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Energetics of B-To-Z Transition In Proc. Nati. Acad. Sci. USA Vol. 80, pp. 6206-6210, October 1983 Biochemistry Energetics of B-to-Z transition in DNA (DNA supercoiling/DNA helical periodicity/two-dimensional electrophoresis/statistical mechanical treatment/structure in solution) LAWRENCE J. PECK AND JAMES C. WANG Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138 Communicated by Robert L. Baldwin, July 11, 1983 ABSTRACT Analysis by two-dimensional gel electrophoresis ternating C-G sequence into the BamHI site of pTR161 (8). of topoisomers of plasmids containing d(pCpG).'d(pCpG). inserts, Plasmids pLP316, pLP324, and pLP342 were constructed by in which n ranges between 8 and 21, shows that the B-to-Z tran- direct ligation of the phosphorylated octamer d(pCpG)4 into sition within the alternating C-G is readily induced by negative the filled-in BamHI site of pTR161. The nucleotide sequences supercoiling and is highly cooperative. The free energy param- of the appropriate insert-containing restriction fragments were eters for the transition in dilute aqueous buffers have been eval- determined according to the procedures of Maxam and Gilbert uated from a statistical mechanical analysis of the data, and these (9). parameters allow prediction of the superhelicities of plasmids at DNA which the transition occurs in alternating C-G inserts over a wide topoisomers containing different linking numbers were range of lengths. In agreement with the crystal structures, the prepared by relaxation of the DNA with calf thymus topoisom- helical handedness of the B structure in solution and that of the erase I in the presence of various amounts of ethidium bromide Z structure are shown to be opposite to each other. Furthermore, as described (6). Several samples with different ranges of link- it is found that the B form of the alternating C-G sequence in so- ing numbers were pooled to give a mixture with a broader dis- lution has a helical periodicity of 10.5 ± 0.1 base pairs per turn, tribution of topoisomers. and the Z form has a helical periodicity of 11.6 ± 0.3 base pairs Electrophoresis. Agarose gel electrophoresis was performed per turn. There also appears to be a significant unwinding of the in horizontal gel slabs immersed in the electrophoresis buffer. right-handed DNA duplex at each of the B/Z junctions. For two-dimensional electrophoresis, 500 ml of melted agarose in TBE buffer (90 mM Tris-boric acid, pH 8.3/2.5 mM Extensive studies of the left-handed Z-DNA structure have been Na2EDTA) was cast over a 1-foot-square (930-cm2) glass plate carried out since the determination of this structure by Rich with laboratory tape around its edges. Two 1/16-inch-square and co-workers (for recent reviews, see refs. 1-3). The ener- (0.16-cm2) sample wells, 6 cm apart, were formed with a Delrin getics of the interconversion between the left-handed Z form template that was held parallel to the top edge of the gel. Six and the right-handed B form under physiological conditions, microliters of a sample, containing a total of about 0.5 ,ug of however, has so far received only cursory attention (4). In the DNA topoisomers plus 3% Ficoll and approximate amounts of present communication, we report a detailed analysis of the tracking dyes, was loaded in each well through a glass micro- dependence of the B-to-Z transition on DNA supercoiling. A pipette with a flame-drawn tip. Electrophoresis in the first di- set of plasmnids containing d(pCpG)n-d(pCpG),, inserts, in which mension was typically 20 hr at 110 V for a 0.7% gel. The gel was n = 8, 12, 16, and 21, has been constructed. For each plasmid, then soaked in a clean tray containing 2 liters of TBE plus chlo- two-dimensional agarose gel electrophoresis has been used to roquine (10); equilibration takes 6-8 hr with gentle agitation. examine simultaneously the mobilities of a group of topological The final concentration of chloroquine was about 1.3 ,uM for isomers (topoisomers) that differ only in their linking numbers. the gels shown. After soaking, the buffer was transferred into The extent of the B-to-Z transition of the alternating C-G insert the electrophoresis tank, and the gel was turned 900 from its in each of the topoisomers can be measured from the two-di- original position. Electrophoresis in the second dimension was mensional electrophoretic patterns. A statistical mechanical typically 18 hr at 100 V for a 0.7% gel. The gel was then soaked analysis of the average number of base pairs that have flipped in several changes of deionized water to remove the chloro- from the right-handed B to the left-handed Z structure as a quine. Staining in water