.I. Mol. Hiol. (1989) 209, 91-100

In Vitro Packaging of Bacteriophage 429 DNA Restriction Fragments and the Role of the Terminal Protein gp3

Shelley Grimes and Dwight Anderson

Departments of Microbiology and Dentistry lTniversity of Minnesota, Minneapolis, MN 55455, U.S.A.

(Received 30 November 1988, and in revised form 9 February 1989)

R&rict.ion fragments of bacteriophage 429 DiXA-gp3 (DSA-gene product 3 complex) were packaged in a completely defined in vitro system that included purified proheads, the DNA packaging protein gp16 and ATP. Roth left and right end DNA-gp3 fragment,s were packaged in this system, in contrast to the oriented and selective packaging of left end DXA-gp3 fragments in extracts; left ends could be packaged quantitatively in the defined system, while the packaging efficiency of right ends was generally about threefold lower. Tn addition, certain internal (non-end) DNA fragments were packaged at efficiencies of about, 10*/h to 15yb. Digestion of the gp3 with trypsin or proteinase K reduced the packaging of whole-length DNA by a factor of 2 or 4, respectively, and removal of the gp3 from whole- length DNA or end fragments with piperidine reduced packaging to the level of internal fragments. Though the terminal protein gp3 was non-essential for DNA translocation in the defined system, it stimulated packaging of left and right) end fragments, and st’abilized packaging of the left end. The packaging of end and internal DNA fragments of the related phage M2Y into 429 proheads was similar to that of 429 DNA fragments, and certain fragments of lambda DNA were packaged at the efficiency of the internal $29 DNA fragments. Selective packaging of DNA-gp3 left ends was restored by the addit’ion of bacterial cell extracts or glycerol to the defined system, and these packaging conditions discriminated between $29 and M2Y DNAs that have distinct terminal proteins.

1. Introduction

In vitro assembly of the Bacillus subtilis bacterio- phage 429 is characterized by simplicity and high efficiency, both in extracts and a completely defined system (Rjornsti et aZ.., 1981; Guo et al., 1986). Two non-capsid proteins, the covalently bound 31,000 M, gp3t of the DNA termini and the 39,000 M, ATP-binding protein gp16, effect the interaction of the prohead and the 19 kb DNA t,o initiate t.he DNA packaging phase of 429 assembly (Bjornsti et al., 1983; Guo et al., 1986, 1987a,b). In extracts, only left end DNA-gp3 restriction frag- ments are packaged, and removal of most of the gp3 with proteinase K treatment blocks DNA packaging (Bjornsti et al., 1983). Utilizing the defined in vitro system that includes purified proheads, DNA-gp3, gpl6 and ATP (Guo et al., 1986), the present study demonstrated that, surprisingly, both the left and right DNA/protein end fragments of $29 and of its relat,ive M2Y were packaged. In addition, some

t Abbreviations used: gp. gene product; kb. IO3 base-pairs.

prot*ein-free fragments of (629 and other phage DNAs were packaged, albeit at a lower efficiency. Thus, the DNA terminal proteins stimulated and stabilized packaging but were not essential in the defined system. Addition of cell extracts or glycerol to the defined reaction mixture restored the selec- tive packaging of 429 DNA-gp3 left ends but markedly reduced packaging of the distinct left end DNA/protein complex of M2Y.

2. Materials and Methods (a) Chemicals, enzymes and isotopr

Piperidine was from Aldrich Chemical. The restriction endonucleases HpaI. BstEII. EcoRI. PvulT and Hind111 were from Bethesda Research Laboratories, a,nd BstNI w-as from New England Biolabs. Trypsin was from Sigma Chemical Co., and proteinase K was from EM Science. DNase I was from Calbiochem: 13H]thymidine was from New England Nuclear.

(b) Purification of proheads, DNA@ and gp16

Proheads were isolated from B. subtilis SpoA12 (sup-) infected with the mutant SW 16(300)-sus 14(1241) that is

defective for the production of the DNA packaging protein. as described by Wichitwechkarn et al. (1989). 429 [3H]DNA-gp3 was isolated from the sup- host infected with the mutant sus4(369)-RusS(22) that is defect,ive for production of gp4 (the regulator of late transcription) and gp8 (the shell protein); gp16 was overproduced as lumps in Escherichia coli and solubilized in guanidinium chloride (Guo ef nl.. 1986). Purification of gpl6 was complet,ed by high-pressure liquid chromatography in a Spherogel TSK 3000 SW rolumn (Beckman) equilibrated with 7.5 M-urea. 1 m&f-dithiothreitol. 10 mM-Tris-acet’ate (pH 5.2).

