Studies on the mechanism of pathway choice during repair of ionizing radiation induced DNA double strand breaks

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Ionizing radiation (IR) generates adverse effects, such as cell killing or carcinogenesis, by inducing DNA double strand breaks (DSBs). DSBs are severe lesions eliciting in cells the "DNA damage response (DDR)": a network of signal transduction pathways that extensively modify cell metabolism and set the stage for DSB processing, cellular adaptation, programmed cell death or senescence. DSBs are processed efficiently in cells. Yet, DSBs are linked to severe biological effects because their processing is associated with mutations and chromosomal translocations. The present project investigates how the DSB processing apparatus underpins these effects. Four pathways, operating on distinct principles, process DSBs: Homologous recombination repair (HRR), canonical non-homologous end joining (c-NHEJ), alternative end joining (alt-EJ) and single strand annealing (SSA). Surprisingly, only HRR is able to faithfully restore the genome while all remaining pathways, including c-NHEJ that is enzymatically dominant, tolerate/catalyze mutations and translocations. Thus, the choice of pathway determines whether genomic alterations accompany DSB processing. Here, we formulate and test a novel hypothesis regarding DSB repair pathway choice promising to advance our understanding regarding logic and necessities inherent to DSB processing. The central question is: How cells choose between c-NHEJ and HRR? We provide evidence that Ku translocation away from the DNA end and mediated by DNA-PK autophosphorylation in the initial steps of c-NHEJ, changes Ku protein conformation and induces an interaction with Mre11 that triggers resection and HRR. We show that under certain conditions well over 50% of DSBs may be processed by HRR making such a mechanism highly relevant to genomic integrity. A large set of promising preliminary experiments guide the development of three work packages testing interactions between Ku and Mre11 in vitro and in vivo, and analyzing their connections to the c-NHEJ- and HRR-apparatus, as well as to checkpoint responses. The proposed molecular characterization of the DSB processing apparatus will advance our understanding of genomic instability sources and will accelerate the development of strategies to improve radiation protection and radiation therapy.