Structural and Functional Characterization of Non-Homologous End Joining Factors

Abstract

DNA double strand breaks represent the most toxic form of DNA damage. In mammals, non-homologous end-joining (NHEJ) is the primary DNA repair pathway for such damage, preventing both carcinogenesis and accelerated aging. Structural understanding of this repair pathway has received considerable attention, but has been significantly limited by the inability to obtain structures of higher order nucleoprotein complexes. A main obstacle in this respect has been difficulty in obtaining highly purified proteins, sufficient for structural determination. Improved protein expression and purification methods developed in this thesis permitted several NHEJ complexes to be selected for structural studies. Among these, Ku70-DNA and Ku70-DNA-PAXX yielded promising preliminary results. In depth optimization for crystal growth was performed and resulted in a full-length PAXX homodimer structure as well as low-resolution diffraction data for a novel Ku70-DNA complex. The PAXX structure confirmed prior suggestions that the C-terminal region of PAXX is highly disordered