(c) Defined system fwr in vitro packaging

[3H]I)NA-gp3, stored at, 4°C‘ BS fractions from isopycnic C&l gradients, was dialyzed over a membrane filter (PO25 pm. Millipore) against, TMS buffer (50 mw-Tris. HCI (pH 7.8). 10 mM-MgCl,, 0.1 M-N;a(:l) for 40 min at room temperature. gpl6 was dialyzed over a filter against 10 mw-Tris.HCl (pH 75), 4 mM-KC1 for 40 min at 4°C just prior to use. Proheads were in MMS buffer (5 mM-maleic acid/NaOH (pH 6.5). 15 m&r-MgCl,. @l M-NaCl; 2 mM-NaN,). For packaging, 200 pg of [3H]D?;A-gp3/ml, 100 pg of gpl6/ml, lOI proheads/ml and reaction buffer (10 mM-ATP, 6 mM-spermidine. 3 mM- 2-mercaptoethanol in TMS buffer) were mixed in the proportions 10 : 7 : 3 : 3 (by vol.) and incubated for 30 min at room temperature (Guo et nl.. 1986). To assay for particles containing [3H]DNA-gp3, the reaction mixturr was diluted to 100 ~1 with TMS buffer, loaded onto a linear ,596 to 2096 (w/r) sucrose density-gradient containing TMS buffer and centrifuged in a SW50.1 rotor at 35.000 revs/min for 30 min at 20°C.

(d) Packaging of IlKA fragm,ents in the

defined system.

[3H]DNL1-gp3 was dialyzed against TMS buffer for 60 to 90 min and digested with restriction enzymes as recom- mended by the manufacturers. The digest’ed DN1Zs were packaged rn the defined system. and the part’iallp filled part’icles were isolated by sucrose dens&y-gradient centri- fugation as described in section (c). above. To ext)ract the DNA from the particles, pooled sucrose density-gradient, fractions were dialyzed against 0.01 x TE buffer (TE buffer is 50 mx-Tris. HCI (pH 7.8), 10 mM-EDTA) for 60 min at room temperature and concentrated to 30 ~1 by evaporat,ion at 60°C: then 0.1 vol. 10 x TE was added, and the DNA was heat-ext,racted for 20 min at 70°C. After treatment with 500 pg of proteinase K/ml for 60 min at 55°C to remove most of the terminal protein gp3. the DATA was run on a 1:/b (w/v) agarose gel in TBE buffer (89 mw-Tris, 89 miv-boric acid (pH 8.3). 2.5 mM-EDTA). Alternatively, DNA packaging was assayed by electro- phoresis in agarose. After 30 min incubation, t)he pack- aging mixtures were treated with 20 pg of DNase I/ml for 20 min at room temperature to digest t’he unpackaged DNA: then @l vol. 10 x TE was added, and the packaged DNA was heat-ext,racted from the particles for 20 min at 70°C. The samples were treated with 500 pg of proteinase K/ml for 60 min at 55°C. reduced to 15 ~1 in a Speed \:ac Concentrator (Savant) and run on a 1 o{> (w/v) agarose gel.

(tz) Partial a,nd complete removal of thP terminal proteirt gp3 from qb2.9 D*v‘4-gps

To digest gp3, purified DNA-gp3 in TM8 buffer was treated for 60 min with either 20 pg of trypsin/ml at 37 “(

or 500 pg of proteinase K/ml at 55’(‘. The I)XA was then purified ;,n a CsCl step gradient) containing N\‘c‘P hufGr (10 rnM-sodium &rate. 80 mM-K,HPO,. 40 mM-KH,l’O, (pH 7.4)). The procedure for complete removal of the terminal prot,ein has been described (Garcia ut nl.. 1984). Briefly. 20 pg of 429 DNA-gp3/ml was dialyzed against TE buffer. digested with 500 pg of protsinase K/ml for 60 min at 55°C’. and t’hen rxtractrd with phenol. Thr remaining peptidr was rrmovc~l It! twwtmrrrt it1 05 M-piprridine fm 2 h at 37”( I. ‘1’111~ l)i})t,ridin,s \I as removed. anti the l)SA \vas anncalrd 1)~. tlialysis agaitrst IO mnl-Tris’ H(‘I (pH X.0). 1 II~.M-EI)TA. O..i ~l~-Na(‘l OV(:I’~ night at 65’(‘. The 11X.4 was then extra&ad \vith phc~n~l. diatyzt,tl as drsclribed in se(ation ((a). ahovt~. antI uscxd ii1 the drfinrcl s~sttw.

Yl\ilp\’ DNA was extracted from phage in ‘1’E:S butfrar (50 mw-Tris. HCl (pH 7.8). 10 mM-EDTA. 0.1 nl-Sat ‘I) containing 4 31-guanidinium cahloride for 60 min at O’Y’. The USA was purified in a (‘~(“1 step gradient csontaining NCP buffer and used in the defined system.

I’reparat,ion of extra& of 429 nlutant int’rc*ttad culls has been described (Bjornxti it nl.. 1981 ). BrirflJ-. 0. su1dili.s SpoAl2 (“ALP-) was infected with the ~su~s7(6ll)-~s~~~sX(i6!~)- sus 16(300)-sus 14( 1241) mutant’ (defective for the scaf- fold, shell and USA packaging proteins), and inc~uhat~ccl for 80 min at, 37”(‘. Protoplasts were produced from t,hr infect,ed cxells and concentrated %)-fold for Iysis in rcact,ion buffer. The extract and the definrd system were mixed in equal volumrs.

