Supercoiled DNA folded by nonhistone proteins in cultured mouse carcinoma cells.
Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with gamma-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.
D015112:4-Nitroquinoline-1-oxide / Q000494:pharmacology; D000167:Acriflavine / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000378:metabolism* / Q000528:radiation effects; D004278:DNA, Superhelical / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D004996:Ethidium / Q000494:pharmacology; D005720:Gamma Rays; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism; D051379:Mice; D009690:Nucleic Acid Conformation; D011402:Pronase / Q000494:pharmacology; D011485:Protein Binding
D002868:Chromosomal Proteins, Non-Histone; D004273:DNA, Neoplasm; D004278:DNA, Superhelical; D003609:Dactinomycin; D000167:Acriflavine; D015112:4-Nitroquinoline-1-oxide; D011402:Pronase; D004996:Ethidium