Molecular determinants of Ku function in non-homologous end-joining and DNA damage signaling pathways

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Ku is an abundant, ubiquitous factor that has been remarkably conserved throughout evolution as it has been identified in all organisms from bacteria to man. Ku is a complex of two proteins that plays an essential role in the repair of double-strand DNA breaks, a form of DNA damage in which both strands of the double helix break, leading to chromosome rearrangements and, ultimately, genomic instability. While it is understood that Ku is essential for the proper repair of DNA, its mode of action is still unclear. It is known that Ku interacts with a number of other factors that play roles in the repair of DNA, however, the details and the outcome of these interactions are poorly understood. Using the crystal structure of Ku that was recently made available, we can predict regions of the protein that are accessible to contact other proteins. Thus we have made mutations in several of these accessible regions to test if they alter Ku DNA repair functions. We have identified two Ku mutations that alter its ability to function in response to DNA damage. We found that these mutations result in different defects suggesting that the mutated regions are involved in distinct functions. We propose to identify the proteins that interact with these Ku motifs and elucidate how they function together in the repair of DNA. In the long-term, these analyses will enhance our understanding of the functions of Ku in the mechanisms that are set in place to deal with double-stranded DNA breaks.