Some structural properties of the DNA molecule from bacteriophage alpha

VIROLOGY

44, 371-382 (1971)

Some

Structural

Properties

of the

Bacteriophage W. D. SUTTON’ Department

DNA

Molecule

from

Alpha G. B. PETERSEN”

AND

of Chemistry and Biochemistry, Massey University, Palmerston Xorth, Xew Zealard, D.S.I.K., Applied Biochemistry Division, Palmerston North, ,Vew Zealand

and

Accepted December 28, 1970 The phage (Y DNA molecule has a molecular weight of 33 million, as estimated by sucrose gradient sedimentation. When prepared by phenol extraction of purified phage, the DNA contains approximately one single-strand break per molecule, but the frequency of single-strand breaks can be reduced by changing the conditions of DNA release. The residual breaks are randomly distributed between the pyrimidinerich H strand and the purine-rich L strand, and do not occur at, any fixed distance from the ends of the molecule. The complementary H and L strands of cy DNA can be prepared by MAK chromatography3 followed by self-annealing and hydroxylapatite chromatography, but this procedure results in strand breakage. Caesium chloride gradient centrifuging in the presence of poly G gives a more efficient separation of the intact strands. Contrary to previous reports, both strands of Q!DNA contain poly G binding sites, but those in the L strand appear to be confined to a small segment having a buoyant density similar to the H strand. INTRODUCTION

The DNA molecule of bacteriophage (Y, a temperate bacteriophage infecting Bacillus tiberius, was one of the first known to have separable complementary strands (Aurisicchio et al., 1961; Cordes et al., 1961a). The DNA has been shown to have a molecular weight of 30-40 million (Aurisicchio, 1966; Davison, 1966). Chemical studies have shown that it contains only the four normal bases, with a G + C content of 42.7 % (Aurisicchio et al., 1961). This base 1 Present address: Department of Zoology, University of Edinburgh, Scotland. 2Present address, to which requests for reprints should be sent: Department of Biochemistry, Universit,y of Otago, Dunedin, New Zealand. 3 Abbreviations used: MAK, methylated albumin adsorbed on kieselguhr; poly G, polyriboguanylic acid; SSC, 0.15 M NaCl, 0.015 M disodium citrate; Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediamine tetraacetate; DDC, sodium diethyl dithiocarbamate.

composition is in good agreement with that expected from the melting curve of 01Dh-A in SSC buffer (Rlarmur and Cordes, 1963) and the buoyant density of cy DNA in caesium chloride gradients (Aurisicchio et at., 1961; Marmur and Cordes, 1963). Complementary strand fractions have been prepared by MAK chroma.tography (Cordes et al., 1961a; Aurisicchio, et al., 1964) and used to demonstrate an apparent polarity of transcription of cy DNA both in. OLIN (Tocchini-Valentini et al., 1963) and ire vitro (Geiduschek et al., 1964). It has been reported that only the H strand contains polv G-binding sites (Sheldrick and Szybalski, 1967). Since CYDNA seemed a suitable molecule for studying the distribution of short nucleotide sequences between the two complementary st,rands (W. Sutton, Ph.D. Thesis, 1969) we have reinvestigated the molecular structure. Our results are not, in complete agreement with t.hose of previous 371

372

SUTTON

AND PETERSEN

workers. In particular, we find that both complementary strands cont,ain poly G-binding sites, a result which must, cast some doubt) on the conclusion that only the H strand is involved in transcription. MATERIALS

AND METHODS

Growth and purification of phage. The clear mutant aC3 and its host B. tiberius were obtained from Dr. E. P. Geiduschek. The phage was grown in layers of soft nutrient agar at 30” and released by homogenising with an equal volume of phage buffer (0.5 Al NaCl, 0.01 AZ RgS04, 0.01 M phosphate, pH 7.0). The soft agar fragments were removed by centrifuging at low speed, and the phage collected by precipitating with 2.5 % (w/v) polyethylene glycol (L. Light and Co., Ltd., Colnbrook, England; molecular weight 20,000) and centrifuging for 5 min at 15,000 rpm in the SS-34 rotor of a Sorvall RC-2B cent.rifuge. The phage pellets were combined and resuspended in a small volume of phage buffer, made 1.5 % in polyethylene glycol, and recentrifuged to remove remaining agar and bacterial fragments. The phage was again precipitated with 2.5 % polyethylene glycol, and resuspended in phage buffer. Such preparations were shown by electron microscopy to be completely free of bacterial debris and to contain a high proportion of intact phage. No traces of bacterial DNA were detected by caesium chloride density gradient centrifugation, The &60:&~,~ ratio of purified phage was 1.54 =t 0.02 (mean f SE for 12 batches). Polyethylene glycol precipitation was found to give near-quantitative (90 %) recovery of phage 01 as judged by both ultraviolet absorption and plaque-forming ability. However, it was noticed that the titer of plaqueforming units was consistently 100-fold less than the titer of phage particles deduced from the yield of DNA. For the preparation of radioactively labelled phage CV,carrier-free 32P-orthophosphate (Radiochemical Centre, Amersham, England) or 33P-orthophosphate (lSew England Nuclear Corp., Boston, Massachusetts) was added to the soft agar growth layers. Phage T7 and Escherichia coli strain W3110 were obtained from Dr. 31. G. Smith. 3H-labelled phage T7 was grown in a nu-

