Bacteriophage T4 head morphogenesis

VIROLOGY

61, 43S442

(1973)

Bacteriophage V. The Role of DNA Synthesis RONALD Department

T4 Head in Maturation

B. LUFTIG

Morphogenesis of an Intermediate

AND

NANCY

in Head Assembly

P. LUNDH

of Microbiology and Immunology, Duke University Durham, North Carolina $7710

Medical

Center,

Accepted November 9, iQY2 On the basis of metabolic-inhibitor studies it was found that maturation of T4 head intermediates, which had accumulated in tsC9 (gene 49) mutant infected cells, was apparently dependent on continued DNA synthesis. This requirement for DNA synthesis in head maturation was confirmed by using a gene 43 mutant (tsP36) blocked in the enzyme, T4 DNA polymerase. In temperature shift-up experiments with tsP36infected cells it was found as previously reported (Luftig and Ganz, 1972) that 300s particles accumulated when enzyme synthesis was halted. These latter structures appear identical to the gene 49-defective intermediates since, when isolated, they both: (i) had an average sedimentation coefficient of 310 f 20s and a nucleoprotein density of 1.34 g/cc; (ii) had t,he major capsid polypeptide in a cleaved state; (iii) were quantitatively rescued (to a >50$$ level in 40 min) after the mutant-blocked function was recovered, and (iv) showed a reduced ability to convert to phage when an inhibitor of DNA synthesis such as fluorodeoxyuridine (FUdR) was added at the time the function was rescued. The maturation of rapidly labeled 300s wild-type (T4D+) head intermediates was also found to be sensitive to inhibitors of l)NA synthesis. Upon isolation these heads had the same properties as the above gene 49 and gene 43 mutant-defective interfor mediates. This strongly suggests that DNA synthesis is a normal requirement maturation of a T4 capsid intermediate, whose properties are defined above.

for head maturation which involved the introduction of at least some of the phage DNA into the head after completion of the capsid structure. On the basis of additiona. st,udics (Luftig and Ganz, 1972), a more specific scheme for head maturation was postulated. This scheme involved: (i) the formation of a capsid intermediate not completely filled with DNA, yet still associated with the 1000s DNA concatenate; (ii) the synthesis of DNA, at a site distinct from the packaging site, providing the energy for filling the head, and (iii) the action of a nuclease, apparently under the control of gene 49, which cleaves a “head-full” of DNA from the concatenate. In this paper we have extensively investigated the second postulate, viz., the requirement of DNA synthesis for head maturation.

INTRODUCTION

The problem of how a full complement of phage T4 DNA is packaged into the head capsid has been under study in several laboratories (Kellenbcrger et al., 1959; Kellenberger et al., 1968; Granboulan et al., 1971; Laemmli, 1970; Lacmmli et al., 1970; Luftig and Wood, 1969; Luftig et al., 1971; Luftig and Ganz, 1972; Simon, 1972; Frankel et al., 1971). Experiments of Frankel et al. (1971) and Luftig et al. (1971) presented cvidencc that gene 49-defective mut’ant infected cells accumulated both (i) a 1000s concatenate of DNA containing more than one phagc-size DNA equivalent, and (ii) defective head structures which served as intermediates in phage maturation. This led to the proposal of a model 432 Copyright All rights

@ 1973 by Academic Press, of reproduction in any form

Inc. reserved.