3. Results

are packaged when unfractionated rrst~rictiori enzyme digests of thr DSA are incubated in extracts capable of assembling $29. Part,ic*lrs containing [ 3H]I)NA fragmentIs are isolated by sucrose density-gradient) centrifugation, whrre t,trc> sedimentation rate of t)htb particles is a linear fun+ t’ion of DNA cont,ent (Hjornst’i rt nl.. 1983). Thr packaging of restriction enzyme fragments of @!3 DNB has now been investigated in the defined in uitro system. Unexpectrdty. both left and right end fragments of 429 DIVA digested with HxtEI 1 or EcoRI were packaged in thtt defined system, in contrast to the highly selective pacakaging of left ends in extracts (Figs 1 and 2; Isjornsti of al.? 1983). Particles sedimenting t’o fractions 1 1 or 17 of the sucrose density gradient (Fig. 1 (a)) contsined thts DXA left (BstETT-A. 12.5 kb) or right (/ZstETT-13. 6.8 kb) end, respect’ively (Fig. 2. lanes 2. 3 and 4). Particles sedimenting to fractions 1X or “0 (Fig. l(b)) contained t’hc DSA Icft (&oRr-A. 9.9 kb) or right’ (&xIRI-(‘, 1.9 kb) end. respectivc~l~ (Fig. 2. lanes 5. fi and 7). The distribution of la,bc~l in

Role of gp3 in $29 DNA Packaging -

BstEII I A

I _ 0,

EcoRI , A 0 ED C 1 I

HpaI , A FHE C DG B 1

bp I I I I I 0 5000 10,000 15,000 20,000

81 (a 1

6-

: 0 x .E E 4-

\ m t

n=

2-

(b)

0 IO 20 30 40 Fraction no.

the sucrose density gradients and the gel analyses showed that both the left and right ends were packaged quite efficiently. The label centering on fraction 5 (Fig. l(a)) represents packaging of undi- gest,ed (whole-length) DNA. The label centering on fractions 36 (Fig. l(a)) and 35 (Fig. l(b)) represents unpackaged DNA. The label centering on fraction 32 (Fig. l(a)) and fractions 30 to 31 (Fig. l(b)) consists of DNA/prohead aggregates; these could be initiation complexes (Guo et al., 1987~~). Approxi- mately one-half of the DNA added was packaged stably.

The analysis of packaging of 429 DNA fragments generated with HpaI is shown in Figure l(c). More than half of the [3H]DNA added appeared in par- ticles sedimenting to fractions 16 or 20 of the sucrose density gradient. The particles centering on fraction 16 contained mainly the HpaT-A left end fragment (Fig. 2, lanes 8 and 9), and the particles centering on fraction 20 contained the HpaT-B right end fragment or the internal (non-end) fragments C. D, E, F and G (Fig. 2, lanes 8 and IO). These latter results were unexpected, in that previous studies with extracts had demonstrated that digestion of the covalently bound terminal protein gp3 with proteinase K results in the loss of DNA packaging (Bjornsti et al., 1983).

(11) Packaging of 429 DNA fragments wa<s assayed directly by electrophoresis in a,garose

Analysis of DNA fragment packaging by elect,ro- phoresis in agarose was utilized as a rapid alterna- tive to sucrose density gradients. After DNase I digestion of unpackaged DNA, EDTA was added. and the packaged DNA was released from particles by heat and analyzed by electrophoresis. Approxi- mately half of the whole-length 429 DNA-gp3 added was packaged (Fig. 3, lanes 1 and 2). With an EcoRI digest about half of the left (EcoRT-A) and right (EcoRI-C) ends were packaged. as well as EcoRI-D and a small amount of EcoR’I-B (lanes 3 and 4). With a HpuI digest’, again the left (HpaI-A) and right (HpaI-B) ends were packaged efliciently: the internal fragments C and 1) (or I(:) were pack- aged also. albeit, at a lower efficiency. and packaging of the fragment,s F and G was barrly detectable (lanes 5 and 6). With a &~tN 1 digest- the left. end (n&NT-B) was packaged quantitatively, while pack- aging of the right end (LMNT-A) was anomalous

Figure 1. 429 DKA-gp3 lefi and right) ends as well as internal restriction fragments were packaged in the defined s,vst.em. [3H]DNA-gp3 digested w-ith /LstlXl (a). BcwRI (b) or HpaI (c) was incubated w-ith proheads. gl)lti and ATP for 30 min, and the partially filled heads were isolated on sucrose density gradients. Sedimentation is from right to left. The fast sedimenting partirles (centered on fractions 11 (a). 13 (b) and 16 (c)) containrd left end fragments. Particles centered on frac+ons 17 (a) and PO (b) contained right end fragments, while particles ~-en- tered on frac-tion X0 (c) contained right ends and internal fra.gments (Fig. 2).

