Template supercoiling during ATP-dependent DNA helix tracking: studies with simian virus 40 large tumor antigen.
Incubation of topologically relaxed plasmid DNA with simian virus 40 (SV40) large tumor antigen (T antigen), ATP, and eubacterial DNA topoisomerase I resulted in the formation of highly positively supercoiled DNA. Eukaryotic DNA topoisomerase I could not substitute for eubacterial DNA topoisomerase 1 in this reaction. Furthermore, the addition of eukaryotic topoisomerase I to a preincubated reaction mixture containing both T antigen and eubacterial topoisomerase I caused rapid relaxation of the positively supercoiled DNA. These results suggest that SV40 T antigen can introduce topoisomerase-relaxable supercoils into DNA in a reaction coupled to ATP hydrolysis. We interpret the observed T antigen supercoiling reaction in terms of a recently proposed twin-supercoiled-domain model that describes the mechanics of DNA helix-tracking processes. According to this model positive and negative supercoils are generated ahead of and behind the moving SV40 T antigen, respectively. The preferential relaxation of negative supercoils by eubacterial DNA topoisomerase I explains the accumulation of positive supercoils in the DNA template. The supercoiling assay using DNA conformation-specific eubacterial DNA topoisomerase I may be of general use for the detection of ATP-dependent DNA helix-tracking proteins.
Yang
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Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Jessee
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Lau
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Zhang
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Liu
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics* / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011092:Polyethylene Glycols / Q000494:pharmacology; D013539:Simian virus 40 / Q000235:genetics*; D013698:Templates, Genetic
D000952:Antigens, Polyomavirus Transforming; D004278:DNA, Superhelical; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D004264:DNA Topoisomerases, Type I