T4 HEAD

MATURATION

AND

DNA

SYNTHESIS

433

Duke. The same results were obtained with FUdR, A grade, obtained from Calbiochem. L-[U-‘~C] lysine monohydrochlor119 minimal medium (119); RI9 supple- idc and L-[U-14C]leucinc at about 300 mCi/ mcnted with a mixture of 1X I) ,L-amino mmole; thymidine [mcthyl-3H] and deoxyacids (319 A); H brot’h; EHA top and bot- guanosine [%3H] at’ 6 Ci/‘mmole was obtom agar; T’O, buffer containing 2 X 1OP’ tained from Schwarz/iVann. J1 MgSO, (0.1 X IjO,-?\lg buffer) has been previously describrd (Luftig and (e) General Procedure for Infection Ganz, 1972). For grow-th of E. coli B.3, a E. coli Bb or B3 was grown in a bubbler thyminc-requiring auxotroph, RI9A medium tube with vigorous aerat,ion at 41.5”C to ~ZZ supplementctd wit,h *5pg/ml of thymidine 4-6 X lOa cells,/ml (microscope count and (P-L Bioch(lmicals). agar plating) in 20-40 ml of A19 or &ISA (1,) Escherichia coli Host StrairLs from an original inoculum of $& vol of a Strains Hb, and B3 obtained from Drs. freshly grown overnight cult,urc. The infectivity and labeling procedure of these S. I’. Champe and D. Hall, respcct’ively, cells for a tcmperat,urc shift,-down cxperihavt: hecr~ dr>scribcd (Luftig et al., 1971; ment is described in E’ig. 1. To verify that Luftig and Ganz, 1972). Both are rcstrican efficient infection had occurred, samples tivc for a,/l mutants. Strain S/6/5 also w-ere removed at 5 min postinfection and rcbstrictivc for ar/7 mutants was used for plated to determine infective centers, t,hr plating and making st’ocks of ts mutants. percentage of infected bact’eria and bacStrain Cl163 was used for plaqucl assays terial survivors. Thcsc were, respectively, and making stocks of anz mut,ant#s. 3.5 X 108/ml; 73 %-; and 1 T; as averaged over all experiment’s reported. In tcmperaturc shift’-up t>xpcrimt:nts inThe phagcx mutants used in this st#udy fection occurred as in E’ig. 1; however, at arc tsC9 (gmc: 49), tsl’36 (gene 43), and the time of shift 20 ml of culture was placed atnS76 (gene 21). They arc from the Cal into a 70°C hot wat)er bath for 15 set and Tech phage collection of W. B. Wood and then transferred t,o a 41.5”C bat,h. have: hem described previously (Epstein Preparation of rxtracts with chloroformet al., 1953; Luftig and Ganz, 1972). E’or DBase, and zone ccntrifugat,ion of the exall isot,ope-incorporation cxpclrimcnts, phage tract on lo-30’:;’ (w/v) linear sucrose stocks grown in 319 or hI9A were purified gradients has previously been described bv two or thrccl cycles of differential cen(Luft’ig et al., 1971). Fractions of 15 drops fiigugation and suspended in 0.1 X P04were collected and t’richloroacetic acid Rig buffer, with rccovcbry of >50 “; of the (TCA)-precipitable counts were assayed in infectious particles. a Beckman liquid scintillation counter. For CsCl equilibrium centrifugation, the pooled (d) Reagents 300s peaks from the sucrose gradients were Chloroform was B & A analytical grade. used. Conditions for centrifugation and Sucrose was from Fisher. Crystalline DBase assay of fractions have been previously dcI (R?u’ase fret) was obtained from Worthscribed (Luft#ig et al., 1971). ington. Chloramphenicol, a gift of Dr. I?. (f) Polyacrylamide Gel Electrophoresis Harriman, was obt’ained from the Duke University Hospital. Nalidixic acid (YAL)’ Acrylamide (7.5 ?l)-SDS gels were used made up in 0.1 N KaOH, was from according to Smit’h et al. (1969). The gels Schwarz/Mann. The fluorodcoxyuridine were run for 20 hr at 3.7 mA/gel in 13.5 X (P’UdR) initially used was a gift of D. Hall, 0.6.cm glass tubes. The samples were boiled 2 min in a 1: 1 mixture with 10 M ’ Abbreviations: Nalidixic acid (NAL) ; triurea, 2 % SDS, and 2 C,L P-mercaptoethachloroacetic acid (TCA) ; fluorodeoxyuridine nol. Autoradiography of such gels were by (FUdR). MATERIALS

AND

(a) Xeclia and Buglers

METHODS

434

LUFTIG

AND LUNDH

Mutant (fsC91 4,,50

Temperature -

,

C'2

Amino Acid chase ct4 Amino Acid jtfecjd.