94 24. Grimes and D. Anderson

A

ECORI L A B ED C

I I,, ,

IHPOI , A FHE C DG B

I II I I ,I I

BstNI, B , A

1

GKHMI E J D HindimI B III., II t

A NF CL I# I u

I3 C

W F

G

n

Figure 2. DNAs extracted from particles isolated on sucrose density gradients (Fig. 1) were analyzed by rlrc- trophoresis in agarose. The lanes contained: 1. whole- length 429 DNA; 2, BstEIT digest of 429 DNA; 3, frac- tions 10 and 11 of Fig. I(a); 4, fractions 16 and 17 of Fig. l(a); 5, EeoRI digest of 429 DNA; 6, fractions 12 and 13 of Fig. l(b); 7, fractions 20 and 21 of Fig. I(b); 8, HpuI digest of 429 DNA; 9, fractions 16 and 17 of Fig. l(c); 10. fractions 20 and 21 of Fig. l(r).

(lanes 7 and 8); the smear below the BstNI-A frag- ment, suggested that this right end, about 840/;, of the genome size. may have been packaged partially or unstably, and thus susceptible to a varying extent to 1)Nase 1 digestion (lane 8). With a HindTII digest, the left end (HindIII-B) was pack- aged quantitatively, while nine of the largest internal fragments visible on the gel were packaged at an efficiency of about, 10% to 1576 (lanes 9 and 10). The small right end (IlindIII-I,) was not resolved on the gel. Taken together, the results showed that left and right end fragments were pacak- aged as well or better than whole-length DNA, and certain internal fragment,s were packaged, although at reduced efficiencies; the internal fragments

I 2 3 4 5 6 7 8 9 10

Figure 3. Packaging of $29 DNA left and right, ends and internal fragments was a,ssayed directly bv rlrrtro- phoresis in agaroye. Restriction enzyme fragments of &El DN;A were packaged in the defined system. and the r’rac- tion mixtures were treated with DIVase I to digest unpackaged DNA. After the addition of EDTA, the pack- aged l>XAs were heat-extracted. treated with p&ein- asr K to remove moat’ of t,he terminal protein and analyzed by electrophoresis in agarose. Odd-numbered lanes COW tainrd the DNAs added to the packaging reaction mix- tures: these served as controls for quantifying the packag- ing of the DNAs observed in the even-numbered lanes. The lanes contained: I 1 whole-length DNA; 2. packaged whole-length DNA; 3. EcoRT digest; 4, packaged Eh:coRT digest; 5, HpaI digest; 6. packaged HpnI digest; 7. BsfNI digest,: 8. packaged BstNI digest: 9, Hind111 digest: IO. packaged Hind111 digest.

EcoRl-B (lane 4) and IlyaI-F, G and H (lane 6) were not recognized as well by the I)?U’A packaging machine.

Although three proheads per genotnc equivalent of digested DNA were used in these rxperirnents (Figs 1, 2 and 3). the same result,s were obtained with 6 and 12 proheads per genomr equivalent of DNA. where the DNA packaging prot,ein gp 16 was also increased proportionally with proheads. End and int’ernal DNA fragments were packaged with t’he same kinetics, and packaging was complete in eight minut,es (data not shown).

Role of gp3 in 429 DNA Packaging 95

(c) DNA-gp3 left ends were packaged more e&iently than right ends in the defined system

The packaging of left and right ends of various sizes was assayed by the use of sucrose density- gradient’ centrifugation (Table 1). Whereas the efi- ciencies of packaging of left and right end fragments 6.8 kb or smaller were comparable, the packaging efficiencies of left and right end fragments 12.5 kb or larger were quite different. Specifically, only 8% to 18% of the 13.1 kb ClaI right ends were retained in particles, compared to 28% or 35 y. of the 12.5 kb RstEII left ends. Similarly, only 2% of the 16.5 kb BstNI right ends were retained in particles, compared to 22 “/b or 25% of the 19.3 kb whole- length $29 DNAs. However, when BstNI fragment packaging was measured by the gentler method of agarose gel electrophoresis, the long right end was packaged at about 25% to 50% efficiency (Fig. 3; other data not shown). Although the efficiency of packing was higher in general when assayed by electrophoresis, there was a notable disparity in the packaging eficiencies of long left ends versus long right ends between the two methods (Table 1; Fig. 3). While long left ends were about) twofold under-represented in gradients, the packaging effi- ciency of the longest right end (BstNI-A) was 12 to 25-fold lower than whole-length DNA. This indi- cated that’ particles with long right ends were more unstable and therefore easily disrupted by the more stressful centrifugation procedure. Further, this suggested that the left-end DNA-gp3 complex stabilized packaged DNA.

