.I. Nol.

Hiol. (1979) 131, 21-40

Preparation and Characterization of Form V DNA, the Duplex DNA Resulting from Association of Complementary, Circular Single-stranded DNA I’. H. STETTLER~, H. WEBER~, TH. KOLLER~ AHD CH. WEISSMANS~ ’ Institut j&r Molekularbiologie I, C’niversittit HBnggerberg, 8093 Ziirich, Switzerland 2 Institut

f Rewived 21 July

f&r Zellbiologie,

Ziirich

ETH-HCinggerberg

8093 Ziirich,

Switzerland

1978, ami

revised form 29 January

~TL

1979)

(~ornplcrnentary circular single stmnds of plasmid PgG or bacteriophage PM2 DNA but not of single-stranded +X174 DNA associate under hybridisation conditions, giving rise to a two-stranded complex. This DNA, which we call form V. llas well-defined physico-chemical properties. It sediments as a sharp peak ilt neutral sucrose gradients; its electrophoretic mobility in agarose gels is between t,hat of covalently closed (form 1) and denatured DNA. In the electron microscope form V appears as highly folded duplex molecules indistinguishable from form I. However, increasing concentrations of ethidium bromide which lead to relaxation a.nd recoiling of form I DNA liavc no comparable effect upon form V. At 260 nm forln V P/?G DNA has a hypochromicity of l&6%, as compared to 23.4% in the ca,se of P/?G form II DNA and IO.5 Tb in t,he case of single-st,randed $X174 DNA. Tile t,llerma,l melting of form V is non-cooperative with gradual increase in absorbance similar to that of single-stranded DNA. The circular dichroism spectrum of form 1’ DNA differs from that of form I, circular nicked (form II) and singlestranded +X 174 DNA in that it shows a negative band at 295 nm and a shift for the main positive band from 273 to 266 nm. We propose that form V consists of right-handrd Watson-Crick type double-llelices which are compensated by an t’cltzal numbrr of left-handed duplex turns and negative supercoils. WC cannot, decide wllethcr left-handed duplex t,llrns are stabilised by base-stacking and hytlrogerr bondillg, as for example irt the models described by Rodley et al. (1976) or Sasisekharan & Pattabiraman (1976), or whether t,hr:y are merely compensatory tllrlls withorlt inllercnt stability.

1. Introduction Many genomcs occur as supercoiled. closed circular double-stranded DNA molecules, designated form I DNA (for a review see Helinski & Clewell, 1971; Bauer & Vinograd. 1974). Under denaturing conditions the ordered double-helical structure of form I DNA is disrupted. however, the strands cannot separate, and a compact collapsed structure, called closed denatured DNA. den DNA 1 or form IV DNA results (Grossman Pt al.. 1974). Intzoduction of a single nick into one strand of form 1: DNA converts it int’o thr open circular or form II DNA, which shows normal denaturation behaviour. as does form III DNA. the linear molecules resulting from t’he cleavage of both strands of t,he circular DNA. Kasamat)su 8: TVu (1976) observed by electron microscopy that upon annealing of

2’ 2

U

H

ST ET’1 1,K I{ k,’ 7’ .-I I,

a preparation containing about 70 y,;, circular single-stranded simian virus 40 (SFT40) DNA, partially double-stranded circular molecules appeared with a frequency of about) 40C!/o. A similar electron microscopic observation \vas macich for Co1 E, by Broker et nl. (1977), who in addition found that cleusturation and reamwaling of DKA containing inverted duplications separat)ecl by non-invwtetl sequences oRen led to the appearance of “underwound loops”. i.rb. rrpions of incompletely renaturecl single strands in an otherwise tlouble-strandrtl molecule. 1n both cases. incomplrt~~ rcnaturation was attributed to topological constraint,s. Evcary right-handed \Vat,sorlCrick double-helical turn formed by t’n-o circular D&*4 sbrands (or thcbir topological equivalent) must) be compcnsatc~tl by r~ithw ii rwgativc~ suprrt\\ist or a Itaft,-handed duplex turn which may or may not involve ~ornplt!nlt:nt,ar?l base intwactions. 7’h~ physical nature of such left,-handed duplex turns is unkno~vn. ‘L’hc turns could btt associated with stable physical interactions br~t\vccn the) component strands. as is thtl case in t,he right-handed Watson-Crick turns. or tjlrtby uoulcl he unstructutwl. without inherent st’ability, tlur only to t~opological constraints. Rotll~y rf al. (1976) hav(a proposed a model in which tM.0 DKA stjrantls art’ m~~sh~cl sick,-bJ,-sitlc,. with short altrrnating left, and right-handed turns. Sasiwktiarail 8~ l’attabirarnan ( 1976) and Sasisekharan ef al. (1977) came to thtl uonclusion that hot’11 right and l(bft-handed double-helices are stereochemically possible for pol~~luclcotic~es. This vicll- was supported b,y calculat,ions of the interaction (w~rgit~s for acljacent basw as well as triplets stacked in a right or left-handed arrangemrnt (Gupt,a 6.1Sasisekharan. 1978). LVc have, investigated sonw aspect s of the structuw of t tir moleculw result,ing from the association of circular. coniplc,nic.rrtal.~,, singlr-stranded DfiL%s;. Circular. single-stranded DNA prepared from plasmitl 1’/3l-Tris.HCl (pH 8), 1 mar-Na,EDTA which was overlaid with 5% sucros~~. 0. I Vi1 sodium dodecyl sulphate using an SW40 Spinco rotor. Fractions containing form I DNA wvrc pooled. dialyscd against. 30 mm-t,riethanol“W(‘. amine-acetate buffer (pH 7.5) and stored at