trient broth culture supplemented with 2 &i/ml thymidine-3H (Radiochemical Centre, Amersham, England), and purified by differential cent,rifuging. Preparation oi DNA. Redistilled, watersaturated phenol was stored in the dark, and buffered to a pH of 8-9 immediately prior to use by shaking with an equal volume of 0.1 M NaOB407. Concentrated phage suspensions were dialysed overnight at 4’ against 0.1 M Tris-HCI (pH 8.0) containing 0.5 dl NaCl and 0.001 M DDC, and the phage DNA was released by gentle shaking with an equal volume of chilled phenol. After removal of the phenol layer, this process was repeated, and the final aqueous layer was dialysed at 4” against several changes of 0.05 M KaC1, 0.001 M EDTA, 0.01 M Tris-HCI (pH 8.0). The A260:A280 ratio of such preparations was 1.85-1.95, and the A260:A230ratio 2.25-2.45. Boun.dary sedimentation of DNA. Boundary sedimentation was carried out by the usual procedures using a Beckman Model E ultracentrifuge and cells fitted with Iiel-F centrepieces with a 30-mm light path. Native DNA was sedimented at pH 7-8 in buffers containing 0.15-1.0 M KaCl. Denatured DNA was sedimented in 0.1 M NaOH, 0.9 II1 NaCl. To avoid any possibility of shearing, concentrated native DNA was carefully pipetted into preassembled cells after removal of the upper window, and the appropriate neutral or alkaline solutions were introduced by syringe after reassembly of the cell. The extent of strand breakage was estimated by the method of Davison and Freifelder (1966), and the general precautions against shearing described by these authors were followed throughout this work. Sucrose gradient sedimentation. Zone sedimentation was carried out at 5” in the SW 39 rotor of a Beckman Model L centrifuge, using linear 5-20 % sucrose gradients containing l-2 M NaC1. Alkaline gradients contained 0.1 M NaOH, 0.9 M NaCl, and were checked before use to ensure that the pH was > 11.0. The total amount of DNA layered onto each gradient was usually less than 1 pg. Molecular weights were calculated from the relative distances sedimented according to the relationships D1/Dz = (AI,/ M&O.35 for neutral sucrose gradients and

STI:UCTURAL

PROPERTI15S

D,/D, = (;\11/A\I,,‘1.40 for alkaline sucrose gradierns (Burgi and Hershey, 1963; Studier, 1965). Denaturatsorr am1 renssociatio?i of’ DXA. DSA was denatured with alkali and reneutralised by the method of Davison (196G). To form poly (:kDSh complexes, high molecular weight, poly (: (I\Iiles laboratories Inc., Elkhart, Indiana, minimum molecular lveight 100,000) VW added to the DKA at a n-eight ratjio of 1 : 4, prior to denaturation. Iirassocintion 1v:r.s carried out by the low temperature formamide method of Bonrier et al., (1!?67) in order to eliminate st,rand breakage by hydrolysis: O( DxL’h at, Z-100 pg/ml \vas dialysrd against 20 volumes of 30 5 (v/v) formamide containing 0.15 AI NaCl, 0.015 J/ sodium citrate. After reassociation of the DSA at 4” to a Cot (product of initial DKA concentration and time) of riot, less than 3.0 set moles/litre, the formamide was removed by dialysis against 0..7 dl SaCl, 0.01 J/ phosphate (pH 7.0). d/AK ch,wlbatogl,aphy. I\Iethylated nlbumin absorbed 011 kieselguhr was prepared by the method of AIandell and Hershey (1960) except that mrtllylation was allowed to proceed for 2 wk. Ii:arly experiments followed in every detail t,he stepwise elution procedure described by Cordrs (8. Cordes, Ph.D. Thesis, Brand& I’niversity, 1963). In other experiments, t81ieprocedure was modified b? t,hc use of refrigerated columns and salt gradient elution (Roger, 1968). 11yrho:cylapafitr chromatography. Crystalline hydroxylapatite was prepared from brushite (Tiselius et al., 1956) and stored at 4” in 0.01 A1 phosphate buffer (pH 7.0). Care n-as taken to avoid breakage of the crystals. Of several independently prepared batches, no two had exactly the same properties for the chromatography of DNA. However, in every case phosphate gradient elution as described by Bernardi (1965) readily separated single-stranded DNA from native DNA. Tile most useful batch of hydroxylapatite gave approximately 73 % recovery of high molecular weight singlestranded DNA, and 95-100 o/o recovery of native DNA, from columns eluted at room t,emperature. Demity gradietlt centrifugation. Analytical den&y gradient, centrifugation was carried

OF XLPIIA

I )?;.I

out at 20-2.5” and 14,i70 rpm in a Heclinian JIodel I’, centrifuge, using cells of 12 mm liglit-path with Kel-11‘ centrcpieccs in :I lplace An-I’ rotor. Analar grade c:tc~sinm chloride was used without further l)uritic:ltion, and mixtures \\-ith an initial densit>- of 1.71-1.72 g/ml were made up from :I cow centrated stock solution of cnrsium chloridt~ containing 0.01 .II Tris-HCI (pH X.0) :incl 0.001 AqI I
374