Samples removed

‘jse

‘/jthym

i

1

’

J

i

1 I

I 0

8 Mmutes

15

21

23

25

post - InfectIon

27 0

IO

30

50

Minutes post -shift FIG. 1. Labeling and incubation regimen for pulse-chase, temperature-shift experiments wit,h t.sCY, in the presence of drugs that inhibit DNA synthesis. Eschcrichia coli Bb or B3 cells were grown as described in Materials and Methods. Then infection and superinfection with tsC9 were performed at a multiplicity of about 4 phage per cell. A pulse of [‘%]lysine (0.2 &i/ml culture) was followed 6 min later by a l,OOC-fold excess of unlabeled amino acid. On the average, SOY6 of t,he label was incorporated into 5%; cold TCA-precipitable counts by the time of the chase. Cultures were divided in half at 22 min postinfection and one half was treat,ed with a drug, viz., FUdIl (200 pg ml) or nalidixic acid (100 ~g ml). Note that the same amount of drug solvent was added to the control culture. Then [“Hlthymidine (4-5 &i/ml) was added at the time of temperature shift-down to the treated and untreated cultures in order to monitor DNA synthesis. The temperature shift-down was accomplished by placing the cells into an ice bath for 1.5-2 min to lower the temperature to 25°C and then transferring it to a 25°C bath. Samples (5 ml) were removed at various times postshift, and extracts were prepared as described in the text). In some experiments an additional drug dose was added at 10 min after the first dose, with essentially no differences obtained in the results. -

the technique of Fairbanks Exposure was for 72 hr. (g) Electron

et al. (1965).

Microscopy

Techniques for negative staining of extracts have been described (Hamilton and Luftig, 1972, in press). Grids mere examined in a Philips Ehl 300 electron microscope. Images were recorded on Kodak estar base 70-mm film. RESULTS

Previous results of Luftig et al. (1971), Frankel et al. (1971), and Luftig and Ganz (1972) have implied that gene 49 plays an important role in T-2 head maturation. These results showed that Wdefective heads, isolated from mutant infected cells

grown under restirictivc conditions, had the following properties: (i) thcx\- banded at a nucleoprotein density and contained on the average an 18 ?;I phage equivalent picce of DNA, and (ii) they were presumably attached to the cell membrarw through a DNase-sensitive linkago. ‘l’hcwforc~, gcnc 49 mut’ant infected cells appear t,o haw bcgun t,he DNA packaging prows, but the question of how it is finished remains un answered. Our previous studies rcvcalcd that pulse-chase tttmpc>raturo-shift, expcrimerits with t&9, a tctmpc~raturt~-scrlsitive mutant of gene 49, would be useful in analyzing packaging rcquiremcnts (Luftig et al., 1971). The experiments described bt+w have used this technique to study the: role of DNA synthesis in phagct mat,uration.

T-l HEAD

MATURATION TABLE

AND

DNA

SYNTHESIS

435

1

EFFI:(T OF FUdRQ AND N~LIDIXIC ACID* ON THE REDISTRIBUTION OF [WILYSINE COUNTS FROM300s P.IRTICLJGSTO 1000s PHAGE~ IN kC%INFEcTED CULTURES Minutes postshift 0 10 30

Control ~~~

1000s

300s

129,000 63,250 27,000

+ FUdR

(1%) (34%) (777;)

300s

1,900 36,000 90,000

123,200 106,800 92,000

+ NAL 1000s

(0%) (10%) (20%)

13,o: 27,000

300s

1000s

76,000 113,000 66,000

(42%,) 38,000

a As assayed by either [3H]thymidine or [3H]deoxyguanosine incorporation added 2 min after FUdR, 1)NA synthesis was decreased to a 6-fold level of the control by 30 min postshift. No change occurred at that, time in the amount of [‘%]lysine incorporated. 4 For NAL, [sH]thymidine incorporation was only slightly decreased (75$& that of the control) at 30 min postshift. c The parentheses indicate the percentage of counts in the phage peak from an average of two experimcnts.

D.VA

Inhibitor

h’tutlies

with

tsC9 Defectice

It had hwn previously shown that two drugs which arc potent inhibitors of bactclrial DKA synt’hesis are FUdR (Cohen ef al., 195s) and KAL (Goss et al., 1965). Sinw thcsc: drugs also blocked T4 phage DSA synthesis to some extent (Mathews, 19W; Baird et al., 197%), we have utilized them in inhibitor experiment’s with kC9 as illustrated in Fig. 1. In the first set of expwimcnts I;CdR or NAL was added several minutrs before shift-down. Samples w’r(’ rcmowd at the times indicated, g;c>ntly troatcld to lysc the cells, and then analvzcd by zone sedimentation in sucrose gradkts as described in Materials and 1Icthods. Under t’hese condit’ions the label \vas dist’ributt>d either in a 300s (gene 49dcfwtivt: head particle) or 1000s (phage) peak. ,1s shown in Table 1, both inhibitors gave a marked reduction of counts flowing from 300s partjiclcs to 1000s phage suggesting that DP;A synthesis is required for maturation of these particles. Since FUdR was the better inhibit’or (Table 1) it has been used in subsequent studies. In one such study we examined the effect of FUdR