CHARACTERIZATION

OV

FORM

V DIVA

‘33

DNA from hact.eriophage +X174 was a gift from Dr P. D. Baas, or was prepared front phage +X 174 (Miles) by extraction wit11 phenol. Lysozyme grade IV chloride was purchased from Sigma and elrctrophoretically purified DNaseI from iVort,hington. Ethidinm bromide and agarose were obtained from Sigma and all other reagents (analytical grade) from Merck. t3AC (ber~zyldimethylalkylamrnonium ctlloride, CIzHzs (C,O?h) and C, 4H29 (40%)) \vas ts gift frown Hayer. t,r\-erkusen, Germany.

.A total of 500 pg of form 1 DNA ill 3 ml elf 0.3 rng rtllidium bromide/ml, 0.1 ,\I-NaCI. albmnin were iii15 rnnl-M&l,, 20 rnhr-Tris.HCl at pH 8 and WO5S, (\v/v) hovine serum cubated for 20 rnitr at 30 ‘C with 0.2 pg of DNase I freshly dissolved in 50 rn;\z-Tris. HC’I Before eactl experiment DNase 1 \pH 8). 5 rnwMy(‘l,, 500 We b ovirrfx scrLUG albumin/ml. g electropl~oreticall~~ t’he amount of conversion of form I DNA was tested by detcrminin lrlto form Il. The reaction was stopped by coolitlg to 0°C’ and adding 120 ~1 of 0.5 RI-EDTA. Aft,cr extraction with phenol (3 times) ttrc nicked DNA was precipitated with ethanol, IYsllspended 11110 m\r-Tris. HCl (pH 8). and dcnatluwl by adding an equal vol. of I.6 31.NaCI 0.4 auNaC)H. 0.02 .M-EDTA, 0.1 >f-Tris (pH 13). The sample was centrifuged through a 5:,, t,o 2Oqb s~~crosc’ gradient it1 0.8 nl-NaCl, 0.2 wNaOH, 0.01 wEDTA. 0.05 wTris (pH 13) at :15,000 revsimin altd 15°C in an SIY40 Spinco rotor’. Centrifugation time was 15 11for P/3(: iirltl 10 It for PM2 DNA. Fractiorrs (0..4 1111)\vvrv collrc+d from ttlv top rwing an IHCO 630 ttrnsity gradient fractionwt0r.

a11 ‘I’tlc fractions from the alkaline sucrose gradierlt \vwe adjusted to pH 8.5 by adding (~t11a1 \~olurn~~ of 1 >I-‘l‘ris.HCl (pH 7.5) atld a sample of ractl \vas analysed by agarose get l,lrctroptrorc~sis. TIke fractions enriched irl circular single-stranded molecules (see Results) \vere pooled and annealed at 60°C for 20 rtlill. Tile DNA was prwipitated with ethanol and tlissolwd irl 10 rn>I-Tris.HCl (pH 8). 0.5 mwI-EDTA. 111 some experiments the DNA was precipit,at,ed with c%tianol without, prior aiinralin g at WY. In bottl procedures the sampler \vere heat,t,d to 50 C (for P/3(:) or 55°C (for PM2) f i,r 10 rnin in order to disrupt tleterothrougll a 5’%, tiisperw aggregat)ex. After quenchill g on ice tht, samplrs were rrntrifuped tc, I’.3’j()!0 SLICTOS~ gradient in 10 rnhl-Tris H( ‘1 (pH 8) at 54,000 revs/rnirl and 15°C in an S\l’(iO Spinco rot,or. Ttle c*entrifugat,ion time \vas 135 min for P/3(: DNA arld 60 min for PM2 DNA. Peak fractions were pooled and stored at ~ 2O’C. For ttrc analyses by gel electrophorcsis they were either lised wittiollt any cha.rige of the solvent or. as in ttie case of LI.~. spectroscopy and circular dichroism. the samples \vere dialyscd against 1 x SSC (SW is 0.15 w HCI (pH 8). 0.5 ~~I-EDTA. In sorw YaCl. 0.0 I5 11.sodi\un citrate. pH 7.5 ) *)I’ 10 InwTris. (‘ases tlw DNA \vas precipitated with rthatrol and dissolved in one of those buffers. Samples \vllictr tlad beer1 prccipitatc,d u itll et,ttarlol arid redissolvc>d as wrll as samples which lIntI tbfbf,il stl,rwl frozc~n \verc Iic~atcvl to 50°C’ for 10 lriitl prior to art>- analysis irl ordtlr t20 tlisrllpt iqgrfyatcx.

