concept/ in biochemi~ try
Edited by: WILLIAM M. SCOVEU Bowling Green State University Bowling Green. Ohio 43403
Supercoiled DNA William M. Scovell Bowling Green State University, Bowling Green, OH 43403
Watson and Crick proposed the structure of DNA ovel three decades ago, yet i t has been only within the past few years that details of the secondary structure of the double helix have been resolved. If there is an overriding principle that guides our thinking about DNA, i t is that its structure is very flexible and exhibits a wide variety of polymorphic states that include the A-, B-. and Z-forms of DNA. The malleability of these secondary structures is influenced by 110th the primary and tertiarvstructure of DNA. In addition. the dire& inhiaction of small molecules, ions, or DNA: binding proteins can affect the topological state of DNA or influenee the structure within restrict& regions or domains of DNA. The genetic material in both prokaryotir and eukaryotic' cellsexhihita a remarkably common tertiary structurecalled su~erroiline. ". which is involved in DNA uackaeine and influences the informational "readout" of the genetic message. These double-stranded DNA's are found to be topologically constrained within the cell as a result of either of two conditions. This constraint can result from the DNA occurrine" in a closed, circular form such as in plasmid, phage, or viral DNA or in many bacterial genomic DNA's. Alternatively, the genomic DNA in eukaryotic nuclei may be complexed with proteins and other cellular molecules a t at least two points and in such a way that the ends are restricted and are not free t o rotate. As a result, these DNA domains, of many kilobases, are topologically constrained
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Topology and Tertlary Structure ol DNA In a covalently closed, circular ("c3") DNA, the topology and the resultant tertiarv structure will be determined bv [he lopolopir.ni linhing nirnber. t * . This quantity is equal t; the total number of times the two strands of DNA are interwound about each other when DNA is constrained to a plane. For a hypothetical DNA of 360 base pairs (h.p.), in which there is one helical turn per 10 b.p., as in the WatsonCrick double helix, the topological linking number is 36. This DNA is said to be relaxed, with a" designating the topological parameter for the torsionally unstrained DNA. Recent findings indicate that there are 10.5 b.p. per turn in DNA, hut the former value will be used in this illustration. The c3 DNA isolated from cells, however, is not relaxed, but is under torsional stress because the topological linking number is not equal to a",or N h.p.110 b.p./turn, where N i s the total number of base pairs in the DNA. In all the isolated
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Prokaryotic cells are those, such as bacteria, that have an illdefined nucleus; eukaryotic cells have a well-defined, membrane limited nucleus.
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DNA's, the value of a is always less than a ' . That is, the strands of the DNA are intertwined fewer times than would be the case in a Watson-Crick double helix. This results in more base pairs per turn and decreases the angle between them. This underwound form of DNA experiences torsional stress and will manifest the topological windings in DNA as an admixture of the duplex winding number (P) and supercoiled turns (7).This results in a tertiary structure associated with the supercoiled DNA. The relationship between a,8, and 7 is governed by a=@+r
(1)
where P is the number of turns in the DNA duplex and r is the number of times the duplex axis winds about the superhelix axis when this axis is constrained to a plane. The value of a is restricted to only integers, while both P and 7 may be noninteger in value. Although this description of the topology and the three-dimensional arrangement of DNA is adequate for naked DNA, it sometimes falls short in dealing with DNA assemblies, such as when DNA is complexed with proteins. An alternate description of DNA topology and DNA ter-
Figure 1. Two topological isomers for a covalently closed, circular DNA with base pairs. Topoisomer A is in the relaxed fwm. while topolsomer B Is negatively supercoiled and represented in two alternative forms. The DNSs are characterized by the topological invariant parameters, a or L. Aa or AL. and o,end me geomenlcai variables. @ or Tand r or W. Nicking of the DNA backbone in topoisorner B changes the topology and relaxes it. (Adaptedfrom adiagram by M. Gellert.) 360
