Immunodetection of DNA-protein crosslinks by slot blotting.
Ultraviolet light, formaldehyde, cis-diamminedichloroplatinum(II), chromate (Cr6+), or chromium chloride (Cr3+) under the appropriate conditions caused the formation of DNA-protein crosslinks in intact Chinese hamster ovary (CHO) cells or in cell nuclei. The DNA-protein crosslinks were isolated, applied to nitrocellulose filters, and reacted with antibodies to nuclear proteins. An antiserum to a 97-kD nuclear protein detected p97-DNA complexes in CHO nuclei and cell cultures treated with UV light, cis-Pt and formaldehyde. Exposure to Cr3+ induced p97-DNA crosslinks only in isolated nuclei, while chromate (Cr6+) treatment resulted in significant crosslink formation only in intact cells. Analysis of western blots with the p97 antiserum indicated that crosslinks induced by formaldehyde or ultraviolet light required DNAase I digestion of DNA for migration of the p97 complexes into the gel. In contrast, the 97-kD antigen from the metal-induced crosslinks was released from DNA and resolved in the gel when 2-mercaptoethanol was included in the electrophoresis sample buffer. Assay of slot blots with an antihistone monoclonal antibody indicated that formaldehyde, but not cis-Pt or chromate, crosslinked histones to the DNA. These results illustrate the utility of immuno-slot blots in detecting and characterizing DNA-protein complexes induced by diverse chemical and physical agents.
Miller
C A
CA
Department of Environmental Medicine, New York University School of Medicine, NY 10016.
Costa
M
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D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002840:Chromates / Q000494:pharmacology*; D002945:Cisplatin / Q000494:pharmacology*; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents; D004247:DNA / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D005260:Female; D005557:Formaldehyde / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000378:metabolism*; D010053:Ovary; D011506:Proteins / Q000378:metabolism*; D014466:Ultraviolet Rays
D002840:Chromates; D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D011506:Proteins; D005557:Formaldehyde; D004247:DNA; D002945:Cisplatin