(d)

~lectrop}lore.sis

I1:l~~ctl~optlorrsis Leas performed on 0.4 (wi \ 8 cm x IO cm slabs of 1 S; (w/v) agarcw* iti 50 mar-Tris.HC‘t (pH 7.8), 20 m>1-sodium ac&ate, 2 m>I-EDTA. For the analysis of ttlcl frac%iorls of ttle neutral sucrose gradient, portions containing 0.1 to 0.2 pg of DNA wert’ tlirectly applied to each slot. Electrophowsis was at 35 to 40 mA for 45 rnin in ttLr case of F’fi(: DNA a,nd for 60 min in t,he cast of PM2 DNA. Gels were stained for 15 min in electrot~tlorcsis buffer rotitaininp 4 pcw ettlidiurn bromide/ml. Stained gels were pllotographetl llndcr Il.\..-illrlmination (254 nm), usin g Kodak Wratten gelatine filter no. 9, on HP4 film plate from llford (10.2 cln ‘.: 12.7 cm) tlrrollgh a filt,cr wit,ti it clrt-off hrknv 560 nm. (e)

Electron.

microscopy

111the BAC hypophase method (Vollenweider et al., 1975) 5 ~1 of a solution containing 2 &ml of DNA, 80 mM-triebhanolamiue-acetatt~ (pH 7.9), 2% (v/v) formaldehyde, 2.4 x lW30/, BAC were spread 0x-w a hypol’hase of cold redistilled water. The DNA/BAC film

24

IT. H.

STETTLEH

E ‘I‘ x4 I,

was adsorbed to electron microscope grids co\~cwtl \rittl a frwtl carbon filn~. For the production of carbon films. t,he carboll rod used was tlipped for 5 min iu to it solutiolI containing 0.01 to 0.1 >r-NaCl. After air drying, t,hv rod was mowlted 111RII vl~~Aron gq~r~. Evaporation of carbon into freshly- cleaved mica \YRS prodr~cod atf :l vacnu~n of a,bollt 2 x 10m6 Torr. In the BAC droplet techniqnr~ (Vollenwcidrr et a./.. 1976) 50-~1 droplets containing 0.5 &ml of DNA, 30 nll~l-t,rietttallola~~lil~~-~~c~t~~t,(, (pH 7.9), I Innr-sodinln acetate, 2 x lW4Yi, BAC were plawd on a shwt of Parafilm (Arnwicar~ (‘an (‘I).). .4ft’rr 10 min the surfaw of the droplet \vits briefl>- torlc~lwd wit11 t tic\ calrctroll rnicroscopt~ grids described aborr. For spreading of part,islly denatrwed form V I>NA molecules t,hc samples \vere heated in the presence of 25% formamide, 25 mnr-N&l, 2 m>I-Tris. HCl (pH 8) to 50’(’ for 10 mill IO rnwTris.HCl (pH 8) to 70°C for or in the presence of 100 mM-N&l. 100 m3i-sllrrose. 10 min. After qnenchin~ in ice the sample was spread by. tjilcl 13.4C trypoplla~w rnot9110ti as described above. After adsorption of ttlc DNA tnoltwdes ttlri prids \\‘(w \l-astwd in redistilled water for 10 min. Unless stated ottlerwise ttlry wew stained for 10 s it1 a solutiorl containing 90”,, (\~/v) ethanol. 1 mix-many1 acetate, 1 m&l-HCI. Tllr PXCC’SSst,aining solutiotl \vas remowd by briefly washing the specimrns irr ct,lratrol ant1 dr>?rlg ttlem OII filter paper. Finally t,llr% grids were rotaq- shadowed at an ar~glr of 7” from an elcctrotr gun wit11 1000 Hz carbonj platinum as measnrecl on a thin film morlit,or (Halwrs). A Hienwns Elmiskop 101 electroll microscope operating at, 100 kV accelrmtirry voltapca \vas 11set1. Micrograplls \VPI’P taken at optical magnificatiorl AS tlrtc~rlnitlcd OII w carhor~ gra?ting wplica, from 27,000 x electroll Ralzers Union. Lirchtor:stein.

3. Results (a) Yrepwation, Purified extracted

form

I DKA,

either

from

bacteriophage

the hybrid PM2.

of ,fomn~ V DNA plasmid

PflG

(Maniatis

u-as treated

with

DNase

et al.. 1976) or DNA 1 to convert

SOY; to

form II: the preparation thus contained on average 1.6 nicks per duplex. This DNA was then denatured in alkali and centrifuged through an alkaline sucrose gradient.

The absorbance profile at 260 nm of such ‘a gradient with PfiG DNA showed tjwo sharp peaks (Fig. 1). Samples of these fractions were examined by electron microscop> using the BAC hypophase method which allows good spreading of single strands (Vollenweider et al.. 1975). The top peak (fractions 18 to 20) consisted mainly of’

Fraction PIG.