tiary structure which is similar, hut perhaps of broader utility, is represented by in which the parameters are L, the topological linking number; T, the helical twist of the DNA helix; and W, the writhe. The terms L and T are identical to a and .4.. resoectivelv. . Although writhe may be equivalent to the number of titratable supercoils as in the previous formalism. it is conceptually a more general des&ption of topology that ma; also include alternative arrangements of DNA in space, such as in toroidal coils, which can occur in lieu of sup&coils. Figure 1shows a 360-b.p. DNA in the relaxed form (A) and in a negatively supercoiled form (B) in which the torsional strain is distributed in two extreme forms. The first form manifests four supercoils while the alternate form represents the strain artificially in the form of disrupted base~ a i r i n that a extends over what was four turns of the duolex. This dkmonstrates that although n (or L)is a topolo&al invariant, 0 (or 7')and T (or W) are geometrical parameters. An example of the utility of W, instead of 7,todescrihe the three-dimensional character of DNA in space occurs when the DNA is an integral part of nucleosomes. Figure 2 shows a hypothetical supercoiled DNA ( r = W = -6), which, when wrapped around octamers of core histones, forms a minichromosome with six nucleosomes. The DNA has converted supercoils into writhe (W = 6, r = 0) or curvature around the protein assemblies. Removal of the proteins from the minichromosome would reveal that the DNA reverts back to
having six supercoils which were represented as writhe within the minichromosome. The to~oloaicallinkine number is a to~oloeicalinvariant tl~ as long as the DNA hackhone r e m a i n s c ~ v ~ e nclosed. Nicking of the supercoiled DNA renders i t relaxed. However, in prokaryotic cells, enzymes called topoisomerases act on DNA to convert i t from one tonoisomer, in to~oloaical . state a,,to another topoisomer, in btate az,where 02 # q . This is accomplished by cutting the DNA, increasing or decreasing the number of turns and resealing the DNA hackbone. Topoisomerase I can relax supercoiled DNA or increase n , while yvrase,a topoisomerase lT,ran decrease a and thereby inrreasc the exwnt of negative supercoiling in an AT['-dr~endentreaction. It is thoueht that the l e d of superhelicity found in DNA within pGkaryotic cells is set by the continuous action of these two DNA to~oisomerases.On the other hand, the geometrical parameters may change due to environmental conditions, such as temverature. ionic strength or the binding of small molecules or proteins. As an evident from ea 1. anv change in the value of B . requires . equal and opposite change i n t h e value of r. Perhaps the most useful way of representing the level of superhelicity or the superhelical density is in terms of the specific linking difference, a, ~
~
where ru - a" is the linking difference. The utility of this expression lies in defining the superhelicity independently of the size of the DNA. For example, -0.05, while the value for isolated E. coli DNA has a a most plasmids and phages isolated from E. coli lies in the range of -0.05 t o -0.08. The value of ofor the strained DNA in Figure 1is readily calculated to he -0.11.
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Figure 2. me complexation of a supercoiled DNA with octamers of core histones to produce a minichromosome. DNA actually wraps arouKl each nuCleOSOme about two times. This would formally restrain more than slx supercoiis. However, a change in the twist compensates for this.
Energetics and the Torsional Tension In Supercoiled DNA Supercoiled DNA is under torsional strain and is therefore a higher energy form than is relaxed DNA. The free energyof supercoiling, AG,, is proportional to the square of the numher of supercoils in the DNA (eq 4). K is estimated to be about 1100 RT, where R is the gas constant and T is the temperature in degrees Kelvin, while N is the number of base pairs.
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Figure 3. Electron micrograph showing linear DNA, relaxed, circular DNA, and Supercoiled DNA. (Figure kindly provided by S. 0. Freyiag.)
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Figure 5. Schematicrepresentation of the unwinding of negatively supercoiled covalently closed, circular, supercoiled). DNA prduced by me binding of increasing amounts of egem (c3s
F i e 6. h strmtwe of planar ethidium bromide Figure 4. Gel electrophoresis of a sample of @X174 RF DNA in which the distribution of topoisomers is observed. Electrophoretic mobility is from lefl to right. The densitometer tracing of the photographic negative reveals 19 distinct bands, corresponding to 19 tapoisamers. The topological difference between adjacent peaks corresponds to AL = Aa = 1.
