Analysis by two-dimensional gel electrophoresis of topoisomers of plasmids containing d(pCpG). 'd(pCpG). inserts, in which n ranges between 8 and 21, shows that the B-to-Z transition within the alternating C-G is readily induced by negative supercoiling and is highly cooperative. The free energy parameters for the transition in dilute aqueous buffers have been evaluated from a statistical mechanical analysis of the data, and these parameters allow prediction of the superhelicities of plasmids at which the transition occurs in alternating C-G inserts over a wide range of lengths. In agreement with the crystal structures, the helical handedness of the B structure in solution and that of the Z structure are shown to be opposite to each other. Furthermore, it is found that the B form of the alternating C-G sequence in solution has a helical periodicity of 10.5 ± 0.1 base pairs per turn, and the Z form has a helical periodicity of 11.6 ± 0.3 base pairs per turn. There also appears to be a significant unwinding of the right-handed DNA duplex at each of the B/Z junctions.Extensive studies of the left-handed Z-DNA structure have been carried out since the determination of this structure by Rich and co-workers (for recent reviews, see refs. 1-3). The energetics of the interconversion between the left-handed Z form and the right-handed B form under physiological conditions, however, has so far received only cursory attention (4). In the present communication, we report a detailed analysis of the dependence of the B-to-Z transition on DNA supercoiling. A set of plasmnids containing d(pCpG)n-d(pCpG),, inserts, in which n = 8, 12, 16, and 21, has been constructed. For each plasmid, two-dimensional agarose gel electrophoresis has been used to examine simultaneously the mobilities of a group of topological isomers (topoisomers) that differ only in their linking numbers. The extent of the B-to-Z transition of the alternating C-G insert in each of the topoisomers can be measured from the two-dimensional electrophoretic patterns. A statistical mechanical analysis of the average number of base pairs that have flipped from the right-handed B to the left-handed Z structure as a function of linking number has enabled us to determine the free energy parameters for this transition. We have also applied the band-shift method (5, 6) to study the helical structure of the alternating C-G inserts; the results indicate that negative supercoiling of the DNA changes the alternating C-G sequence from a right-handed helix with 10.5 base pairs (bp) per turn to a left-handed helix with 11.6 bp per turn under physiological conditions. MATERIALS AND METHODS Plasmid Construction. Inserting 32 bp of alternating d(pCpG) into the filled-in BamHI site of pBR322 to generate the plasmid pLP32 has been described (7). The plasmid pLP332 was constructed by inserting the BamHI fragment containing the alternating C-G sequence into the BamHI site of pTR161 (8) (0.16-cm2) sample wells, 6 cm apart, were formed with a Delrin template that was held paralle...

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Negative supercoiling of plasmid DNAs containing 24--42 base pairs of alternating d(pCpG) inserts is shown to cause the flipping of the helical hand of the inserts from right to left under physiological conditions. For a negatively supercoiled DNA with a fixed linking number, this flipping reduces its superhelicity and, therefore, is accompanied by a shift of its electrophoretic mobility in agarose gel. Quantitation of the mobility shifts indicates that essentially the whole stretch of contiguous alternating d(pCpG) flips to the left-handed helical form when the negative superhelical density (specific linking difference) is greatest than or equal to 0.03.

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