Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA

J. Mol. Biol. (1990) 215, 287-299

Role of Gene 6 Exonuclease in the Replication and Packaging of Bacteriophage T7 D N A Philip Serwer, Robert H. Watson and Marjatta Son

Department of Biochemistry The University of Texas Health Science Center San Antonio, T X 78284-7760, U.S.A. (Received 8 January 1990; accepted 15 M a y 1990) When bacteriophage T7 gene 6 exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed. By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made. (1) Most degradation of intracellular DNA requires the presence of T7 gene 3 endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene 3 and gene 6 products are absent. (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene 6 exonuclease. (3) Fragments produced in the absence of gene 6 exonuclease are linear and do not further degrade; precursors of the fragments are non-linear. (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication. On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene 6 exonuclease is caused by cleavage at branches. The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene 6 exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene 6 exonuclease during a wild-type T7 infection. Observation of joining of genomes in the absence of gene 6 exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication. The observed selective shortening of the left end indicates that gene 6 exonuclease is required for formation of most, possibly all, mature left ends.

1. Introduction After its replication in an infected host (in vivo), the linear, double-stranded DNA of bacteriophage T7 is found to be polymerized end to end; the polymer formed is called a concatemer (Kelly & Thomas, 1969; Schlegel & Thomas, 1972; Langman et al., 1978). Subsequently, monomers in the concatemer are packaged in the protein outer shell (capsid) of the bacteriophage and cut to a monomeric DNA (sedimentation coefficient, s=32; Studier, 1965) that has unique ends (non-permuted) and a 160 base-pair terminal repeat (Ritchie et al., 1967; Dunn & Studier, 1983). The capsid that packages DNA changes several of its physical characteristics during packaging. These changes include a 0022-2836/90/180287-13 $03.00/0

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decrease in electrophoretic velocity during agarose gel electrophoresis (for a review, see Serwer, 1989). The concatemerization of T7 D N A could, in theory, occur by replication on a circular template, end-to-end joining of linear DNA, or possibly both of these alternatives (see Kelly & Thomas, 1969). Although in vitro T7 DNA packaging extracts cyclize T7 DNA (Son et al., 1988), attempts to find circular DNA formed in vivo before replication have yielded no circles (Striitling et al., 1973; FrSlich et al., 1975). Electron microscopy reveals that at least the initial replicating molecules are linear (Dressier et al., 1972; Wolfson et al., 1972; Wolfson & Dressier, 1979; Tamanoi et al., .1980). Before packaging begins, replicated DNA concatemerizes (Kelly & Thomas, 1969; Schlegel & Thomas, 1972). During

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packaging, most replicated DNA in wild-type T7 (T7wtt)-infected cells is either part of a concatemer, part of a more rapidly sedimenting, branched DNA (100 S + DNA), shorter than mature DNA or packaged (Serwer, 1974; Serwer et al., 1987). The production of concatemers is temporally associated with changing of the preferred replication origin from t h a t at position 15 (a position of T7 DNA is labeled by the percentage of the monomerie DNA length from the left end), to several secondary origins (Tamanoi et al., 1980; Dunn & Studier, 1983; Fuller et al., 1983; Studier & Dunn, 1983; Fuller & Richardson, 1985a,b; Rabkin & Richardson, 1988). Secondary origins have been found at positions 1, 47, 68 and 98. To understand the relationship of known in vitro biochemical activities to in vivo function, investigation is made of the changes in intracellular DNA that are caused by genetically removing specific proteins from infected cells. In previous studies, removal of p3:[: (an endonuclease; deMassy et al., 1984, 1987) was found to cause the accumulation of rapidly sedimenting DNA ( s = 3 3 to 400). Initially, this DNA was assumed to be concatemeric (StrEtling et al., 1973; FrSlich et al., 1975; Miller et al., 1976; Langman et al., 1978). However, subsequent studies revealed this DNA to be both highly branched and with reduced levels, if any, of unbranched concatemers (Serwer et al., 1987). This latter result is in agreement with the observation of debranching activity for p3 both in vitro (deMassy et al., 1984; 1987; Dickie et al., 1987) and in vivo (Panayotatos & Fontaine, 1987). In the absence of p6 (a 5 ' - , 3 ' exonuclease) in vivo, an increase in <32 S DNA has invariably been found, sometimes accompanied by no DNA sedimenting more rapidly than at 32 S (FrSlich et al., 1975). Alternatively, reduced levels of > 3 2 S DNA were observed, but this > 3 2 S DNA was degraded intraceilularly to < 3 2 S DNA (Miller et al., 1976). Previously presented models propose t h a t p6 has an essential function in DNA replication (Shinozaki & Okazaki, 1977; Engler & Richardson, ]983), recombination (Roeder & Sadowski, 1978; Ogawa et al., 1978), concatemerization (Lee & Sadowski, 1985) and packaging (Sadowski, 1977; White & Richardson, 1987). In the present study, a more comprehensive analytical procedure than t h a t previously used, including centrifugation followed by either restriction endonuelease analysis or field inversion gel electrophoresis (FIGE; Carle et al., 1986; Olson, 1989), has been used to determine the effect in vivo of removing,o6 alone, p6 and p l 9 (p19 is needed for DNA packaging, but is not needed for 1"Abbreviations used: T7wt, wild-type T7; 100 S +, rapidly sedimenting, branched DNA; FIGE, field inversion gel electrophoresis; kb, l03 base-pairs; PFG, pulsedfield agarose gel; 2d-AGE, two dimensional agarose gel electrophoresis. ~: Proteins encoded by T7 genes, will be indicated by p, followed by the number of the gene from Studier & Dunn (1983).

either DNA replication or capsid assembly; Roeder & Sadowski, 1977), or p6 and p3. Implications of the results for understanding T7 DNA replication, concatemerization and packaging are discussed.