The highly character of supercoiled DNA is of - - poised importance in a t least two respects. Any reaction on the strained DNA substrate which reduces AG, will he thermodynamically favored. For example, processes that involve the separation of the polynucleotide strands will be preferred with supercoiled DNA. Consequently, interactions occurring on DNA which require an input of energy, such as those associated with the initiation of DNA replication or transcription, will he favored. It has been estimated that for a DNA with a specific linkine difference of -0.06. an increase in a by one unit (i.e., less strained) is favored by ahout 9 kcallmol. Therefore. these processes should have an energetic advantage on the strained substrate relative to the relaxed DNA in which there is no stored free energy.
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Physlco-Chemlcal Characteristics DNA molecules with linking numher differences have a different topology and tertinry structure. At the two extremes, highly supercoiled DNA exhibits a compact structure, while the relaxed form is an open, extended structure. This can he directlv \.isualized on the electron microeraoh - . in Figure 3. Gel electro~horesisDrovides a simple. . . vet - eleeant. .. . method of determining the distribution of topoisomers in a sample. The mohilitv of each toooisomer is a futjction of the linkine: number diffkrence, the corresponding numher of supercoils~ and the resultant com~actnessof each topological form. Under appropriate con&tions, each topo~o&~isomer can he resolved on n grl, with each exhibiting a different electrophoretic mobility. Figure 4 shows s u c h a gel and the densitometer tracing of the hands for a sample of @XI74RF DNA. At least 19 topoisomers are resolved, with the mobility 584
Journal of Chemical Education
of each being dependent on the linking numher difference. With the careful choice of conditions, this procedure can be utilized to isolate individual topoisohers by extracting the appropriate band out of the gel. The supercoils in DNA can he titrated out by a variety of agents referred to as unwinding agents. These agents produce an effective decrease in the duplex winding number ((3) and a corresponding decrease in the numher of negative su~ercoils.This interaction with the DNA mav occur hv anv numher of modes, including direct covalent hindihg to the bases, electrostatic interaction, or intercalation of planar ring structures between the stacked bases. In all cases, however, the linking number difference is reduced leading t o a corresponding decrease in the number of supercoils and a lowering of the torsional strain. However, these reductions in Aa result not from a change in a,which is a topological invariant, hut because of an effective decrease in the value of an. ~ i ~ u 5r depicts e the action of an unwinding agent on negatively. supercoiled DNA (a < a'). Negative supercoils . are progressively titrated out to produce the intactrelaxed form (a = a'). Further hinding continues to unwind the DNA backbone and leads to the production of supercoils of the opposite handedness, or positive supercoils ( a > a'). The extent of unwinding of the DNA can he conveniently monitored by gel electrophoresis or alternatively by sedimentation analvsis. For a ~ l a n a dve. r such as ethidium hromide (Fig. 6), which intercalates inio and unwinds the DNA, the sedimentation coefficient changes as shown in Firure 7. The compact DNA, with either positive or negative supercoils, exhibits a relatively large sedimentation coefficient, while c3 DNA, which is partially unwound or fully relaxed, is value. , ~ The characterized by a correspondingly smaller S Z ~ findings from both techniques parallel each other in that the electrophoreticmobility changes in the same way as does the value for linear Svedherg coefficient. Note also that the 820, DNA (or nicked circular DNA) is not markedly influenced by ethidium hinding.
ofs
(6-6
Figure 7. The sedimematlon coefficient of closed circular DNA and linear DNA (- - -) complexed with varying amounts of ethidiurn bromide(Em0r). The approximate values of o and r for the closed circular DNA are shown at m e top ol m e plot. (Adapted from a diagram by J. Vinograd.)
various means for relieving this torsional tension within living cells may be operative. Of particular concern, it has been determined that the bulk of the suoercoiled DNA in eukaryotic cells is not torsionally strained. This stems from the DNA wraonine . . .. about histone octamers to form nucleowmes, which introduces a stable writhe and effecrivelv reducesor eliminates thr linking numher difference.Thissituation is exemplified in ~ i ~ u 2.r eAn alternative means to reduce torsional strain may occur when a segment of underwound DNA undergoes a transition in secondary structure from the B-form of DNA to the Z-form. For example, the conversion of one right-handed turn of B-DNA to a lefthanded Z-DNA will remove approximately two supercoils and reduce the free energy of supercoiling. In both of these cases, just as with the binding of unwinding agents to DNA, the free energy decrease results not from a change in cr, but by a decrease in the effective value of cro in the linking number difference. Recognizing now the general nature of supercoiled DNA, it is not surprising that it exhibits characteristics of singlestranded DNA. This was originally demonstrated from findings that it was a substrate for S1 nuclease, a single-strand specific nuclease. In a number of plasmids, such as E. coli plasmid Col El, a unique sequence was shown to be hypersensitive to S1 nuclease cleavage. This indicated that the single-stranded reeion in this olasmid is hiehlv localized and is i o t a general destabilization distribute; throughout the DNA duplex. In addition, single-strand binding proteins have been shown to hind strongly to supercoiled DNA, while exhibiting little or no affinity for relaxed DNA.