(d) cleavage or removal of the terminal protein gp3 decreased th,e packaging of whole-length

DNA in the de$ned system

Tn extracts, trypsin-treated $29 DNA-gp3 is packaged about half as efficiently as DNA-gp3 (Bjornsti et al., 1982), while proteinase K-treated DNA-gp3 is inactive (Bjornsti et al., 1983). Trypsin

Table 1 DNA-gp3 left ends were more eficiently packaged in

the defined system than right ends

Restriction End Packaging fragment Sizr (kb) (left or right) efficiency (O/o)?

HindIlI-R 2.9 L 100 EIWRI-C I.9 R 100,70

HpaI-A 6.X I, 69, 71 RstEII-13 88 R 52, 57

EcoRI-A 9.9 L 56, 51

BstEII-A 12.5 L 28, 35 ClaI-A 13.1 R IO. 8. 18

B*tNT-A 16.5 R 2, 2

429 DNA 1 9.3 I, 25, 22 193 R ?

7 The packaging efficiency was the fraction of the total [‘HIDNA-gp3 in the sucrose density gradient that appeared in part~ially filled or filled particles.

I2 3 4 5 6 7 6

Figure 4. Cleavage or removal of the terminal prot,ein gp3 decreased DNA packaging in the defined system. The gp3 of 429 DNA-gp3 was digested with trypsin OI protrinase K. or completely removed with piperidine. The DIZ’As were packaged in the defined system, and packag- ing was assayed as described in the legend to Fig. 3. The lanes contained: 1. whole-length I)NA: 2. packaged whole- length DNA: 3, trypsin-treated I)I$VA; 1. packaged trypsin-treated DXA; 5, proteinast: K-treated DNA; 6. packaged proteinase K-treated DNA: 7. gp3-free Dh’A: 8, packaged gp3-free DIVA.

digestion leaves 25 of the 266 amino acid residues of gp3 attached to 429 DNA, and proteinase K leaves ten residues (Hermoso et al., 1985). DNA-gp3 was digested with trypsin or proteinase K, or treated with piperidine to complet’ely remove the gp3 (Garcia et al., 1984); proteinase activity was verified by electrophoresis, since gp3 inhibits the migration of DNA-gp3 into an agarose gel (Ito et al., 1976). The packaging of these DNAs was evaluated in the defined packaging system (Fig. 4). The packaging of trypsin-treated DNA-gp3 was about half as effi- cient as untreated DNA-gp3 (lanes 1 through 4), while the packaging of proteinase K-treated DNA-gp3 was about one-quarter as efficient (lanes 5 and 6). About 15% of the gp3-free DNA was packaged, roughly the efficiency of packaging of internal restriction fragments (lanes 7 and 8; results in (b), above).

(c) gpd-free ends and internal restriction fragnlents of 429 DNA were packaged equally

in the de$ned system

Piperidine treatment was used to prepare gp3-free 429 DNA, and fragments generated by digestion with HpaT were packaged in the defined system. Packaging of the left (HpaI-A) and right (HpaT-B) ends and the internal fragments Hpal-C and D (or E) was equivalent at an efficiency of approximately

96 S. Grimes and D. Anderson

Figure 5. Left and right end protein-free HpaI frag- ments of $29 DNA were packaged with the same &i- ciency as internal fragments. The gp3 of 429 DR;A-gp3 was removed with piperidine and the DNA digested with HpaI (lanes I and 2). A portion of the DNA was extract’ed with phenol and packaged in the defined system (lanes 3 and 4). The lanes contained: 1, HpnI digest; 2, packaged HpaI digest; 3, phenol-extracted HpaI digest,; 4. packaged phenol-extracted Hpal digest.

157; (Fig. 5, lanes 1 and 2). The packaging of the other HpaI fragments was not detected. The pack- aging of HpaI-digested gp3-free DNA treated with proteinase K and subsequently extracted with phenol was approximately the same (lanes 3 and 4), showing that the presence of the restriction enzyme did not affect the packaging of fragments.

M2Y B C A D Pvuls L I I I I

x KDLJNHG B C EMI FO A HP01 ” II II I I I I II I I, I

,M

I 2 3 4

Figure 6. Certain fragments of M2Y and lambda IJIVi1s were packaged in t,he defined system. MdY and lambda DI$As were digested wit)h PvuIT and HptrI. respectively. and packaged into 429 proheads; packaging was assayed as descsrihrd in t~he legend t,o Fig. 3. The lanes contained: 1, PvuII digest, of M2Y 1)&A: 2. packaged /‘~nlI digest of M2Y DNA: 3. Hpal digest, of lambda I)XA; 1. packaged Hpa,I digest of lambda DNA.