SUTTON

AND

PETERSEN

amounts of single-st,rand breakage in several bacteriophage DNA molecules. Their prepThe Molecular Weight of O( DNA arations of (YDNA contained only about 50 % The sedimentation coefficient of the Q intact strands. Freifelder (1966) has reDNA molecule was measured by Cordes ported that a! DNA prepared by another (S. Cordes, Ph.D. Thesis, Brandeis Univermethod (heat release at SO’) contains 70% sity, 1963), who obtained an soZo,W value of intact strands. During our early experiments, 35 from boundary sedimentation measure- in which crude Q: phage prepared by difments of OLDNA at a series of DNA con- ferential centrifugation was used, we found centrations. Using the relationship between that the phage DNA, although intact in the sedimentation coefficient and molecular native form, contained S-10 single-strand weight derived by Studier (1965), this cor- breaks per molecule as assayed by either responds to a molecular weight of 32.4 X 106. zone or boundary sedimentation in alkaline Others have deduced a molecular weight of buffers. More intact DNA could be pre3440 X lo6 by boundary sedimentation pared if the phage preparations were dialysed and autoradiography (Aurisicchio, 1966). overnight against 0.001 M EDTA, or puriDavison (1966) deduced a molecular weight fied by selective precipitation with polyof 30 X lo6 from the szo,W of the intact ethylene glycol, prior to the phenol treatment. single strands of a DNA. In our experiments, This latter method routinely gave preparamixtures of 3H-labelled T7 DNA and 32P- tions of (Y DNA containing 45-60% intact labelled a! DNA were cosedimented through strands (Fig. 2), with or without dialysis of sucrose gradients (Fig. 1). The ratio of the the phage against EDTA. No evidence of distances travelled by the two sedimenting double-strand breakage could be detected zones was measured in two independent (Fig. 2b). experiments, giving values of 1.098 f 0.020 During attempts to obtain a DNA with and 1.097 f 0.020. Taking the ~~20,Wfor T7 an even higher degree of strand intactness, DNA to be 32 & 0.8 (Studier, 1965), the it was found that overnight dialysis of the s%, w for (Y DNA is found to be 35.1 f 0.9. phage against a buffer containing 0.5 ill If the molecular weight of phage T7 DNA NaCl and 0.001 M DDC prior to phenol is taken as 25 X lo6 (Studier, 1965; Richardson, 1966; Leighton and Rubenstein, 1969), the molecular weight of a: DNA is calculated to be 32.9 X 106. RESULTS

Single-Strand Breaks Davison and Freifelder (1966) have reported the occurrence of characteristic

RADIAL

FIG. 1. zone sedimentation of native ti DNA. A mixture of BH-labeled T7 DNA (0.3 pg) and SeP-labeled LYDNA (0.5 pg, prepared in this case from crude phage) was layered on to a 5 ml neutral sucrose gradient and sedimented for 6 hr at 35,000 rpm. The radioactivity was measured in 3-drop fractions: O---C, aH counts; 0-0, 32P counts.

DISTANCE

FIG. 2. Boundary sedimentation of native and denatured O( DNA. Phage a DNA, prepared from purified phage dialysed against 0.5 M NaCl, 0.001 M EDTA, 0.1 M Tris-HCl (pH 8.0), was assayed for intactness by boundary sedimentation in (a) 0.1 M NaOH, 0.9 M NaCl; (b) 1 M NaCl, 0.001 M Tris-HCl (pH 8.0); (c) DNA prepared from a portion of the same phage preparation after dialysis against 0.5 M NaCl, 0.001 M DDC, 0.1 M Tris-HCl (pH 8.0) was sedimented in 0.1 M NaOH, 0.9 M NaCl. Rotor speed 37,020 rpm, DNA concentration 25 fig/ml. Ultraviolet photographs taken at S-min intervals were scanned with a Joyce-Loebl recording microdensitometer.

STRUCTURAL

PROPEKTIISS

treatment resulted in DNA containing 7075% intact strands (Fig. 2~). This increase in strand intactness was observed repeatedly with portions of the same a phage preparations which gave result,s similar to Fig. 2a. No further increase in the degree of strandintactness of (Y DNA was achieved by increasing the DDC concentration to 0.01 M, by including S-hydroxyquinoline at a concentration of 1%) in the phenol, by carrying out, the DNA release procedure with a 1: 1 mixture of phenol and chloroform, or by heat-shocking the dialysed phage (1 minute at 100”) immediately prior to phenol treatment. Further phenol trcatmcnt of a preparation of purified a: DSA containing 70 70 intact’ strands did not result, in any detectable increase in strand breakage, nor did storage of t.he DSA for several days at 4”. Since no preferred fragment size was detected either by boundary sedimentation or by zone sedimentation, it was concluded that the strand breaks were not located at any fixed distance from the ends of the a! DXA molecule. The following experiment was performed to measure the distribution of strand breaks between the complementary H and L strands. “2P-labelled intact strands of a DNA4 were prepared by sucrose gradient sedimentation (Fig. 3), combined with unfractionated 331’-labelled (YDNA and poly G, and separated into H and L strands by caesium chloride gradient centrifugation (Fig. 3b). If breaks were mainly in one strand, the 32P-labelled DNA should have been enriched for the other strand, and this would be reflected in a reduced 33P:32Pratio for the more intact strand. As shown in Pig. Xc, no preference n-as detected. The measured distributions of label were : 33P, 50.4 %I L strand, 49.6% H strand; 321’, 47.5 %’ L strand, 52.5 % H strand. It was concluded that the location of single-strand breaks in this preparation of a. DNA was essentially random. Separation

of the Complem.entary

Strands

The complementary H and L strands of a: DNA have been reported to be separable by MAK chromatography at pH 6.8 using a stepwise elution method at room tempera-