lmpllcatlons for Blologlcal Functlon With a growing understanding of the nature of supercoiled DNA, one is faced with the issue of its importance to the biology of living organisms. In both prokaryotic and eukaryotic cells, two maior questions have attracted a great deal of current interest. i)l s t h e DNAduplex under torsional stress in vivo? 2) If so, does torsional stress influence such processes as DNA repliration, transcription, and other viral processes? The first question was addressed by Sinden et al.. who used a DNA cross-linking agent, psoral&, to determine the level of torsional strain within cells. The basis of the measurement relied on the greater affinitv of osoralenfor underwound DNA than fur ;he relnxed form. hey measured the rate of ns(,ralen nhotnbindinr before and after nirkine the in rays and found tbat the DNA& provivo DNA by karvotic cells was suoercoiled. On the other hand, they detected virtually no difference in the DNA in eukaryotiecells before or after gamma rav treatment indicatingtbat the bulk . of the DNA w& formall; relaxed. An enormous amount of work on prokaryotic cells has shown that torsional strain plays a significant role in hiological processes. For example, replication in E. coli, not only of the genomic DNA, but also of plasmid and some bacteriophage DNA's, is affected if DNA gyrase activity is inhibited. In addition, E. coli RNA polymerase transcribes supercoiled DNAmoreefficiently than arelaxedsubstrate. However, the transcriptional response to genes in supercoiled DNA in vitro shows tbat the amount of RNA produced varies greatly and is dependent on the promoter (sequence) of each gene. While some genes are maximally expressed on highly supercoiled DNA, other genes, such as the gene for gyrase, are transcribed most efficiently at low superhelicity. Despite findings that the bulk of the suriercoiled DNA in eukarjolic cells iS formally relaxed by coriplexntion in nuclwsomes and with perhaps uthrr nurlear factors, a number of recent observations suggest that a level of torsional stress may be associated with transcriptionally active genes within domains in chromatin. A number of investigators have shown that transcriptionallv active genes are verv accessible to and exhibit a hvoer-. sensitivity to cleavage by a number of nucleases, including S1 nuclease and DNase I, while transcriptionallv inactive genes are not. S1 nucleasecleaves in regions of non-B-form DNA and these sites may thus reflect an altered conformation of the DNA, usually mapped a t the 5'-end of the active genes. Coupled with this, i t was found that these same gene sequences, such as the active a- and 0-globin genes, when cloned in plasmids, are also sensitive to the same nuclease nrobes when thev are in sunereoiled olasmids but not relaxed plasmids. his suggest's that locai torsional strain may be the drivineu force for the altered conformation in eukarvotic chromatin and the resulting transcriptional selectivity for the eene seauences. Other evidence alona these same lines intimating a possible role for torsional strain in transcriotional activitv sunport this hmothesis. For examole. theievel of transciiption of the herpes simplex virus thymidine kinase gene cloned in supercoiled and linear plasmids was determined after injection into Xenopus oo&tes (i.e., frog eggs). The supercoiled plasmids were transcribed 5001000 times more efficiently than the linear DNA. These few examples cited here provide only a glimpse of the support for the role of torsional strain in DNA metabolism. The second part of this article will develop this area more fully.
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(11 Bauer, W.R:Criek,F.H.C.; White J. H.Sci. Amar
1980,243,118.
(21 Harland. R. H.: Weintrauh. H.: MeKnieht. S. L. Nolure 1983,302.38.
17) Wsng. J. C. Ann. Rsu. Biochsrn. 1985.54.665. (8) Zubay. G.In "Biahemistry"; Addison-Wedey; Reading, MA, 1983.
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