on phagc-synthesized

de nouo after

the shift-don-n of a t&9-infected culture. Prom previous studiw \vith chloramphenico1 added at the time of shift-down (Luftig et nl., 1971), it \vas found that’ only 30’.0 of thn phagc produced at 30 min postshift

FIG. 2. Burst size of a Kg-infected culture after rescue of the gene 49 function in the presence (-----) or absence (p) of FUdR. Samples (0.05 ml) were removed from the infected cells at the indicated times into 5 ml of dilution medium (
came from head pwcursors that had accumulated at the restrictive temperatuw. Hence, if FUdR did not affect tie now synthesis of phagc, \I-P would expect a burst size of about 70 ‘,( of the control, untreated culture. Instead, after treatment with FUdR, we found that the burst size was one-third that of the urltreated culture (l:ig. 2). This could be due either to: (i)

436

LUFTIG

AND

LUNDH

FIG 3. Extracts prepared from tsC%infected cell cultures at 30 min postshift (a) with FUdR or (b) withot it FUdR, as observed in the electron microscope. Arrows point to eit,her (a) empty-appear ,ing heads, or (b) phage particles. The same distribution of particles appeared when the infected cells w‘ere sponta meously lysed on the grids. X86,000.

T4 HEAD

MATURATION

a lower phage DNA replication rate (as evidenced by the decrease in t3H]thymidine incorporation) coupled with a decrease in conversion of 300s particles (Table l), or (ii) the production of fragile phage. This latter alternative is unlikely, since as seen in Fig. 3a, large amounts of unfilled heads wchre observed in both spontaneously lysed cells and freshly made extracts of FUdRtreated cells at 30 min postshift,. When quantified by electron microscopy over sevtral fields (300 total particles) it \vas found at this time that SO?; of the head &ucturcs w’crc unfilled. In t)he control without FUdR (Fig. 3b), 60 ‘,( of t,hc structurtls appeared as phage. Hence, the decreased burst size after FUdR addition is most likely due>to the lowered rate of DXA synthesis. This small amount of DNA synthesis that occurs in the presence of 1’UdR evidently also explains the rescue of a fraction of the 300s particles (25’L of control) obscrvcd in Table 1. Such a content’ion is further supported by the fact that these rcscucd, 1000s particles appear normal in the cl(lctron microscope and arc identical to wild-type phagc> with regard to specific infcctivitg, viz., 0.S8 PlW/‘A,,,,,,, with FUdR and 1.03 lvithout FUdR. This rules out, thcx possibilit?T that these phagc particlcs have been productad by an aberrant pathwax, as is probably the cast for bacteriophagct T5-infclctcd E. coli in th(L prcsr~~rc of E’UdR (ZwcGg et al., 1972). Although the above cbxpc+mcnts suggest TABLE

2

OS THE MATURATION OF @-DEFECTIVE HERDS IN THE PRESENCI. OF Esc~:sa THYMIDINE"

EFFECT

OF FUdR

Minutes postshift

0 10 30

GEXE

Percentage of [WIlysine countsh in 1000s peak relative to total ~__ -FUdR +FUdR I<;, 307,; ac:;,

4Li;

4% 82%

Q The experiment was performed as in Table 1, with an addition of thymidine (1.8 mg/ml) at 15 min postinfection; FUdlt was added 8 min later. I, Over all peaks the average number of [‘“Cllgsine counts was 22,000 * 5,000.

