Local Unwinding of DNA during RNA Synthesis in vitro

Abstract

IT is not possible to use conventional methods to determine whether the DNA template unwinds during transcription of RNA, because they would not be sensitive enough to detect the very small amounts of denaturation which would be expected. We have therefore used a recently developed sensitive method which is based on the unwinding of DNA which takes place with formaldehyde^1,2. For DNA with locally denatured regions or defects, that is segments with locally disrupted stacking interaction between the base pairs, the kinetic curve of DNA unwinding under the action of formaldehyde is a straight line when plotted against the axeswhich follows the equationv is the fraction of the hydrogen bonded base pairs at time t, c is the concentration of defects (c=1/n̄ where n̄ is the mean number of base pairs in the helical region interposed between two adjacent defects), v and v are the kinetic constants of the reaction of DNA with formaldehyde. Plotting the experimental data on the above axes gives the intercept I=2vc. The constant v is determined from special calibration experiments with shear degraded DNA of known molecular weight. The defect concentration c can then b readily obtained from the relation c = I=/2v.