2. Materials and Methods (a) Strains of bacleria and bacteriopluwes Bacteriophage T7 amber mutant in gene 3, T73§ (mutant 29), T7 s (mutant 28), T76 (mutant 233), T719 (mutant 10) and T7wt were received from Dr F. W. Studier and were described by Studier (1969). Double mutants were prepared by use of genetic crosses and identified by complementation (Studier, 1969). Lysates and the contents of lysates obtained by infecting a nonpermissive host with T7 amber mutants are identified by the symbol for the mutant bacteriophage. For example, a lysate obtained by use of T76 is called a T76 lysate. The host for T7wt and the non-permissive host for amber mutants was Escherichia coli BB/1. The permissive host for amber mutants was Escherichia coli O-1 l'. (b) Buffers and reagents Standard/G buffer contained 0"15M-NaCI, 0"05MTris" HCI (pH 7"4), 0"005 M-EDTA, 100~ug gelatin/ml. Cultures for radiolabeling of intracellular T7 DNA were grown in M9 medium (Kellenberger & S~chaud, 1957). Seakem LE agarose (Marine Colloids Division of the FMC Corporation, Rockland, ME) was used for tl~e eiectrophoresis of DNA and capsids. IsoGel agarose (from Marine Colloids) was used for the electrophoresis of bacteriophage T7 (see Serwer et al., 1983). 3H-labeled thymidine (40 to 70Ci/mmol) and 14C-labeled algal hydrolysate were purchased from ICN Biomedicals, Inc. (Irvine, CA). Nyeodenz was purchased from Accurate Chemical and Scientific Corporation (Westbury, NY). All restriction endonucleases were purchased from New England Biolabs (Beverly, MA). (c) Infection, radiolabeling and lysis of cells To detect and characterize either T7 DNA or T7 proteins in lysates of E. coli infected with either T7wt or a T7 amber mutant, E. coli BB/1 was grown in log phase to 4-0x 108/ml in M9 medium at 30°C with aeration. The cells were infected (multiplicity of infection of 15) and incubation was continued at 30°C with aeration. At the time after infection indicated, either intracellular DNA was labeled by addition of [3H]thymidine or intracellular proteins were labeled by addition of 14C-labeled algal hydrolysate, and incubation was continued at 30°C. By l0 to 12 min after infection, the level of host synthesis is at least 20-fold less than the level of T7 synthesis of both DNA (Langman & Paetkau, 1978) and proteins (Studier, 1972). During kinetic labeling experiments, labeling was stopped, while continuing the infection, by adding 0"5 mg/ml (final concentration) of unlabeled thymidine to the culture. Cultures were quenched by dilution into an equal volume of an ice-cold solution of 50°/o sucrose, 0"3 M-NaCI, 0"l M-Tris" HCI (pH 7"4), 0"01M-EDTA, 0"008 M-KCN. For analysis of DNA, quenched cells were § T7 amber mutants are labeled in subscript by the number(s) of the mutant gene.

Replication a n d P a c k a g i n g of T 7 D N A pelleted and lysed with lysozyme and the ionic detergent, Sarkosyl NL97 (see Serwer, 1974; Serwer et al., 1987). This detergent inactivates nucleases that, otherwise, would interfere with the restriction endonuclease analysis of intracellular DNA. For analysis of 14C-labeled capsids, the same procedure of lysis was used, except that the nonionic detergent, Brij sS, was used. Sarkosyl disrupts one of the T7 capsids (capsid I; see Results). Neither of the |ysis procedures disrupts mature bacteriophage T7, and both lysis procedures avoid extraction procedures (e.g. extraction with phenol) that can cause selective loss of the particles in lysate. (d) Fractionation by centrifugation Initial fractionation of lysates was performed by centrifugation in a biphasic gradient of sucrose (on top) and a radio-opaque compound (on bottom). For analysis of DNA, the radio-opaque compound was Nycodenz (Serwer et al., 1987). For analysis of capsids, the radio-opaque compound was sodium iothalamate; the gradient and procedure also have been described (Serwer, 1980). The percentage of total trichioracetic acid-precipitable aH in each fraction of a sucrose-Nyeodenz gradient was determined by the procedure of Serwer et al. (1987), and is presented as a function of fraction number. After initial fractionation, capsids were fractionated further by use of buoyant density centrifugation in a metrizamide density gradient (Serwer, 1980). Capsid-DNA complexes were fractionated by centrifugation in a cesium chloride step gradient (Serwer & Watson, 1981).

(e) A garose gel electrophoresis of D N A : one-dimensional To resolve by length DNA molecules that were obtained from a sucrose-Nydocenz gradient and t h a t had lengths as great as 200 kb, agarose gel electrophoresis was performed by use of FIGE. To prepare DNA from a sucrose-Nycodenz gradient for FIGE, a portion of a fraction was heated to 75°C for l0 min (to extrude DNA packaged in capsids) and sometimes diluted in standard/G buffer, as indicated in the Figure legends, before 3-fold dilution with electrophoresis buffer that contained 400 #g bromphenol blue/ml. Twenty p] was layered in sample wells of a 1"5°/o (w/v) submerged agarese gel and F I G E was run for 30 h at 3 V/cm, 15°C in 0"01 M-sodium phosphate (pH 7"4), 0-001 M-EDTA. A 4-port device for FIGE, obtained from DNAStar (Madison, WI), was used. The forward electrophoresis/reverse electrophoresis time ratio was 3 : l, ramped 6 to 18 s forward. Buffer was circulated at /> 100 ml/min through a constant-temperature bath that maintained the temperature + 2 ° C at the surface of the gel. DNA length versus mobility for T7 concatemers was calibrated with either a collection of concatemers of 48"5 kb bacteriophage ~ DNA (Anand, 1986; Cantor et al., 1988) or a mixture of the linear DNAs of mature bacteriophages T7, T5 (120 kb) and T4 (170 kb) (Son et al., 1988). Additional length standards were 3H-labeled mature T7 DNA and restriction endonuclease BglII fragments of mature T7 DNA (below). During pulsed-field agarose gel (PFG) electrophoresis, as the length of the DNA increases, eventually resolution by DNA length is lost (for reviews, see Cantor et al., 1988; Olson, 1989). The result is the migration of those linear DNAs with length above a critical range in one compressed zone (the zone of compression; see also Vollrath & Davis, 1987; Mathew et al., 1988). The zone of compression, for the separations by F I G E used here,

