Nucleic Acids Research, 1980, Vol. 8, No. 5 1145-1165
© 1980
Articles |
The early melting of closed duplex DNA: analysis by banding in buoyant neutral rubidium trichloroacetate
Department of Microbiology, Health Sciences Center, State University of New York at Stony Brook Stony Brook, NY 11794, USA
Received November 13, 1979.
Aqueous RbTCA permits the buoyant banding of both native and denatured DNA at room temperature and neutral pH. A unique property of this solvent is the buoyant resolution of closed circular, underwound DNA (I) from the corresponded nicked (II) species. Conditions are reported here in which PM-2 DNA I is physically resolved from native PM-2 DNA II, the buoyant separation being 1.27 ma/ml in 3.3 H RbTCA at 25°C. The separation between nicked and closed DNAs increases with temperature up to 35.5°C, at which PM-2 DNA II cooperatively melts and subsequently pellets. The isothermal buoyant density of a closed DNA increases linearly as the linking number (Lk) of the closed DNA decreases. The early melting of closed DNA may be monitored with high precision by buoyant banding in RbTCA, it being possible to detect the disruption of as few as 40 base pairs in PM-2 DNA (10,000 base pairs). The constraint that the linking number be conserved in closed DNA requires that a change in duplex windina be accompanied by a compensating change in suoer-coilina. We estimate the linking number deficiency of PM-2 DNA I to be 0.094 turns per decibase pair. This result permits the estimation of the EtdBr unwinding angle, ø, by comparison with alternative determinations of the linking number deficiency which depend upon the value of ø. The result obtained here is that ø = 27.7° ± 0.5° and is approximately independent of temperature over the range 15° – 35°.
Present address: Department of Biochemistry and Biophysics, University of California Medical Center, San Francisco, California 94143.