AND

DNA

SYNTHESIS

437

that I!‘UdR blocks phage rescue by inhibiting DNA synthesis, it is still possible that the block in phage rescue observed with FUdR was due to another more indirect reason. For example, the irreversible inhibition of thymidylate aynt’hetase (Cohen et al., 1958) could have prevented utSilization of this enzyme as a, structural component in T4D+ phage (Mathews, personal communication) therefore prevent,ing maturation. This possibility is unlikely since it was found that if a large (about lO,OOO-fold) excess of thymidine was added before FUdR treat’ment, no inhibition occurred. This is indicated in Table 2, where the flow of label from the 300s to the 1000s positions at various times postshift was shown to be independent of t’hc addition of FUdR. The results reported thus far suggest that DNA synt’hesis is needed for the completion of head maturation. To furthrr examine this contention wc looked at head maturation under conditions whercl the function of T4 DNA polymcrase was blocked late in infection. Tenlperature Mift

Experimeuts with tsPS6

tsP36, a temperature-sensitive mutant in gene 33, the T4 DNA polymerasc, is extrcmely efficient in shutting off DNA synthesis at the restrict& tcmperaturc (Riva et al., 1971). WC confirmed our previous result,s (Luftig and (ianz, 1972) which showrd that a pulst>-lab&d culture of tsl’36infrcted E. coli Bh, shifted from 25°C to 31.5”C late in infection, yieldt:d vc’r?; few labeled phagt> (Table> 3). Howevclr, in the control cult’urc left at 25”C, most of the label flowed from the 300s peak to the 1000s peak (Table 3). Isolation and examination of t,hc tsP3G 300s particles accumulated at 43 min postjinfection in the shift-up experiment showed t,hey had the same physical propert,& as gcrlc 49.dchfect’ive heads, viz., p = 1.34 g/cc and SzO,, = 310 f 20. Furt,her, polyacrylamidc gcbl clectrophoretic autoradiograms showed that in both particles the major capsid polypcaptide was in a cleaved state (Fig. 5a, h). Since the tsP36 DNA polymerase activity can bc recovered after a shift down from

438

LUFTIG TABLE

AND

3

THE DISTRIBUTION OF [14C]L~~~~~ LABEL BETWEEN 300s AND 1000s PARTICLES AFTER A TEMPERATURE SHIFT-UP OF A tsP3fY INFECTED CULTURE Minutes postshift

15

25

25’

1000s

300s

1000s

27,000

1,000

22,500

35,500+

(4%) 2,000 (5%)

4,600 (17%) 20,300 (52%)

1

L

Unshifted

Shifted

300s

LUNDH

CAM itA

c’2

19,000

a The culture was infected, pulse-labeled, and shifted up to 42°C as shown in Fig. 4; at 38 min after infection, however, the culture was not shifted down and no CAM and FUdR were added. [“H]Thymidine at the time of shift-up showed no incorporation into TCA-precipitable material, confirming the block of enzyme activity. As a control, one half of the infected culture (unshifted) was left at 25°C throughout the experiment. Zone centrifugation was performed on extracts of samples removed from both cultures at times equivalent to 15 and 25 min postshift. The parentheses indicate the percentage of counts in the phage peak. * The pooled peak of 300s particles at this time was run on a CsCl equilibrium gradient under previously described conditions (Luftig el al., 1971). The average density was 1.34 g/cc.

the high temperature (Riva et al., 1971), we now asked if tsP36-defective heads which had accumulated as in the previous shift-up experiment, could be rescued. The shiftdown part of the experiment outlined in Fig. 4 was performed. As seen in Fig. 6a, b, c, the tsP36-defective heads were rescued, but at a slower rate than for MXinfected cultures (Table 1). When the peaks such as in Fig. 6a, b, c, were quantified, we found on the average over two experiments that 0, 7, 18, and 53 % of the label was converted from the 300s to the 1000s position at 0, 10, 20, and 40 min postshift, respectively. Thus, not, only do defective structures isolated as 300s particles accumulate in the absence of DNA polymerase activity but in addition, these are intermediates rather than abortive structures. This agrees with our previous results which suggested that FUdR blocked head maturation by inhibiting DNA synthesis. Further, if FUdR

