In vitro packaging of covalently closed circular monomers of bacteriophage DNA

J. Mol. Biol. (1975) 98, 465-478

In Vitro Packaging of Covalenfly Closed Circular Monomers of

Bacteriophage DNA GAu, J. PRUSS, JAMES C. W ~ G t

AND

RIChArD CAL~TDAR

Molecular Biology Department and Chemistry De~artmen~ University of California BerbelJy, Calif. 94720, U.S.A.

(Received 11 April 1975) Extracts from ooliphage P2 or P4-infected cells can package exogenous P2 or P4 DNA into viable phage particles. This in vit~o system was used to determine the structure of the I)NA substrata for the packaging reaction. Results obtained with covalently joined linear ooncatemers, eovalently closed circles, and mature phage DNA as the substrates indicate that DNA packaging in the phages P2 and P4 does not involve the cutting of mature phage DNA from a concatemerio precursor. Instead, the substrata of the in vitro DNA packaging reaction appears to be either a covalently closed circular monomer of the phage DNA or the mature phage DNA itself. Sedimentation analysis of P2 phage DNA synthesized in cells infected with a P2 head mutant showed that covalently closed circular DNA molecules accumulate in such cells. Together, the in vit/roand ~ v~voresults suggest that covalently closed monomer circles are the substrates of the P2 and P4 DNA packaging reactions.

1. Introduction We have reported previously on an in vitro DNA packaging system for temperate coliphage P2 and its satellite phage P4 (Pruss e~ al., 1974). These DNA phages possess linear duplex chromosomes of unique sequence which terminate in single-stranded complementary ends (Tnman & Bertani, 1969; Tnman e~ al., 1971). The base sequences of the cohesive ends of the P2 and P4 genomes are identical (Wang et al., 1973); however, overall homology between the two chromosomes is less than 1 ~ by DNADNA hybridization (Lindqvist, 1974). The P4 genome has a molecular weight of 7.5 • 10e (Wang et al., 1973). I t is, therefore,, only about one-third the size of the P2 genome (22• 10e; Tnman & Bertani, 1969). Satellite phage P4 requires the produets of all the known P2 late genes for synthesis of phage particles. As seen under the electron microscope, satellite and helper phage are very s!mflar: the P2 and P4 tails are identical (T~men et al., 1971); the P4 head, however, has only one-third the volume of the P2 head (Gibbs et al., 1973). The requirement of P4 for P2 late genes suggests that many steps in phage assembly, DNA packaging in particular, might be very sjmiI~r for the two phages. We have found, in fact, that P4 DNA can be packaged in extracts prepared from P2-infected cells (Pruss e~ al., 1974). Experiments in which P4 DNA was packaged in a P2-infected cell extract produced 465

466

G. J. P R U S S , J. C. W A N G A N D R . C A L E N D A R

P4 plaque-formers contalnlng three copies,~Jof the P4 genome. This production of phage carrying multiple copies of the P4 chromosome suggested t h a t concatemeric D N A might be the 9substrate for the in vitro packaging reaction. The role of concatemeric D N A as a precursor to m a t u r e phage D N A has been demonstrated in vivo for a number~of diverse phage species (Botstein & Levine, 1968; Skalka, 1971; T h o m a s et aJ., ~96~; l~rankel, 1968). I n addition, Syvanen (1975) has shown t h a t the D N A substrate for phage h packaging in vitro is a covalently linked concatemer. I n the case of P2 and P4, however, in vivo'experiments have indicated t h a t the phage D N A is present in infected cells as covalently closed circles (Lindqvist, 1971 ; Lindqvist & Six, 1971). This p a p e r reports the results of in vitro and in vivo experiments performed to determine w h a t form of D N A serves as substrate for P2 and P4 packaging. The in vitro packaging system was nsed to test directly different forms of P2 and P4 D N A for their ability to serve as packaging substrate. The in vivo studies consisted of sedimentation analysis of lysates of cells infected with a P2 head m u t a n t and were used to ascertain what form of phage D N A accumulates under those conditions. 2. M a t e r i a l s a n a M e t h o d s

(a) OhemicaZs [Mcthyl-aH]thymldlne (spee. act. about 75 Ci/mmol) was purchased from ICN Pharmaceuticals, Inc. Special enzyme grade sucrose was purchased from SchwarzfiViann. Mitomycin C was obtained from Sigma Chemical Company. Ethidium bromide was purchased from Calbioehem Corporation. (b) Bae~riophagc strains The two P2 strains used for infection carry the v~rl mutation, which prevents the establishment of immunity but does not affect sensitivity to immunity (Bertani, 1957). P2 vixl am12 is defective in cell lysis (Lindahl, 1971); P2 virl amQ34 carries a mutation affecting head synthesis (Lengyel et 02., 1973; Sunshine et 02., 1971). The 3 P2 strains from which DNA was extracted for use in the in vitro packaging reactions all carried the virs2 mutation, which confers insensitivity to P2 immunity (Bertanl, 1975). The stra.ln~ were P2 v/r22; P2 virz2 tsLav, which carries a temperature-sensitive mutation affecting head synthesis (Lindahl, 1969; Lengyel et 02., 1973); and P2 v/r22 amA12~, which carries an amber mutation in a gene required for P2 DlqA synthesis (Lindahl, 1970; Lindqvist, 1971). P4 vir 1 areA1, an amber mutant unable to synthesize I)NA under non-permissive conditions (Gibbs et 02., 1973), was used as the infecting phage~in making P4-infected cell extracts. P4 DNA was extracted from P4 v/r 1, a clear plaque mutant (Lindqvist & Six, 1971). (c) Batter/02 attain8 The bacterial strains used are shown in Table 1 ; all are derivatives of Escherichia coli C (Bertani & Weigle, 1953). (d) Media Super TPG medium (Pruss et 02., 1974), which is a highly enriched version of the Trisbased minimal TPG-CAA (Lindqvist, 1971) was used to grow strains C-la and C-1766 for the in vitro DNA packaging experiments. TPG-CAA medium supplemented with either thymidine or thymine as indicated was used to grow strains HF4704 and C-1760 for [SH]thymidine labeling experiments. T P G adsorption medium is TPG-CAA medium minus glucose and amino acids but containing 2.5 gg thymine/ml. LB (Tryptone/yeast extractbased rich medium) medium (Bertani, 1951) was used to grow the indicator strains. (e) _Prcpa,ra~r~ of ex~ruet~for in vitro D N A loa~kagmg These extracts are a 3000 g supernatant fraction derived from lysates of concentrated infected cells. P2-infeeted cell extracts were prepared from C-la infected with P2 virl

