To maintain genomic stability, cells have developed an elaborate DNA damage response (DDR) system, which is responsible for sensing DNA damage, halting the ongoing cell cycle, and repairing DNA damage. Failure to detect and repair DNA damage leads to genomic instability, which in turn can drive tumorigenesis. Many human genetic cancer predisposition syndromes are linked to defective DDR. For example, mutations in the BRCA1 gene were found in about 50% of familial breast cancer cases. Because individual tumors often have unique defects in the DDR pathway, insight into the basic mechanisms by which cells repair different DNA lesions could also guide individualized therapy. A promising example is the use of PARP inhibitors in cancers with BRCA1 and BRCA2 mutations. On the other hand, many studies suggest that overexpression of DNA repair factors contributes to resistance to radiochemotherapy. Therefore, studying this pathway has important implications in cancer pathogenesis and cancer therapy. UFM1 is the most recently identified Ub-like proteins. The cellular function of ufmylation remains unclear. Here we show for the first time that Ufm1 signaling is involved in the DNA damage response. We found that the E3 ligase UFL1 is important for ATM activation and Ufl1 deficiency sensitizes prostate cancer cells to radiation. In addition, UfSP2, which cleaves Ufm1 from target proteins, inhibits ATM activation and its overexpression sensitizes cells to DNA damage. Based on these Preliminary Data, we hypothesize that UFL1 and UfSP2 are two new factors in the DNA damage response. UFL1 promotes ATM activation and DDR, while UfSP2 has a opposite function. Because UFL1 and UFM1 are deleted in 20% of prostate cancer, we further hypothesize that defective UFM1 signaling would sensitize prostate cancer to radiation. In this application, we will further explore how UFL1 and UfSP2 regulates NBS1, ATM activation and DDR. In addition, we will examine how UFL1 and UfSP2 themselves are regulated. We will also test the role of UFL1 and UfSP2 in radiosensitivity using clinically relevant models. Our Specific Aims are: Aim 1. Investigate the regulation of ATM signaling by Ufmylation; Aim 2. Study the regulation of UFL1 and UfSP2 by DNA damage signaling; Aim 3. Investigate the role of UFM1 signaling in prostate cancer therapy. Our studies will comprehensively reveal a novel function of UFL1 signaling in the DNA damage response and radiosensitivity. In addition, it will reveal a new therapeutic strategy based on synthetic lethality in treating prostate cancer cells, especially those with deletion of UFL1 or UFM1. !