1. Zonal

sediment&ion

of’ nicked,

no

alkali-denatured

digested with DNaso I in the presence of et,hidium conversion of form I to form II. The sample (100 pg) 20% sucrose gradient. (See Materials and Methods for at 260 nm. The fractions (0.4 ml) were collected from

Pj3G L)NA. P/X DNA (form I) was bromide under conditions leading to 80?;, form II DNA as judged by agarosc gel elect’rophoresis. Fractions from the bot’tom peak (peak c) in Figure 3 showed a band with a mobility between that of form TI and form 1, i.e. substantially less than t’hat of form \- (lane c. Fig. 4). Further analyses of t)he DNA from peaks b and c are described below. To find out whether form I’ DNA can originate from circular single st’rands of the strands, bhe behaviour of same sequence. i.e. in the absence of complementary bacteriophage @174 DNA. which consists of single-stranded circular plus strands (Hayashi et nl.. 1963) was studied. Form 1’ PPG DNA and 4X174 DKA were heated in parallel t’o 80°C for ten minutes in 90~1~formamide. 10 mM-Tris. NC1 at pH 8. This treatment led t)o the denat’uration of form V P/3G DNA (cf. Fig. 5, lane b). A portion of each heat’ed sample was adjusted to 0+5 M-x&l and 5Oq: formamide at pH 8. and kept for 24 hours at room temperature. A s shovl-n in Figure 5 (lanes a and b). M

a

b

c

d

annealing of heat-denatured P/3G DNA led to a mobility characteristic for form LY, \v.hereas the mobility of $X174 DNA remained unchanged (lanes c and d) and was similar to that of denatured PgG DNA (lane b). Since +X174 DEB consists of only plus strands this experiment suggests that complementary single strands are reyuired for the formation of form V DNA. (b) Electron

microscopic

ch~aractwisation

oJ’fom

I7 DNA

Electron microscopy was first carried out by the RAC hypophase method on DNA samples heated for ten minutes to 50”(! in 25 mM-NaCl and 25% formamide, or to

“8

I-.

H.

STE’l’T1.BK

i”:T

.-II,.

70°C in 100 mM-EaCl and 100 m&x-sucrose. Form 11 DlUA examined under such conditions appeared as well-spread circular filaments wit#h a width characterist’ic for double-stranded DNA, and showed no signs of denaturat,ion (data not shown). A preparation of P/?G DNA containing about iO”/O form V (peak b, Fig. 3) showed mainly tangled filaments beside molecules with the typical appearance of form II. About one-third of the tangled molecules could be t,entatively traced despit,c frequent crossings of the strands. As described for the products arising from annealed circular single-stranded SV40 or Colicin E, DNA (Kasamatsu & Wu. 1976: Broker rl al.. 1977) they consisted of thick stretches, typical for double-stranded DEB. alternating with loops composed of thin filaments typical for single-stranded DNA. WC will call the thick stretches two-stranded (whereby t,wo-stranded does not imply a particular structure) and the thin &retches single-stranded. Two selected molecules are shobvn t,he calculated in Figure 6(a) and (b). The histogram b of Figure 7 demonstrates single strand contour lengths of such selected form \’ P,PG DNA molecules. ‘I’hc values were obtained by adding the lengths of the singltl-st,randed segmcnt~s to t*wice the lengths of t,hc tn.o-stranded segments. In doing so w disregarded a possible difference in the internucleotide distance of the bwo-stranded and the singlr-stranded filaments. The average contour lengths determined in this fashion were about twice those found for fully denatured circular P/3G DNA (histogram a. Fig. 7). From this wt. conclude that’ form V DNA is composed of two, most likely circular. singhstranded filaments. A preparation of form V PflG DNA spread by the BBC hypophasc method wit’hout being subjected to denaturing conditions revealed essentially thr same picture, however, form V DPI’A showed smaller and fewer denaburat’ion bubbles and a higher degree of tangling. Only about lO’$& of the molecules \\We as wf>ll spread and analysable as the one illustrated in Figure 8. A histogram indicating thca proportion of two-stranded segments in a set, of analysable form V DXA tnolt~cules is shown in Figure 9. It should be noted that- a bias may have been introduced hi this selection. We assume that the high degree of tangling of form V DNA molecules is due to supercoiling. We therefore counted the number of filament crossings on t,he same well-spread molecules which were used to obtain the data of Figure 9. Crossing of the t,wo branches of a single-stranded loop by a thick filament was counted as ant crossing. Figure 10 shows a rough correlat,ion between t,hr proportion of two-stranded regions and the number of filament crossings. The number of such crossings represents an upper estimate for the number of superhclical turns. This value lies betwcrn 15 and 37 superhelical turns for molecules containing 80 to UO(iA two-stranded regions. The rapidly sedimenting material from peak c in Figure 3 was prepared fhr electron microscopy under the partially denaturing conditions described above. Most’ of the molecules ( >90°/,) were not analysablo because of tangling. ‘I’hc~ botal single strand contour length of a few traceable molecules (Fig. K(c)). measured in t’hc same \!‘a> as for form V DNA (cf. histogram c, Fig. 7). indicated that such complexes arcs composed of three single-stranded P/ZIG DNA molecules. This form of DSA was not investigated further. The occurrence of these molecules is explained by the fact that renaturation of DNA strands at, high concentrations oft’en results in t\vo homologous strands independently pairing with and competing for a single complementary st,rand. Zn order to determine whether form I7 consis& of two circular single strands, or whether it also contains single-stranded linear molecules. a portion of a DNA preparation which had been shown by electron microscopy to contain 28($; form 11 was