(f) DNA fragments 01 phages M2 Y and lambda were packaged in the 429 dqfined system

The packaging of gp3-free restriction fragments of $29 DNA suggested that fragments of other DNAs could be packaged in the defined system. The DNAs of phage M2Y (a relative of #29) and the coliphage lambda were digested with PvuII and HpaT. respectively, and the fragments were packaged in the 429 defined system (Fig. 6). The M2Y left) end ( PvuTI-R) was packaged quantitatively, and about, one-third of the right end (PwuTT-D) was packaged (lanes 1 and 2); these fragments have a terminal protein that is distinct from the gp3 of 429 DNA (Yoshikawa Pr Tto, 1981). The large internal frag- ment PvuII-A was packaged efficiently, while pack- aging of the internal fragment PvuII-C was barely detectable. With lambda DNA also, which has no t.erminal prot.ein, a selectivity in fragment pack- aging was observed; the HpaT fragments A, IS, D (or E) and J were packaged more efficiently than frag- ments C, F, G, H and T (lanes 3 and 4). This selec- tivit,y resembled the resuhs with the Hpal fragments of $29 DNA (sections (b) and (e). above) and was highly reproducible.

(g) Selectiw left end packaging irk thr defined system ulas restored “,y eztrnct

or glycrrol addstaon

The detined system was c~omplementrd with ext.rarts of infected (7 -X- 16 14 -) ot’ uninfrc%cStl cells to determine whether a phage or hact,erial factor was responsible for the exclusive packaging of left end restriction fragments observed in extract,s (Bjornsti et al., 1983). Addition of an extract of infected cells restored the orientation of packaging (Fig. 7(a)). Only the 3H-labeled BsfEII-A (left end) fragment was packaged. in contrast t,o the pa,(:k- aging of both ends in the rvntrol. Tht, same result was obtained with t,he addition of either glycerol (Fig. 7(b)) or an extracat of uninfec+d cells (data not shown). Packaging of the left end c.onrplex w-as strongly favor& under these conditions. indicating t’hat t,he left and right end prot)ein-I)NA c~omplexrs were distinct. The same results were obt,ained when packaging of the fragments was assayed I)?- rlrc*t,ro- phoresis in agarosc (data not. shown): thus. the effect of glycerol was not due t,o unstable pwka@ng of right ends.

Role of gp3 in 429 DNA Packaging 97

r

20 Fraction no.

Figure 7. Addition of either glycerol or extracts of $29- infected cells restored the oriented packaging of $29 DE&gp3 left ends in bhe defined system. [3H]BstEII fragments were packaged in the defined system (a) with (0) or without (a) the addition of an extract’ of T-8-lti-I4--infected cells or (b) glycerol (17% final), and packaging was analyzed by sucrose density-gradient, cen- trifugation. The fast-sedimenting particles (centered on fract,ion 10) observed when an extract or glycerol was added contained the BstEII-A (left end) fragment (see Figs I(a) and 2). Packaging efficiencies for left ends were 329” (a) and lOoi, (0) in (a) and 18% (0) and 13% (0) in (b). Radioactivity not shown: 4860, 8167 and 4787 cts/ min in fractions 31, 32 and 33, respectively, of (a) with extract: 3292 and 4193 cts/min in fractions 34 and 35 of (b) control: and 4924 and 3947 cts/min in fractions 34 and 35 of (b) with glycerol.

(h) Addition of glycerol to the dejined system reduced the recognition of M2Y DNA

by the 429 packaging machine

Addition of glycerol or extracts of &her infected or uninfected cells to the defined syst’em restored the selective packaging of 429 DXA left ends that, is observed in extracts (section (g), above; Bjornsti et al.. 1983). Whole-length 429 and M2Y 13H]DNAs were packaged in the defined system with and without’ glycerol to further evaluate the role of the covalently bound terminal proteins. The terminal proteins of 429 and M2Y are distinct (Yoshikawa & Tto, 1981), while 40 to 60 nucleotides at the DNA left end are identical (Yoshikawa et al.. 1985). Addi- tion of glycerol had little or no effect on t,he pack- aging of 429 DNB-gp3 in t’he defined system (Fig. 8(a)), and a significant fraction of the label was centered at fraction 6 of the sucrose density gradients, the position of filled heads (Hjornsti et al., 1981). Tn contrast, the glycerol dramatically reduced the packaging of M2Y DXA into 429 proheads (Fig. 8(b)). Tn the presence of glycerol, the bulk of the M2Y DNA remained at the top of the gradient, the position of unpackaged DNA (Guo et al., 1987a), suggesting a defect in initiat,ion of pack- aging; without’ glycerol, the free M2Y DNA centered on fraction 30. the position of prohead-DNA complexes (Guo et aZ., 1987a). Thus, under the more st’ringent packaging condition imposed by the addi- tion of glycerol, the $29 packaging machine st’rongly favored 429 DNA-gp3 over the M2Y DNA-prot,ein complex.

