Steven M. Stirdivant, Jan Klysik and Robert D. Wells- Energetic and Structural Inter-relationship between DNA Supercoiling and the Right- to Left-handed Z Helix Transitions in Recombinant

8/3/2019 Steven M. Stirdivant, Jan Klysik and Robert D. Wells- Energetic and Structural Inter-relationship between DNA Super

1/7

THE OURNAL F BIOLOGICALHEMISTRYPrinted in U.S A .Vol. 257, No. 7, Issue of September 10, p. 10159-10165, 982

Energetic and Structural Inter-relationship betweenand the Right- o Left-handed Z Helix Transitions nPlasmids*DNA SupercoilingRecombinant

(Received for publication, February 16, 1982)

Steven M. Stirdivant$, Ja n IQysikg, and Robert D. Wells8From the Departmentof Biochemistry, University of Wisconsin, Madison, College of Agr icu ltu ral andLife Sciences,Madison, Wisconsin 53706

We have evaluated the B to Z conformational tran-sitions in supercoiled recombinant plasmids containingdifferent lengths of (dC-dG) described in the precedingpaper. The sodium hloride-induced right- t o left-handed transition in a small segment of the plasmidscaused a relaxation of (-) supercoils which was moni-tored by electrophoretic mobilityhanges of individualtopoisomers on agarose gels containing NaCl at con-centrations up t o 5.0 M. The number of supercoils re-laxed was proportiona l to the length of the (dC-dG)segment in the plasmid in good agreement with theo-retical values. A sho rt B/Z junction region (


2/7

10160 Left-handedD N A in Plasmidsof buffer-equilibrated phenol at an appropriate ime interval. AUreaction products were extracted with phenol and dialyzed exhaus-tively uersus 10 r n ~ris-HC1 (pH 8.0), 0.1m~ EDTA.Isolation of Topoisomers-Individual topoisomers of the variousplasmids were isolated by eluting the topoisomer bands from agarosegelsusing the chaotropic agents NaI or KI (15, 16). Topoisomerpopulations prepared as described above were electrophoresed on a2% agarose slab gel in a buffer of 40 r n ~ris, 20 mM sodium acetate,2 mM EDTA (final pH 8.3). Exposure of the plasmid DNA in the gelto ethidium bromide and UV irradiation caused significant nickingand linearization. Therefore, the two outside lanes of the gel were cutoff, and the DNA was visualized by ethidium bromide staining. Th emigration positions of the topoisomers were marked and used to alignthe cutt ing of the unstained portion of the gel. The DNA was elutedby dissolving the agarose in saturated NaI or KI (3 l/g of agarose),and the mixture was filtered over Whatman GF/C filters (16). NaI(Mallinkrodt Chemical Works) gave less nicking than KI (Mallink-rodt) in this procedure, but the yields were approximately equivalent(20-509) with both salts.The DNA was eluted from the GF/C filterswith 5 mM Tris-HC1 (pH 8.0), 0.5 mM EDTA. The topoisomers weredialyzed into 10m~ Tris-HCI, 0.1 mM EDTA. Fig. 1shows the resultsof a topoisomer isolation of pRW751. In general the different samplescontain only one topoisomer or one dominant topoisomer and one ortwo minor contaminants. Nicking and linearization are minimal. 2-5pg of a single topoisomer can be isolated from one agarose slab gel bythis procedure.Agarose Gel Electrophoresis a t Hig h NaCl Concentrations-l-2% agarose gels (Sigma Type I) were prepared in 1.0-5.0 M NaCl (asindicated under Results), containing 80 m~ Tris, 40 m~ sodiumacetate, 4 m~ EDTA, (final pH 8.3). The gels were 0.5 mm thick tominimize the high current and heat generation which was inherentunder these high ionic strength conditions. For gels containing con-centrations higher than 3.0 M NaCI, the agarose was prepared in 3.0M NaCl, and thegels were pre-electrophoresed for 24 h in the desiredNaCl concentration before the samples were loaded. The gels withNaCl concentrations below 3.0 M were prepared at th edesired NaClconcentrations and were pre-electrophoresed for 2-3 h before loading.The ionic conditions and pH were maintained by circulating largequantities of buffer (4 liters) through t he system; the 4 iters ofcirculating buffer were replaced every 24 h. DNA samples wereprepared for loading by lyophilization to dryness and were resus-pended in 3 pl of electrophoresis buffer with NaCI04 substituted forNaCl at the indicated molar concentrations. Samples were equili-brated in the loading buffer for 1-2 h before loading. Electrophoresistimes ranged from 24 tc 120 h, depending on th e NaCl concentrationand the concentration of agarose. After the NXE,the gels were stainedby desalting with distilled water for 1 h and then stained with 10 pg/ml of ethidium bromide and visualized under UV irradiation. The

