include enzymes involved in eukaryotic DNA replication and repair, an area which relates more directly to cancer. We are particularly interested in enzymes involved in initiation of DNA replication as well as those involved in progression of the replication fork. We are also beginning to look at some of the enzymes involved in DNA repair processes.