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starts at approximately the position of a pentamerie T7 concatemer. Any DNA found closer to the origin than the zone of compression is assumed not to be simple linear DNA. This latter DNA might be branched, circular or attached to a non-DNA object. Near the zone of compression, a comparatively weak band of DNA released from mature bacteriophages was sometimes observed (arrow with an asterisk in Fig. 2). This latter band is formed by a complex of a T7 eapsid with its extruded DNA, a particle previously characterized (Serwer, 1974). Most of the capsid-DNA complexes are dissociated by the elevated temperature used to extrude T7 DNA; these complexes are undesirable for the studies performed here, but do not obscure any significant features of the data. At 3 V/cm, the use of PFG electrophoresis improves the quality of bands formed by capsid-DNA complexes; a study of this phenomenon is in progress. Electrophoresis of restriction endonuclease fragments was performed by bringing digests (see section (h), below) to 100 to 200/~g bromphenol blue/ml and performing invariant-field, horizontal agarose gel electrophoresis at 0"5 V/cm in 0-7 %o agarose gels. The eleetrophoresis buffer contained 0"05M-sodium phosphate (pH7-4), 0'001 MEDTA. The length of fragments was determined by reference to standards, by use of procedures previously described (Serwer el al., 1987). Alter either PFG or invariant field electrophoresis, to detect all-labeled DNA the gel was dried and subjected to fluorography, by use of sodium salicylate as the fluor (Serwer et al., 1987). To detect 32P-labeled T 7 DNA hybridized to DNA fractionated by agarose gel electrophoresis, DNA in the gel was transferred to nitrocellulose and subsequently hybridized, by the procedure of Southern (1975). After hybridization, the gel was subjected to autoradiography. T7 DNA was labeled with 32p by nick translation (Rigby el al., 1977), by use of a kit purchased from Boehringer-Mannheim. Kodak SB diagnostic X-ray film was used for both fluorograms and autoradiograms. Unlabeled DNA used as a length standard was detected by staining with 1/~g ethidium bromide/ml. The distances migrated by the unlabeled DNA were scaled to the distances migrated by labeled DNA by use of the mature T7 DNA present as a standard among both the all-labeled and the unlabeled DNA standards. To detect and characterize the capsids in lysates of E. coli BB/I infected with either T7wt or a T7 amber mutant, the 14C-labeled proteins in fractions of a sucrosemetrizamide gradient were diluted into 2-9 parts of 400/~g bromphenol blue/ml, 3 % (w/v) sucrose, 0"01 M-sodium phosphate (pH 7-4), 0"02 M-MgCI2, 50/~g DNAase I/ml. After incubation for 30 min at 30°C, 20/~l of this mixture was subjected to electrophoresis in a 0-9% agarose gel for 16h at 1V/era, room temperature (25(_2)°C) in 0"05 M-sodium phosphate (pH 7-4), 0-001 M-EDTA (Serwer, 1980; Serwer et al., 1983). The 14C-labeled capsids were detected by autoradiography performed at room temperature with Kodak SB diagnostic X-ray film. For eleetrophoresis of bacteriophage T7, the EDTA in the electrophoresis buffer was replaced with 0"001 ~-MgCI 2.

(f) Two-dimensional agarose gel electrophoresis Two procedures of 2d-AGE were used for the analysis of DNA: (1) electrophoresis through a 0"2% gel, followed by orthogonally oriented electrophoresis through a 1"5 % gel, both performed by use of an invariant electrical field (Serwer et al., 1987}; (2) FIGE, performed as described above, followed by orthogonally oriented invariant field eleetrophoresis at 7 V/em for 0"54 h. After 2d-AGE, DNA

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not radiolabeled was observed by use of staining with ethidium bromide; all-labeled DNA was observed by use of fluorography. (g) Densitometry and reproduction of fluorograms Densitometry of fluorograms was performed by capture of video images of gels, followed by processing for densitomerry by use of a Macintosh II computer and the program, IMAGE. The software and hardware have been described (O'Neill, et al., 1989; Griess & Serwer, 1990). Figs I, 2 and 5 were reproduced by conventional photographic means. Figs 3, 4 and 6 were collated after capture

Analysis of intracellular T76 DNA by use of centrifugation in a sucrose-Nycodenz gradient, followed by F I G E , has increased knowledge of the types of DNA present. In addition, comparison of the results obtained with T76 with those obtained with T76, x9 has revealed the effects of blocking DNA packaging. After centrifugation, the profile of acid-precipitable tritium revealed a net loss of s value for both T76 (Fig. l(a)) and T76, x9 (Fig. l(b)) intracellular DNA, when compared with the profile obtained for T7wt (Fig. l(c)) and T719 (Fig. l(d)). F o r T76 and T76,19, most DNA formed a broad peak at 20 to 45 S. In addition, the percentage of DNA t h a t co-purified with mature bacteriophages was reduced for T76, T76, 19 and T719 (Fig. 1, fractions 2 to 4). Thus, the effects of removing p6 on the mean s value of unpackaged DNA are not detectably altered by blocking DNA packaging (i.e. removing p19). However, effects on more subtle characteristics of unpackaged intracellular DNA are altered detectably (see below). The results for T76 and T719 are in agreement with those previously obtained (FrSlich et al., 1975; Miller et al., 1976; Langman et al., 1978; Serwer et al., 1987). The one-dimensional procedure described for Figure 1 yielded more information when enhanced b y analysis in a second dimension, by use of F I G E . After F I G E of DNA in each fraction of Figure l(a), most 20 to 45 S T76 DNA was found (Fig. 2(a)) to migrate as either a linear monomer or shorter DNA. A band at the position of both monomeric T7 DNA ( + 10%) and linear DNA 0"3 times as long as monomerie T7 DNA m a y be seen in Figure 2(a) (fractions 20 to 28). This latter band is broad enough to be formed by DNA heterogeneous in length. Analysis of specific intracellular cleavage of DNA was, therefore, performed after restriction endonuclease