CIRCULAR DNA PACKAGING T~B~

407

1

~ a r ~ e r ~ l s~r~n~ Strain designation

Relevant genotype~

C-la C-1055 C-1197

supsups u p - (P2 +)

C-1757 C-1758

supD + supD + (P2co~)

C-1760

sup- ~hy- u~r.A - (P2 +)

C-1766 C-1900 HF4704

supD + (P2 cox4) sup- (P2 tsL~) s ~ - $hy- uvrA-

Reference or origin Sasaki & Bertani, 1965 Wiman e~ a/., 1970 From C-1055 by lysogeniza~ion (Pruss e$ a/., 1974) Stmshinc e~ aL, 1971 From C-1757 b y lysogenization (Gibbs e$ a/., 1973) From HF4704 by lysogenization (Gibbs e~ al., 1973) Gibbs e~ a/., 1973 From C-1055 by lysogenization Lindqvist & Siusheimer, 1967

Abbreviations used: supD +, amber suppressor D, according to T~ylor & Trotter's (1972) nomenclature; thy-, thymidine or thymlue requiring; ~ r A - , u.v.-svusitive, fails to repair u.v. damage. amxs. P4-infeeted cell extracts were p r e p a r e d from C-1766 infected with P 4 v~rl areA1. E x t r a c t p r e p a r a t i o n followed t h e procedure given in Pruss et al. (1974), except t h a t large quantities of concentrated infected cells were p r e p a r e d a t one t i m e a n d stored for up to 2 m o n t h s a t --64~ On t h e d a y of an experiment, a n a p p r o p r i a t e volume of the cells was thawed, a n d the remaining steps in e x t r a c t p r e p a r a t i o n were carried out. (f) I n vitro D • A packagin~y reaction The reaction conditions were the same as those described in Pruss e~ aZ. (1974): 50 pl of e x t r a c t were i n c u b a t e d for 60 mln a t 37~ in the presence of 5 rnm-phosphoenolpyruvate, 1 m ~ - A T P , 1-3 ~g p y r u v a t e kinase, a n d exogenously a d d e d P2 or P4 DNA. I n t h e stand a r d reaction, P2 virs2 or P4 virx DI~A was a d d e d as t h e packaging substrate. I n 2 experim e n t s designed to test whether genetic recombination p l a y s a role in in v~ro packaging, a m i x t u r e of P2 v~r22 tsL87 a n d P2 v/rs2 amAls7 D N A was a d d e d as the packaging substrafe. Depending on the particular experiment, the concentration of exogenous D N A in t h e reaction v a r i e d from 0.01 to 6.5 pg/ml. The final reaction volume was 65 pl; each reaction was performed in duplicate. The reaction was s t o p p e d b y transferring to 0~ a n d diluting 10-fold w i t h P-buffer conta.~nlng 50 /~g pancreatic DNAase/ml. P.buffer is 10 m~-Tris.HC1, p H 7"2; 1% , . m ~ o n l u m acetate, a n d MgC12 a t 10 m ~ for P2 or 80 m ~ for P4. Packaging of the exogenous D N A into viable phage particles was assayed b y plating the diluted reaction mixture on suitable indicator bacteria. Strain C-1197 was the s t a n d a r d indicator used to detect packaging of p 2 vira~ or P4 v/r 1 D N A into p.f.u.~. Since this indicator carries a P2 prophage b u t no a m b e r suppressor, neither t h e ~rnrnunity sensitive P2 a m b e r m u t a n t phage n o r the P 4 a m b e r m u t a n t phage used for infection can grow on it. To detect t h e t o t a l p.f.u, produced b y packaging P 2 v~ra2 tsL87 p l u s P2 v/r22 amAla7 D N A , t h e diluted reaction m i x t u r e was p l a t e d on C-1758 a t 30~ This strain contains a P2 prophage; therefore, the i m m u n i t y sensitive P2 phage strain used for infection is unable to grow on it. I n t h e same experiment, P2 v~r22 p.f.u, produced b y packaging D N A molecules arising from recombination between the P2 v~r22 ~sL37 a n d P2 v/r22 amAx27 D N A were detected b y plating on C-1900 a t 42~ This strain cont~in~ a P2 tsL87 prophage b u t lacks an a m b e r suppressor. Consequently, i t does n o t allow growth of the P2 infecting phage strain, which is sensitive to i m m u n i t y a n d which carries a n Abbreviation used: p,f.u,, plaque-formlng units.