FIG. 1. Electrophoretic analysis f ndividual topoisomers ofpRW751. pRW751 topoisomer samples isolated as described underMaterials and Methods were electrophoresed on a 1.5% agarose gelin 40 m~ Tris-HC1, 20 mM sodium acetate, 2 r n ~DTA (pH 8.3).The two outside lanes contained a full population of pRW751 topo-isomers asmarkers.

results obtained were independent of the NaCI04 concentration inwhich the samples were loaded. Reproducibility of results is excellentin all NaCl concentrations employed. However, the visual quality ofthe gels declines with higher NaCl concentrations and longer electro-phoresis times.RESULTS

Total Relaxa t ion of (dC-dG)-containing Plasmids-Neg-atively supercoiled ovalently closed plasmids containinglengths of (dC-dG ) sequen ce will undergo a chan ge in thelinking difference, and thu sa loss in the superhelical density,if the (dC-dG) sequences undergo a conformational changefrom a B t o a Z structure. The oss of (-) supercoils shou ld bepropor t iona l to the lengthf the (dC-dG) sequence presentnthe plasmid. T o de te rmine the numb erof supercoils lost perunit length f (dC-dG ), ndividual topoisomersof the plasmidspRW751, pRW755, pRW756, and pRW757 were electropho-resed in low and high NaCl conc entratio ns. The chan gen t helinking difference is determin ed by co mp aring the differencein the num ber of supercoils between the (dC-dG)-containingplasmid and a control plasmid at bo thow salt concentrat ionsand at concentrat ions above the cooperative transit ion. Thecontrol plasmids are approxim ately the same size and havevir tually the same sequence (>99.5% identical) as the (dC-dG)-containing plasmids but contain no stretchesf (dC-dG).Fig. 2 show s the relative migration positions of th e (dC-dG)-con taining topoisom ers RW751, pRW755, pRW756, andpRW757 relativeoontrolopoisomers pRW451 andpRZ4032 when electrophoresed in 20 mMNa. Fig. 3 showsthe relat ive m igration osit ions of the sam eplasmid topoiso-mers electrophoresed in 4.5 M NaC1. Comparison of the dif-ference in the nu mb er of supercoils present in the (dC-dG)-containing topoisomers relat ive to the control topoisomera tboth high and low NaCl concentrations gives the maximumsupercoils relaxed du e to th e - t ransition in the (dC-dG)segments. T he nu mb er of supercoils relaxed for pRW751 is10.5; pRW755, 4.7; pRW756, 5.7; and pRW757, 0 upercoils.At 4.0 M NaCl the same extent of relaxation was observedindicating that themaximum relaxation had been reached.Table I summ arizes these data and comp ares the experi-A B C D E F G H I J

FIG. 2. Electrophoretic mobility ofplasmid s c ontaini ng dif-feren t leng ths f (dC-dG)under normal alt condition s. Individ-ual topoisomers and marker topoisomer populations were electropho-resed on a 1.58 agarose gel at 25 C in 40 mM Tris-HCI, 20mM sodiumacetate, 2mM EDTA (pH 8.3) to determine the difference in numberof supercoils in control and (dC-dG)-containing plasmids. The lanescontain: A, pRW751 isolated topoisomer; B , pRW451 marker popu-lation; C, pRW451 isolated topoisomer; D, RW755 isolated topoiso-mer; E, G, nd I , pRZ4032 marker population; F, pRZ4032 isolatedtopoisomer; H , pRW756 isolated topoisomer; J, pRW757 isolatedtopoisomer. Some (+) supercoiled topoisomers are seen between (-)supercoiled ones in Lanes E , G , and I .