Replication and Packaging of T7 DNA I

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Figure 2. FIGE of intracellular DNA. DNA from fractions of the sucrose-Nycodenz gradients indicated in Fig. 1 was subjected to FIGE-fluorography (T7 genotype, followed by the extra dilution for fractions 1 to 8 and the time of fluorographic exposure in days): (a) T76 (none; 5); (a') T7~ (none; 45); (b) T7~, 19 (none; 5); (e) T7wt (8 x ; 15); (d) T719 (2 × ; 45). The filled vertical arrows indicate the positions of integral coneatemers in the sucrose-Nycodenz gradients; the numbered horizontal lines at the left indicate the positions of the linear DNAs whose length is indicated(the unit of length is a mature genome). The filled arrowheads indicate the origins of electrophoresis; the open arrows indicate the directions of centrifugation (horizontal) and electrophoresis (vertical). Lane m ((c) and (d)) has a mixture of both mature T7 DNA and a restriction endonuclease BglII digest of mature T7 DNA; only the smaller of the 2 BgIII fragments is observed here; the larger fragment was not separated from the mature T7 DNA. The curved arrow in (b) indicates fractions that were accidentally reversed.

cleavage (see section (f) below). Weaker bands further from the origin and a significant, background of DNA t h a t did not form a band were also observed. The latter bands are difficult to see in Figure 2(a). The profile of bands observed for T76 in Figure 2(a), fractions 20 to 28, was also observed for T76, 19 in Figure 2(b), but not for T7wt (Fig. 2(c)) and T719 (Fig. 2(d)). I n contrast to the T76 and T76,19 profiles in Figures l and 2, the T7wt (Figs l(c) and 2(c)) and T719 (Figs l(d) and 2(d)) profiles showed DNA sedimenting more as concatemers than as smaller-sized particles. In the case of T7wt, but not T719, bands formed by integral concatemers (2-mer, 3-mer and 4-met) are clearly seen in Figure 2. This latter observation has been made at lower resolution by use of electrophoresis with an invariant field (Serwer et al., 1987). Although DNA sedimenting as concatemers was depleted in the T76 and T76.19 profiles of Figure 2, it was not completely absent. A comparatively weak band at the position of a dimeric concatemer is visible for T76, 19 (Fig. 2(b)) and also for T7¢ in longer exposures of the gel in Figure 2(a) (not shown). In addition, some DNA t h a t is between monomeric DNA and the origin of electrophoresis

and t h a t has a continuously variable migration rate is present for both T76 and T76.19, though more was observed for T76, 19. This latter DNA extended through the zone of compression to the origin, a characteristic of non-linear DNA. Although some concatemer-sedimenting DNA accumulated at t h e zone of compression for T7wt (Fig. 2(c)) and T719 (Fig. 2(d)), no such accumulation was observed for either T7~ (Fig. 2(a)) or T76, 19 (Fig. 2(b)). To test for the presence of non-linear DNA, 25 S to 4 5 S T76,19 DNA was subjected to 2d-AGE. After 2d-AGE by use of either procedure (1) or procedure (2) (see Materials and Methods, section (f)), more than 92~/o of the 3H-labeled DNA was observed on a single line, when DNA with an s value of either 35 to 45 or 25 to 34 was analyzed. This line was coincident with a line formed by unlabeled marker DNAs, of 4 to 170 kb size, known to be linear (data not shown). In the first dimension, the remaining 3H-labeled DNA was either caught at the origin of electrophoresis or distributed continuously from the origin to the position of a linear dimertrimer. In the second dimension of procedure (2), neither of these latter two types of D N A moved. The conclusion m a y therefore be drawn t h a t these

P. Serwer et al.

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Figure 3. DNA packaging by T76: analysis of capsids. A culture of E. coli BB/1 was divided into 3 separate portions. The three portions were infected with, respectively: (a) T76; (b) TTwt; (c) T75. The infected cultures were labeled with 14C-labeled amino acids, quenched, lysed and fractionated by centrifugation in a sucrose-sodium iothalamate gradient (see Materials and Methods). After incubation with DNAase I, the capsids in portions of fractions indicated in (a) to (c) were analyzed by agarose gel electrophoresis-autoradiography (right-hand panels). The mature bacteriophage in the fractions indicated were then analyzed by use of electrophoresis in IsoGel agarose (left-hand panels). The positions in the biphasic density gradient of mature bacteriophage (~) capsid I (CI), DNA-free capsid II (CII) and capsid II-DNA complexes (CII-DNA) are indicated at the bottom. Positions in the gel are indicated at the right. The arrowheads indicate the origins of electrophoresis; the arrows indicate the directions of centrifugation (horizontal) and electrophoresis (vertical). The broadly distributed 14C at the right in (b) and (c) is in unassembled proteins. This material is missing in (a) because more fractions were collected for (a) (i.e. the drop size was smaller) and the pattern for (a) is therefore shifted to the right. latter DNAs are not uncomplexed linear DNAs. They are possibly branched, bound to a non-DNA object, circular, or some combination of these. Because procedure ()) has been shown to discriminate linear DNA from both branched (Bell & Beyers, 1983; Welsh & Cantor, 1987) and open circular (Levene & Zimm, 1987; Serwer et al., 1987) DNA, the DNA t h a t comigrates with linear DNA in both dimensions will be assumed here to be linear DNA. In the case of T76, analysis of fractions 2 to 4 revealed a significant peak of packaged DNA-containing particles (Fig. 2(a'); Fig. 2(a) is a 9 x weaker exposure of the same gel). Even when the fluorographic exposure was three times the exposure in Figure 2(a'), neither the T76, 19 nor the T719 gradient yielded this peak (not shown). The percentage of packaged T76 DNA in Figure 2(a') was only 1/50 to 1/100 times the percentage of packaged TTwt DNA in Figure 2(c). The reversion rate to wild-type of the T7~ inoculum used for Figure 2(a) was low enough (]0 -5) t h a t the presence

