Inhibition of UV-damaged DNA recognition activity in HeLa cells by calcium ionophore A23187: potential involvement of cytosolic proteins.
A UV-damage recognition protein (UVDRP) was detected from HeLa nuclear extracts using an in vitro DNA-binding assay. Treatment of cells with a Ca2+ ionophore (A23187) caused a dramatic inhibition of the UVDRP binding activity from nuclear extracts. In the absence of EDTA, addition of cytosolic extracts to the binding reaction effectively blocked the UVDRP binding activity, associated with an increased nuclease activity. However, pretreatment of cytosolic extracts with proteinase destroyed a majority of the inhibitory effect on UVDRP binding activity. Using extracts from A23187-treated cells, a similar conclusion was drawn except that the inhibitory effect of cytosolic extracts on UVDRP binding was completely diminished by proteinase K. In addition, inclusion of the cytosolic extracts in the binding reaction did not alter the UVDRP binding dissociation pattern by specific competitors. These results suggest the potential involvement of cytosolic proteins, probably through a "all-or-none" action mechanism, in the recognition of UV-damaged DNA of HeLa cells.
Chao
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CC
Department of Biochemistry, Chang Gung Medical College, Taoyuan, Taiwan, Republic of China.
Huang
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SL
D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004249:DNA Damage; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014466:Ultraviolet Rays
D015342:DNA Probes; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000001:Calcimycin; D004492:Edetic Acid; D002118:Calcium