OF ALPHA

::j!j

I)NA

L IL 1.4 ; 1.0 20.6

FRACTION

NUMBER

FIG. 3. The distribution of strand breakage between the H and L strands of phage LY .l>NA. (a) %?-labeled intact strands of it: I>NA were prepared by zone sedimentation of alkali-denatured DNA (3.5 pg) through a neutral sucrose gradient for 3 hr at 38,000 rpm. (b) The two peak fractions from (a) were dialysed and combined with poly G and unfractionated 33P-labeled a DNA (3.2 pg), and the complementary strands were separat.ed by raesium chloride density gradient ceni,rifugation. Radioactivity was measured in 5-drop fract,ions: O----O, B3P radioactivity; @---a, 32P radioactivity; (c) O-----o. the 331’/32P percentage ratio in each .5-drop fraction.

ture (Cordes et al., 196lb). The complementary nature of the H and 1, strands was proved by measurement of the base compositions and by the ability to reassociate in mixtures, but not separately, to give a product with a buoyant density and hypochromicity approaching that of native DXA (Marmur and Cordes, 1963). A4urisicchio et al., (1964) also fractionated heat-denatured LYDPI’A by ll’IAK chromatography, but found their method suitable only for the production of an L strand fraction. After many experiments, using several of the published variations on the MAK chromatography procedure (S. Cordes, Ph.D. Thesis, Brandeis University, 1963; Aurisicchio et al., 1964; Roger, 1968; Rudner et al., 1968), we concluded that for the fractionation of high molecular-weight (Y DNA the most consistent results were obtained by salt gradient elution from refrigerated RIAK

376

SUTTON

AND

w 0.2 U 3 g 0.1 2

m d

IO FRACTION

20 NUMBER

FIG. 4. MAK chromatography of denatured a: DNA. Alkali-denatured 01 DNA (530 fig) was fractionated on a 3 cm X 1 cm diameter MAK column at 4”, using linear NaCl gradient elution (Roger, 1968) in 0.05 41 phosphate buffer (pH 6.8) : ultraviolet absorbance at 260 nm (7.ml frac-, concentration. Fractions tions), - - - -, NaCl were assayed for H and L content by analytical caesium chloride gradient centrifugation, with the following results: fract,ion 9, 1007, L; fraction 11, 60% L, 407, H; fraction 13, 30% L, 70% H; fraction 15, 20y0 L, 80% H.

columns (Fig. 4). DNA fractions eluted early from such columns were highly enriched (90-100%) for L strands. Later fractions were usually enriched for H strands, but always contained a significant L strand component. To obtain purified strand preparations, L- or H-enriched fractions from MAK chromatography were concentrated by adsorption to hydroxylapatite, then self-annealed in 30 % formamide; the reassociated fraction of the DNA was removed by hydroxylapatite chromatography. L strands prepared by this method contained no H strands detectable by analytical caesium chloride gradient centrifugation, and a second cycle of self-annealing followed by hydroxylapatite chromatography gave less than 2 % of the radioactivity in the reassociated fraction. It was, however, found that both L and H strand preparations obtained in this way consisted mainly of halfand quarter-Iength fragments (Fig. 5). Strand breakage appeared to be a consequence of MAK and hydroxylapatite chromatography of the dissociated strands. In view of the possibility of intrastrand heterogeneity in base-composition and secondary structure, such preparations could not be assumed to represent fully the sequences

PETEliSEN

found in the intact complementary strands of a DNA. Preparative density gradient centrifugation was used to give a more efficient separation. Although the buoyant density difference between the H and L strands in neutral caesium chloride, 0.005 g/ml (Aurisicchio et al., 1961), was found to be greatly increased in caesium sulfate gradients in the presence of mercuric ions (n’andi et al., 1965), the poly G-binding method (Kubinski et al., 1966; Sheldrick and Szybalski, 1967) was found to be more convenient for routine use. The increased density separation of H and L

FRACTION

NUMBER

FIG. 5. Zone sedimentation of purified H and L strand fractions of 01 DNA. 32P-labelled H and L strands of 01 DNA were prepared by MAK chromatography followed by self-annealing and hydroxylapatite chromatography as described in the text, then 2-3-pg portions of (a) L strands and (b) H strands were sedimented through 5-ml alkaline sucrose gradients for 4 hr at 35,000 rpm: 0-0, 32P radioactivity (IO-drop fractions). The arrows indicate where intact strands were found in identical gradients run at the same time as these experiments. (c) 33P-labelled H strands and 32P-labelled L strands of cy DNA were prepared by two successive cycles of caesium chloride density gradient centrifugation as described in Fig. 7. A mixture of the two preparations was dialysed against 0.01 ii/I Tris-HCl (pH S,O), 0.001 Af EDTA, and a l-fig portion sedimented through a 5 ml alkaline sucrose gradient for 3.5 hr at 38,000 rpm: O-O, 33P-radioactivity; $?P radioactivity ((i-drop fractions). O-0,

IBUOYANT

DENSITY

Fro. 6. ISffcct of poly (; on 1he buoyant densit) of drnat,urrd N I jN.4. Portions of denatured LY T)NA (557; strand-intact) were centrifuged lo eyl~ililr)rir~m ill c:tesi\un chloride density gradiellts. The density marker is native 01DNA. Ultraviolet photographs wcrc scanned with a Joyce-Lo&l rnicrodctrsitonlei~r. recordilrg Ciu-ve a, No poly G; bz poly (;/I )XA = 14. complexes formed at 20 pg I)NA/ml; C, poly G/DNA = !d, complexes forrnpd at 120 pg I)NA\i’rnl; tl. poly (;/I)SA = “i.

strands result,ing from polg G-binding is shown in Icig. 0. The separation obtained in preparative crntrifugings is shown in Fig. 7. Radioactive H and L strand fractions usually showd lcw than 3 % contamination with the complementary strand when assayed bv recentrifugation with added now radioactive OLDSA and poly G (Fig. ‘ib). The accuracy of t’his assay was established by an appropriatr control experiment. Wllfm cart was taken to minimize shear damage during handling of the separated strands, a substantial fraction of intact, strands was maintained even after two cycles of dtwity gradient, crntrifugation (Fig. k).

