Development of Novel Technologies to Investigate DNA Double-Strand Break Repair Uncovers a Role for the ATM Kinase in Error-Free NHEJ with Implications for Neurodegenerative Diseases

Abstract

DNA double strand breaks (DSBs) are considered to be the most lethal genotoxic lesion because they can result in chromosomal translocations or result in a major loss of genetic information if repaired incorrectly. To preserve genomic integrity, mammalian cells have evolved a set of complimentary and redundant repair pathways that faithfully repair DSBs. Consequently, eukaryotic cells utilize an evolutionary conserved set of protein kinase signaling pathways that recognize and respond to DNA damage by pausing cell cycle progression and recruiting DNA repair machinery to ultimately determine the fidelity of DSB repair. Mutations and/or acquired defects that compromise the function of DNA damage response (DDR) pathways result in enhanced mutagenesis and underlie the development and progression of cancer and neurodegenerative conditions. How cells chose which DSB repair pathways to use when fixing a DSB in order to maximize repair fidelity is incompletely understood. To better understand how cells decide which repair pathway to use when fixing DSBs and to specifically investigate protein kinase signaling that coordinates DSB repair pathway selection, we developed a set of multicolor fluorescent reporter systems, named DSB-Spectrum and DSB-Prism. DSB-Prism is uniquely designed to report on the choice between DSB repair via error-free non-homologous end joining (EF-NHEJ), mutagenic end joining (mut-EJ), alternative end joining (alt-EJ), homologous recombination (HR), and single strand annealing (SSA) at a single break created within individual cells by CRISPR-Cas9. We demonstrate that DSB-Prism robustly reveals patterns of DSB repair pathway compensation following chemical inhibition or genetic perturbation of DDR repair factors. We report that the majority, but not all, EF-NHEJ repair requires DNA-PKcs. We observed that DNA-PKcs kinase activity is essential for its function in EF-NHEJ repair, while autophosphorylation of DNA-PKcs on the previously mapped ABCDE phosphorylation site cluster plays only a minor role in this process primarily through the Ku80 DNA-PKcs long range synaptic complex. We utilized DSB-Prism to uncover a novel role for the ATM kinase in promoting EF-NHEJ repair at highly transcribed genes. We show that ATM promotes EF-NHEJ repair via two genetically distinct pathways independently of DNA-PKcs kinase signaling. First, ATM promotes EF-NHEJ through a phosphorylation-dependent interaction between 53BP1 and RIF1 independently of the Shieldin and CST complexes, which we propose serves to physically hold DSB ends together in a redundant manner with the core NHEJ-mediated end synapsis machinery. Second, we propose that ATM promotes EF-NHEJ via promoting R-loop resolution by both SETX and ERCC6L2. We show that the role of ATM in promoting EF-NHEJ is largely independent of MRN-dependent ATM activation, and completely independent of ROS-dependent ATM activation. We discover a novel N-terminal set of positively charged residues that we propose directly interact with the negatively charged DNA phosphate backbone adjacent to DSBs in order to activate ATM. These N-terminal positively charged residues, in combination with MRN, promote binding of ATM to chromatin but are particularly important for ATM’s function in promoting EF-NHEJ repair within the DSB-Prism reporter. Finally, we characterized a cohort of ATM patient mutations and observe that the ability of ATM mutants to promote EF-NHEJ perfectly correlates with patient clinical A-T disease severity. We propose that this loss of EF-NHEJ repair is a major mechanistic cause of Purkinje cell death and cerebellar neurodegeneration observed in A-T patients.