4. Discussion

Two methods were used to assay DNA packaging in the defined system: (1) sucrose density-gradient, isolation of particles with packaged 13H]DNA; and (2) agarose gel electrophoresis of the reaction mixture after release of the packaged DNA from particles. With method (l), generalI>: 200/,, to 25~o of the whole-length DNA added sedimented as filled heads (Fig. 8; Table l), and more than 307, of the left, end restriction fragments were packaged (Fig. 1; Table 1). Wit,h method (2) , the packaging of whole- length DNA-gp3 was generally 500/,,. and the pack- aging of left end fragments was oft.en quantitative (Figs 3 and 4). Therefore, some filled heads lost their DXA in sucrose density-gradient centrifugation, resulting in underestimates of packaging efficiency. Agarose gel analysis gave a more reliable quantifica- tion of DXA packaging.

Whereas only DNA-gp3 left ends are packaged in extracts (Bjornsti et al., 1983), both left and right ends of DNA-gp3 as well as some. but’ not all. internal fragments were packaged in t,he defined system. Thus substrate recognition was less strin- gent in the defined system than in extracts. which resemble more closely t)he environment of the infected cell. For DNA-gp3 left ends, packaging efficiency was inversely related to fragment size (Fig. 3). The packaging efficiencies of the 2.7 kb

98 8. Grimes and Il. Anderson

(bl I I I

20 30 40 Fraction no

Figure 8. Phage MZY DNA packaging was sensitive to the presence of glycerol in the defined system. (a) 429 or

(h) MZY DKAs were packaged in the defined system wit,h (0) or without (0) glycerol (17 y0 final), and the filled heads (centered on fraction 6) were isolated in sucrose density gradients. Both 429 and M2Y DNAs were pack aged efficiently in the defined system. but the packaging of M2Y DNA was reduced dramatically in the presence of glycerol. Radioactivity not shown: 5481. 5222 and 3463 cts/min in fractions 34,35 and 36, respectively. of(b) with glycerol.

BstNI, 2.9 kb HindIII, 6% kb HpaI and 9.9 kb EcoRT fragments decreased progressively from quantitative to about 50%. The packaging of

l)n’A-gp3 right ends was generally about t)wo- to threefold less efficient than t,he packaging of left ends in analyses by electrophoresis (Fig. 3), and this difference was augmented in sucrose densit>- gradient, analyses (Table 1). These data suggested that the DNA-protein complexes of the left arid right ends were dist’inct and that the gp3 of the left end stabilized DNA packaging. gp3 is purport,edl> t,he same at. both DNA ends, since gp3 isolat,ed from the purified 1 35S]DNA-gp3 by piperidine treatment produces a single ba,nd in SDS/polyacrylamide gel electrophoresis (Prieto et u.l., 1984). Howcavrr. ‘in attempt,s to characterize the terminal gp3 of isolated left and right end restrict.ion fragments. t,he protein of the right end was repeatedly lost to proteolysis (our unpublished results); thus. t,he gp3 of the left and right ends must differ at, least in conformation if not in amino acid sequence. The multifunctional gp3 primes the replication of 429 DNA-gp3 from both ends, but not simultaneously (Inciarte ef al.? 1980; Harding H: Ito, 1980). The DNA left and right ends show inverted t~erminal repeats of S’-AAAGTA, and 35’$o of the first 200 bases are identical (Yoshikawa et al.. 1981; Escarmis & Salas, 1981). The gp3 is linked t,o the first base A of the DNA t,hrough serine at, position 23% (Hermoso et al.. 198.5); and interactions of t.he gp3 with differing IeR and right end nuc*leotidr sequences may dictate gp3 and DNA cdonformat ion and, consequently. sensitivity to prot,eolysis as well as recognition by the prohead and/or t,he IJNA packaging protein gpl6. In addition. I hr apparrnt unstable packaging of the long right ends such as the 16.5 kb HstNl-A fragment (Fig. 3: Tahlr 1) suggested that the packaging process for left wrsws right ends or fhe t.opology of these pa&aged complexes may be different.

Digestion of I)SA-gp3 with trypsin or prot,eill- ase K resulted in a tlrcarease in packaging of whole- length 429 DNA by factors of about 2’ and 3, respect,ively. Complete removal of gp3 wit.h piperi- dine resulted in parity in the packaging of whoIt,- length DXA. left and right ends, and internal frap- ments: on the average, only about 109;, to Is?;, of any protein-free DNA was packaged (Figs 3. 1. 5 and 6). The results showed that the effects of thtk terminal protein in packaging were dramatic. repro- ducible, and hierarchical: DKA-gp3 Ief% ends would be packaged quantitatively, depending upon sizr: DNA-gp3 right ends. trypsin-treated whole-length DNA: and prot,einase K-treated DSA were pack- aged less efficiently. in that order: a,nc-I finally. the packaging of protein-free ends, internad fragments and whole lengths was generally limited to about 10% to 15O/o of the molecules a.dded.