VI conclude t’hat form V DNA is composed of circular single-stranded molecules. Further electron microscopic analysis was carried out using the BAC droplet method under conditions which minimise stretching artifacts during adsorption of the molecules to the grids (Vollenweider et al., 1976). Figure 11(a) shows a typical micrograph of a specimen of the form V PgG DNA preparation used above, containing about. 30% form II. A tot’al of 76 out of 259 unselected molecules analysed (30%) appeared as circular, relaxed filaments with the typical appearance of form II, while 183 (70%) of the molecules were folded, resembling form I molecules. Form I DNA. however, was known to be absent in this preparation, as determined by electrophoretic analysis. These folded filaments are assumed t,o be form V DNX. For comparison, Figure 11(b) shows a mixture of form I and form II P/3G DNA and +X174 DNA. Under these conditions single-stranded +X174 DNA appears as crumpled strands which are barely visible (see arrows in Fig. 11 (b)) because bhe specimen was not stained prior to shadowing. 4X174 DNA is clearly different~ from forms T. IT and V DNA. (c) Effect of ethidium~ bromide err. di$ferent forms of circular

DNa-l

Since the electron microscopic appearance of t’he molecules thought t’o be form V DNA was almost indistinguishable from that of form I DNA it was of interest to examine the effect of ethidium bromide on the two species. It has been shown that

c

e

I’Ic. X. Electron micrograph of form V PgG DNA spread onto a hypophase under non-denaturing wnditions. A solution containing 4 pg DNA/ml in 10 mMa-Tris.HCl (pH 8), 100 mu-NaCl, 20/,, f’ormamid~ , 3.5 x 1W3% BAC was spread on a hypophane of re~listillrd wabr. Tnterpretat,iorl 01 rhtz image of r,ne of the molrxxdas is shown in the inset.

32

LT. H.

STETTLER

E’/’

,I],.

6 t

too

90

00

60

70

50

40

30

20

10

Two-stranded regions (%)

. IO

, .. . . .

.

f

I-

Ll 100

s 90

11 00

11 70

Two-stranded

" 60

" 50

" 40

1

regions (%)

FIG. 10. Number of filament, crossings in form V UNX muleculrs ati a f’uctitm of the lm>portwn of two-strandedness. Crossing of the two bran&w of a single-stranded loop by a thick filament was oount~ed as one crossing. For the analysis t.hr moleculrs WPT~ use11 which served for thrl data of Fig. 9.

if form I DNA is treated with an increasing amount of ct~hidium bromide its supercoiled structure is first relaxed and then supercoiling in the reverse sense ensues (Bauer & Vinograd, 1968). In order to determine whether form V DNA shows a comparable transition, a mixture of forms I. Tl and V of PM2 DKA vvas subject’cd to agarose gel electrophoresis in the presence of increasing concentrations of ethidium bromide (%spejo $ Lebowitz, 1976). As slu~wtr in Figure 1,*) t,he absolute mobilit’y of form II decreased slightly vvith increasing cthidium bromide concent)ration (see also Fig. 1 of Espejo & Lebowitz, 1976). The mobility of form I DNA showed CL dramatic

Fin. 12. Agarose gel electrophoresis of forms I, 11 and V PN2 I)Bd in thffercnt concentrations of ethidium bromide. Electrophoresis was carried out as described by Espejo & Lebowitz (1976). The 1 o/0 agarose gels (13.5 cm x 0.5 cm x 0.45 cm) in 0.05 xx-Tris-acetatr (pH 7.8), 0.02 M-sodium acetate, OfJO2 nXaZEDTA with ethidium bromide at the concentrations indicated below were formed in a Savant Instrument Inc. HGE-1312 slab go1 apparatus. The gel was subdivided with Plexiglass spacers into 18 lanes containing the following ethidium bromide concentrations (pg x 10Z/ml), from left to right: 0, 3,6,9. 12, 15. 18, 21, 24. 27, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 0. To each slot 0.1 pg each of form I, II and V PM2 DNA was applied in 14O/, glycerol and 0.020/, bromphenol blue. Eleotrophoresis was at about 35 mA for 4 h. Bp this time hromphenol blur migratrd to about 0.6 of the length of thr gel. 11. I and V dpnotr th(x hands c~orrcxpr,ntliI~g to th(* 3 DX.4 forms.

P

I

CHARACTERIZ.-\TION

OF

FORM

V

I)S,\

3.5

dependence on the dye concentration. It, q mobility relative to that of form II DN;\ decreased from about 2.4 in the absence of ct,hidinm bromide to 1 at dye concentrations between 0.16 to 0.22 pg/ml and then increased again to reach about it’s initial value at, 1 pg/ml, as described earlier (Espejo & Lebowit,z, 1976). In cont’rast to the behaviour of form I, the mobilit’y of form V relative tjo that of form TI changed only at dye concentrations above 1 pg/ml where the relative mobility decreased but did not reach that of form T. It is striking that the band of form 1’ DKA broadened at high dye concentrations, while those of forms 1 and 11 did not. (d) Optical

absorbawe

of ~ttabiuemd dertatured jiimt

IT DNA

In order to determine the hypochromicity of form V relative to form IL DNA thtb ultraviolet absorption of native and alkali-denatured PfiG DNA form 11 and V was tlrt~crminrd (Table 1). The hypochromicity of form 11 DNA at 260 nm was found to TABLE

Relative ~ultraviolet

I

absorbance of P/3(: f arm V, P/3(: forwl, II, ad +X174 DNA at alkaliwe atzd n,eutral pH

-&3l

-4 280

(%)