plus ethidium bromide at 1 ,ug/ml for function of linking number has enabled us to determine the free 2 hr was followed by extensive destaining in several changes of energy parameters for this transition. We have also applied the water before photographing. band-shift method (5, 6) to study the helical structure of the A smaller 8-inch-square (413-cm2) gel was used for gels run alternating C-G inserts; the results indicate that negative su- in TBE plus either sodium chloride or hexammine cobalt(III) percoiling of the DNA changes the alternating C-G sequence chloride. The electrophoresis buffer was recirculated through from a right-handed helix with 10.5 base pairs (bp) per turn to a 4-liter external buffer reservoir. In addition, the gel was cooled a left-handed helix with 11.6 bp per turn under physiological by running tap water underneath the Plexiglas platform under conditions. the gel. The electrophoresis voltage for 1.7% gels was about twice as high as that for 0.7% gels. MATERIALS AND METHODS Plasmid Construction. Inserting 32 bp of alternating d(pCpG) RESULTS into the filled-in BamHI site of pBR322 to generate the plasmid Dependence of the B-to-Z Transition on Supercoiling as pLP32 has been described (7). The plasmid pLP332 was con- Revealed by Two-Dimensional Gel Electrophoresis. Resolu- structed by inserting the BamHI fragment containing the al- tion of DNA topoisomers of different linking numbers by two- dimensional gel electrophoresis (11) was carried out by first The publication costs of this article were defrayed in part by page charge running a mixture of topoisomers along one edge of a slab agar- payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: bp, base pair(s). 6206 Downloaded by guest on September 23, 2021 Biochemistry: Peck and Wang Proc. Natl. Acad. Sci. USA 80 (1983) 6207 ose gel. Electrophoresis in the second dimension was carried hundred base pairs, the definition of ao needs modification be- out after soaking the gel in the electrophoresis buffer contain- cause it deviates significantly from the average linking number ing an appropriate concentration of chloroquine (10). Fig. 1 Left of the completely relaxed DNA. This point will be discussed illustrates the results of such an experiment with a control plas- elsewhere. The linking difference of each topoisomer under mid pTR161. The topoisomers are resolved into distinct spots the second-dimension conditions can also be evaluated, but this that lie along a smooth curve. As shown in Fig. 1 Left, the link- quantity is not needed here.) The apices of the two arcs in- ing numbers of the spots decrease progressively as their enu- dicated by arrows I and II in Fig. 1 Left correspond to the min- meration increases: the linking number a of spot 6, for ex- ima in electrophoretic mobilities in the first and second di- ample, is lower than that of spot 5 by 1. mension, respectively. Apex I represents the position of a relaxed The two-dimensional analysis is particularly convenient for DNA under first-dimension electrophoresis conditions (in the examining DNA supercoiling-induced structural changes. Fig. TBE buffer at room temperature). The spots to the right of I 1 Right illustrates the pattern obtained with plasmid pLP332, are positively supercoiled during the first-dimension electro- which is pTR161 containing a d(pCpG)16&d(pCpG)16 insert. The phoresis, whereas those to the left are negatively supercoiled. negative supercoiling-induced B-to-Z transition of the inserted The quantity a - a' for spot 6, for example, is -2.5. alternating C-G sequence is revealed by a sharp break, around The second essential quantity that can be measured is the spot 17, in the curve that traces through the topoisomer spots. change in the twist number, ATw, when a transition, such as This break is due to the presence of base pairs in the left-handed the B-to-Z transition studied here, occurs. As we have done helical conformation within the C-G insert in the more nega- previously (7), in the region where the topoisomers differ in tively supercoiled members of the mixture during electropho- ther gel mobilities we assume that a pair of topoisomers with resis in the first dimension. For a given negatively supercoiled the same gel mobility has the same writhing number Wr. It then topoisomer, the flipping of the helical hand of the C-G insert follows from the relationship a Lk = Tw + Wr, in which Lk reduces its negative superhelicity and hence its gel electro- is the linking number (15, 16), that ATw = Aa for two topo- phoretic mobility (4, 7, 12). Upon binding of chloroquine prior isomers of the same Wr. The pair of spots 12 and 17 in Fig.
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