468

G. J . P R U S S , J . C. W A N G A N D R . C A L E N D A R

a m b e r m u t a t i o n , or of P2 ~r~z amAxzT; t h e incubation temperature, 42~ is non.permissive for P 2 ~ r ~ t~Lsv. I n addition, P2 v~r~ ~ s ~ v cannot give rise t o plaques on this strain v/a recombination with t h e prophage, since t h e prophage itself carries t h e t~Lsv mutation. P2 ~ r ~ amAl~v does n o t initiate replication in this strain; thus m a r k e r rescue of amAx~7 + from the prophage is v e r y rare ( ~ 10-~).

(g) Preparation of DNA D N A was e x t r a c t e d w i t h phenol from purified P2 r~r22, P2 v/r2~ t~Lnv, P2 vir~ areA127, a n d P 4 v/r 1 phage stocks (Thomas & Abelson, 1966!. This D N A consists of double-stranded molecules possessing single-stranded cohesive ends a n d is referred to as m a t u r e phage DI~TA. Covalently closed circular D N A forms were p r e p a r e d as follows: P2 or 1>4 D N A e x t r a c t e d as described was diluted in 0.1 ~-I~TaC1, 0.01 ~ - E D T A to ~12eo ~ 0.2 to 1.0. The per cent of oligomerie circles increases w i t h increasing D N A concentration. I t was t h e n h e a t e d a t 75~ for 10 to 15 rnln a n d cooled quickly to 0~ N e x t , t h e D N A was allowed to anneal either b y incubation for 2 h a t 45~ or b y dialyzing overnight a t r o o m t e m p e r a t u r e against high salt (2 M-NaCI, 0-01 M-EDTA). A f t e r anne~llng, t h e D N A was concentrated b y dialysis against sucrose followed b y dialysis agains~ 10 m ~ - T r i s (pH 8), 2 rn~-MgCl~, 1 mME D T A . Ligase closure was performed a t 30~ for 30 mi~ in t h e presence of 10 -5 to 10 - 4 z~D P N a n d 50 pg bovine p l a s m a alb~mln/ml. E. coZi D N A ligase was purified according to Olivera & L e h m a n (1967). The products of t h e ligase reaction were b a n d e d in e t h i d i u m bromide/CsC1 (Radloff e~ aJ., 1967), mid t h e closed circles were isolated from t h e gradient. E t h i d i u m bromide was r e m o v e d b y n-butanol e x t r a c t i o n after which t h e D N A was dialyzed against high salt followed b y dialysis against 10 mM-Tris (pH 8), 2 mM-MgC12 or 10 mM-Tris (pH 8), 50 m~r-NaC1. Covalently joined linear concatemers were p r e p a r e d in a slm~lar m a u n e r : P2 or P 4 DI~TA was d/luted to Aae0 -~ 5 in 100 mM-NaC1, 1 ~ - E D T A , 10 m~-Tris, p H 8, a n d was allowed to anneal a t 45~ for 2 h. Ligase closure was t h e n performed as described except t h a t t h e reaction was carried out a t 37~ for 1 h. Separation of linear D N A from the m i n u t e a m o u n t s of covalently closed circles also formed was achieved according to t h e procedure a l r e a d y described. To separate covalently closed P2 ~ r ~ or P4 v~rl circular monomers from d~mers, 2 pg of aH-labeled circular P2 v / r ~ or P 4 v~r~ D N A was l a y e r e d onto a linear 5o//o to 20% (w/v) n e u t r a l sucrose g r a d i e n t eontaln~ng I0 m ~ - T r i s (pH 8), ~0 m~r-l~TaC1, 0.i ~r-EDTA, a n d centrifuged a t 40,000 revs/m~u in a n SWS0.1 r o t o r for 210 rnln a t 4~ Drops were collected from t h e b o t t o m of t h e tube. Distribution of label was determined b y counting a portion from each fraction in Aquasol (New E n g l a n d Nuclear) in a P a c k a r d model 3320 liquid scintillation spectrometer. The p e a k fractions of each species were pooled n d dialyzed overnight against 10 mM-Trls (pH 8), 50 m~r-NaC1, 0"1 ~ - E D T ~ .

(h) Preparation of aH-~bded phage (i) aH-to,b e ~

P2

HF4704 was grown a t 37~ with aeration to a d e n s i t y o f a b o u t 2 • 108 eells/ml in T P G . C A A m e d i u m s u p p l e m e n t a l w i t h 10 pg thymidlne/ml. The culture was t h e n har. vested b y centrifugation a t 10,000 g for 20 mln a n d resuspended in t h e same volume in w a r m e d T P G - C A A m e d i u m supplemented w i t h 2/~g t h y m i d i n e / m l a n d 0.4 ~Ci [ S H ] t h y m i dine]ml. The resuspended cells were allowed to resume growth at. 37~ w i t h aeration. A f t e r 5 rn~n of aeration, t h e culture was inoculated with P2 v~r~s p h a g e a t a m u l t i p l i c i t y of a b o u t 8. A t 22 rnln after infection, which Was shortly before lysis was expected to occur, E D T A was a d d e d to a final concentration of 4 n ~ . A t 55 m i n after infection, b y which t i m e lysis should h a v e been complete, t h e culture was centrifuged a t 10,000 g for 20 m~n to remove bacterial debris. P h a g e were isolated from t h e s u p e r n a t a n t b y precipitation w i t h 70//0 polyethylene glycol (PEG) followed b y D N A a s e t r e a t m e n t of t h e precipitate as described in Lengyel e~ a/. (1973) a n d in B a r r e t t e~ aL (unpublished data). T h e r e s u l t a n t phage p r e p a r a t i o n was further purified b y 2 successive bandings in a CsC1 gradient.