(‘YJ

-4 mo/~ 280

sin,gle-strclndrtl

Hypochromicity at 260 urn (%)

PflG form

V alkaline neutral

100 81.4

54.5 41.0

I.83 I.99

18.6

Pj3G form

II

alkaline ncut i-al

100 76.6

55.8 40.0

1.70 1%

“3.4

alkaline neu t-ral

100 89.5

5X.2 60.7

1.72 1.77

10.5

4x174

Samples of DNA in 1 x SW, at concentrations between 0.2 and 0.4 rl,,,, unit per ml, wew denatured by adding 0.04 vol. of 5 .n-NaOH. Absorbances was measured on a Gilford spectrophotometer 2400 before and after denaturation of the DXA. Absorbance values were corrected for the dilut,ion by t,he added N&OH. Form V contained 5 “/A form I1 molecules as determined b> electron microscopy. The data shown ape the average of measurements on 3 samples of form 11 and on 2 samples each of form V and of sir@-strantlnd 4X 174 DNA. Variations between replicatca samples never exceeded 1%.

be 23.4%. the corresponding value for single-stranded $X174 DiYA was l@fio/,. Under, identical conditions a solution of form V DNA with 5% contaminating form II (as determined by electron microscopy) showed 18.6 yh hypochromicity. From these data (and assuming that single-stranded P/3G DNA has the same hypochromicity as +X174 DR’S) we calculated the contribution of two-stranded ordered structure to the hypochromicity of z)ure form V to be 61 yO of the value for form LI DNA. If each hypochromicitp measurement is associated with &to+0/h error, then the limiting values for the percentage ordered duplex structure are 49 and 74. During thermal denaturation (Fig. 13) of form V DNA nicking of one strand during the rather long heating process (about 1 11) could lead to a conversion of some of the form V into form II. Therefore. attention should be mainly directed to the

20

25

30

35 40

45

50

55 60 65 70

75 00 85 90

95 100 110

Temperature (“C)

shape of the \vith form II denaturat,ion and 95°C as

melting curves rather than to bhe ext,ent of hyperchromicity: whereas P/3G DNA the well-knoxvn (e.g. see Mandel Bt Marmur. 1968) co-operative was observed, the absorbance of form V increased gradually between 50 in the case of single-stranded +X174 DNA (Sinsheimer. 1959). (e) Circular

dichroim

spectroscopy

of form,

V DNA

The circular dichroism spectra (Fig. 14) of form I and I I P/3G DNA in 1 x SK’ are similar except, for t,he magnitude of the positive band around 270 nm and the trough around 250 nm as discussed in d&ail by Mae&e & Wang (1971). In agreement, with Johnson & Tinoco (1969) the double Cotton effect typical for DNA is weaker in the case of single-stranded 4X174 DNA. However, form V DNA shows a clearly different circular dichroism spectrum with a negative band at 295 nm and a shift for the main positive Cotton effect from 273 nm to 266 nm. This st,rongly suggests that’ 1 j< SSC form V DNA has a different conformation than form in I, II and singlestranded DNA.

4. Discussion Vinograd et al. (1968) have characterised the topological properties of closed duplex DNA by the parameters LX(topological winding number), /3 (duplex winding number) and 7 (superhelix winding number), where 7 = a - /3. It is clear that any molecule

::;

I

220

230

240

250

260

270

280

290

300

310

320

330

340

Wavelength (nm) FIG. 14. Circular dichroism spectra of forms I, II, III and V 1’fiG I)NA and of +X174 DNA. Circular dichroism spect,ra of DNA samples in 1 x SSC at, concentrations between 0.14 and 0.4 were recorded with EL Jobin-Yvon [Dichrogwph Mark III. Several measurrment,a .-I 260 unitj/rnl were lat,rr repeatetl, with similar results, on a Jasco J-500C instrument,. Single-st,randd $X Ii4 IlN.4 (. .): P/X IIN. form I (-~ -): form TI and form IIJ (.~ -. ~-.-): form \ (~-~ ~~~ ),

put together from two separate, covalently closed circular DKAs must have the topological winding number GC= 0. because /3 and 7 of the original components art’ zero. If duplex formation is based at least in part on the formation of Watson-Crick helices, the introduction of p, (p R :> 0) right-handed turns must be accompanied by the concurrent formation of /IL (flL < 0) left-handed turns and/or 7 negative supercoils, such that cc = pR + /IL + T = 0. Right-handed and left-handed turns ma! occur in a few large or many small domains. In the one ext,reme there is but oncx domain each of right-handed and left-handed turns, which confront each other in t,wo points. Towards the other extreme right-handed and left,-handed domains may alternat’r every five base-pairs, as in the SBS (side-by-side) model of Rodley et al. (1976). but int’ermediate, possibly irregular domain sizes are possible. In any of thescb cases. superhelical turns come about when lpRl # l&J. For t’he SBS model this inequality is given because five bases in a right-handed helical conformation produce a 180” t’urn. while the left-handed structure has a corresponding value of -1145”; bhis leads to an additional full right-handed turn for about every 100 base-pairs. Tn thta case of P/3(; this would result in about, 50 to 60 negative (right-handed) superhelical t,urns. Even if the rotation per base-pair were the same for right and left-handed helices. the number of base-pairs involved in right and left-handed helices may differ because of differences in the stability of these structures. Another extreme possibilit), \lould be a structure lacking left-handed duplex turns altogether, in which right,handed Watson-Crick turns are compensated exclusively by negative superhelical t’urns. Such a molecule would have a superhelix density (J = r//3 = -1, which means that if a major portion of it consisted of Watson-Crick helix it would have to contain several hundred negative superhelical turns.

