Purification of nuclear proteins that bind to cisplatin-damaged DNA. Identity with high mobility group proteins 1 and 2.
The biochemical processes responsible for the recognition and repair of cisplatin-damaged DNA in human cells are not well understood. We have developed a damaged DNA affinity precipitation technique that allows the direct visualization and characterization of cellular proteins that bind to cisplatin-damaged DNA. The method separates damaged DNA-binding proteins from complex radiolabeled cell mixtures and further resolves them into individual polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique is complementary to gel retardation and Southwestern blotting analyses that have been previously used to identify cellular components that specifically bind to cisplatin-damaged DNA. Using this technique, we have characterized a set of HeLaS3 nuclear proteins of 26.5, 28, 90, and 97 kDa that specifically bind to cisplatin-DNA adducts. Competition studies with soluble cisplatin-damaged DNA confirmed these findings. The major cisplatin-damaged DNA-binding proteins of 26.5 and 28 kDa recognized adducts of DNA modified with cisplatin but not with its trans-isomer or with UV radiation. These proteins were purified 450-fold to near homogeneity by ion-exchange and cisplatin-damaged DNA affinity chromatography. Amino-terminal sequence analysis showed that the 26.5- and 28-kDa proteins were identical to high mobility group (HMG) proteins HMG-2 and HMG-1, respectively.
Hughes
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Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Engelsberg
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BN
Billings
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PC
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002945:Cisplatin / Q000633:toxicity*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D006609:High Mobility Group Proteins; D009687:Nuclear Proteins; D004247:DNA; D002945:Cisplatin