CIRCULAR D N A PACKAGING

489

(ii) aH-/~bel~ P 4 C.1760 was grown and infected with P4 ~ r l accorfllng ix) the procedure described above for 8H-labeled P2. Since the lyric cycle of P4 is considerably longer t h a n t h a t of P2, E D T A was added at 50 mln after infection, and the culture was centrifuged to remove bacterial debris at 90 rain after infection. Phage were concentrated from the lysate supernatant b y precipitation with 6% P E G followed b y DNAase treatment of the preoipi. rate as described b y Gibbs e~ a/. (1978) and b y Barrett e~ a~. (unpublished data). The phage were further purified b y banding twice in CsC1. (i) D27~4 ntaf/wraNon ecaperlmenta The conditions for infection were adapted from Lindqvist (1971). E. col~ HP4704 was grown at 87~ with aeration to a density of about 2 x 10e cells/ml in TPG-CAA medium supplement~d with 10/Lg thymino/ml. At this point, the cells were transferred to an opaque flask and incubated at 37~ for 10 mln without aeration in the presence of 60 /~g mitomycin C/ml. Treatment of uvr~4- cells with mitomycin C before P2 infection selectively suppresses host D N A synthesis. The mitomycin C-treated cells were concentrated b y collection on a Milliporo filter, washed with TPG-CAA adsorption medium, and resns. pended in one-tenth volume of t h a t medium. These cells were inoculated with P2 v~r1 o/ral2 or P2 v~r1 watQ.q4 at a multiplicity of about 8 and incubated at 37~ for 10 mln without aeration to allow phage adsorption and injection. A portion of the cells was left uninfected, b u t otherwise treated identically. The cells were finally diluted with nine-tenths volume TPG-CAA medium containing 2"5/~g thymlno/ml and allowed to resume aeration at 37~ At 9 rain after growth with aeration was resumed, [aH]thymidine was added to the infected cells to a final concentration of 20/tCi/ml. Two mln later, unlabeled thymidine was added to a final concentration of 650/~g/ml to prevent further incorporation of radioactivity. A t various times during the chase, 200-/L1portions were transferred to t.~bes oonte~nlng an equal volume of lysis mixture. The composition of lysis mixture and the procedure for lysis are from Botstein (1968) except t h a t lysates were stored at 0~ and analyzed within 2 h of preparation. I n each experiment, a lysate cont,.inlug mature, all-labeled P2 D N A was prepared as a r-arker. The marker lysates were made b y mi~ing uninfected, unlabeled cells with all-labeled P2 ~ r l phage and lysing t h e m as above. This all-labeled P2 virl was a gift from Dr J a n e t Geisselsoder. The lysates were analyzed b y sedimentation through 5% to 20% alkaline sucrose gradients made up in 0.25 M-NaOH, 0.75 ~-NaC1, 0.005 M-EDTA. All gradients were poured on top of a 0.3 ml shelf of 60% alk,.llne sucrose to allow recovery of very rapidly sedimenting material. The procedure for applying samples to the gradients was adapted from Botstein (1968): 100 /~1 of a solution 0.35 M in N a O H and conta~nlng 1% (w/v) Sarkosyl was layered on the gradient, followed b y application of 100 /~1 of lysate. The lysate~ were layered using an Eppendorf micropipet with a wide bore tip (obtained b y cutting about $ r, ra off the end of a standard Eppendorf tip). Centrlfugation was carried out at 35,000 revs/r-l- for 90 m~n in a SWS0.1 rotor at about 4~ Drops were collected from the b o t t o m of the tubes. A n 80/L1 portion from each fraction was pipetted onto a 2.5-cm disk of W h a t m a n 3 MM filter paper and allowed to dry thoroughly. The filters were then soaked twice in cold 10% triohloroaeetic acid, washed once in cold water and once in 95 % ethanol, air dried, and counted in toluene based scintillation fluid in a Packard model 3320 liquid scintillation spectrometer. 3. R e s u l t s To prepare t h e forms o f D N A tested i7~ ~itro, m a t u r e p h a g e D N A was annealed u n d e r conditions w h i c h f a v o r e d f o r m a t i o n of either linear cone~temers or circles. These h y d r o g e n - b o n d e d molecules were t h e n eovalently sealed l~ing ~/. coli D N A ligase. I n t h e rest o f t h e paper, covalently joined linear coneatemers will be referred t o simply as linear concatemers, a n d covalently closed circular molecules will be referred t o as circles.

470

G. J. P R U S S , J. C. W A N G A N D R. C A L E N D A R (a) P 2 D N A forms paclcaged in an extract of P2-infected cells

Table 2(a) compares t h e packaging efficiencies o f m a t u r e phage D N A , linear coneatemers, a n d circles in vitro. Of t h e three D N A forms tested, circles are p a c k a g e d m o s t efficiently and co~catemers least efficiently. T h e circular D N A ~is~d in t h e experiments in Table 2(a) was ~ m i x t u r e o f m o n o m e r s a n d etlmers with a b o u t t e n times as m a n y monomerie molecules as dimeric ones. To determine which of these forms was responsibl e for t h e packaging a c t i v i t y observed, 8H-labeled circular P2 D N A was sedimented t h r o u g h a 5~/o t o 2 0 ~ neutral sucrose gradient (Fig. 1). The m o n o m e r a n d dimer peaks were t h e n tested separately in t h e packaging reaction. As can be seen in Table 2(b), the m o n o m e r circle was t h e b e t t e r substrate.