3/7

Left-handed D N A in Plasmids111

10161I

A B CI1

A B CB C D E

TABLEMaximum relaxation for plasmids with different lengths of (dC-dG)The reproducibility indetermining the experimental relaxation was

Plasmid Length of dC- Calculated relax- Experimental re-kO.1 supercoil turn.

dG) tract ation laxationbP A supercoils A supercoils at 25 "CpRW751 58 10.4pRW755 26 4.7pRW756 32pHW757

10.54.75.7 5.71.8 0mentally determined relaxation with the theoretical relaxationbased on he number of (dC-dG) residues in the homopolymerblocks. The calculated relaxation assumes tha t all of the (dC-dG) residues in the homopolymer segments are converted toa Z conformation while all other sequences in the plasmidremain in a B conformation. With the exception of pRW757,the observed number of supercoils removed by the B -* Ztransition agrees with that predicted. This extent f relaxationalso rules out the possibility that the relaxation is due tocruciform formation (17,18) n the (dC-dG) blocks sincecruciforms would remove only a little more than half thatnumber of supercoils. The fact tha t pRW757, containing only10bpof perfectly alternating (dC-dG), does not undergo arelaxation at 4.5 M NaCl and at the superhelical densitiestested demonstrates that shorter (dC-dG) stretches are lesslikely to convert from B toZ in a plasmid.

Effect of Temperature on the Total elaxation-The totalrelaxation of pRW751 varied with the temperature of theelectrophoresis gel. Table I1 lists the total relaxation observedfor pRW751 n4.5 M NaCl at four different temperaturesobtained by varying the wattage of the electrophoresis or theambient temperature. The temperatures are nly approximatefor the gels run a t 2.8 and 4.0 watts; however, it is clear thatincreasing the temperature decreases the number of supercoilsrelaxed a t 4.5 M NaCl. A 5.0 M gel run at 35-40 "C showed thesame extent of relaxation as the 4.5 M gel run at the sametemperature. Furthermore, the topoisomer used in this set ofexperiments (16 supercoils at 3.0 M NaCl if no B -* Z transi-tion) is completely relaxed at 3.0-3.5 M NaCl at 25 "C (seeFig. 8). It has been observed that the temperature effect ismore pronounced at NaCl concentrations close to the mini-mumnecessary to complete the full relaxation. Thus, the

FIG. . Maximal relaxations ofplasmids containing differentlengths of (dC-dG) in high NaCl. Thesame plasmids and topoisomer popula-tions shown in Fig. 2 were electropho-resed in 4.5 M NaCl on 1.5% agarosegelsat 25 OC.The lanes are:GelZ,A,pRW751topoisomer;B , pRW451 marker popula-tion; C , pRW451 topoisomer. Gel II,A ,pRW755 topoisomer; B , pRZ4032marker population;C , pRZ4032 topoiso-mer. GelZIZ,A, pRW756 topoisomers;B ,pRZ4032 marker population; , pR7A032topoisomer; D, pR7A032marker popu-lation;E , pRW757 topoisomer.

TABLE1Effectof temnerature on the maximum relaxation of p RW751Ge l NaCl concen- Watts Approximate Supercoils re-tration temperature laxed

M "CA 4.5 4.0 45-50 9.7B 4.5 2.8 35-40 10.1C 4.5 2.1 25 10.5D 4.5 2.0 4 10.6E 5.0 3.0 35-40 10.2

effect does not appear to be a simple case of shifting theequilibrium of the B+ Z transition.NaCl Concentration Necessary to Initiate the B + ZTransition as a Function of Superhelical Density-Right-handed (-) supercoils contribute a favorable free energy toany process which involvesn unwinding of the primary helix.Therefore, increasing (-) superhelical density should lowerthe NaCl concentration necessary to cause the B+ Z struc-tural transition. To study thiseffect, we have used gel electro-phoresis at different NaCl concentrations to determine thenumber of supercoils necessary to facilitate the start of thetransition for pRW751 at a given NaCl concentration. Deter-mining the minimum NaCl concentration necessary to causea relaxation for an individual topoisomer is a technical prob-lem since a large number of gels would be equired. Therefore,populations of pRW751 topoisomers were run a t differentNaCl concentrations. A t a given NaClconcentration, the morehighly supercoiled topoisomers will be relaxed while thosewith an insufficient number of supercoils will not undergo arelaxation. This determines the minimum number of super-coils necessary o begin the B -* Z transition at a given NaClconcentration.An example of this type of analysis using populations ofpRW751 is shown in Fig. 4. These data also validate the useof topoisomer populations. Identical DNA samples were elec-trophoresed in 2.0 and 2.33 M NaCl. A population of pRW751obtained by relaxation of the plasmid by nick-closing enzymein 2.0p g / d of ethidium bromide was electrophoresed in LaneB of both gels. This relaxation results in a Boltzman distri-bution of 8 observable topoisomers. In the presence of 2.0 or2.33 M NaCl, the more highly supercoiled topoisomers werepartially relaxed. We define the start of the B -* Z transitionas a loss of a t least 0.5 supercoil resulting from the presenceof the (dC-dG) sequences. In the 2.0 M gel, the most highly