Amtno Acid chase ,

Sompies removed 1

lntecllon Is, 2nd

FIG. 4. Labeling and incubation regimen for pulse-chase, temperature-shift experiments with tsP36 in the presence of FUdR. The mode of infection and labeling is the same as given in Fig. 1. At 27 min postinfection, t,he tsP36 culture was shifted up to 41.5% for about 10 min. The label was then chased out, and chloramphenicol (CAM) was added 1 min later to prevent further synthesis of labeled head precursor structures. FUdR was added at this time to test the effect of inhibiting DNA synthesis on the rescue of labeled gene 43defective heads. In some experiments an additional dose of 200 pg/ml FUdR was given 10 min later with no change in the results obtained. [3H]Thymidine was added to monitor the level of inhibition. Five-milliliter samples were removed as described in Fig. 1 and extracts prepared as described in the text.

was added at the time of shift-down of tsP36-infected cells (Fig. 3), the rescue was markedly reduced on the average over three experiments to 0, 4, 15, and 28 % at 0, 10, 20, and 40 min postshift, respectively (Fig. Gd, e, f). This is again consistent with the postulate that DNA synthesis is required for head maturation. Experiments with T4D+ Intermediates In order to show that the a.bove results were not due to some unlikely mutant sideeffects, the conversion of rapidly labeled 300s particles to phage was examined in a T4D+ wild-type infection. In this experiment FUdR (200 pg/ml) was added at 15 min postinfection followed by a I-min pulse of [‘4C]leucine or lysine at 16 min.

T4 HEAD

MATUIlATIOZ;

ilND

DNA

SYNTHESIS

of i.5’ji polyacrylamide-SDS gels of various capsid FIG. 5. Autoradiogram These samples are: (a) Kg-defective particles; (b) IsP3Gdefective particles; amN7G (gene 21) chloroform-DNase extract; (d) T4D+ rapidly labeled 30% were isolated from chloroform-DNase extracts by zone centrifugation. In the major capsid polypeptide (top arrow) is uncleaved (Laemmli, 1970) with a MW this as a control value, the capsid polypeptides of (a), (b), and (d) have a MW arrow) which makes them equivalent to the cleaved form (Laemmli, 1970).

As seen in Fig. 7, FUdR efficiently blocked the conversion of rapidly labeled 300s particles to phage. At’ 32 min postinfection, in the absence of FUdR, 90 ! ( of the label was in the 1000s posit’ion while only 12 Y; of t’he label had shifted to the 1000s position in thcl presence of E’UdR. A similar affect was found with KAL (100 pg/ml), n-here only 40 ‘I; of the label had shifted to the 1000s position at 33 min postinfection. This laxer efficiency of inhibition for KAL, compared to FUdR, is prwumably dw to a

439

intermediate particles. (c) the pellet from an part.icles. The particles gene 21 extract (c), the of 56,000 daltons. Using = 4G,OOOdaltons (lower

dccreascd inhibition of [“Hlthymidine incorporation, i.e., 50 L,b vs 15 9b of control, rcspectivcly, at 19 min p&infection. When analyzed, the T4D+ 300s particles (rapidly labeled in a I-min pulse), banded in CsCl at a nuclcoprotein density of 1.34 g/cc, and had the major capsid polypeptide in a cleaved state (lcig. 5d). WC conclude that t,hc requirement for Di\A replication prcviously discussed for tsC9 and fsP36 involvcs a normal step in the complrtion of head packaging.

440

LUFTIG

3.

ICI 40 In,”

(b) 20 min

IO 1 0 mm (-FlJdR)

AND

r

IO-

Ol

IO

20

IO Froct~on

20

, IO

20

Number

FIG. 6. Zone centrifugation of extracts from a tsP36-infected culture, prepared as described in Fig. 4. After the shift-up at 28 min and labeling of the 300s heads, the shift-down was performed 10 min later without (a, b, e) or with (d, e, f) the addition of FUdR. [3H]Thymidine incorporation was blocked to 50% of the control at 40 min postshift. Five-milliliter samples were removed at the times indicated and extracts prepared as described in Fig. 1.

