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. Author manuscript; available in PMC: 2010 Jun 25.

Published in final edited form as: Oncogene. 2010 Apr 19;29(24):3509–3518. doi: 10.1038/onc.2010.108

The SIM peptide inhibits NHEJ. (a) The SIM peptide sensitizes WT and BRCA1-deficient (BRCA1^−/−) mouse ES cell lines, but not a Ku-deficient (Ku70^−/−) cell line, to radiation. Cells were transfected with WT-SIM-2r, SC-SIM-2r or a vector cDNA, and irradiated (10 Gy) or not irradiated. Cell viability was detected 72 h after treatment and normalized to cells transfected with an empty expression vector. (b, c) Schematic representations of fluorescence-based assays for measuring HR or NHEJ DSB repair. (b) After DSBs are generated by I-SceI in cells carrying the DR-GFP reporter, HR uses the downstream GFP sequence (truncated GFP) as a template, which restores the coding sequence of functional GFP. The panel on the right shows the results of repair of DR-GFP in GFP-positive cells from mouse ES cells co-transfected with an I-Sce I expression vector and one of the following: WT-SIM-2R, SC-SIM-2r or expression vector (JS74). (c) EJ5-GFP contains a promoter that is separated from a GFP coding region by a puro gene that is separated by two I-SceI sites. Once DSBs are generated by I-SceI and the puro gene is excised by NHEJ repair, the promoter is joined to the rest of the expression region, leading to restoration of functional GFP. The graph on the upper right panel shows the frequency of repair of EJ5-GFP (resulting in GFP-positive cells) in WT and Ku70^−/− ES cells co-transfected with an I-SceI expression vector and one of the following: WT-SIM-2r, SC-SIM-2r or vector control. P-values are indicated. The graph on the lower right panel shows the comparison of the effect of a single-SIM and double-SIM constructs on DNA DSB repair by NHEJ using a GFP-based chromosomal reporter in WT ES cells. (d) The WT-SIM-GFP (WT) or the VI-SIM-GFP (VI) mutant control peptide was immunoprecipitated by an anti-FLAG antibody, followed by western blotting with an anti-Ku70 antibody. A band corresponding to the molecular weight of SUMOylated Ku70 was repeatedly identified. The lower panel shows 10% of the input for immunoprecipitation, stained with Coomassie blue. DSB, double-strand break; ES, embryonic stem; GFP, green fluorescent protein; HR, homologous recombination; NHEJ, non-homologous end joining; SC, scrambled; SIM, SUMO-interaction motif; SUMO, small ubiquitin-like modifiers; WT, wild type.

The SIM peptide inhibits NHEJ. (a) The SIM peptide sensitizes WT and BRCA1-deficient (BRCA1^−/−) mouse ES cell lines, but not a Ku-deficient (Ku70^−/−) cell line, to radiation. Cells were transfected with WT-SIM-2r, SC-SIM-2r or a vector cDNA, and irradiated (10 Gy) or not irradiated. Cell viability was detected 72 h after treatment and normalized to cells transfected with an empty expression vector. (b, c) Schematic representations of fluorescence-based assays for measuring HR or NHEJ DSB repair. (b) After DSBs are generated by I-SceI in cells carrying the DR-GFP reporter, HR uses the downstream GFP sequence (truncated GFP) as a template, which restores the coding sequence of functional GFP. The panel on the right shows the results of repair of DR-GFP in GFP-positive cells from mouse ES cells co-transfected with an I-Sce I expression vector and one of the following: WT-SIM-2R, SC-SIM-2r or expression vector (JS74). (c) EJ5-GFP contains a promoter that is separated from a GFP coding region by a puro gene that is separated by two I-SceI sites. Once DSBs are generated by I-SceI and the puro gene is excised by NHEJ repair, the promoter is joined to the rest of the expression region, leading to restoration of functional GFP. The graph on the upper right panel shows the frequency of repair of EJ5-GFP (resulting in GFP-positive cells) in WT and Ku70^−/− ES cells co-transfected with an I-SceI expression vector and one of the following: WT-SIM-2r, SC-SIM-2r or vector control. P-values are indicated. The graph on the lower right panel shows the comparison of the effect of a single-SIM and double-SIM constructs on DNA DSB repair by NHEJ using a GFP-based chromosomal reporter in WT ES cells. (d) The WT-SIM-GFP (WT) or the VI-SIM-GFP (VI) mutant control peptide was immunoprecipitated by an anti-FLAG antibody, followed by western blotting with an anti-Ku70 antibody. A band corresponding to the molecular weight of SUMOylated Ku70 was repeatedly identified. The lower panel shows 10% of the input for immunoprecipitation, stained with Coomassie blue. DSB, double-strand break; ES, embryonic stem; GFP, green fluorescent protein; HR, homologous recombination; NHEJ, non-homologous end joining; SC, scrambled; SIM, SUMO-interaction motif; SUMO, small ubiquitin-like modifiers; WT, wild type.