LiR-OP NUMBER

FIG;. 7. S(Lparatiott :rl~d assay of that II and 1, strands of 01 IjN.4 1)~ drllsity gradient ernt,riflqqtion (a) Alkali-derratllrrd :S~l’-lahellrd CY I jX-4 (50 pg) comhincd with poly (; (12.5 rg) was nc11tralised hy the addition of a half-volumr of 0.3 .21 KHZPOlr 0.1 JI KBITt’04, and separated into F1 and I, strands I)y crnt.rifLlging for 12 hr at 38,Oi)O rpm in a caesirmr chloride detlsity gradient with a preformed conrrntratioll step: ::Yl’-rtrdio:lCtivit~was measllred itr i- :llltl 5.drop fracl ions. (hi Pooled L strand fractions werf’ dialgsed againar 0.1 32 Tris-IICl (pH 8.0), 0.031 31 lCl)T.4. A 2-p~ portion was ~ombi~~rcl \vit h Ilonradioact ivcs C( IjN.4 (20 pg) and l~bly (; :III~ scparatt>d into II and L strand fracliolls as above (7.drop) frac’t ions 1.

-I

Tllr results of Cordes (S. Cordes, Ph.D. TIlesis, Ekandcis Iinivcrsity, 190;3) suggest#eda r:lthr>r homogeneous distribution of b:Lsc-clomI)ositiou \vithin the O( DNA molecult. So 1~etcrogc~nrit-y in buoyant densit) was found after slwaring the native DKA to quarter-length I’ragments, and denaturation gave OI~;V the t\vo bands, H and L, chxracterist,ic of the int,act, denatured DNA. We have confirmed this observation, malting use of tmlrc3 grwter rwolution obbaincd on cacsium

BUOYANT

DENSITY

FIG. 8. I&~nsity gradic~t11 ~c,tllt.ifl~g:tli(,tl r~l mercaru-ic complexes of dcnat ltrcbd 01I )SA. l’ort.iolls (2 pg) of hea-drnat Ilred O/ I )X.4 wrre crtri rifuged to equilibrirun in racsilurl slrlfa(r density gradictlls (initial density I.59 g?‘rnl) containing IIgs(I I ! ions at 12, = 0.3. (‘tirvf, n, 20”; strand-illtact CY DPiA, h. fragnrc~nted CY I)N;A, mt~an Irlolccrllal weight 3.5 X 106. 1~liraviolr~ photographs w(‘r(’ scanned with a Joycc~-l,ocThl rcc*ordillg rrric*rodensitomrtrr.

37s

SUTTON

BUOYANT

AND

DENSITY

FIG. 9. Effect of poly G on the buoyant

density of purified L strands. L strands of a DNA, prepared by MAK column chromatography as in Fig. 4, were centrifuged and scanned as in Fig. 6 but using a Beckman Analytrol densitometer. Curve a, no poly G; b, poly G/DNA = x.

sulfate gradients in the presence of mercuric ions (Fig. S). The idea of a simple molecular structure received support from the conclusion that in CYDNA only the H strand contains poly G-binding sites (Sheldrick and Szybalski, 1967). Our own experiments suggest a more complex structure. As shown in Fig. 6, the buoyant density of the L strand as well as the H strand was found to be significantly increased by low concentrations of poly G. At higher poly G:DNA ratios, both H and L strands were carried completely outside the usual density range (Fig. 6, curve d). Furthermore, when the poly G-DNA complexes were formed at high DNA concentrations (loo-150 pg/ml) a third band was sometimes found at a density intermediate between the H and L strands (Fig. 6, curve c). We interpret this as representing complexes containing both strands bound together by poly G. In support of this interpretation, when the middle band was isolated from a preparative caesium chloride gradient and recentrifuged after dissociation and reformation of the poly G-DNA complexes at a lower DNA concentration, only the normal H and L bands were obtained. It was found that purified L strands prepared by MAK chromatography mostly retained their poly G-binding capacity (Fig. 9), showing that the interaction of L strands with poly G does not require the presence of H strands. In later experiments, however, it was noticed that an L strand preparation that had been subjected to hydroxylapatite chromatography and several cycles of freezing

PETERSEN

and thawing had lost the ability to bind poly G. Density gradient centrifugation in the absence of poly G showed that the preparation now appeared to contain a small amount of DNA with a density approaching that of the H strand. This unexpected result was interpreted as suggesting that the intact L strand might contain a small segment of pyrimidine-rich DNA, perhaps 5000-10,000 nucleotides long. If this “heavy segment” contained all the L strand poly G-binding sites, then fragmentation of the strand would give rise both to “heavy” DNA and to pieces of L strand unable to bind poly G. This interpretation was supported by an experiment in which 32P-labelled (YDNA was sheared to approximately quarter-length fragments by repeated passages in the alkaline form through a No. 22 syringe needle, then combined with poly G and highly strand-intact nonradioactive cx DNA and centrifuged in a caesium chloride gradient. It was found that most of the sheared L strand formed a sharp band at the density expected for intact L strands in the absence of poly G (Fig. 10). Most of the sheared H strand retained the heavy density. In another experiment, 32P-labelled L strands and 33P-labelled H strands were each purified by two cycles of caesium chloride gradient centrifugation. In a further density gradient the poly G complexes of the two intact strands were clearly separated (Fig.