The DNA packaging schemes ot’ the phages lambda. P2, T3: T7, P22 and T4 ut,iliee a DNA- binding protein (small terminase subunit) that is the functional equivalent of t,he c*ovalent.ly bound gp3 of 429 (for reviews, see l%lack. 1988: Serwrr. 1989). The T3 counterpart, gpl8, is required in t,he defined in vitro system (Shibata c:f al., 1987). The T4 termi- nase protein gpl6 stimulates packaging of mature

Role of gp3 in qM9 DNA Packaging 99

T4 DNA by 10 to IOO-fold and concatemeric DNA by 50 to 875-fold in the defined system (Rao & Black, 1988). The lambda small terminase subunit gpNu1 binds to DNA @hinder & Gold, 1988) and mediates interaction with the larger subunit gpA that binds to the procapsid (for a review, see Feiss, 1986). Changes in the P22 small terminase subunit alter the substrate recognition in packaging (Jackson et al., 1982; Casjens et aE., 1987). The packaging of protein-free fragments of $29, M2Y and lambda DNAs in the present study showed that the small protein was not required for DNA translo- cation, although packaging of the DNA-protein complexes of 429 and M2Y was often more efficient by an order of magnitude (Figs 3, 4, 5 and 6). Thus, the gpl6 of 429, which initiates the 429 DNA-gp3 packaging cascade by binding to the prohead (Guo et al., 1987a), must have the capacity for both DNA binding and packaging.

The select,ivity of the 429 defined system pack- aging machine for certain internal (non-end) restric- tion fragments was inexplicable. Packaging of the smallest HpaI fragments, F, G, and H (1.7, 1.6 and 0.7 kb, respectively), was very inefficient (Figs 3 and 5) but the equally inefficient packaging of the EcoRI-B fragment of 5.9 kb (Fig. 3) augered against fragment size as a determinant. Fragment selec- tivity was observed with enzymes that generated sticky (EcoRT) or blunt (HpaI) ends. Considering DNA sequences. the 44 kb ElindIII-A fragment was packaged substantially better than the 5.9 kb EcoRI-B fragment that’ includes all of the IlindIII-A sequence (Fig. 3). However, nine internal HindIII fragments that comprise 96% of the internal (non-end) sequences were packaged at similar efficiencies (Fig. 3).

The packaging of DNA fragments of the phages M2Y and lambda into 429 proheads was consistent with the 429 DNA results. Phage M2Y DNA has substantial sequence homology with 429 DNA at both the left and right ends (Yoshikawa et al., 1985), but’ the terminal proteins of the two phages are distinct (Yoshikawa & Tto, 1981). The M2Y left and right end t%uII DNA fragments were packaged in the same manner as the left and right ends of $29 DNA, and only one of two internal fragments were packaged efficiently (Fig. 6, lanes 1 and 2). The difference in the $29 and M2Y terminal proteins is based on two-dimensional peptide maps (Yoshikawa & Ito, 1981) but, whatever the difference in sequence and folding, the 429 packaging machine recognized the M2Y DNA as self in the normal defined system. With lambda DNA, again certain NpaI-generated fragments were selected by the 429 DNA packaging apparatus (Fig. 6, lanes 3 and 4). The maximum packaging efficiencies were of the order of lo’+; to 15% of the fragments added (Fig. 6), resembling the packaging of gp3-free HpaI fragments of 429 DNA (Fig. 5). About half of the lambda fragments were recognized effectively by the 429 packaging machine.

Remarkably, the selective packaging of left end 429 DNA-gp3 fragments observed in extracts

(Bjornsti et al., 1983) was rest’ored in t,he defined system by the addition of glycerol or extracts of either infected or uninfected B. subtilio (Fig. 7). The effect could be the result of increased viscosity of the medium or alteration (even repair) of a com- ponent of the defined system. While glycerol addi- tion had no effect on the packaging of whole-length 429 [3H]DNA, the packaging of M2Y [3H]DNA was markedly reduced in the presence of glycerol (Fig. 8); this more stringent packaging condition thus revealed a mismatch of t,he distinct M2Y t’erminal protein for the $29 packaging machine that was not detected in the basic defined system. Very little M2Y [3H]DNA appeared at, the position of DNA-prohead complexes in the sucrose density gradient’ (Fig. 8; Guo et al., 1987a), suggesting that packaging initiation was blocked.

429 utilizes a DNA packaging mechanism in which substrate recognition in extracts (and prob- ably in &ro) is dependent upon the terminal protein gp3. This requirement was obviated for DNA trans- location in the highly purified defined system, although the gp3 still stimulated and stabilized packaging. Attempts are being made to reconstruct t.he TINA packaging machine in. vitro from purified proteins in the hope of defining the constituents and events of substrate recognition. ATP hydrolysis and DNA translocation.

We acknowledge the technical help of’ Charlene Peterson, Stephen Erickson, Mary-Ann Gomes, Maria- Jose Martin, Sarah Turnquist and Jesdawan Wichitwechkarn. We thank Bernard Reilly for helpful discussions and Charlene Peterson for preparing the manuscript. The work was supported by grants DE-03606. DE-08515 and GM-39931 from the National Instit’ubes of’ Health. S.G. is a graduate studrnt in Genrtics and Cell Biology at the ITniversity of Minnesota.

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Edited by M. F. Moody