of revertants cannot be the cause of the limited T76 DNA packaging observed. (b) D N A packaging by T76 The data of section (a), above, indicate that, at reduced levels, DNA packaging goes to completion for T76. To determine the level of T76 DNA packaging t h a t does not go to completion, agarose gel electrophoresis of capsids in a T76 lysate was used to probe for the previously demonstrated (Serwer, 1980) TTwt DNA packaging-associated change in the electrophoretic mobility of the capsid (i.e. the conversion of capsid I to capsid II). Agarose gel electrophoresis of capsids in a T76 lysate, after previous centrifugation in a sucrose-iothalamate gradient and post-centrifugation digestion of attached DNA by DNAase I, revealed conversion of capsid I to both DNA-free calSsid II and a capsid I I - D N A complex (right-hand panel of Fig. 3(a)). In the case of T75 from the same experiment, only a much smaller a m o u n t of DNA-free capsid II and no

Replication and Packaging of T7 D N A capsid II-DNA complex was observed (right-hand panel of Fig. 3(c)). During other experiments (not shown), no capsid II of any kind was observed for T75. In a separate experiment, the results for T719 were indistinguishable from the results for T7 s (not shown; results for the gene 19 mutant of the T7-related bacteriophage, T3, were shown by Serwer et al. (1985)). By comparison with results, also obtained in the same experiment, for T7wt (right-hand panel of Fig. 3(b)), for T7a the conversion from capsid I to both DNA-free capsid II and capsid II-DNA complexes was found to be 0"3 to 0"4 times as efficient as it was for T7wt. To probe for the formation of T76 mature bacteriophage, IsoGel agarose was used for the agarose gel electrophoresis of particles in the first ten fractions of the gradients used for Figure 3(a) to (e). No mature bacteriophage was observed for T75 (~b in the left-hand panel of Fig. 3(c)). As found in Figure 2(a), a significant fraction of the capsid-associated 14C was found in mature bacteriophage for T76 (left-hand panel of Fig. 3(a)). However, this fraction was only 0-02 times that found for T7wt (left-hand panel of Fig. 3(b)). Thus, for T76 the formation of mature T7 is more inhibited than the capsid I to capsid II transition. Some of the DNA-free capsid .II observed in TTwt-infected cells is metrizamide permeable (MHD capsid II) and some is metrizamide impermeable (MLD capsid II) (Serwer, 1980). Both MHD and MLD capsid II were found among the DNA-free T76 capsid II shown in Figure 3(b). The impermeable/ permeable capsid II ratio was 0"60, compared to 1.1 for T7wt (data not shown). Some of the DNA of T7wt capsid II-DNA complexes is concatemeric (Serwer & Watson, 1981). However, comparatively few of the previously described (for a review, see Serwer, 1989) capsid II-concatemeric DNA complexes were found for T76. The T7 capsid II-concatemeric DNA complexes were 35O/o of the total capsid I I - D N A complex for T7wt, but only 15~o for T76 (data not shown). This deficiency of concatemers in capsid II-DNA complexes is paralleled by the deficiency of all concatemers in T76 lysates (i.e. Figs 1 and 2).

(c) The effect of removing p3 The studies described in the previous two sections reveal that DNA packaging is not required for most of the degradation of T7 DNA caused by removal of P6. To test for the possibility that p3 (T7 endonuclease) is required, the experiment illustrated in Figure 1 was also performed for T73.6. As previously found for T73 (Serwer et al., 1987): (1) more than 90% of the total acid-precipitable 3H-labeled DNA was found to be 100S + DNA (Fig. l(e)), and (2) most of this 100S + DNA was arrested near the origin during agarose gel electrophoresis (data not shown). Thus, the conclusion is drawn that p3 is required for the degradation of T76 DNA.

293

(d) Kinetics of T76 DIVA The data given in Figures 1 to 3 were obtained by analysis of DNA at only one time after infection. To determine the kinetics of the various forms of DNA observed in Figure 2, a T76 culture was labeled with [3H]thymidine from 13"0 to 14"5 minutes after infection. After stopping labeling by adding unlabeled thymidine, portions of the culture were quenched at 14"5, 16"0, 19"0 and 22.0 minutes after infection. Analysis of the intracellular DNA was made by centrifugation, followed by FIGE (i.e. as in Fig. 2). At 14"5 minutes, most DNA was found in a continuous distribution that extended past the zone of compression for linear DNA (Fig. 4(a)). As the time increased to 16"0 and 19"0 minutes (Fig. 4(b) and (c)), the relative amount of this latter DNA decreased as the amount of monomeric and shorter, linear DNA increased. However, the profile at 22-0 minutes (Fig. 4(d)) did not significantly differ from profile at 19.0 minutes (Fig. 4(c)). That is, after the initial conversion to monomeric and shorter DNA, further change did not occur. As found in the experiment illustrated in Figure 2, no discrete-length concatemers longer than a dimer appeared at any time for Figure 4. The dimer first appeared at 19-0 minutes (Fig. 4(c)) and its amount appeared to decrease at 22"0 minutes. This decrease was not observed for T76, tg, but the other aspects of Figure 4 were observed for T76, 19 (data not shown).