IO 20 30 FRACTION NUMBER

FIG. 10. Density gradient centrifugation of sheared and intact LYDNA in the presence of poly G. A preparation of 32P-labelled (x DNA (266 pg/ ml) in 0.1 1M NaOH was sheared by ten passages through a No. 22 syringe needle. A portion (4 pg) of the sheared DNA was combined with 106 pg of nonradioactive a DNA (75% strand-intact) and 25 rg of poly G, denatured in alkali and reneutralised, and centrifuged for 45 hr at 36,006 density gradient: rpm in a caesium chloride @--a, 32P radioactivity @-drop fractions); C-0, 260 nm absorbance (after dilution).

STRUCTURAL

PROPISRTIF:S

OF AT,l’tlA

1)xA

:
sayed for their content of t,he light segment by densit,y gradient cent’rifugation. DNA fragments with a folded secondary st)ruct uw would be expected t’o have an increawd sediment ation coefficient at neutral pH and would thus appear mainly in the Itaadingedge fractions (Studier, 1969). However, both fractions had a normal content of tlw light segment

FRACTION

NUMBER

FIG. 11. 1)ensity gradient, centrifugation of sheared and intact II and L strands in the presence of poly G. 32P-labelled L st.rands and 33P-labelled H strands of 01DNA were prepared by two cycles of density gradient centrifugation as in Fig. 5. (a) Portions of JJ and L were combined with nonradioactive o( DNA (50 pg) and poly G (15 pg) and recentrifuged: 0 -~-O, 33P radioactivity: 32P-radiosct.ivit,y (7-drop fractions o----o, colmted in a tol~lr,rre/Triton-X100 scintillator mixture). (b) A similar experiment after the radioactive strand mixture had been sheared to (Ittarter-length fragments as in approximately Fig. 10 (&drop frac2 t ioIls).

11)) although an incipient, shoulder, perhaps due t,o the ratther high content of half fragments, was found on the heavy side of the L strand peak. A mixt,ure of the same skand preparations sheared to quarter-lengt’h fragmen& centrifuged in anot,her caesium chloride gradient, showed a shoulder of heavy DKA derived from the I, strand and a shoulder of light DNA, presumably purinerich, derived from the H strand (Fig. lib). It could be argued that the reduced buoyant, density of the “light segment” of the H stmnd, :md its reduced capacity to bind poly G, is a reflection of secondaq structure rat,hrr t hart base composition. A single experiment suggested that this was not the case. The sheared 33P-labelled H strand preparation described above was fract,ionated by zone sedimentation through a neutral sucrose gradient. Leading-edge and trailing-edge fractions were then as-

It is clear from our results and from thcw of others (Davison and Freifelder, 1966; Preifelder, 1966), that the DKA of phage 0~. as isolated by phenol treatment, contains randomly located single-st,rand breaks. Similar breakage has been reported in t)hr DXA molecules of phages PBS1 and SP50 (Yam:igishi, 1968 ; Reznikoff and Thomas, 1969). Two general classes of explanation may br invoked : tait her the st rand breaks are alread! present in the DNA when it is pack:~ged into phage particles (k’ig. la), or else thcl!. are introdlwd during DSA release from the phage by endonuclease a&on or 1)~. metal-ion ca talysrd hydrolysis. Thcl Iat t et mechanism can scarcely explain our own results, since our purifit~d phage prrp:lr:ltiorrs

NUCLEASE

FIG. 12. Possible explanations for the origin of single-strand breaks in purified bacteriophage DNA. (a) I)NA packaged in nicked form inside the phage particle. (b) DNA packaged in st,randintact form inside t,he phage particle, but attacked during phenol release by an endonuclease preselr t as an integral component of the phage.

380

SUTTON

AND

were dialysed overnight agianst strong chelating agents prior to phenol treatment, and further phenol treatment, of the purified DNA did not result in any detectable increase in strand breakage. A single experiment in which strand-intact DNA from phage Xb2b5 was incubated with intact and osmotically lysed preparations of phage LY, and t)hen sedimented through alkaline sucrose gradients, failed to demonstrate any endonuclease activity. However, no serious at,tempt was made to optimize the conditions for t’his assay. We feel that an explanation of the type shown in Fig. 12b, where an endonuclease is present as an integral component of the normal phage part’icle, has not yet been ruled out. Such an enzyme might be very labile once released from the phage particle, as is the RNA polymerase found inside Reovirus particles (Kates and McAuslan, 1967). It, seems to us not unreasonable to propose the existence of a phage-enclosed endonuclease, since such a component might be required to release phage DNA molecules from concatenated phage-precursor DNA (Smith and Skalka, 1966; Frandel, 1966).

