Endonuclease TEIICHI Department
of T4 Ghosts MINAGAWA
of Botany, Faculty of Science, Kyoto University, Kyoto, Japan Accepted August 23,1976
An endonuclease was found to associate with purified T4 particles. Its activity was low with intact phages but became distinct when phage particles were disrupted by osmotic shock, and more distinct when they were disassembled by guanidine. The enzyme cleaved all DNA species tested so far, including glucosylated and nonglucosylated T4 DNA, T3 DNA and fd DNA. The cleavage was more efficient with doublestranded DNA than with single-stranded DNA. Defective capsids of mutants of genes 4, 10, 14, 16, and 49 had enzyme activity which was comparable to capsids of the wild type, suggesting that the enzyme is located on capsids. The nucleolytic activity was barely detectable, if at all, with four out of five mutants of gene 17. INTRODUCTION
Recent investigations have revealed that there is a close relationship between the maturation of the precursor DNA of T4 phage and assembly of capsid components. From genetic analysis, Streisinger et al. (1976) proposed a model for the formation of T4 DNA in which it was postulated that the precursor DNA, which is longer than the unit length of phage DNA, is cleaved according to the capacity of capsids. If a part of precursor DNA is encapsidated and cleaved at a site close to the base of the capsids, DNA strands of defined length would be formed. This model is substantiated by the following facts. First, replicating DNA is different from particle DNA in its structure and involves longer molecules than those in phage heads (Frankel, 1966, 1968a). Second, replicating DNA is a precursor of particle DNA. Thus, conversion of precursor to particle DNA does not proceed in cells infected with mutants defective in capsid formation, where no or aberrant particles are formed (Frankel, 196813; Minagawa and Fujisawa, 1968a,b; Fujisawa and Minagawa, 1971a). This is also true with mutants of genes 16, 17, and 49 (Minagawa and Fujisawa, 1968a,b; Fujisawa and Minagawa, 1971a1, which permit formation of normal-looking but empty 234 Copyright All rights
8 1977 by Academic Press, Inc. of reproduction in any form reserved.
heads (King, 1968; Luftig et al., 1971). Finally, the length of DNA in phage particles is proportional to the size of the capsid (Eiserling et al., 1970; Doerman et al., 1973; Fujisawa and Minagawa, 1971a; Mosig et al., 1972; Uhlenhopp et al., 1974). If the headful model is really applicable for the maturation of T4 DNA, the cleavage of the precursor DNA should be catalyzed by an enzyme. If such an enzyme exists it should cleave T4 DNA endonucleolytitally. Conversely, the enzyme should recognize completion of encapsidation of DNA. A possible way for this to occur would be that such an enzyme resides on the base of the capsids, though one cannot rule out that the enzyme might leave after it functions. With these considerations in mind, I examined the nuclease activity of, T4 ghosts and defective capsids and found that endonucleolytic activity exists in ghosts and defective capsids but not in those of mutants of gene 17. MATERIALS
Strains Escherichia coli strain 1100 (endonuclease I-) obtained from Dr. M. Sekiguchi was used as a host for preparation of phages and defective capsids, and W4597 (UDPG-PPase-) from Dr. T. Fukasawa
was used as a host to prepare 32P-nonglycosylated T4 DNA. B and B3 (thy-) were used to prepare [“‘PI- and L3HlT4 DNA, respectively. K38 was used for preparation of fd and spheroplast assay of fd DNA, and CR63 for the preparation of am mutants of T4. All mutants of T4D were gifts from Drs. R. S. Edgar and W. B. Wood. Am x 5 is a multiple mutant of DNA negative genes 41(am N81), 42(am Nl221, 43(czm B221, 44(am N82), and 45(am ElO). Media Components of M9 and low P-TG were described previously (Fujisawa and Minagawa, 1971b). PGY used for growth of E. coli K38 and fd contained 10 g of pepton, 3 g of NaCl, and 1 g of glucose yeast extract in 1000 ml of demineralized water, and pH was adjusted to 7.4. Suspension buffer contained 5 volumes of 1 M sucrose and 3 volumes of 0.1 M Tris-HCl at pH 8.5. Dilution buffer for spheroplasts was composed of 0.1 M NaCl, 0.005 M MgS04, 0.001 M CaCl,, and 0.01 M Tris-HCl at pH 8.5. PEG buffer was 8% (w/v) polyethylene glyco1 in dilution buffer. Soft agar-sucrose contained 0.005 M MgS04, 0.001 M CaCl,, 27 g of sucrose, and 1 g of agar in 200 ml of PGY. Solutions of NaCl used for preparations were buffered with 0.01 M Tris at pH 7.4. Preparation tures