4/7

10162 Left-handedD N A in Plasmids2.0 M NaC l 2.33 M N a C l

A B C D A B C D

supercoiled topoisomer migrated at 8.5 supercoils referencedto the pRW451 marker population. Thus, we deduce that 9supercoils are the minimum necessary to achieve a partialrelaxation at 2.0 M NaC1. At 2.33 M NaCl, the most highlysupercoiled topoisomer migrated at 7.5 supercoils. Therefore,8 supercoils are required to induce a relaxation.To confm that theesults obtained with these populationswere valid, individual topoisomers were electrophoresed inLanes A and D on both gels. If no B+ Z transition occurred,the major topoisomer in Lane A will migrate a t 7 supercoils,while the minor contaminants migrate at 6 and8 supercoils atthese ionic strengths. The topoisomer electrophoresed inLaneD migrated at 8supercoils in the absenceofa B+ Z transition.In 2.0 M NaCl, the topoisomers in Lane A show no relaxation,migrating a t 6, 7, and 8 supercoils. However, at 2.33 M NaC1,the number 8 topoisomer was relaxed and co-migrated withtopoisomer 7 in the 7 supercoil position. In Lane D at 2.0 M,the topoisomer migrated at 8 supercoils demonstrating thatno relaxation occurred. At 2.33 M NaCl, this number 8 topo-isomer migrated ashaving 7 supercoils, clearly confuming theobservation in Lane A. Since the individual topoisomers dem-onstrated that a plasmid with 8 supercoils was partially re-laxed at 2.33 M, he result obtained with the population oftopoisomers is validated.Fig. 5 shows the results obtained from analyses performedas n Fig. 4 with pRW751 topoisomer populations electropho-resed a t different NaCl concentrations from 1.25 to 4.5 M.Forthe range of superhelical densities examined, there is a linearrelationship between the superhelical density and theln[NaCl] necessary to begin the B+ Z transition. The equa-tion for the line is: 7 = 10.6 ln[NaCl] + 16.7 (7 equals thenumber of supercoils).

NaCl Titration of the B + Z Transition-To determinethe extent of relaxation due t,o the B to Z transition, singletopoisomers of pRW751were electrophoresed at differentNaCl concentrations. The extent of relaxation was determinedby comparing the migration of pRW751 to themigration of apRW451 control topoisomer. Typical da ta atone of the NaClconcentrations tested (1.85 M ) are shown inFig.6. ThepRW751 topoisomer in Lane A, which would contain 12.6supercoils if no B to Z transition had occurred, lost 4.7 super-coils. The topoisomer in Lane E, which would have 9.6 super-coils if no B to Z transition had occurred, lost 0.8 supercoil.

FIG.4. Determination of the min-imum number of supercoils neces-s a r y to cause a partial relaxation ata specific NaCl concentration. Iden-tical plasmids and topoisomer popula-tions were electrophoresed in 2.0 and2.33 M NaCl at 25 "C. For both gels thelanes are: A, pRW751 topoisomers mi-grating at 6, 7, and 8 supercoils at theseNaCl concentrations in the absence of aB + Z transition; B , a population ofpRW751 obtained by reacting with nick-closing enzyme in the presence of 2 pg/ml of ethidium bromide; C, a completepopulation of pRW451 topoisomers; D,apRW751 topoisomer migrating at 8supercoils at these NaCl concentrationsin the absence of a B + Z transition.

I n [ N o C I ]FIG.5. Relationship of plasmid superhelical density and theNaCl concentration necessary to initiate the relaxation ofpRW751. A series of pRW751 topoisomer populations were electro-phoresed at different NaCl concentrations at 25 "C. nalysis of the

resultsas n Fig. 4 ields the relationship shown. Th ey axis representsthe minimum numberof supercoils which must be present in order toobtain a relaxation at the NaCl concentration of the electrophoresis.The line through the points is a least squares fit of the data.Thus, the topoisomers still migrate as distinct bands in theseintermediate states of relaxation although the bands are notquite assharp as hose found when he relaxation is at its endpoint. Similar determinations wereperformed on isolatedtopoisomers of pRW751 at NaCl concentrations from 1.25-5.0

Fig. 7 shows the number of (-) supercoils present in 1topoisomer of pRW451 and 4 topoisomers of pRW751 atdifferent NaCl concentrations. The pRW751 topoisomersshow a decrease in the number of supercoils as the NaClconcentration increases. The control pRW451 topoisomer hasa slight increase in the number of supercoils present between

M.