TABLE 2

.Relative pacIcaging e~ciencies of linear and circular P2 D N A in a P2-infeoted cell extract Form of DNA

(a) Mature Linear conoatemers Circles (b) Monomer circles Dimer circles

Relative p.f.u./~g DNA

1 0.22• 2.95• 1 0.17 4- 0.02

(a) The data shown are the average of 4 separate experiments. Three different preparations of extract and one preparation of DNA were used. The absolute packaging efficiency of the mature DNA varied between 1 • 104 and 3.6 • 105 p.f.u./~g DNA depending on the particular extract used in an experiment. (b) The data shown are the average of 2 experiments. Two different preparations of extract and one preparation of DNA were used. The abolute packaging efficiencies varied from 8.7 • 104 to 1.0• 106 p.f.u./~g DNA. Dimer circles were tested at a final concentration of 0.05 ~g]ml; monomer circles were tested at 0.25 ~g]ml. However, the packaging efficiency of mixed circles has been shown to be constant over the range 0.06 to 5.4 ~g DNA/ml (data not shown). Estimated standard errors are indicated in both (a) and (b). (i) Recombination i8 not required for paclcaging monomer circles I t is conceivable t h a t t h e observed packaging of m o n o m e r circles was the result of packaging molecules which h a d first recombined with other P2 D N A molecules to f o r m oligomers. Such a m e c h a n i s m has been d e m o n s t r a t e d for the in vitro packaging of m o n o m e r circles o f ~ D N A (Syvanen, 1974). A l t h o u g h this possibility was lmlil~ely in t h e case o f P2, since m o n o m e r circles are a b e t t e r packaging substrate t h a n dlmer circles, experiments were performed ~o determine w h e t h e r recombination was a prerequisite for packaging m o n o m e r circles. To test for recombination o f m o n o m e r circles with endogenous P2 D N A , we t o o k a d v a n t a g e o f the fact t h a t the P2 p h a g e used for infection carries an a m b e r m u t a t i o n a n d is sensitive to i m m u n i t y , whereas t h e exogenous P 2 D N A is a m * a n d carries a n immunity-insensitive marker. R e c o m b i n a t i o n between D N A molecules o f these two genotypes followed b y packaging should give rise to i m m u n i t y insensitive, a m -

CIRCULAR

DNA PACKAGING

471

monomer

300

u

,~ 200 Z r.,. o,,I

Q.

o I00

4

0

I 5 Boftom

I I0

= 15

20

25

Froclion no.

30

35

40 Top

FzG. 1. N e u t r a l sucrose g r a d i e n t s e d i m e n t a t i o n of SH-labeled circular P 2 D N A . S e d i m e n t a t i o n was carried o u t as described in Materials a n d Methods. F r a c t i o n s 16, 17, a n d 18 were pooled, dialyzed, a n d thereafter referred to as P2 m o n o m e r circles. F r a c t i o n s 9 a n d 10 were pooled a n d dialyzed to yield t h e D N A referred to as P2 dimer circles.

and immunity sensitive, am + recombinants. Therefore, the products of the in vitro packaging reactions to which monomer circles of P2 DNA had been added were assayed on two different indicator strains, C-1758 and C-1055. Strain C-1758 is permissive for immunity insensitive, a m - P2 phage; strain C-1055 is permissive for immunity sensitive, am + P2 phage. The infecting P2 phage cannot plate on either of these indicators, but p.f.u, having the genotype of the exogenous DI~A can plate on both of them. 51 of the plaques which arose on C-1758 and 51 of those on C-1055 were picked into wells containing LB broth and then replica tested for the ability to plate on C-1197, ,which allows only immunity insensitive, am + P2 p.f.u, to plate. Of the 102 plaques picked, all 102 were able to plate on C-1197; therefore, all must have had the genotype of the exogenous DNA. Consequently, recombination of monomer circles with endogenous DNA is not required for packaging. To test for recombination of exogenous circles with each other, a mixture of circular P2 vir22 tsL87 and P2 vir22 amAz2~ DNA was used as substrate for the packaging reaction. The A (early) and L (head) genes are separated by the P2 attachment site (art) and are far from one another on the P2 genetic map (Fig. 2). The reaction products were assayed for p.f.u, of the parental genotypes (vir22 ts- and vir~2 a m - ) and for p.f.u, of one of the recombinant genotypes (vir22 t8 + am +) as described in Materials and Methods. Phage having the genotypes of the exogenous DNA were found in 10S-fold higher concentration than phage having the recombinant genotype. Therefore, recombination of the exogenous circular DNA molecules with each other is not required for packaging. Thus, covalently closed circular monomers of P2 DNA are an efficient substrate for packaging in vitro. The packaging of monomer circles implies, moreover, that the enzyme responsible for generating the cohesive ends of mature phage DNA requires only one cohesive end site to act. The term "cohesive end site" refers to the specific site on the phage chromosome where the staggered cuts which produce the cohesive ends are made.

472

G. J . P R U S S ,

J . C. W A N G

AND R. CALENDAR

Necessary for cleavage Capsid (cleaved)

0

P

0 N M L

Head

K

t Lysis

aft

Tail genes

v/~ I

BA

Early

F i e . 2. Genetie m a p of phage P2 (Lindahl, 1969,1970,1971; Sunshine aS aL, 1971) w i t h emphasis on markers used in this study. Transcription units are indicated b y horizontal arrows.