and Phage Struc-
(1) For preparation of phages and defective structures of T4, E. coli 1100 was used as a host unless otherwise mentioned. Cells were aeroated at 37” in M9 supplemented with 0.1% casamino acid and 0.5 pg/ml of thiamine to a density of 5 to 10 x 10H cells/ml, infected with phage at an input ratio of 3, and superinfected 5 min after infection at the same input ratio. Aeration was continued for 2.5 hr and chloroform was added to lyse cells. The lysate was filtered through a thin layer of Supercel. To sediment phages from the lysate, solid NaCl (0.5 M) and polyethylene glycol (lo%, w/v) were added and allowed to stand in the cold room for 3 days (Yamamoto et al., 1970). The supernatant was
discarded by syphoning and the sediment was collected by lowspeed centrifugation. The collected sediment was suspended in 0.1 M NaCl and centrifuged at 16,000 rpm for 1 hr. Phage was purified by 3 cycles of differential centrifugation at 6000 rpm for 10 min and at 15,000 rpm for 1 hr. Finally, 3 to 5 ml of a phage suspension was loaded onto the top of a step gradient of Angioconray (methylglucanine iodamide, Daiichi Chemical Co.), sucrose (3 ml of 48%, 2 ml of 40%, and 2 ml of 32% Angioconray, and 3 ml of 10% sucrose from the bottom in the tube), and centrifuged in a Hitachi RPS 25A-3 rotor at 23,000 rpm for 2.5 hr. Phages were banded at the position of interphase between 48 and 40% Angioconray. The band was collected by syphoning and was dialyzed against 0.1 M NaCl for 20 hr with several changes of the buffer. A ts mutant was grown at 30 or 42”. Am x 5 was prepared by the same method except that CR63 was used as a host. To purify defective capsids, the sediment of polyethylene glycol as described for phages was suspended in 0.1 M NaCl and centrifuged at 22,000 rpm for 1 hr. The pellet was resuspended and contaminating phage particles were removed by adsorption to heat-killed cells (at 70” for 30 min) of E. coli 1100. Differential centrifugation at 10,000 rpm for 10 min and at 22,000 rpm for 1 hr was repeated three times. (2) For preparation of fd, E. coli K38 was aerated in PGY to a density of 5 x lox ml at 37” and was infected at an input ratio of 0.1 after addition of CaCl, to 0.002 M. Aeration was continued for 3 hr, and cells were removed by centrifugation. Phages were precipitated from supernatant by the addition of solid polyethylene glycol (10%) and NaCl (0.5 MI, and were purified by differential centrifugation at 40,000 rpm for 1 hr and at 10,000 rpm for 10 min. Preparation
Solid NaCl was dissolved in a phage suspension containing 5 X lOI particles per ml to 5 M. Twenty volumes of distilled water was dumped in and stirred vigorously (Minagawa, 1961). Ghosts were sedimented by centrifugation at 22,000 rpm for 1 hr, washed with 0.02 M NaCl by centrifu-
gation three times, in 0.1 M NaCl. Disassembling
A suspension containing 1 x 1013 phage particles per ml was mixed with 1 M TrisHCl at pH 7.4 to 0.1 M, mercaptoethanol to 0.002 M, and guanidine-HCl to 6.5 M, and was incubated at 37” for 1.5 hr after sonication. After incubation it was dialyzed against 0.3 M NaCl-0.002 M mercaptoethanol in the cold to remove guanidine. DNA was removed by adding l/3 volume of 5% streptomycin, and phage protein was precipitated by ammonium sulfate at 80% saturation. The precipitate was dissolved in and dialyzed against 0.1 M NaCl-0.002 M mercaptoethanol and was called disassembled phage. In some experiments, ghosts prepared by osmotic shock were disassembled similarly, but streptomycin precipitation was omitted. Preparation
of Cell Fraction