:1x

C’.

H.

STETTLEK

E’/’

.-lL.

\Ce have shown that the annealing of covalently closed, complementary strands ot E’,9G or PM2 DNA leads to the formation of a molecular species, form V. with defined electrophoret’ic mobility and sedimentation behaviour, which on denaturation again gives rise to single-stranded, covalently closed. circular DKA. The association leading to form V must, be based on sequence complcmentaritg, since single-stranded. circular $X174 plus strand DNA does not give rise to form V upon annealing under conditions used for complementary circular DNAs. The circular dichroism spectrum of form T’ DNA is different from that’ of douhlr and single-stranded Dh’A. It is quite similar to t,he spectrum of poly(dG-dC) m . very high salt (Pohl & ,Jovin. 1972). There is also a certain similarity to RKA spectra (Tinoco & Cantor, 1970) which do not,, however, show the strong negative difference in ellipticity at 246 nm. Tt has been suggested that the differences in circular dichroism between DNA and RNA are due to the tilted base-pairs present in RK.A but not in the B-form of DNA. which has the base planes perpendicular t,o t’he helix axis (Yang & Samrjima, 1968; Tinoco. 1968: Johnson & Tinoco, 1969). Although the circular dichroism spectrum of form V cannot be interpreted at present, it’ clearly suggests t,hat in one and the same solvent form V has a different structure than single and double-stranded DNA. Upon heating. the absorbance of form \’ increased gradually and did not show tilt, co-operative melting behaviour typical for form II D1V$. Several explanations could account for this apparent lack of co-operativit,y : the ordered arrays of stacked bases could be heterogeneous, or short, compared to those of form II or linear DNA. Furthermore, t,he stability of the Watson-Crick helical domains may he inversely related to hhr den&y of super-helical t,urn s and/or left-handed helical t,urns. so that the melting temperature rises as the molecule unwinds. From hypochromicity measurrments, t,he contribution of ordered duplex structure to the hypochromicit.y of form V was estimated to be about 50 t,o 75(:;; of tIllat of form Il. If wc assume that only right-handed helices increase hypochromicitg then form C DNA would contain 50 to 75yb of this type of structure and 25 to SO?, Irutat,ivr lefthanded turns. If. on the other hand, putative left-handed helices increased hypochromicity to about t,ha same extent as right-handed ones, then as litt,lc as 50 to 750,:, of form V would have an ordered right, and left’-handed duplex structure. In tjhr cast’ of a side-by-side structure as proposed by Rodley et al. (1976) at least 50 to 7.5y0 of the form V molecule would be double-stranded, unless the contribution to hypochromicity of this form of ordered structure were great,er than that, of the Watson-Crick helices (which wc assume constitute the st,ructurr of normal double-stranded DKA). fn short. t,he simplest interpretation of the hypochromicity data makes it, unlikely that form V .DSS has less than 50 to 75% ordered duplex st~ruct,urr or more than 50 t’o 757; right-handed Watson-Crick helices. Electron microscopic examination showed that 60 to ROO/” of the form V molecule consisted of thick filaments. The approximate agreement of this range with the amount of ordered structure estimated from the hypochromicity data suggests that a large proportion of t,hr thick stretches rrprcsrnt regions of ordered st,ructure. Form V DNA of bobh PflG and PM2 resembles form I in electron microscopic appearance. in particular as regards the degree of folding and the width of the filament. This suggests that form V contains many superhelical twists. The high electrophoretic mobility of form V, greater even than that of form 1 is also compatible with the compact structure of a highly supercoiled molecule. We have estimated the number of suprrt,wists by counting the number of double-strand crossing points in individual