5/7

Left-handedD N A in PLasmids 101631.85M N a C l

A B C D E

- N

FIG.6. Typical electrophoresis of pRW751 topoisomers re-vealing ntermediate elaxation tates. Twoopoisomers ofpRW751 and pRW451 control topoisomers were electrophoresed in1.85M NaCl at 25 "C. Lane A, pRW751 topoisomer 10 Lanes B and0,ull range of pRW451 topoisomers; Lane C, pRW451 topoisomer5; Lane E , pRW751 topoisomer 7. The topoisomers are named by thenumber of (-) supercoils which would be present at 3.0 M NaCl if noB + Z transition had occurred. N designates the migration positionof nicked plasmid.

\\

I I I I I2.0 3.0 4.0 5.0

[NaCI ] MFIG.7. Number of supercoils in pRW751 and pRW451 topo-isomers at different NaCl concentrations. pRW751 topoisomerswith different numbersof supercoils were electrophoresed along withpRW451 control topoisomers at 25 "C at different NaCl concentra-tions as in the legend to Fig. 6. The number of (-) supercoils in the

sitions to th e complete ladder of pRW451 topoisomers. 0,pRW751individual topoisomers w as determined by comparing migration po-topoisomer 16;A , pRW751 topoisomer 13;0,pRW751 topoisomer 10,0 ,pRW751 topoisomer 7;.,RW451 topoisomer 5. Topoisomers arenumbered by the scheme described in the legend to Fig. 6.1.0 and 3.0 M NaCl. Beyond 3.0 M, no further increase wasobserved. Thus , the numberf supercoils present for RW751a t a given NaCl concentration reflects the exte nt of th e B +2 transition in the (dC-dG) blocks as well as the small non-sequence specific ionic effect on thewhole plasmid.The number of supercoils relaxed at various NaCl concen-trations due to theB --., 2 transition in the (dC-dG) blocks in

different pRW751 topoisomers s presented in Fig. 8. Th e zerorelaxation data poin ts are derived from Fig. 5. These pointsrepresent theNaCl concentration at which a topoisomer withn supercoils would begin the B4 transition if in fact it hadn + 1 supercoils. It is assumed that this is the highest NaClconcentration at which the topoisomer with n supercoils hasnot begun the transition.

The most striking featuref the titrati on datas the biphasicnature of th e curves. The first plateau of the trans itionoccursafter a loss of 5.5-6 supercoils. Thus, we conclude th at th e 2-bp (dC-dG) block undergoes the transition before the 26-bpblock he maximum elaxation due o he 32-bp block inpRW756 is 5.7 whe reas the relaxation resulting from th e 26-bp block in pRW755 is only 4.7. The fa ct that opoisomers 16and 13 both have the sameaximum relaxation demonstratesthat the tot al relaxation does not vary with the superhelicaldensity.For topoisomers 10and 7, a full relaxation was not obta inedeven at 5.0 M NaCl. Topoisomer 7 does not even begin th esecond phase of the trans ition.Fig. 7 shows that bothopoiso-mers 10 and 7 have a small numbe r of supercoils at 5.0 MNaCl. It would app ear tha t at ow superhelical densities thefree energy of supercoiling is insufficient to obtaina completetransition in bo th (dC-dG) blocks. T hese results, along withprevious observations (1) with pRW751 topoisomers of evenlower superhelical density, demonstrate that theB+ Z tran-sitionwi not induce left-handed+) supercoils in t he plasmid.pRW751 has never been observed to go from a negatively toa positively supercoiled state as a result of the B+ Z transitionregardless of the NaCl concentration or superhelical density.This suggests tha t the energy derived from negative super-coiling is critical to the stability of the Z conformation in acovalently closed plasmid. By the same reasoning, positivesupercoiling must be very unfavorable energetically with re-gard to 2 DNA stability.

[NaCI ] MFIG.8. Extent of relaxation versus NaCl concentration fordifferent topoisomers of pRW751. A series of pRW751 topoiso-mers w as electrophoresed on agarose gels at 25 "C at different NaClconcentrations. Comparison of the migration positions of these to-poisomers relative toa control pRW451 topoisomeras in Fig. 6 yieldsthe results shown. The number of supercoilsrelaxedfor a givenpRW751 topoisomer are plotted relative to he control topoisomer ata specific NaCl concentration.0,pRW751 topoisomer 16;A , pRW751topoisomer 13;0,RW751 topoisomer 1% 0 ,pRW751 topoisomer 7.Topoisomers are numbered by the scheme presented in the legend toFig. 6. Inset, a topoisomer of pRW755 and of pRW756 was electro-phoresed in different NaCl concentrations and the relaxation of (-)supercoils was analysed in the same manner as pRW751above.

pRW755would contain 11.2 (-) supercoilsandpRW75610.7 (-)supercoils at 3.0 M NaCl fno B + Z ransition had occurred. +,pRW756; 0 ,pRW755.