(e) Analysis of total mutant D N A by use of centrifugationJollowed by restriction endonuclease XbaI cleavage Although the blockage of DNA packaging caused by removal of pl9 did not significantly alter the centrifugation profile ofT76 DNA (i.e. Fig. l(a) and (b)), the occurrence of post-initiation steps of the DNA packaging pathway during a T76 infection raises the question of whether subtler characteristics of T76 DNA are altered by the removal of p19. To help to answer this question, DNA in each fraction of a T76 sucrose-Nyeodenz gradient was digested to completion by restriction endonuclease XbaI and the digestion products were analyzed by agarose gel electrophoresis-fluorography. To help to eliminate bands formed by residual host DNA, three host DNA-specific enzymes, EcoRI, BamHI and PstI, were added to the XbaI. The XbaI fragment produced by joining of the left and right ends of mature DNA during concatemerization (Langman et al., 1978; Serwer et al., 1987) was present for T76 and was enriched among DNAs that sedimented as coneatemer (terminally joined, or TJ(A-D) fragment in Fig. 5(a)). However, the integrated intensity of the TJ(A-D) band, when compared to the integrated intensity of the internal B band, was four- to tenfold less in the case of T76 (Fig. 5(a)) than it was in the case of either T76.19 (Fig. 5(b)) or T7wt {Fig. 5(c)). Thus, removal of pl9 {i.e. blockage of packaging) stops loss of the TJ(A-D) fragment

294

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caused by removal of p6, even though a significant change in average s value of the DNA was not observed. The relative intensity of the T76,19 TJ(A-D) fragment from DNA sedimenting as concatemers (Fig. 5(b)) is comparable to that of the T7wt TJ(A-D) fragment (Fig. 5(c)) and two- to threefold less than that of the T719 TJ(A-D) fragment (Fig. 5(d)). Although concatemerization of T7 DNA occurs in the absence of p6 in vivo, a previous study has found that p6 is required for concatemerization in T7 DNA packaging extracts (in vitro) (Lee & Sadowski, 1985). By use of more efficient DNA packaging extracts more recently developed (Son et al., 1988, 1989), requirement for p6 during in vitro concatemerization has been confirmed; concatemers were detected by rotating gel electrophoresis (Son et al., 1988), followed by Southern blotting-hybridization (data not shown). In addition to the depletion of TJ(A-D) XbaI fragment observed in Figure 5(a) (T76), the 25 to 50 S DNA yielded two major XbaI fragments (C* and D*) not observed in Figure 5(c) (T7wt) or (d) (T719). The C* and D* bands were significantly broader than the D band (see below); therefore, the DNA that forms the C* and D* bands is assumed to

be heterogeneous. The mean length of the C* fragment is 17"9% of the mature T7 length; the mean length of the D* fragment is 13"5% of the mature T7 length. The C* and D* XbaI fragments were also observed in the 25 to 50 S region of Figure 5(b) (T76,19), indicating that both of these fragments were produced by an event independent of p19. Neither the T76 100 S + DNA (Fig. 5(a)) nor the T76, 19 100 S + DNA (Fig. 5(b)) released detectable C* and D* XbaI fragments. (This conclusion was drawn from a 3-fold darker exposure of the fluorograms in Fig. 5(a) and (b).) The D (right end) fragment was observed throughout the gradients of T76 (Fig. 5(a)), T76, 19 (Fig. 5(b)) and T7wt (Fig. 5(c)). As previously found (Serwer et al., 1987), this fragment was missing from the gradient of T719 (Fig. 5(d)). The presence of the D fragment in the XbaI profile of the T76,19 gradient indicates that p6 was required for the loss of the D fragment in the XbaI profile of the T719 gradient. However, during other experiments, the D band of T76, 19 was depleted (see Fig. 6(b), below). The cause of this variability is not known. The use of host-specific restriction endonucleases during the digestions shown in Figure 5 minimized the chances of the formation of bands by host DNA.

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apparently variable-length DNA, sometimes including minor bands, was observed (Fig. 5(a)) and confirmed to be T7 DNA by hybridization (Fig. 6(a)). Because this background was also present for T76, z9 (Fig. 5(b)), most of it is not produced by DNA packaging. However, the background for T7wt (Fig. 5(c)) was significantly higher than the background for T7t9 (Fig. 5(d)). This latter observation can be explained by the production of some variable-length DNA during DNA packaging.

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To test this possibility further, an XbaI digest of T76 DNA was blotted and hybridized to a2p-labeled T7 DNA after agarose gel electrophoresis. DNA that formed all of the bands described above hybridized to T7 DNA (Fig. 6(a)), even though no detectable hybridization to two orders of magnitude more E. coli DNA was detected (not shown). In addition to the bands observed after XbaI digestion of 25 to 50 S T76 DNA, a background of

