Thus, we conclude that WRAP53 protects cells against spontaneous DNA damage

Thus, we conclude that WRAP53 protects cells against spontaneous DNA damage. conversation and accumulation of RNF8 at DSBs. In this manner, WRAP53 controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53 impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53 as a novel regulator of DSB repair by providing a scaffold for DNA repair factors. gene encodes a regulatory RNA (WRAP53) that is produced by usage of an alternative start point for transcription. Although this RNA controls the response of p53 to cellular stress, WRAP53 acts independently of WRAP53 and does not play a role in the regulation of p53 (Farnebo 2009; Mahmoudi et al. 2009). Aberrations in WRAP53 have been linked to several genetic disorders. For example, inherited mutations in WRAP53 that affect its WD40 domain name cause dyskeratosis congenita, a disorder involving bone marrow failure, premature aging, and cancer predisposition (Zhong et al. 2011). Iopamidol Moreover, SNPs in or altered expression of the protein itself are associated with elevated risk for a variety of sporadic tumors and radioresistant head and neck malignancy cells, hematoxicity, Rabbit Polyclonal to K6PP and disturbed DNA repair in workers exposed to benzene (Garcia-Closas et al. 2007; Lan et al. 2009; Schildkraut et al. 2009; Mahmoudi et al. 2011; Medrek et al. 2013; Garvin et al. 2014). Furthermore, patients with spinal muscular atrophy, a neurodegenerative disorder that is the leading genetic cause of Iopamidol infant mortality worldwide, exhibit loss of WRAP53 function (Mahmoudi et al. 2010). Intriguingly, neurodegeneration, aging, and cancer are all processes linked to accumulation of DNA damage. Although this suggests a role for WRAP53 in DNA repair, this role remains unknown. It is noteworthy in this context that WRAP53 has been identified in several proteomic and Iopamidol genome-wide siRNA screens designed to detect factors associated with DDR (Matsuoka et al. 2007; Paulsen et al. 2009; Adamson et al. 2012). These links, together with WRAP53s function as a scaffold protein, prompted us to inquire whether WRAP53 is involved in the assembly of repair factors at sites of DNA damage and whether loss of this function impairs DNA DSB repair. Results WRAP53 is usually recruited to sites of DNA damage in an ATM-, H2AX-, and MDC1-dependent manner To elucidate the involvement of WRAP53 in the DDR, we initially laser-microirradiated U2OS cells and observed a rapid relocalization of WRAP53 to DNA lesions. WRAP53 was present at DNA lesions within a few minutes (Fig. 1A), placing this protein high upstream in the DNA damage signaling cascade. This localization of WRAP53 at DNA damage sites was observed in other cell types, including human fibroblasts and H1299 lung cancer cells, and with five different antibodies against WRAP53 (Supplemental Fig. 1A,B). One of the WRAP53 antibodies, mouse monoclonal -WDR79 clone 1F12, revealed formation of WRAP53 foci in response to ionizing radiation (IR) as well as enrichment of WRAP53 in Cajal bodies, confirming its reliability (Supplemental Fig. 1C). Furthermore, the WRAP53 foci clearly overlapped with H2AX, and the staining was specific, since it Iopamidol could be eliminated by siRNA oligos targeting WRAP53 (Fig. 1B). These WRAP53 foci appeared rapidly following exposure to IR and were dissolved gradually over a period of 24 h, a time course similar to that of H2AX foci (Fig. 1C). Open in a separate window Physique 1. WRAP53 accumulates at sites of DNA damage in an ATM/H2AX/MDC1-dependent manner. (= 3; (***) 0.001 as determined by Students and then immunostained for RNF168 and conjugated ubiquitin (with the FK2 antibody). In the case of GFP-RNF8 staining, following treatment with oligonucleotides for 24 h, the cells were transiently transfected with the GFP-RNF8 plasmid for 8 h, exposed to IR (6 Gy), allowed to recover for 1 h, and then fixed and analyzed. (and as the percentage of 200 cells counted in each experiment whose nuclei contained IRIF. In the case of GFP-RNF8, only successfully transfected cells were counted. (= 3; (**) 0.01; (***) 0.001, as determined by Students shows the percentage of 100 GFP transfected cells in each experiment whose nuclei were 53BP1-positive. The error bars depict the SEM. = 3; (*) 0.05, as determined by Students showing the percentage of nuclei containing 10 H2AX foci (= 200). (= 3; (*) 0.05; (**) 0.01; (***) 0.001, as determined by Students gene. Following I-SceI cleavage, the gene was removed, and repair of these DSBs by NHEJ placed a promoter adjacent to the GFP gene.