Date of Award
Doctor of Philosophy (PhD)
Cancer and Developmental Biology
Peter J. McKinnon, Ph.D.
Tiffany N. Seagroves, Ph.D. David J. Solecki, Ph.D. J. Paul Taylor, M.D., Ph.D. Jian Zuo, Ph.D
The DNA damage response (DDR) orchestrates a network of cellular processes such as cell cycle progression, DNA repair, and apoptosis when complex DNA lesions arise to maintain genomic integrity. ATM, ATR, and DNA-PKcs (encoded by PRKDC) are related phosphatidylinositol-3-kinase like serine/threonine kinases (PIKK) that collectively regulate the DDR network. Studies have demonstrated these kinases can phosphorylate many of the same substrates, suggesting a significant potential for functional redundancy. However, deficiencies in these kinases have been linked to distinct neural degenerative and developmental disorders, underscoring their unique functions for maintain genomic integrity during nervous system development.
Here we utilized mouse genetic analyses to identify the functional interplay between these kinases during neurogenesis. DNA-PKcs function is directly involved in and most associated with the non-homologous end-joining (NHEJ) pathway. The function of DNA-PKcs during neurogenesis remains unclear despite evidence linking mutations in related NHEJ factor genes to neurological diseases. For example, deficiency in Ku70, Ku80, XRCC4, or DNA ligase IV (Lig4), but not DNA-PKcs results in defective embryonic neurogenesis in mice. This discrepancy may arise from the fact that ATM and ATR can compensate for the loss of DNAPKcs. For instance, if ATM and DNA-PKcs were capable of functioning redundantly in neural tissue, then DNA-PKcs-null mice would not necessarily have a noticeable phenotype. Determining functional redundancy is difficult since Atr germ line and [Atm;Prkdc] double-null mice are embryonic lethal. To overcome these challenges, we used mice with germ line Prkdc inactivated in combination with conditional alleles for Atm and Atr to assess if these kinases function cooperatively in the DDR during neural development, consequently defining the role(s) of DNA-PKcs during neurogenesis.
We found DNA-PKcs loss sensitized DNA damage induced p53-dependent apoptosis, and exacerbated checkpoint activation after ionizing radiation (IR) in a developmental stage and neural cell type-specific manner, independent of ATM and ATR. Our data suggests, during neurogenesis DNA-PKcs functions as a component of the DNA-PK holoenzyme to maintain genomic integrity in proliferating and non-proliferating neurons. We propose DNA-PK specifically enhances NHEJ DNA double-strand break repair kinetics during murine neurogenesis by acting as a scaffold protein, which is critical to maintain normal nervous development when high levels of genotoxic stress occur. In contrast, we found that ATM and ATR coordinated the DDR during neurogenesis to direct DNA damage induced apoptosis in proliferating and non-proliferating cortical neural progenitors. Furthermore, we found ATR controlled the IR-induced G2/M checkpoint, independent of ATM and DNA-PKcs.
In summary, this work established a basic understanding of DNA-PKcs function during nervous system development with respect to ATM and ATR. Importantly, our data implicates DNA damage induced p53-dependent apoptosis can be activated in the absence of all three PIKK DNA damage-signaling kinases during murine neurogenesis. The human neurodegenerative disease ataxia telangiectasia (A-T) is thought to arise from mutations in ATM preventing the elimination of DNA damaged neurons during neurogenesis, which then fail to function appropriately, resulting in neural degeneration. It is thought murine models fail to recapitulate the neurodegenerative disease observed in humans because mice are more resistant to DNA damage. The observation of a PIKK-independent DNA damage induced apoptotic process occurring during murine neurogenesis suggests murine embryonic neurons posses a pathway to eliminate DNA damaged cells even in the absence of ATM, ATR, and DNA-PKcs. Therefore, murine models may fail to recapitulate A-T, not because mice are more resistant to DNA damage, but because they have an alternative mechanism to eliminate DNA damaged neurons during neural development. The emphasis on mouse genetics to dissect the important processes of these kinases during neurogenesis was an important step to ensure the data collected illustrated the DDR within a biological context. Overall, our work illustrates the divergent functions of these kinases, despite substrate overlap, to show how they play unique and essential cooperative roles during the DDR, underscoring the distinct neuropathology that develops when each is defective.
Enriquez-Rios, Vanessa D. , "Integration of ATM, ATR, and DNA-PKcs Signaling Maintains Genome Integrity During Neurogenesis" (2015). Theses and Dissertations (ETD). Paper 72. http://dx.doi.org/10.21007/etd.cghs.2015.0085.