Uninfected cells of E. coli B or 1100, or cells of 1100 which had been infected and superinfected with T4 and aerated at 37” for 25 min, were collected and washed by 0.1 M NaCl. Washed cells were sonicated, mixed with equal volume of CsCl saturated at 20”, and centrifuged at 23,000 rpm for 16 hr at 20”. The top fraction in the tube was carefully removed, dissolved in 0.1 M NaCl, and dialyzed against 0.1 M NaCl. Preparation
The method for preparation of DNA was described previously (Fujisawa and Minagawa, 1917b). A phage suspension was mixed with ethylenediaminetetraacetate (EDTA, 0.01 M) and sodium dodecyl sulfate (SDS, 0.4%), and was heated at 70” for 10 min, then shaken with phenol three times and dialyzed against buffer containing 0.1 M NaCl and 0.0005 M EDTA overnight with several changes of the buffer. SDS was not removed completely by dialysis; however, the endonucleolytic activity of sonicated T4 ghosts was higher with this DNA than with DNA prepared in the absence of SDS. The reason for this is not known. For denaturation of T4 DNA, 1 M NaOH
was gently mixed with DNA in a dialysis tube to a final concentration 0.3 M, and kept at room temperature for 10 min. NaOH was removed by dialysis against 0.1 M NaCl. Low-P TG and M9 supplemented with 0.1% casamino acid were used for labeling DNA with 32P04 and [3H]thymidine, respectively. Labeled T3 DNA was prepared by Dr. H. Matsuo, and labeled fd DNA was prepared by Dr. Y. Ryo of this laboratory. Assay for the Nucleolytic
To assay the endonucleolytic activity, sedimentation analysis of 32P-labeled DNA was performed after incubation with protein. A reaction mixture contained, in 0.2 ml, [32PlDNA, 0.005 M MgS04, 0.04 M cacodylate buffer at pH 6.0 unless otherwise mentioned, and phage preparation or cell fraction. The mixture was incubated at 37” for 30 min. The reaction was terminated by adding 0.1 ml of 0.2 M EDTA and 0.02 ml of 8% SDS, and [3H]DNA was added as a reference. An aliquot was applied onto the top of a 4-ml neutral or alkaline linear sucrose gradient (5 to 20%) and was centrifuged at 40,000 rpm for 1.5 hr at 16” for T4 DNA and for 3 hr for T3 DNA, and at 24,000 rpm for 16 hr for fd DNA in a Hitachi RPS 40 rotor. After centrifugation, drops were collected on paper disks from the bottom by syphoning, dried, washed by 5% trichloroacetic acid three times and acetone once, and then dried to count radioactivity in a Packard Liquid-scintillation counter. Neutral sucrose gradients were made in a buffer containing 0.9 M NaCl, 0.001 M EDTA, and 0.01 M Tris buffer at pH 7.4, and alkaline gradients were made in a solution containing 0.9 M NaCl, 0.001 M EDTA, and 0.3 M NaOH. In some experiments, the endonucleolytic activity was measured by transfection of fd DNA. The reaction mixture contained 0.25 pg/ml of fd DNA, 0.005 M MgS04, 0.04 M sodium phosphate buffer at pH 6.0, and an appropriate amount of phage protein, and was incubated at 37”. After incubation, the reaction mixture was diluted to l/40 with saline. When infective phages were contained in the mixture, the diluted
mixture was heated at 85” for 10 min before plating. By this heat treatment, efficiency of transfection decreased to 60%. Either 0.05 or 0.1 ml of the diluted reaction mixture was mixed with 0.5 ml of dilution buffer and 0.1 ml of spheroplasts, and was incubated at 37” for 10 min, and then plated after being mixed with 2 ml of melted soft agar-sucrose. Efficiency of transfection of fd DNA was 10e6 per molecule. The numbers of plaques decreased linearly to about 20% when plotted against either protein concentrations in the reaction mixture or reaction times. Preparation
The method of Melechen et al. (1971) was modified slightly. Cells of E. coli K38 were grown in 100 ml of PGY to a density of 1 x log/ml, collected, and suspended in 1 ml of suspension buffer. Cells were lysed by the addition of 0.017 ml of 0.2 M EDTA and 0.014 ml of 5 mg/ml of egg white lysozyme at room temperature for 1 min. Lysis was terminated by the addition of 0.014 ml of 1 M MgSO, and dilution by 9 ml of icechilled PEG buffer. Spheroplasts could be stored in the refrigerator without loss of activity for 10 days. Measurement
Protein was measured according to Lowry et al. (1951). One phage equivalent
corresponded to 2.5 serum albumin.