(b) Packaging of P4 D~VA in extracts of P4-infected, P2 lysogen8 Table 3 compares the packaging efficiencies of mature P4 phage DNA, linear concatemers, and circles in vitro. As is the ease with P2, circular molecules are packaged more efficiently t h a n linear concatemers. The preparations of circular P4 DNA tested in the above experiments were mixtures of monomers, dimers, and trimers in the a p p r o ~ m a t e proportions given in Table 3. The preparation having the highest proportion of monomer circles was packaged most efficiently, suggesting t h a t monomers might be the preferred substrate, To test this hypothesis directly, 3H-labeled circular P4 Dl~A was sedimented through a 5 % to 20% neutral sucrose gradient to allow separation of the different sized species (Fig. 3). The packaging of monomer and multimer fractions was then tested in vitro. As can be seen in Table 4, multimeric circles were consistently packaged with lower efficiency than monomer circles. TABLE 3

Rela$ive packaging,e~ciencie8 of linear and circular P4 DNA in an extrac~ prepared from Pg-infected, P2-1ysogenic cell~ F o r m of D N A

The

Relative p.f.u.]~g D N A

Mature Linear eoncatemer8

1 0.37

Circles: monomer, dimer :trimer(mass ratios) = 87:11 : 2 monomer 9dimer: trimer(mass ratios) ----72: 21 : 7

9.4 3.4

absolute effieieney of paokaging mature P4 DNA in this experiment was 3.0 • 10s p.f.u./~g

DNA.

Thus, the substrate specificity of packaging P4 DNA in vitro into P4-sized heads appears similar to t h a t of packaging P2 DNA in vitro into P2-sized heads. In particular, monomer circles m a y be the preferred substrate. Moreover, the enzyme which makes the P4 cohesive ends--which is most likely the same enzyme as t h a t which makes the P2 cohesive ends (Wang e$ al., 1973; Six, 1975)--requires only one cohesive end site to act.

CIRCULAR

DNA PACKAGING

473

monl ~rner

300

u

7 O

~t 200

-

dimer expecl expect trimer/'~ j

5 Bottom

I0

15

20 25 Froclion no.

30

35

40 Top

Fro. 3. Neutral sucrose gradient sedimentation of SH-labeled circular P 4 DNA. Sedimentation was carried out as described in Materials a n d Methods. Fractions 20, 21, a n d 22 were pooled, dialyzed, and used as t h e source of P 4 m o n o m e r circles in the packaging reactions. Fractions 15 a n d 16 were pooled, dialyzed, a n d used as the source of P 4 dimer circles. The expected positions of trlmer a n d t e t r a m e r circles were predicted from the position of monomer.

TABLE 4

Relative paclcaging e~ciencles of monomers and multimers of circular P4 D N A in extracts of Pal.infected, P2-1ysogenic cells Size of D N A circles

Monomer Dimer Fraction 13 F r a c t i o n 12 Fraction 11 Fraction 10

Relative p.f.u./~g D N A Expt 1 Expt 2 1 0.22 0.27 0.38 0.46 0.42

1 0.16 0.18 0.26 8.29

Two different preparations of extract were used. I n experiment 1, t h e D N A concentration in the packaging reactions varied from 0.16/~g/ml for monomer circles to 0.01 Fg/ml for fraction 10. I n experiment 2, all D N A forms were tested a t a final concentration of 0.02 pg]rnl. The packaging efficiency of monomer circles in experiment 1 was 2.2 • 106 p.Lu.h~g D N A ; in experiment 2 it was 1.2 • 108 p.f.u./pg DNA. The packaging efficiency of m o n o m e r circles in experiment 2 was the highest ever observed in our system.

(c) F o r ~ of P4 D N A ~ a ~ k ~

in an ~trac~ of P2-infected cel~

I n the previous two situations, a DNA molecule the size of one genome appears to be preferred over a concatcmeric molecule as the substrate for packaging in vitro. One can also ask what is the preferred substrate under conditions in which more Chan one genome is incorporated into a phage head, as occurs when 1)4 D N A is packaged in an extract of P2-infected cells (Pruss et u~., 1974). Is any P4 DNA concatemer of length greater t h a n or equal to three P4 genomes an adequate substrate, or is a

474

G. J . P R U S S ,

J . C. W A N G A N D R . C A L E N D A R

preference shown for a molecule which approximates the size of the P2 genome? To answer this question, we used circular P4 DNA from the sucrose gradient shown in Figure 3 as substrate for packaging in an extract of P2-infected cells. As can be seen in Figure 4, packaging efficiency of the DNA as a function of position in the gradient reaches a peak at the fractions where trimers are expected ;"it does not increase continuously with increasing sedimentation coefficient. Consequently, the preferred substrate is a molecule which approximates the size of a single P2 genome.

expect expect trimer tetramer 1

--g.500

I00

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r-~

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I

]

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15

20 25 Fraction no.

30

35

40 Top

F i e . 4. Packaging effieieney of circular P4 DNA in a P2-infeeted cell extract as a funotion of DNA size. The monomer pool, dimer pool, and fractions 10 to 13 of the D N A from the gradient in Fig. 3 were tested for ability to serve as packaging substrates in a P2-infected cell extract. The relative packaging effieieneies of the different forms are plotted as a function of their position in the grs~lient. For comparison, the gradient profile of Fig. 3 is also shown. - - O - - O - - , SH-labeled P4 DNA, - - 9 - - 9 relative p.f.u./~g DNA.

(d) Ma~ur~ion of D N A in rive Monomer circles are clearly a better substrate for packaging in vitro than linear concatemers. This result implies that monomer circles are the precursors of mature phage DNA. Therefore, ix) obtain further evidence that circles are the DNA packaging substrate, we studied the maturation of I)NA in rive. We expected that P2 phage defective in head synthesis might also be defective in DNA maturation, since a close relationship between these two processes has been demonstrated for several different bacteriophage species (Botstein etal., 1973; Wake et al., 1972; Frankel, 1968). Consequently, cells infected with P2 head mutant phage under non-permissive conditions might accumulate the precursor of mature phage DNA. The head mutant we chose for this study, P2 virz amQs4, makes heads devoid of DNA, as determined by examining thin sections of infected cells under the electron microscope (Lengye] e~ al., 1973). In these experiments, cells infected with P2 lysis-defective or P2 head mutant phage were given a two-mlnute pulse of [SH]thymidine followed by a chase with excess unlabeled thymidine. At various times during the chase, portions were removed from the cultures and lysed gently. Since the lysis procedure also lyses phage, a "marker" lysate containing 3H-labeled mature P2 DNA was prepared by lysing a mixture of 8H-labeled P2 phage and uninfected, unlabeled cells. The lysates were

