Dynamics of eukaryotic junction-resolving enzyme GEN1 - DNA junction interactions

Repair of damage to cellular DNA is extremely important to human health, and one important repair process is recombination. This involves the formation of branched structures in cellular DNA, that are then processed by enzymes. One such enzyme is called GEN1. We have recently solved the atomic structure of the GEN1 protein, and this structure has provided considerable insight into the probable mechanism of action. We now shall extend our knowledge by observation of single molecules of the enzyme as they carry out the processing of junctions, using highly sensitive fluorescence methods. We will be able to dissect the processes by which the protein binds to the DNA and assembles on the branched junction, and how it alters the structure of the DNA. We can then proceed to study the process by which the enzyme cuts the DNA of the junction at specific points, and how this is coupled to changes in structure. Our goal is a full understanding of how this enzyme achieves a well regulated processing of the junction. Our ultimate goal is to intervene in the process using small molecules, which could ultimately result in therapeutic agents.

Our recent crystal structure of the eukaryotic Holliday junction-resolving enzyme GEN1 bound to a product of resolution has provided the high atomic resolution view of this structure, and a clear indication of how a junction is bound by a dimer of the protein. We are now moving on to study the dynamic processes of binding, conformational change and junction resolution using fluorescence resonance energy transfer (FRET) studies of single molecules. GEN1 differs from resolving enzymes of lower organisms primarily in that it is monomeric in free solution. This raises questions with regard to its assembly on the junction, and at what stage conformational chance occurs in the junction. These events raise the possibility of being regulated in vivo. Upon activation of the enzyme by addition of magnesium ions it proceeds to cleave the junction in a very specific manner, in two stages. The structural changes that accompany this are not known, and will thus be studied by the single-molecule FRET. Our hypothesis is that these changes are coupled to the cleavages such that a productive resolution is ensured.

The impact of the work on GEN1 lies in the potential design of novel therapeutic agents, particularly in the anti-cancer area. Structure-selective enzymes are essential in every aspect of nucleic acid function, and we believe these could be significant drug targets in cancer therapy. GEN1 has been reported absent in some ovarian and colon cancer cell lines; these cells are hypersensitive to DNA-damaging agents, suggesting that inactivation of GEN1 could be a sensitizing strategy in therapy. Information emerging from these studies is likely to feed into drug discovery either as direct therapeutic agents, or perhaps more likely as sensitizers to enhance radio- or chemotherapy in cancer patients. These approaches have been discussed with our Drug Discovery unit in Dundee.

In this laboratory we make significant efforts to engage with the public. Professor Lilley is a frequent speaker at public events, such as the Dundee Café Science and the Fife Science Fair. He has also recently given an interview on Radio Scotland concerning the potential for GEN1 is therapeutic development.