Abstract
Damaged DNA-binding (DDB) activity comprises two major protein components, damaged DNA-binding protein 1 (DDB1) and 2 (DDB2). Those are implicated in the repair of ultraviolet (UV) radiation–induced DNA damage. To assess the functional correlation between DDBs and UV-damaged-DNA recognition activity, we identified UV-damaged-DNA recognition activities in rodent cell lines. There is a cell type-dependent expression of DDB1 and DDB2. Rodent cells had less abundant DDBs and lower UV-damaged-DNA recognition activity than did human tumor cells. Interestingly, the profusion of DDBs is associated with UV-damaged-DNA recognition activity in these rodent cell lines. We also discovered rat tissue-dependent expression of DDBs and its functional correlation with UV-damaged-DNA recognition activity. Moreover, the rat DDB1 cDNA (3850 nucleotides) from rat brain cDNA library was isolated. It contained the complete length of the open reading frame that encodes an 1140-amino-acid polypeptide with a predicted molecular weight of 126.8 kDa. The predicted protein size from the rat ddb1 gene resembles that from human DDB1 (127 kDa). Rat DDB1 shares highly conserved sequencing (greater than 98% similarity) with those of mouse, human, and monkey. Rat and fruit fly DDB1 exhibit 62.23% identity and 57.66% homology, respectively. The evolutionary conservation of the DDB1 sequence suggests that DDB1 may play a pivotal role in mammals as well as in other eukaryotes. However, overexpression of DDB1 through a recombinant rat ddb1 adenovirus did not augment UV-damaged-DNA recognition, whereas overexpressing DDB2 through a recombinant human ddb2 adenovirus partly restored the recognition activity of rodent cells. To examine the DDB2-modulated DDB activity involving in DNA repair and UV sensitivity, we established DDB2-overexpressing hamster V79 cell line that was stable transfectted with full-length open reading frame of human ddb2 cDNA. 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 parent and DDB2-overwxpressing cells demonstrated no difference in the reactivation of plasmid DNA carrying UV damage. These results suggest that DDB2 may proficiently involve in repair of bulky genomic DNA damage. DDB2-overexpressing cells also displayed resistance to UV-induced apoptosis and cytotoxicity. Moreover, the possible role of DDB2 as a determinant of cellular sensitivity to UV was investigated. DDB2-depleted cells were established by stable transfection of the resistant cells with DDB2 antisense cDNA. The cells that have depletion of DDB2 protein can restore cellular sensitivity to UV-induced apoptosis. Whereas the extent of UV-induced activation of apoptosis executioners, including DNA fragmentation factor, and caspase-3 were reduced in the UV-resistant cells compared with those apparent in the sensitive cells, depletion of DDB2 from the resistant cells restored the normal activation patterns for these proteins. Our findings indicate that DDB2 potentiates DNA repair and protects cells from UV-induced cytotoxicity and apoptosis. These results also suggest that DDB2 involved in the development of UV resistance. In cisplatin-damaged DNA recognition protein study, we found that high mobility group binding protein 1 (HMGB1) DRP activity was reduced with the extent of cell resistance to cisplatin. Induction of PKC activity by TPA lowered DRP activity of HMGB1 both in sensitive and resistant cells. However, inhibition of PKC activity by sphingosine or staurosporine failed to decrease HMGB1 activity in both cells. The result indicates a possible mechanism, through the phosphorylated HMGB1 reduce its damaged DNA DRP activity that resulted in cells proficient to resistant cisplatin.