PETERSEN

tained numerous single-strand breaks. In view of the likely compositional heterogeneity of the L strand, such fragment.ed preparations cannot be regarded as representative of the entire strand. The conclusion that only the H strand of (Y DXA is transcribed (Tocchini-Valent,ini et al., 1963; Geiduschek et al., 1964) depends on hybridisation experiments which used “L strands” prepared by t,he method of Aurisicchio et al., (1964), and will certainly need to be reexamined. In view of the correlation found between poly G-binding sites and transcribing regions in other DNA molecules (Taylor et al., 1967; Summers and Szybalski, 1968), it would seem quite likely that at least the “heavy segment” of the L strand of CLDNA may also be transcribed. It has been report,ed t,hat the DNA molecules of several temperate phages (Skalka et al., 1968; Falkow and Cowie, 1968) and bacterial sex factors (Falkow et al., 1966) are characterised by a segment,al distribution of nucleotides into regions of distinct base composition. Our results, and those of Cordes (S. Cordes, Ph.D. Thesis, Brandeis University, 1963), do not, suggest a similar pattern for the Poly G-Binding Sites DNA molecule from phage a. The most The observed distribution of poly G- likely structure for cy DNA can be regarded binding sites in the (Y DNA molecule is of as differing from an “ideal” structure, with some interest. Our observation that the in- a uniformly purine-rich L strand and a tact L strand can bind poly G is in disagree- uniformly pyrimidine-rich H strand, by an ment with the conclusion of Sheldrick and inversion of lo-20 % of the molecular length. Szybalski (1967). However, these authors It is not clear that this degree of structural did not,ice that the H and L strands of (Y heterogeneity is any greater than that which DNA were less well separated at high poly can be demonstrated in the “homogeneous” G concentrations than the strands of t,he DNA molecule of phage T4 by poly UG other Bacillus phages studied. ;\Iore im- binding (Guha and Szybalski, 196s). portantly, since poly G-binding sites are ACKNOWLEDGMENT released from most of the L strand by shearing t,o quarter-length fragments, it) is W. D. S. thanks the Director, Applied Biochemistry Division, D.S.I.R. for the generous clearly necessary to study intact rather than fragmented DNA preparat,ions if valid con- provision of laboratory space and facilities. clusions are to be made about the structure REFERENCES and function of 01 DNA. It is interesting AURISICCHIO, S. (1966). The DNA of phage 0~. that the “I, strand” preparat,ions of Aurisicin Nucleic Acid Research” In “Procedures chio et al., (1964) had a mean sedimentation (G. L. Cantoni and D. R. Davis, eds.), p. 562. coefficient of only 13 S, as compared with Harper & Row, New York. 25 S for the heat-denatured whole a! DNA. AURISICCHIO, S., COPPO, A., 110~1~1, P., FRONSince even the latter value is much too low ~MJ, C., GKAZIOSI, F., and TOSCHI, G. (1961). for intact strands of a! DNA in SSC buffer, Rapporli dei Laboratori di Fisica dell’lstituto Superiore di Sanita, ISS., 61, 33. these DNA preparations must, have con-

STRUCTURAL

PROPERTII:,S

C., GAETA, (1964). Chromatographic purificat,ion of one of the complementary strands of D??A of phage (Y. Biochim. Biophys. Acfa 80, 51‘1-51G. lSs~~.u.\nor, (:. (19(S). Chromatography of nucleic acids 011 hydroxyl:tpatiLe. Nature (London) 206, 779-X3. BOKNER, J., KCJXG, G., and BEKHOR, I. (1967). h method for t hr hybridisation of nucleic acid molecules at low trmperature. Biochemistry 6, 3G50-3653. Bnrrxn, C. F., and TImu, V. (1969). Rapid equilibrinnl isopycnic CsCl gradient,s. Biochi,m.. Biophj/s. Ar,/a 179, 136-14-I. JS~RGI, li:., and HERSHEY, A. 1). (1963). Sedimentatiou rate as a measure of molecular weight of I)Nh. Biophfys. .I. 3, 309-321. CLLUSEN, T. (1968). Measurement of 32P activity in a liquid scintillat,ion counter without the use of s~intillalor. Anal. Biochepn. 22, 70-73. CORDES, S., &STEIN, IT. T., and MARMUR, J. (196la). Some properties of the deoxyribonucleic acid of phage CI. Nature (London) 191, 1097-109s. CORDES, S., EPSTEIN-, H. T., and MARMUR, J. (19Glb). Properties of the isolated complement ary strands of the I)NA of phage 01. Fed. Proc., Fed. Amer. Sot. Ezp. Biol. 21, 375. I).\VISON, I’. F. (1966). The rate of strand separaI ion in alkali-treated DNA. J. Mol. Biol. 22, !)i-108. 1’. F., and FREIFELDER, D. (1966). J).\YISON, 1,abilit.y of single-stranded DNA to hydrodynamic shear. J. Mol. Bid. 16, 49(t502. F \LKOw, S., and COME, D. B. (1968). Intramoleclllar heterogeneity of the DNA of temperate Ijactrriophages. .I. Hacleriol. 96, 777-784. F.\LKox\., S., C~T.~I~ELI,.\, II. V., WOHLHIETER, .J. A.. and WAYYNAIIE, T. (1966). The molecular llafllr(, of R-facators. .I. Mol. Biol. 17, 102-116. FII\NKEL, I?. R. (1Mfi). Studies on the nature of replicating DN4 ilr TJ-infected Escherichia coli. ./. .1Ic11.BLOl. 18, 127-143. FHEIVELDEI:, I). (1966). Inactivation of phage 01 1)~ singl+strand breakage. Virology 30, 32&332. GLIIJI~SI.HEK, 14;. l’., To~,~~~I~~I-~.~LEN,~I~I, C;. I’., :nltl S.\ns.\‘l. M. T. (19fiJ). Asymetric synthesis IIf I:?;A in ~ilro: d(~pendence 011 DNA contitlrlity and cclllforrrl:rlii)t1. f’roc. .\-at. ACM.!. Sri. 17.S. 52, -W-193. C;UII.\. A., and Szr 11\I.SKI, W. (1968). Fractionat io11 of the rolnplf,rnrll!‘tl’S strands of coliphage T-l 1)TA based on the asymmetric distribution l)f the }NJly IT :111d poly U.(: binding sites. l~-iro/r,gy X4, (XL3 (ilG. K.\‘I’Es, .I. 1’., and M~A~-sI..\N, B. It. (1967). PoxL4~~~(~~~CCHI~). S., I)oL~E, F., nr~d TOSCHI, (:.

