Ca(2+)-mediated inhibition of a nuclear protein that recognizes UV-damaged DNA and is constitutively overexpressed in resistant human cells: DNA-binding assay.
A nuclear protein that recognizes UV-damaged DNA was detected from HeLa cells using DNA-binding assay. Treatment of cells with Ca2+ ionophore (A23187) caused a dramatic inhibition of the damage-recognition activity. In contrast, in vitro treatment of nuclear extracts with agents that affect protein conformation (such as urea, NP40 and Ca2+) did not significantly affect on the damage-recognition activity. The Ca(2+)-mediated inhibition of UV damage recognition was reconstituted by the addition of the cytosolic extracts, suggesting that the Ca2+ effect does not directly act on the UV damage-recognition protein. The expression of the detected nuclear protein was increased in UV-resistant HeLa cells. In contrast, the level of this protein was dramatically reduced in UV-sensitive xeroderma pigmentosum group A cells. In addition, UV damage-recognition protein is resistant to RNase, and is independent of the previously identified proteins that bind cisplatin-DNA adduct. These findings implied that the recognition of UV-DNA adduct is modulated by the intracellular level of Ca2+.
Chao
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CC
Department of Biochemistry, Chang Gung Medical College, Taoyuan, Taiwan, Republic of China.
Huang
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Lin-Chao
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D001667:Binding, Competitive; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival; D002945:Cisplatin / Q000494:pharmacology; D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D004351:Drug Resistance; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D011487:Protein Conformation; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000001:Calcimycin; D004247:DNA; D002945:Cisplatin; D002118:Calcium