CIRCULAR

DNA PACKAGING

475

then sedimented through alkaline sucrose gradients and the distribution of acidprecipitable radioactivity was determined. Only phage DNA could incorporate label in these exper/ments, since mitomycin C was used to suppress host DNA synthesis (Lindqvist, 1971). Figure 5(a), (b), (c) and (d) shows the results of the control experiment: the maturation of 1)2 DNA in cells infected with P2 lysis-defective phage. Immediately after the pulse, three peaks of radioactivity are found. One is at the bottom of the gradient (on the shelf); the second is at a position corresponding to a sedimentation rate 3.7 times that of the marker; and the third is at the position of marker. The peak which sediments 3.7 times as fast as marker must consist of covalently closed circular duplex DNA, since such molecules are expected to sediment three to four times as fast as single-stranded DNA generated by denaturation of mature DNA (Gellert, 1967). Between 4 and 11 minutes after the pulse, nearly all the label in the duplex circle peak disappears. During the same time period, however, increasing amounts of label appear in the peak which sediments at the position of marker. Normal lysis time corresponds to the ll-m~n chase time point (22 min after infection). By 50 mln after the infection (39 rain chase), virtually all the label sediments at the position of marker. These results agree with those of T,indqvist (1971), who found that circular duplex P2 DNA pulse-labeled late in infection is chased into mature DNA.

Head~" 0 mln chase

(e)

Head'; 4 min chase

(f)

Marker peak

(hi

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25

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30 Top

FIo. 5. Alkaline sucrose gradient sedimentation of pulse-labeled intraoellular P 2 DNA. Sedim e n t a t i o n was carried out as described in Materials a n d Methods. The drop size decreases considerably a t t h e t o p of these gradients. As a result, the top 5 to 6 fractions, those above f r a c t i o n 22, correspond to only 3 to 4 n o r m a l sized fractions. The arrow in (h) indicates t h e p e a k posit/on of m a r k e r D N A in a parallel gradient.

476

G. J. PRUSS, J. C. WANG AND R. CALENDAR

Figure 5(e), (f) and (g) shows the results os parallel infection with the P2 head mutant, P2 virl a ~ 3 4 . Immediately after the pulse, radioactivity is found in the same three peaks.as i~ the control. In this case, however, label disappears from the peak correspondh~/g,to marker and appearsin the circular duplex peak during the chase. By the time lysis occu_~s, duplex circles account for the largest fraction of the labeled DNA. Thus, duplex circles, which have been shown to be a precursor of mature DNA in control experiments, accumulate in cells infected with P2 head mutant phage. 4. Discussion Circular monomers of P$ DNA are packaged more efficiently than linear concatemers in extracts of P2-infected cells. Similarly, circular monomers of P4 DNA are packaged more efficiently than linear concatemers in extracts of P4-infected, P2lysogenic cells. These results imply that concatemeric DNA is not the normal substrafe for DNA packaging for phages P$ and P4. The packaging substrate appears to be a circular DNA molecule the size of one genome. P4 DNA can also be packaged in extracts of P2-infeeted celL~. In this case, particles contaln~ng three copies of the P4 genome are produced (Pruss ~ al., 1974). When monomers and multimers of circular P4 DNA were tested for the ability to serve as packaging substrate in P2-infected cell extracts, the trimer was found to be packaged most efficiently. Since a trimeric P4 DNA molecule approximates closely the size of a m ~ e r i c P2 DNA molecule, these results again support the notion that the normal substrate for DNA packaging for phages P2 and P4 is a unit genome size, circular, DNA molecule. DNA maturation experiments further support the conclusion that circular DNA is the packaging substrate. In cells infected with a P2 lysis-defective mutant, pulse~ labeled phage DNA was observed to chase from covalently closed circular duplex DNA to mature or nicked circular DNA. In cells infected with P2 head mutant phage, however, label accumulated in covalently closed circular duplex DNA. These experiments suggest that circular DNA is the precursor to mature phage DNA. Thus, P2 and P4 appear to be unique among double-stranded DNA phages studied so far in that they use a circular monomer of the phage DNA as packaging substrate. Emcient packaging of circular monomers suggests, moreover, that the enzyme which cleaves the cohesive end site of P2 and P4 DNA requires only one such site to act. Thus the double cohesive end-sites model suggested by Wang & BreT.in~ki (1973) appears to be invalid for the P2 and P4 systems. The identification of circular monomers of the phage DNA as the packaging substrate also provides an explanation for the very low frequency of recombination observed for P2 (Lindahl, 1969). Most recombination products would not serve as an efficient substrate for packaging as they can in the case of ~, but must first be cleaved to generate monomer circles. The experiments presented here indicate that cleavage of P2 and P4 replicative DNA into unit genome lengths occurs independently of head assembly. Previous studies of P2 DNA replication (SchnSs & Tnman, 1971; Kainuma-Kuroda & Okazaki, 1975) have suggested that P2 DNA is replicated via a modified rolling circle mechanism in which the tail of the circle is cleaved once it reaches unit genome length. This cleavage might occur at cohesive end sites, as suggested by Kainuma-Kuroda & Okazaki (1975 i. Monomer circles would be generated by annealing of the cohesive ends followed by ligase closure. We prefer an alternative model, however, suggested