DISCUSSION

In one of the classical experiments of bacteriophage biochemistry, Hershey, Dixon, and Chase (1953) showed that newly replicated T-even phage DNA acDNA pool” cumulated in a “replicative before being removed for head maturation. This significant finding pointed out a basic difference between phage and host DNA replication, since in the latter case newly replicated strands were immediately segregated. Hershey (1953) then amplified his result further to show that the replicative DNA pool was maintained at a constant size of about 40 phage equivalents of DNA in the infected cell. More recently, Frankel (1966; 1968) has characterized the replicating DNA in T4 and found that it was in the form of a concatameric DNA molecule which contained at least 20 phage equivalents of DNA. One explanation for the existence of such a large, relatively constant DNA pool is that the cutting of a DNA phage equivalent is intimately con-

LUNDH

nected with packaging a head full of DNA. This “head-full” model of DNA packaging was originally proposed by Streisinger et al. (1967) and has been supported by experiments on “petite” or 3$ phage particles (Mosig, 1966; Eiserling et al., 1970), as well as with giant-headed phage containing several phage DNA equivalents (Eiserling, personal communicat,ion). The recent experiments of Luftig et al. (1971), Frankel et al. (1971), and Luftig and Ganz (1972) provide the basis for describing the following mechanism by which a head-full scheme may operate. It is postulated that the energy needed to synthesize DNA provides the driving force for packaging a full DNA equivalent into capsid intermediate structures, at a site distinct from the replication point. These intermediates contain some DNA which is associat,ed with the replication pool. When the head is filled, a nuclease controlled by gene 49 cuts the packaged DNA from the replication pool. A corollary to this postulate is that the pool size stays constant, in agreement with the results of Hershey (1953). The results report,ed in this paper provide additional support for such a packaging mechanism. Evidence derived from the use of inhibitors of DNA synthesis (FUdR and NAL), and a mutant in T4 DNA polymerase strongly suggests that DNA replication is required for head maturation. Further, the intermediate structures blocked at this stage of assembly have been isolated under several different conditions of phage infection and appear identical. They contain about 20% of a phage equivalent, of DNA in a nuclease-resistant form, have the major capsid polypeptide in a cleaved state, and in vivo are still presumably associated with the replicating DNA pool. A similar intermediate has been reported by Laemmli (personal communication) . We are currently attempting to isolate the postulated DNA replicative complex with attached capsid intermediate particles. This should make it possible to study the details of DNA packaging in uitro.

T4 HEAD

MATURATION

I9’lWdR)

AND

DNA

25’l+FUdA)

lel

Fraction

SYNTHESIS

441

(1)

Number

FIG. 7. Zone centrifugation of extracts from a T4D+-infected culture, with or without FUdR. The mode of infection at 41.5’C is the same as given in Fig. 1. At 13 min postinfection the culture was split in half; FUdR was added at 2 min and 12 min later to one half of the culture. At 16 min, a 1-min pulse chase of [‘%]leucine was added to each part, as per Fig. 1. In addition, [3H]thymidine (5 &i/ml) was added at, this time to monitor DN.4 inhibition. The inhibition varied from 15-354; of the control at 19-32 min postinfection. Five-milliliter samples were removed at the times indicated in t’he graph and extracts prepared as described in Fig. 1. The percentage of counts in the 1000s phage peak at 19, 25, and 32 min postinfection as averaged over three experiments was 32,79, and 91% (-FUdR) and 3,8, and 12% (+FUdR), respect,ively. A plateau of rescue (about 3O’jL,) is reached in the latter case by 52 min postinfect,ion. This additional increase is presrlmably due to the escape 1)NA synt,hesis t,hat. st)ill occurs in the presence of FUdR. ACKNOWLEDGMENTS We are grateful for the aid and encouragement provided by Dan Hamilton, Carol Ganz, Ken Culbreth, and Y. Ito. We thank U. Laemmli, F. Eiserling, and C. Mathews for communicating experimental results prior to publication. The research was supported by Public Health Service Grants I-ROl-CA-11976 and l-S04-FR06148 from the National Cancer Institute and the Division of Research Facilities and Resources, respectively. REFERENCES BAIRD, J. P., BOUROUIGNON, G. J., and STERNGLANZ, R. (1972). Effect of nalidixic acid on the growth of deoxyribonucleic acid bacteriophages. J. Viral. 9, 17-21. COHICN, S. S., FI~AKS, J. G., BARNEIZ, H. D., LOSB, M. R., and LICHTENSTEIN, J. (1958). The mode of action of 5-fluorouracil and its derivatives. l’roc. Nat. Acad. Sci. USA 44, 1004-1012. EIW;RLING, F. A., GEIDUSCHEK, E. P., EPSTEIN, R. H., and METTER, E. J. (1970). Capsid size

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