6/7

10164 Left-handed D NA in PlasmidsThe cooperativity of the f i t phase of the transitions (Fig.

8) decreases as the initial superhelical density is decreased.However, when plotted on a n[NaCl] scale, the slopes appearroughly equivalent (not shown). The sharpness of the secondphase of the t ransition decreases with decreasing superhelicaldensity even when plotted on a n[NaCl] scale. Thi s indicatesthat there s more than a simple ln[NaCl] versus superhelicaldensity relationship that is occurring in this situation. Thiscomplexity may reflect changes in the free energies of the Band Z conformations, the junctions, and/or supercoiling Overvarying NaCl concentrations.The inset in Fig. 8 shows the NaCl titration results for atopoisomer of pRW755 and pRW756. When there is no B-Z transition, the pRW755 topoisomer has 0.5 more supercoilthan the pRW756 topoisomer. In spite of the fact that thepRW755 topoisomer has a slightly higher superhelical density,it undergoes the transition at a higher NaCl concentrationthan does pRW756. These results demonstrate that the Zconformation is less stable in he 26-bp (dC-dG) block than inthe 32-bp block. Thus, the biphasic nature of the pRW751titration curves can be explained in terms of different stabili-ties of the Z conformation for the two (dC-dG) blocks. This(dC-dG) length effect on Z DNA stability argues in favor ofan all or none transition occurring in the blocks (seeDiscussion). Furthermore, the monophasic transitions forpRW755 and pRW756 rule out the ossibility that the iphasicrelaxation for pRW751 is aresult of cruciform formationfollowed by a transition to theZ conformation.

DISCUSSIONThe isolation of single topoisomers of (dC-dG)-containingplasmids coupled with the ability to electrophorese thosetopoisomers in high NaCl concentrations has allowed a de-tailed study of the properties of left-handed Z DNA in cova-lently closed plasmids. We chose to study theNaC1-mediatedB -+ Z transition since it is the best defined system, although

other perturbantshave a similar effect (2).The experimentally determined NaC1-induced relaxation ofplasmids containing different lengths of (dC-dG) agrees wellwith the calculated relaxation for a B+ Z transition, assuminga junction of zero base pairs. A junction of zero base pairs isdefined as a DNA with entire (dC-dG) trac t in a Z conforma-tion while the base pairs abutting the (dC-dG) blocks are ina B onformation.P NMR (1 ) and laser Raman spectroscopy(10) on the 157-bpBum HI restriction fragment rom pRW751(1) indicates that nearly all the (dC-dG) sequence is in a Zconformation while nearly all adjoining sequences are in a Bor B-like conformation, at least with regard to the phospho-diester backbone. These results, coupled with the supercoilrelaxation data, argue strongly for a very short B/Z junction,probably on the order of 5 bp or less. These da ta also dem-onst rate that the relaxation observed cannot be caused bycruciform formation in the (dC-dG) blocks (see Results).pRW757, containing only 10 bp of (dC-dG), did not undergo a relaxation in NaCl concentrat ions up to 4.5 M and asuperhelical density of -0.015. It is uncertain why the transi-tion did not occur for thi s shor tength of (dC-dG) under heseconditons. One possible explanation is that the activationenergy is too high. There may be a minimum number of basepairs necessary to obtain a nucleation in a (dC-dG) block.Alternatively, the Z conformation may not be stable in (dC-dG) segments of such short length. Crystallographic studies(19) indicated that a region of 4 bp of (dC-dG) abut ting onAATT sequence would not adopt a left-handed structure.An unexpected result was the t emperature dependence ofthe t otal relaxation observed for pRW751. For synthetic ho-mopolymers there is no temperature effect on the equilibrium