(f) Further restriction endonuclease analysis

Comparison of XbaI digests on the same gel revealed that the T76 A band (Fig. 6{b); lanes marked l have T76 DNA; XbaI digests are indicated by an X above the l) was both closer to the B band and also broader than the A band of mature T7 DNA (Fig. 6(b); lanes marked ~ have mature T7 DNA). In contrast, the T76, 19 A band was further from the B band than the mature T7 A band (Fig. 7(b); lanes marked 2 have T76.19 DNA). For T76, 19 and, to a lesser extent, T%, the right-end fragment (D fragment) and the left-end fragment {A fragment} were depleted relative to the internal (B and C) fragments. The XbaI digests of Figure 6(b) were further digested with BcII, a restriction endonuclease whose only recognition site on T7 DNA is within the XbaI A fragment. For T76 DNA, digestion with BclI confirmed the identification of the XbaI A fragment. That is, this latter fragment was cleaved. Two left-end fragments were produced by this cleavage (bracketed region marked A' in Fig. 6(b); digests obtained with both XbaI and BclI are indicated at the top by X over B): a major fragment 1 to 2°/o shorter than the A' fragment and a minor fragment, not detected in all experiments, that was 5 % longer. Only the longer fragment was observed for T76.19 in Figure 6(b). Thus, removing pl9 {i.e. blocking DNA packaging) prevents the appearance of the shortened left end of intracellular DNA observed in the absence of p6. The breadth of the T76 left-end band in Figure 6(b) indicates heterogeneity in the amount of shortening. Because of this heterogeneity, some DNA that forms the left-end A band of an XbaI digest is probably lost in the background of the digest obtained by use of both XbaI and BclI. The right-end BelI fragment of the XbaI A fragment was indistinguishable for all DNAs (A" in Fig. 6(b)). The variable length of T76 and T76, z9 left-end fragments prompted analysis of T719 and T7wt intracellular DNA. In the case of T719 and T7wt, the left-end fragment of intracellular 40 to 50 S DNA was 5 °/o longer than that of the mature DNA (data not shown). A longer than mature-length leftend restriction endonuclease fragment from intracellular T7 DNA has previously been observed after DNA packaging was genetically blocked by use of a mutation in T7 gene 1 (RNA polymerase); the two polynucleotide chains were covalently joined to

296

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Figure 6. Further restriction endonuclease analysis. An Xba] digest (X), an XbaI +BclI digest (x), an XbaI +SfiI digest (x) or an XbaI + EcoNI digest (x) (the digest used is indicated at the top of the Figure) was subjected to invariant field agarose gel electrophoresis, followed by: (a) Southern blotting-hybridization, or ((b) to (c)) fluorography. The lanes are marked by their contents, according to the following: ~, DNA from mature bacteriophage T7; 1, intracellular 30 to 45 S T76 DNA; 2, intraceilular 30 to 45 S T76. 19 DNA. (a), (b) and (c) are 3 separate gels. The filled arrowheads indicate the origins of electrophoresis; the open arrow indicates the direction of electrophoresis. In (d), the T7 genome is represented by map units (position 0-00 is the left end; position 100"00 is the right end). Sites of cleavage are represented for the following restriction endonucleases (see Materials and Methods): BclI (B); EcoNI (E); SfiI (S), XbaI (X). The site that is cleaved in T7~- and T76. 19-infected cells is shown for the C* and D* XbaI fragments. form a hairpin at the left end of this fragment (J. J. Dunn & F . W . Studier, personal communication). This phenomenon will not be described further here. In Figure 6(b), it m a y be seen t h a t neither the C* nor the D* X b a I fragments were cleaved by BclI, indicating t h a t these fragments do not overlap the T7 position, 20.8. To map the site of intracellular cleavage of the X b a I D* fragment, an XbaI digest was further digested with SfiI, a restriction endonuclease whose only site of cleavage is in the X b a I D fragment. When an XbaI digest of T76 intracellular DNA (Fig. 6(b); digests obtained with both X b a I and SfiI are indicated at the top by X over S) was further digested with SfiI, both the X b a I D and D* fragments were cleaved and the larger products (D' and D*' in Fig. 6(b); the shorter products were not visible) were reduced in length by 3-6°/o of the length of T7 DNA. This result places the intra-

cellular cleavage t h a t produced the D* fragment at position 99"3(___0"2) bases (see Fig. 6(d)). To locate the intracellular cleavage t h a t produced the X b a I C* fragment, an X b a I digest of T76 DNA was further cleaved by digestion with EcoNI, a restriction endonuclease whose only site of cleavage is in the XbaI B fragment. When an X b a I digest of T7~ DNA (Fig. 6(c)) was further digested with EcoNI (X over E in Fig. 6(c)), both the X b a I B and C* fragments were cleaved and the expected two products of the B fragment (brackets marked B', B" in Fig. 6(c)) were observed. The cleavage of the C* fragment in Figure 6(c) and the known length of this fragment locate the site of intracellular cleavage at either position 67"9 or position 75-3 (see Fig. 6(d)). This ambiguity is not rigorously resolvable with the left two lanes in Figure 6(c) because the presence of B' and B" fragments derived from

Replication and Packaging of T7 D N A

the B fragment prevents determination of which of these products of EcoNI digestion is produced by cleavage of the C* fragment. Resolution of this ambiguity was achieved by digestion with SfiI alone (S in Fig. 6(c)). The result was production of three fragments that formed bands: the (expected) D and D* fragments and also a fragment with a left end at position 68.1 ___0.2 (band marked with an asterisk in Fig. 6(c)). Thus, the site of intracellular cleavage is at position 67.9+_0.2 for the C* fragment (see Fig. 6(d)). 4. Discussion