E., FR~NTALI,

OF .2LPIlB

::s 1

1)S.Z

virus 1 )NLdependrnt RNA polymcrase. Kaf. Acd Si. U.S. 58, 134-l-21, KUBINSKI, IL\LSKI.

I-I.,

OP\R\-KUHINSK.\,

S.,

I’roc,

:r~~d SZY-

W. (196G). Patterns of interttct,ion between polyribonucleot,ides and individllal l)NA strands tlcrived from several vc~rtcbralcs, bacteria and bacteriophages. ./. .Ilo/. Bid. 20, 313-329. LEIGHTOS. S. H., and RUHENVIEIN, I, (lSfi!) 1. Calibratiotl of molecular weight scales for I)NA. J. Md. Hiol. 46, 313-328. M.\NUELI,, J. I)., and HERSHEY, A. 1). (,19~iO1. A fractionating co1um11 for ailalysis of cinelr,ic* acids. A ncrl. Hiochuar 1, M-77. M.wMUR, J., and <:ORI)ES, 8. (19(i3). Studies on i he complcme~~t,ar~~ strauds of bacteriophagSr1 1)X.4. In “Ilrforrn:rtiolral Macromolecllles” ill. .J. Vogel, 15. Bryson. and J. 0. Lnmpc~n, t&.i, pp. 79-87. Academic Press, New York. N.\mr, u. S., w.ZNG, .J. C., and ~)AYIi>SON, Iv. (1965). Separation of deoxyribonucleic acids by Hg (II) binding and CS&(.)~ density-gradient centrifugalion. Biochemistry 4, 1687.-1691i. PATTERSON, 31. S., and GREENE, 11. (:. (19S3i. Measurement of low energy beta-emitters i 11 aqueous solution by liquid scintillation coln~t illg of emulsions. And. Chem. 37, 854-857. REZNIKOFF, W. S., arid THOM.XF,, C. A., .JIL (t!Ni!Jl, The anatomy of the SP 50 bacteriophage 1)X.\ molecule. Virology 37, 309-317. RICHARDSOX, C. C. (19G6). The 5’.terminal nucleotides of T7 bacteriophage deoxvrihorrllclcic acid. J. ilIo1. Biol. 15, 49-61. ROGER, M. (19G8j. Chromat,ograptiic rf:solut.ion of complementary strands of denatured prre~~mococcal 11X.4. Proc. Sot. A cntl. Sci. I,-.S. 59, 200-2Oi. RUDNER, R., K~RKM, J. I)., and CH.\IUXFV. 1,;. (1968). Separation of B. .subliZis I)XA irrto complementary strands. I. biological properties. 1’roc. Xaf. Acad. Sci. I;.S. 60, KHHiX5. SHELI)RICK, l’.. and HZY~L~LSKI, W. (1967) Distribution of pyrimidine ‘iclllsters” between the complementary DNA strands of c*rrt,ain Bacillus bacteriophages. .J. Mol. Bid. 20, 217~228. SK \I,,< \, ii.. 13riw1, t’., and JlZ4lSHl~:>-, .I. 12. (1968). Segmental distribrltioll of n~~cleot idw in the 1)X.4 of bacteriophage lambda .I. 11~~1. Bid. X4, l-16. SMITH, Al. (;., :ttrd SK.U>L\, A. (1966). jion~t~ ~,r,,prrties of 11X.4 from phage-infected b:tc.tcri:i. J. Gr,r /‘h!/siof. 49, No. 6 Pt. 2, 127-142. Srrr:r,rr.:~<, F. W. (I9G.5). Sedimentat.ion st,titlitxs (,I the siz,cs :III~ shay):) of 1)N.A. -1. 3/01. Niol. II, 37:1 39;).

382

SUTTON

AND

SIJMMERS, W. C., and SZYB~LSKI, W. (1968). Totally asymmetric transcription of coliphage T7 in viva: correlation with poly G binding sites, Virology 34, 9-16. SUTTON, W. D. (1969). The structure of the bacteriophage alpha DNA molecule. Ph.D. Thesis, Massey University, New Zealand. TAYLOR, K. HRADECNA, Z., and SZYBALSKI, W. (1967). Asymmetric distribution of the transcribing regions on the complementary strands of coliphage X DNA. Proc. Nat. Acad. Sci. U.S. 57, 1618-1625.

PETERSEN TISELIUS, A., HJ~RTEN, S., and LEVIN, 6. (1956). Protein chromatography on calcium phosphate columns. Arch. Biochem. Biophys. 65, 132-155. TOCCHINI-VALENTINI, G. P., STODOLSKY, M., AURISICCHIO, S., SARNAT, M., GRAZIOSI, F., WEISS, S. B., and GEIDUSCHEK, E. P. (1963). On the asymmetry of RNA synthesis in wivo. Proc. Nat. Acad. Sci. U.S. 50, 935-942. YAMAGISHI, H. (1968). Single strand interruptions in PBS1 bacteriophage DNA molecule. J. Mol. Biol. 35, 623-633.