of the B - transition (3). The t emperature effect cannotresult from a change in the free energy of supercoiling since itincreases with increasing temperature. This would favor theB * Z-induced relaxation. Thus, the temperatureependencefor the plasmid must be a result of the junction regions inthese molecules. It is possible tha t high temperature shifts theequilibrium position of the junction region away from thenormal B DNA sequences into the (dC-dG) sequences wherethe junction may be energetically more favorable.The ti tra tion f several topoisomers of pRW751 at differentNaCl concentrations reveals that supercoiling contributes afavorable free energy, that the elaxation is biphasic, and tha tstates of partial relaxation exist. One possible explanation forthe partial relaxation states is that the B+ Z transition of(dC-dG) segments in a plasmid is a cooperative all or nonetransition, as for the polymer (dC-dG), (3). Thus, the partialrelaxation states, in the case of an all or none transition,wouldrepresent an averaging of the amount f the time a opoisomerwas relaxed or unrelaxed over the time period of the electro-phoresis. The rat e of interconversion between the B and Zconformation would have to be reasonably fast with respectto the electrophoresis time to give discretebandson theagarose gel. In thi s ituation, the degree of relaxation observedon the gel would eflect the equilibrium of the B toZ transitionat a given NaCl concentration.The second possibility is that partial B -* Z stat es exist inwhich part of the (dC-dG) block exists in a Z conformationand the rest exists in a B onformation with a junctionregionin between. A nucleation of the Z conformation would occurin the (dC-dG) sequence and the conformation would spreadoutward toward the (dC-dG)/B DNA interfaces as the NaClconcentration was increased. The plasmid would be relaxedup to the point where the free energy contributed by super-coiling would no longer offset the unfavorable free energy ofthe Z conformation at a particular NaCl concentration.The fmding tha t the ength of the (dC-dG) block alters thestability of the Z conformation argues strongly for an all ornone transition. If the partial conversion model were true, onewould expect to observe identical extents of partial relaxationoccurring at identical NaCl concentrations. For example, iftwo supercoils have been relaxed in both pRW755 (26-bp (dC-dG)) and pRW756 (32-bp (dC-dG)),approximately 11 bp of(dC-dG) would have been converted to a Z conformation inboth plasmids. In such a situation where the B/Z junctionregion isnot near he ends of the (dC-dG)blocks, the energeticstate of the two plasmids should be nearly identical and thesame extent of relaxation should occur in both plasmids a tthe same NaCl concentration. Since different NaCl concentra-tions are needed to obtain the same relaxation for pRW755and pRW756, it seems highly likely that the entire (dC-dG)block undergoes an all or none transition. Increasing thelength of the (dC-dG) block probably increases Z conforma-tion stability by increasing the cooperativity parameter andthe free energy derived from a greater relaxation of superhel-ical density.If the all or none model holds true, then the intermediatestates of relaxation represent the equilibria of the B -* Ztransition n the (dC-dG) blocks. However, since it seemslikely that conversion of (dC-dG) base pairs next o the nat ura lB DNA interface may not be as favorable as for base pairs inthe middle of the (dC-dG) block, the equilibria may be morecomplex tha n in synthetic polymers.An important question was whether supercoiling is Sufi-cient to facilitate the conversion of the (dC-dG) blocks fromB to Z a t physiological ionic strengths. Fig. 3 shows a linearrelationship between the number of supercoils present in atopoisomer of pRW751 and the ln[NaCl] necessary to begin


7/7

Left-handed D N A in Plasmids 10165the B + Z transition over the NaCl range tested. The freeenergy difference of the B -+ Z transition for polymer (dC-dG) appears proportional to the ln[NaCl] (3 ) while the freeenergy difference of the B + Z transition as a function ofsuperhelical density is not simply proportional to thenumberof supercoils present (20). Therefore, the linear relationshipmay be ortuitous, a result of the limited range of superhelicaldensity examined. It is reasonable, based on calculations byBenham (20), hat the elationship of ln[NaCl] versus numberof supercoils might appear linear over a limited range ofsuperhelical densities. The quadratic component of the freeenergy difference (20) may not become significant until highersuperhelical densities are reached. Unfortunately, the resolu-tion limit of the gel system used here does not allow analysisof more highly supercoiled topoisomers.If the relationship shown in Fig. 4 becomes more quadraticwith respect to superhelical density as theuperhelical densityis increased, extrapolation of the linear relationship to physi-ological conditions should overestimate the number of super-coils necessary to obtain a stable Z conformation. Using theequation of the line of Fig. 4, 34 supercoils or a superhelicaldensity of 0.08 would be suffkient to obtainat least a partialstabilization of the Z conformation a t 200 m~ NaC1. This iswithin the range of in vivo superhelical densities reported fordifferent plasmids in E . coli (21).Thus, the data suggest tha tsupercoiling alone is sufficient to stabilize the Z conformationunder physiological conditions. However, it is uncertainwhether Z DNA or cruciforms (17, 18) are more stable for(dC-dG) sequences a t high superhelical densities and low ionicstrengths.A number of studies (reviewed in Ref. 4) have indicatedthat DNA supercoiling can modulate DNA replication, recom-bination, and transcription. Induction of a Z conformationwould decrease the superhelical stress of a DNA segmentthereby influencing these processes. Thus, the B toZ transi-tion may be an important regulatory mechanism. Also, it isobvious that Z DNA and the B/Z junctions would provideunique protein-binding sites. Furthermore, the Z conforma-tion could be a unique transcription termination sequence,Finally, it has been suggested that alternating purine-pyrimi-dine sequences could serve as a recombination hot spot (22).Since supercoiled plasmids containing (dC-dG) sequencesshowed high recombination frequencies (11) and since super-