The increased DNA degradation of a T7~ infection has been found to fall into two categories. Most degradation depended on the presence of p3 and was independent of p19 (i.e. independent of DNA packaging). Cleavages of this first type were both nonspecific and specific; two sites of specific cleavage were observed. In the second category is a comparatively small amount of cleavage that depends on the presence of p19. The pl9-dependent cleavage was specific for both the joint between genomes in a concatemer and the left end. The formation of wildtype levels of T76, 19 terminal joints indicates a p6-independent mechanism for forming these joints. These and the other observations given in Results may be analyzed in the context of DNA replication, concatemerization and packaging. (a) Replication The accumulation product of a T73, 6 infection is 100S + DNA with properties that suggest the accumulation of branches. The presence of branches is supported by the absence of further degradation of the initially produced T7 o DNA fragments; these fragments were found by 2d-AGE to be linear. That is, the infected cell appears not to have detectable amounts of endonucleolytic activity directed at linear T7 DNA. The heterogeneous, non-linear DNA that converts to linear DNA (see Figs 2 and 4) is a candidate for the branched DNA precursor of the linear T76 fragments. The accumulation of branches in the absence of p3 is consistent with the known (deMassy et al., 1984, 1987; Dickie et al., 1987) debranehing activity of p3. Presumably, p3 cleaves these branches when present. Because p3 does not have sufficient nucleotide sequence specificity to produce bands during digestion of branched DNA, the additional conclusion is drawn that specificity of branch formation (not nucleotide sequence specificity of cleavage) is the cause of the specificity of intracellular cleavage that produces the C* and D* bands. Because of its independence of pl9, the apparent increase in p3-sensitive branches caused by removal of p6 in vivo cannot require DNA packaging. Because genetic recombination is depressed in vivo in the absence of p6 (Powling & Knippers, 1974; Kerr & Sadowski, 1975; Miller et al., 1976) and depression of physical joining of two DNA mole-

297

eules is the cause (Tsujimoto & Ogawa, 1978; Ogawa et al., 1978), the assumption is made that the increased branching does not occur during recombination. Thus, the working assumption is made that the increased branching in the absence of p6 occurs during DNA replication. A possible mechanism by which removal of p6 causes replication-induced increase in p3-sensitive branches is deduced from: (1) the known in vitro activity of p6 exonuclease: 5'-*3' exonuclease active at DNA-ends, DNA nicks (Kerr & Sadowski, 1972a,b) and RNA in RNA-DNA hybrids (RNAase H activity; Shinozaki & Okazaki, 1978); (2) the known effects that removing p6 in vivo has on RNA primers of DNA replication : accumulation of 5'-tetraribonucleotide Primers covalently joined to DNA; Shinozaki & Okazaki (1977). These primers are exonucleolytically degraded by p6 in vitro. The gaps thus created are filled in vitro by form II of T7 DNA polymerase; the completed polynucleotide chain is closed by T7 ligase (Engler & Richardson, 1983). From these data, the following hypothesis is derived. The branches caused by the absence of p6 are formed by displacement of the RNA primer of one DNA segment by the replicating end of a second segment; the second segment is located on the 5' side of the first segment (primer displacement hypothesis). This type of branching has been observed in vitro (Romano & Richardson, 1979; Lechner et al., 1983). The primer displacement hypothesis predicts the formation of p3-sensitive sites at the points of initiation for discontinuous lagging-strand replication and also at any leading-strand initiation site encountered by a growing 3' end. By this hypothesis, the intracellular cleavages that produce the C* and D* fragments observed here are caused by leading-strand DNA replication that initiates at the origins at positions 68 and 98. Selective cleavage near the former origin, but not the latter, has also been observed for unfractionated intracellular T7 DNA (Lee & Sadowski, 1982). The n0n-specific cleavages would be produced at origins of laggingstrand synthesis. {b) Concatemerization The observation (see Results} that p6 is not required in vivo for concatemer-producing, terminal DNA joining is in contrast to two other observations: (1) p6 is required for concatemerization that is observed in T7 in vitro DNA packaging extracts (Lee & Sadowski, 1985; and see Results); (2} terminal DNA joining does not occur in vivo for unreplicated, parental DNA, even in the presence of p6 (Str~tling et al., 1973; FrShlich et al., 1975). An explanation for these observations is that singlestranded cohesive ends necessary for terminal joining are produced in vivo by DNA replication and in vitro by the exonucleolytic digestion of p6; only replicated DNA is observed by the procedures given in Results. Absent or comparatively inefficient semiconservative DNA replication has been found in T7

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DNA packaging extracts, unless deoxyribonucleotide triphosphates are added (Hinkle & Richardson, 1974), and most DNA in these extracts is not branched (Son et al., 1988). A possible mechanism by which semiconservative DNA replication generates a single-stranded end on one of the two termini of a linear DNA is the noncompletion of replication on the parental lagging strand. Subsequent joining of progeny linear, monomeric DNAs via the cohesive ends would produce concatemers directly (Watson, 1972). Alternatively, cyclization via the cohesive end might be followed by concatemerization produced by replication more than once around the circle (for example, see Gilbert & Dressier, 1968). At present, information to distinguish these two possibilities (both m a y occur) has not apparently been obtained. (c) Packaging In the absence of p6, the capsid transformations t h a t accompany DNA packaging are only partially suppressed. The assumption t h a t T76 packaging of concatemers proceeds to the point of cleaving DNA at the joint between genomes explains the increase t h a t occurs in the recovery of the T J fragment, when TTs, 19 is substituted for T7s. During a TTs infection, the cleavage of the joint between genomes was accompanied b y production of a shortened left end and a right end not detectably altered in length. This observation has also been made during the in vitro packaging of a T7 DNA fragment (White & Richardson, 1987) and indicates a difference between the mechanisms for cutting the left and right ends. A possible mechanism for producing this difference is: (1) duplication of the terminal repeat t h a t is associated with the cutting of the left end, but not the right; and (2) blockage of duplication by removal of p6. A more detailed hypothesis was presented by White & Richardson (1987). The authors thank Drs J . J . Dunn and F. W. Studier for communicating unpublished results, Dr Gary A. Griess for assistance with the densitometry, and Linda C. Winchester for typing the manuscript. Support was received from the National Institutes of Health (grant GM24365) and the 1%obert A. Welch Foundation (grant AQ-764).

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Edited by J. K a r n