BACKGROUND: Ultraviolet radiation (UV) damaged-DNA binding (DDB) activity comprises two major components: damaged-DNA binding protein 1 (DDB1) and 2 (DDB2). Although the function of DDB is unclear, mutation on DDB2 is associated with cellular sensitivity to a variety of genotoxic agents including UV. It has been suggested that DDB2 may play a role in UV-induced DNA repair. However, evidence that DDB2 involves in DNA repair and UV sensitivity is lacking. METHODS: To examine the role of DDB2, we established DDB2-overexpressing hamster V79 cell lines, V79ddb2, by stable transfection with full-length open reading frame of human ddb2 cDNA. Cells were irradiated with UV and determined its DNA repair activity by testing the remaining photoproducts on the chromatin and measuring the plasmid reactivation, respectively. UV induced cytotoxicity was determined by the colorimetric assay (MTT assay), and apoptotic cells exhibiting morphological features of chromatin condensation and nuclear fragmentation were counted after 4-diamidino-2-phenylindole (DAPI) staining. RESULTS: DDB activity was increased in DDB2-overexpressing cell lines. Analysis on DNA repair indicated that UV photoproducts were removed in a time-dependent manner and there was greater than 50% of damage removed within 12 h in DDB2-overexpressing cells. In contrast, nearly all the damage remained unrepaired in V79 cells. However, using bacterial CAT gene as a reporter, both V79 and V79ddb2 cells demonstrated no difference in the reactivation of plasmid DNA carrying UV damage. These results suggest that DDB2 may involve in repair of bulky genomic DNA damage. Although a maximum of only 30% of apoptosis was induced, UV irradiation caused a dose-dependent apoptosis and cytotoxicity in these cell lines. V79ddb2 cells displayed resistance to UV-induced apoptosis and cytotoxicity. CONCLUSION: Our findings indicate that overexpression of DDB2 in V79 cell potentiates DNA repair and protects cells from UV-induced cytotoxicity. These results also suggest that DDB2 may be involved in the development of UV resistance.
BACKGROUND: Ultraviolet radiation (UV) damaged-DNA binding (DDB) activity comprises two major components: damaged-DNA binding protein 1 (DDB1) and 2 (DDB2). Although the function of DDB is unclear, mutation on DDB2 is associated with cellular sensitivity to a variety of genotoxic agents including UV. It has been suggested that DDB2 may play a role in UV-induced DNA repair. However, evidence that DDB2 involves in DNA repair and UV sensitivity is lacking. METHODS: To examine the role of DDB2, we established DDB2-overexpressing hamster V79 cell lines, V79ddb2, by stable transfection with full-length open reading frame of humanddb2 cDNA. Cells were irradiated with UV and determined its DNA repair activity by testing the remaining photoproducts on the chromatin and measuring the plasmid reactivation, respectively. UV induced cytotoxicity was determined by the colorimetric assay (MTT assay), and apoptotic cells exhibiting morphological features of chromatin condensation and nuclear fragmentation were counted after 4-diamidino-2-phenylindole (DAPI) staining. RESULTS: DDB activity was increased in DDB2-overexpressing cell lines. Analysis on DNA repair indicated that UV photoproducts were removed in a time-dependent manner and there was greater than 50% of damage removed within 12 h in DDB2-overexpressing cells. In contrast, nearly all the damage remained unrepaired in V79 cells. However, using bacterial CAT gene as a reporter, both V79 and V79ddb2 cells demonstrated no difference in the reactivation of plasmid DNA carrying UV damage. These results suggest that DDB2 may involve in repair of bulky genomic DNA damage. Although a maximum of only 30% of apoptosis was induced, UV irradiation caused a dose-dependent apoptosis and cytotoxicity in these cell lines. V79ddb2 cells displayed resistance to UV-induced apoptosis and cytotoxicity. CONCLUSION: Our findings indicate that overexpression of DDB2 in V79 cell potentiates DNA repair and protects cells from UV-induced cytotoxicity. These results also suggest that DDB2 may be involved in the development of UV resistance.