Abstract
NHEJ is a major pathway of the repair of DNA double strand breaks. There are seven important factors involved in NHEJ. In the original model, Ku70/Ku80 heterodimer binds to DNA break ends and DNA-PKcs/Artemis cleans up the DNA overhanging ends. After XRCC4 is recruited and as a template for Ligase IV binding, Ligase IV joins the two DNA break ends. XRCC4 forms dimer or tetramer in the cell and the dimerization of XRCC4 is required for Ligase IV binding. XLF was identified recently. XLF has been shown structural similarity to XRCC4. As XRCC4, XLF also form homodimer and improves the DNA recognizing ability in the NHEJ. The hypothesis now favors the XRCC4, XLF and Ligase IV form a complex for DNA binding instead of the original proposed XRCC4/Ligase IV complex. In this thesis, I have overexpressed specific proteins by transfection of plasmids into 293T cells and tested the binding activity by co-immunoprecipitation and western blotting. The result shows that the formation of the homodimer of both XLF and XRCC4 does not compete with the formation of the XLF/XRCC4 heterodimer. The minimal sequence of XRCC4 critical for XRCC4 dimerization and XLF binding has been mapped at the region of first 134 amino acids of XRCC4. Further deletion loses both XRCC4 dimerization and XLF binding activity. This result suggests that the XRCC4 dimerization and XLF binding are concomitant. SIRT2 controls cellular proliferation and is a mitotic checkpoint protein that functions in the early metaphase to prevent chromosomal instability. SIRT2 blocks the entry of mitosis in the presence of the cyclin B/cdc2 activity. XRCC4 binds to SIRT2 primary at the G2/M phase (Lee et al, submitted). In this thesis, the result shows that XLF also binds to SIRT2. The binding between XRCC4 and SIRT2 does not disrupt the binding of XRCC4 to XLF. Furthermore, SIRT2 enhances the binding of XLF to XRCC4. Therefore, SIRT2 might bind to XLF and XRCC4 as a complex and provides a regulatory point between the cell cycle control and NHEJ repairing machinery.