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Purpose: Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy.We hypothesized that inhibiting DNA damage repair would sensitize tumors to radiation-induced DNA damage.Experimental Design: A novel strategy for inhibiting DNA repair was tested.We designed small DNA molecules that mimic DNA double-strand breaks (called Dbait), trap DNA-PK complex and act by disorganizing damage signaling and DNA repair.Results: We show that stable 32bp long Dbait, induce pan-nuclear phosphorylation of DNA damage response (DDR) components such as H2AX, Rpa32, Chkl, Chk2, Nbsl and p53.However, cellular response to Dbait differs from the response to irradiation by different ways: (i) it depends only on DNA-PK kinase activity and not on ATM, (ii) the phosphorylation signal lasts several days and (iii) it distributes in the treated population in an "all-or-none" response, depending on a Dbait concentration threshold.Irradiation of Dbait treated cells impaired localization of Nbsl, BP53 and Rad51 at repair foci and inhibited non homologous end joining as well as homologous recombination.Altogether, our results suggest that the hyperactivation of DNA-PK by Dbait induces a "false" DNA damage signaling that fools DNA repair system.In vivo, Dbait administration induces regression of radioresistant head and neck squamous cell carcinoma (Hep2), melanoma (SK28 and LU1205) and Glioblastoma (U87) tumors.The combination of Dbait treatment and fractionated radiotherapy significantly enhanced the therapeutic effect.Tumor growth control by Dbait molecules depends directly on the dose and is observed with various irradiation protocols.The induction ofH2AX phosphorylation in tumors treated with Dbait suggests that it acts in vivo through the induction of"false" DNA damage signaling and repair inhibition.Conclusions: This new strategy of inhibition by "false" damage signal molecules could provide a new method for enhancing radiotherapy efficiency in radio-resistant tumors.