coiling may be sufficient to induce the Z conformation underphysiological conditions, it is possible that the left-handedconformation may be present in E . coli. for these (dC-dG)-containing plasmids.REFERENCES

1. Klysik, J., Stirdivant, S. M., Larson, J. E., Hart, P. A. , and Wells,2. Zacharias, W., Larson, J. E., Klysik, J., Stirdivant, S . M., and3. Pohl, F. M., and Jovin, T. M. (1972) J. M ol . Biol. 67,375-3964. Wells, R. D., Goodman, T. C., Hillen, W., Horne, G. T., Klein, R.D., Larson, J. E., Muller, U. R., Neuendorf, S. K. Panayotatos,N., and Stirdivant, S. M. (1980) Prog. Nucleic Acid R es. Mol.Biol. 24, 167-2675. Wang, A. H.-J., Quigley, G. J., Kolpak, F. J., Crawford, J. L., vanBoom, J. H., van der Marel, G., and Rich, A . (1979) Nature6. Behe, M., and Felsenfeld, G. (1981) Proc. Natl. Acad. Sci. U . S.7. Patel, D. J., Canuel, L. L., and Pohl, F. M. (1979) Proc. Natl.8. Mitra, C. K., Sarma, M. H., and Sarma, R.H. (1981) Biochemistry9. Pohl, F. M., h a d e , A,, and Stockburger, M. (1973) Biochim.Biophys. Acta 335.85-9210. Wartell, R. M., Klysik, J., Hillen, W., and Wells, R. D. (1982)Proc. Natt. Acad. Sci.U . S. A . 79,2549-255311. Klysik, J., Stirdivant, S. M., and Wells, R. D. (1982) J.Biol.Chem. 257 , 10152-1015812 . Struhl, K., Cameron, J. R., and Davis, R. D. (1976) Proc. Natl.Acad. Sci. U. S. A . 73, 1471-147513. Hardies, S. C., Patient, R. K., Klein, R. D., Ho, F., Reznikoff, W.S., and Wells, R. D. (1979) J. Biol. Chem. 254, 5527-553414 . Dynan, W . S., Jendrisak, J. J., Hager, D. A. , and Burgess, R. R.(1981) J. Biol. Chem. 256 , 5860-586515. Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad . Sci. U.16. Yang, R. C.-A., Lis, J., and Wu, R . (1979) Methods Enzymol. 68,17. Panayotatos, N., and Wells, R. D. (1981) Nature 289,466-47018 . Lilley, D. M. . (1980) Proc. Natl. Acud. Sci. U. S. A . 77, 6468-647219. Wing, R., Drew, H., Takano, T., Broka, C., Tanaka, S., Itakura,K., and Dickerson, R. E. (1980) Nature 287, 755-75820. Benham, C. J. (1981) J. Mol. Biol. 150, 43-6821 . Bauer, W. R . (1978) Annu. Re v. Biophys. Bioeng. 7,287-31322. Shen, S.-H., Slightom, J. L., and Smithies, 0. 1981) Cell 26,191-

R. D. (1981) Nature 290,672-677Wells, R. D. (1982) J. Biol. Chem. 257,2775-2782

282,680-686A . 78, 1619-1623Acad. Sci. U. S. A . 76,2508-251120,2036-2041

S. A . 76,65615-619176-182

203

Documents

Steven M. Stirdivant, Jan Klysik and Robert D. Wells- Energetic and Structural Inter-relationship between DNA Supercoiling and the Right- to Left-handed Z Helix Transitions in Recombinant