CHARACTERIZATlON

OF FORM

V I)N.-\

39

P/3G DNA molecules. This number increases clearly with increasing two-strandedness of the molecule. suggesting, by extrapolation, the presence of approximately 30. maximally 80, supertwists in a completely double-stranded molecule. The higher value was arrived at by adding to the 30 supertwists found for a 900/A two-stranded molecule the 50 supertwists which would result if the residual 1Oo/o of the molecukb were wound up into Watson-Crick turns. This number of supertwists is in the same order of magnitude as the 50 to 60 required by the side-by-side model, but clear]? far below t,he number required if all thick-stranded regions seen in the electrou microscope (60 to 90%) consisted of Watson-Crick helices compensated by superhelical turns. From our experience with electron microscopy of single-stranded DNA we consider it extremely unlikely that under the conditions used non-complementary singlest)randed DNA could associate to form thick filaments by some unspecific interactions. It would thus seem that a certain proportion of the thick filament must be formed by left-handed turns. These turns could be either merely compensatory, without inherent) st,ability, or they could consist of an ordered: possibly base-paired structure. The exact amount of ordered structure in these thick st’retches cannot be estimateci at present, since (1) the contribution of a putative left-handed double-helical ordered st’ructure to the hypochromicity is unknown, and (2) t’he errors associated with thcb estimation of hot’h the hypochromicity and t,he proportion of t,hiok stretches seeu in the electron microscope are too large. Ethidium bromide affects form I and form V in a strikingly diflerent fashion: as discussed above. the negative supercoils of form I are tit,rated out, as increasing amounts of the dye intercalate in the right-handed helix leading to a strong reduction of c+ctrophoretic mobility. In t’he case of form C a reduced elect’rophoret,ic mobilit? could be observed only at very high ethidium bromide concentrations. A possibk explanat’ion could be that the unwinding of the Watson-Crick helix by ethidium bromide is first compensated by unwinding of left-handed rat,her t,han superhelical turns. lcaving the number of superhelical turns approximately constant. \Vhat. in summary, is the structure of form V DNA? We should consider tjwo basically different models. (1) About 50% of the molecule consist of a right-handed Wat,sorl-Crick type double-helical structure. In the remainder of the molecule thcl single strands form left-handed compensatory turns with neither hydrogen bonding between t~he component strands nor the base-stacking associated therewith. A minor proportion (about 10 to 20%) of the right-handed turns is compensated by negativta suprrhelical turns. (2) Between 50 and 75y0 of the molecule consist of ordered righthanded and left-handed, double-helical structures. A special case of such an arrangement is the model proposed by Rodley et al. (1976) or Sasisekharan et al. (1977). Because of difftlrences in stability or st,ructure of right-handed and left,-handrcl duplex turns their number may be unequal. and the difference in numbers bvould havca to b(b compfansated by superhelical turns. Our estimate of the extent of ordrrccl structure is not sufficiently reliable to dist’inguish bet\\,een the t\vo possibilities, MOIYover. it is clear that intermediates between thr two models are also possible. Ll’r tltark 13~ W. Simon, ETH, their collaborators for the circular Zixicll. for llis llelp in drtermininp

Ziirictk, dichroism

Dr (2. WagniGrr. Ullivctrsity specta and Dr M. Hirnstiel,

of Ziiricll, University tile DNA melting profiles; nr F. Poll, University Konst,;rne, for discussion and advice in interpreting t,ltr ciwnlar tlichroism spwtra: Mr \I’. Roll for pn~parinp the form I P/3(: DS.4.

atltt of of ;tncl

40 This work was \~issr,nsctlaftlictlrrl

Agwo, Bauer, Baucr,

U. H. suppolted F’orschung

STICTTLER

by tllc Schweizrrisctlrr (gratlt no. 3.1590.73).

,Y’1’ ;1 I,. Nat,ionalfontts zur F;iirtlcrung atltt by ttle KalIt~ort of Ziirirlr.

dot

M.. Dow, D. KS Brorltali, C. ( 1969). Hio@ys. ./. 9, I281 13 I 1. W. R. & Vinograd. J. (1968). ./. ,Wo/. Bid. 33, I4 I 17 I. L%‘. R. & Vinoprad, .J. (1974). Basic F’ri&~/es irt ‘\;rrc/e,i~ dcitl C!/zemistry (‘J’s’O. I’. 0. I’., cd), vol. 2. pp. 265-303, Academic Press. New York. Broker, T. K., Soil, L. b%Chow, L. T. (1977). .I. .lIo/. Hid. 113, 579 58’3. Currier, T. C. & Nester, E. W. (1976). 4nal. Biochena. 76, 431 441. Espe,jo, H. T. & Ca,nelo. IX. S. (1968). l’irolnyy, 34, 538 747. Espejo, R. T. & Lebowitz, J. (1976). Anal. Biocheva. 72, 95 103. (:rossmnrl, I,. 1.. Wnt,sotr. R. & Vinograd. .J. (1954). ,I. Jlol. &in/. 86. 271 283. (iupta. (:. & Sasisrkharatl, V. (1978). R’uc/. .-tcids Rev. 5. 1655 1673. Hayashi, M.. Hn~ashi. M. FC. KS Spirpplrnan. S. (l!Xl). I’roc. A\‘at. ;2cu.r/. Sri.. 1 ‘,,Y.=l. 50, 664 -672. Hrlinski, D. K. & Clrw~ll, I>. B. (l!)il). A4rc~!,r. /\\-itt. IC. M. ( 1976). I’roc. ,Yat. .-lcat/. SC?.. I’.,S.A. 73, 2959. 2963. Sasisektraran. V. B Pattahira,man, X. (1976). C’crrr. Sci. 45, ii!) 78 I Sa.sisrkhararr, V.. Pattahiramarr, p\‘. K- (:llpt)a. (:. (l!lii). (‘/WV. ,Sci. 46, i(i3 SW. Sinstlcinwr. K. L. (1959). .I. Mol. Uiol. 1. 43 55. Tinoco, I.. .Jr (1960). .I. .Am.er. Chem. Sot. 82. 4586 47!)0. ‘I’inoco. 1.. *Jr (1968). .J. Chim. /‘hp. 65. 91 07. ‘I’irrono. I.. .Jr Rr Ca,nt,or, (!. R. (l!fTO). Nethods I~iorhew. =I~ul. 18. XI ~%O:i. Vinograd, .J _. Lebo\vitz, .I ., Kdloff, K . . LVatscJrl. I