CIRCULAR DNA PACKAGING

477

to us b y D. Botstein. I n this model, the c/~-aeting product of P2 gene A, which is located near the replication origin (Fig. 2), promotes a site-specific recombination event to cleave the tail and produce monomer circles. B o t h models predict t h a t immediately after intracellular phage DNA is pulse-labeled, radioactivity should be found in a peak which sediments in alkali like mature DNA; this is what we observed (Fig. 5). B o t h models also predict t h a t label should be transferred from mature (or nicked circular) DNA to covalently closed duplex circles at early times during the chase; at later times, as DNA starts being packaged, label should transfer back again to mature DNA. I n cells infected with the P2 head m u t a n t P2 virl amQ84, we observed a chase of label from material sedimenting like mature DNA to duplex circles, as predicted. This chase was not observed in the control infection, however. Perhaps, under normal conditions, DNA packaging m a y begin before the production of duplex circles terminates, thereby obscuring the pathway. We thank P. Anderson, D. Botstein and J. Geisselsoder for stimulating discussions. This investigation was supported by Public l=Iealth Service research grants AI-08722 from the National Institute of Allergy and Infectious Diseases, G1K-14621 from the National Institute of General Medical Sciences, and CA-14097 from the National Cancer Institute, by training grant GIRL01389 from the National Institute of General Medical Sciences, and by research grant B1KS-74-19607 from the National Science Foundation.

REFERENCES Bertani, G. (1951). J. Bact~ol. 62, 293-300. Bertani, G. (1975). Mol. •en. Genet. 136, 107-137. Bertani, L. E. (1957). Virology, 4, 53-71. Bertani, G. & Weigle, J. J. (1953). J. Bacb~ol. 65, 113-121. Botstein, D. (1968). J. Mol. Biol. 84, 621-641. Botstein, D. & Levine, M. (1968). Cold S~ring Harbor Syrup. Q ~ n t . Biol. 38, 659-667. Botstein, D., Waddell, C. H. & King, J. (1973). J. Mol. Biol. 80, 669-695. Frankel, F. R. (1968). Gold S t r r i ~ Harbor Syrup. Quant. Biol. 33, 485-493. GoUor~, 1K. (1967). Proo. N~..4cad. Sc~., U.S.A. 57, 148-155. Gibbs, W., Goldstein, R. N., Wiener, R., Lindqvist, B. & Calendar, R. (1973). Virology, 53, 24-39. Tnm~n, R. B. & Bertani, G. (1969). J. MoL Biol. 44, 533-549. Inman, R. B., SehnTs, iK., Simon, L. D., Six, E. W. & Walker, D. H. (1971). Virology, 44, 67-72. K&inuma-Kuroda, R. & Okazakl, R. (1975). J. Mol. Biol. 94, 213-228. Lengyel, J. A., Goldstein, R. N., Marsh, M., Sunshine, 1K. G. & Calendar, R. (1973). Virology, 53, 1-23. Lindahl, G. (1969). Virology, 39, 839-860. Lindahl, G. (1970). Virology, 42, 522-533. Lindah]~ G. (1971). Virology, 46, 620-633. Lindqvist, B. H. (1971). Mol. Gen. Genet. 110, 178--196. Lindqvist, B. H. (1974). Prec. Nat. Acad. Sci., U.S.A. 71, 2752-2755. Lindqvist, B. H. & Sinsheimer, R. L. (1967). J. Mol. Biol. 28, 87-94. Lindqvist, B. H. & Six, E. (1971). Virology, 43, 1-7. Olivera, B. M. & Lehman, I. R. (1967). Prec. Nc~. Acad. Sci., U.S.A. 57, 1426-1433. Pruss, G., Goldstein, R. N. & Calendar, R. (1974). Prec. Nat. Acad. Sci., U.S.A. 71, 2367-2371. Radloff, R., Bauer, W. & Vinograd, J. (1967). Prec. Nat. Acad. Sci., U.S.A. 57, 1514-1521. Sasaki, I. & Bertani, G. (1965). J. Gen. Mi~robiol. 40, 365-376. Sehn~s, M. & Tnm~m, R. B. (1971). J. Mol. Biol. 55, 31-38.

478

G . J . PRUSS, J. C. WANG AND R. C A L E N D A R

Six, E. W. (1975). Virology, in the press. Skalka, A. (1971). I n The Bac~ricpho~e Zwtnbda (Hershey, A. D., ed.), pp. 535-547, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Sunshine, M. G., Thorn, M., Gibbs, W., Calendar, R. & Kelly, B. (1971). V~rology, 46, 691-702. Syvanen, M. (1974). Pror 27a~. Acad. Sci., U.S.A. 71, 2496-2499. Syvanen, M. (1975). J. M~L BICL 91, 165-174. Taylor, A. L. & Trotter, C. D. (1972). Bac~4,ol. Rev. 36, 504-524. Thomas, C. A., J r & Abelson, J. (1966). In Procedure,8 in Nucleic Acid Re.search (Cantoni, G. L. & Davies, D. R., eds), pp. 553-561, Harper &Row, New York. Thomas, C. A., Jr, Kelly, T. ft., J r & Rh0ades, M. (1968). Cold S~'t~ng HwtSor Syrup. Quant. Biol. 33, 417-424. , Wake, R. G., Kaiser, A. I). & Tnrnan, R. B. (1972). J. Mol. Biol. 64, 519-540. Wang, J. C. & Brezinski, D. P. (1973). _Proc. Nc~. Acad. Sci., U.S.A. 70, 2667-2670. Wang, J. C., Martin, K. V. & Calendar, R. (1973). Bicchemistrry, 12, 2119-2123. Wiman, M., Bertani, G., Kelly, B. & Sasaki, I. (1970). Mol. Gen. Oenet. 107, 1-31.