Small Molecule And Protein Modifiers of The NHEJ Pathway

Non-Homologous End Joining (NHEJ) is a pathway for the repair of DNA doube-strand breaks (DSBs) that is active throughout the cell cycle and does not use a template to direct the repair. NHEJ is dependent upon the Ku70/80, DNA-PKcs,XRCC4, Ligase IV, and XLF proteins, and deficiency in any one of these factors results in failure of DSB repair by NHEJ. Additionally, proteins and small molecules have been shown to contribute to NHEJ. Deficiencies in these so-called “accessory” factors generally results in reduced NHEJ efficiency, rather than loss of NHEJ function. This dissertation examines the roles of two types of NHEJ accessory molecules, and the work presented here represents two studies. The first study focused on the small molecule myo-inositol hexakisphosphate (IP6), which has previously been shown to contribute to NHEJ in vitro. A biotinylated version of IP6 (IP6-bio) was developed and characterized as a possible tool to study the role of IP6 in NHEJ and other cellular processes. IP6-bio was similar to native IP6 in its ability to contribute to NHEJ in vitro and interacted with the required NHEJ factor Ku70/80, which had previously been reported to bind IP6. IP6-bio was not bound by other NHEJ factors. We concluded from the se data that IP6-bio can be used as a tool to study the role of IP6 in NHEJ. IP6-bio was also shown to interact with the known IP6-binding protein casein kinase 2 (CK2). Binding of IP6-bio by established IP6-binding proteins Ku70/80 and CK2 indicated that the critical determinants for IP6—binding were present in IP6-bio, and suggested that IP6-bio might be used to identify new IP6-binding proteins. Competition experiments using IP6 and less phosphorylated inositol phosphates as competitors showed that IP6-bio-binding proteins were preferentially competed off by IP6 and not by other inositol phosphates. These data indicate that proteins that bound IP6-bio may be bona fide IP6-binding proteins. Along these lines, one protein from the HEK293 extract that bound IP6-bio was the nucleolar protein nucleolin, which has been shown to participate in chromatin remodeling to facilitate DSB repair. Taken together the results of this study demonstrate that IP6-bio can by synthesized and used as a tool to investigate the role of IP6 in cellular processes, and to identify new IP6-binding proteins. The second study investigated the role of the small proteins encoded by the Cell Cycle Regulator of NHEJ (CYREN) gene in NHEJ. CYREN-deficient cells had reduced end-joining and increased sensitivity to agents that cause DNA damage that requires NHEJ for repair. Additionally, extracts prepared from CYREN-deficient cells had reduced NHEJ activity in vitro. CYREN-encoded proteins were found to physically interact with the required NHEJ protein XLF. Further, CYREN-XLF interaction impacted DNA-binding by XLF, and reduced XLF-XRCC4-DNA interactions. We interpret these data as a role for CYREN in regulating the number of DNA ends that are processed during NHEJ to minimize error. Overall, this study identifies CYREN-encoded proteins as participants in NHEJ and proposes a mechanism for the biochemical role played by CYREN-encoded proteins.