Genomic instability (GI) and The Ku proteins

Genomic instability (GI) is a constant threat to all organisms. DNA double strand breaks (DSBs) are particularly damaging as they can lead to mutations and chromosome abnormalities. Non-homologous end-joining is a crucial mechanism to deal with DSBs. The Ku proteins have been shown to be key contributors in this pathway. Ku70 and Ku80 are DNA binding proteins which repair DSBs and are of central importance to genome integrity and safeguarding. These proteins have been shown to form heterodimers in vitro using pull-down assays. However, no studies have been carried out living cells under physiological conditions to determine the dimerization process. There are clear disadvantages to using pull down assays, as they lack information about the spatiotemporal kinetics of DNA repair proteins in real time and in living cells. In the next three years, the research will involve investigation of Ku70 and Ku80 homo- and hetero- dimerization in live cells, the influence of other DNA repair proteins and the chemicals/inhibitors that affect this process at physiological conditions. The studies will involve use of Fluorescence Lifetime Imaging Microscopy Forster Resonance Energy Transfer (FLIM-FRET), gene editing CRISPR/Cas9 technology, BiFC (bimolecular fluorescence complementation) and anisotropy to investigate Ku dynamics and interactions in live cells.

The proposed research addresses the following BBSRC priority areas: Lifelong Health and Wellbeing and Healthy ageing across the life course. Ku deficiencies and deregulation are linked to premature aging, various types of cancer and immune system disorders. A better understanding of Ku70/80 interactions at the cellular level has implications for the study and the treatment of aging and many human diseases. By studying how and when Ku interactions occur, we aim to gain an insight into the conditions and molecular mechanisms behind these diseases.


WUB, ENWW