lo-‘O pg of bovine
The Nucleolytic Activity of T4 Ghosts When [ZsaP]T4 DNA was incubated in the presence of 4.5 x 10” phage equivalents per milliliter of sonicated ghosts at 37” for 30 min and centrifuged through a neutral sucrose gradient, a portion sedimented a little more slowly than the reference [:‘H]T4 DNA, as shown by surve a of Fig. 1A. The slower sedimentation of ]:@PlT4 DNA was more striking when the same concentration of disassembled ghosts was used (curve b). Figure 1B shows the sedimentation patterns of these DNAs through alkaline sucrose gradients, showing that slow sedimentation of [:‘2P]T4 DNA was again more distinct with disassembled ghosts and that more singlestrand breaks were inserted than doublestrand breaks on the DNA after incubation. The sedimentation of [:‘2P]T4 DNA varied depending on the concentrations of ghost preparations or periods of incubation. After prolonged incubation, the average segment length of polynucleotide chains was shorter than one-hundredth unit length of T4 DNA. In some experiments, the nucleolytic activity was conveniently expressed by the ratio of the difference between the running distance of
FIG. 1. The endonucleolytic activity of ghosts. [32PlT4 DNA (2 pg/ml) was incubated with 4.5 x IO” phage equivalents per ml of sonicated (a) or disassembled (b) ghosts of T4. The reaction mixtures were centrifuged through neutral (A) or alkaline (B) sucrose gradients at 40,000 rpm for 1.5 hr. IsHIT DNA was added as a reference (c). The three curves were superimposed by calculating the fraction of maximum L3HlDNA as 0 and the fraction of the meniscus as 1.
[32PlDNA and 13HlDNA to the running distance of 13HlDNA. When the average relative length of r3*P]T4 was estimated from this and other experiments according to the equation of Burgi and Hershey (1963) and Abelson et al. (1966), the ratio of the numbers of single-strand breaks to those of double-strand breaks was approximately 10 to 15. In these experiments, acid-insoluble 32P-activity was measured before and after incubation. This and another observation that single-stranded circular fd DNA became slowly sedimented as will be seen later, indicate that cleavage of’ [32PlT4 DNA is not exonucleolytic but endonucleolytic. As the control experiments, the nucleolytic activity of cell fraction, prepared as described in Materials and Methods, of uninfected E. coli B and 1100, and from T4-infected E. coli 1100, was assayed under the same condition. As seen in the alkaline sedimentation patterns of [32PlT4 DNA after incubation (Fig. 21, there was a slight or no endonucleolytic activity in cell fractions of uninfected E. coli 1100, while strong activity was seen in cell fractions of E. coli B. This is compatible with the fact that E. coli 1100 is an endo I- and E. coli B is an endo I+ strain; the nucleolytic activity of 1100 was detectable, however, at a higher concentration, 5 x 1Ol2 phage equivalent protein per milliliter. Unexpectedly 90% of the radioactivity remained as acid-insoluble after incubation with cell fractions of E. coli B, suggesting that exonucleolytic and endonucleolytic activities other than endonuclease I of host cells were not detectable under the present conditions. The nucleolytic activity of ghosts was also examined after the treatment with CsCl, since such treatment was used in the preparation of cell fractions. The activity was slightly lower than that of disassembled ghosts but higher than that of sonicated ghosts. The nucleolytic activity was not detectable in cell fractions of E. coli 1100 infected with T4 and subsequently aerated at 37” for 25 min. This may be understood if it is assumed that the nucleolytic substance was not synthesized in sufficient quantities by 25 min after infection. The above experiments suggest that the endonucleo-
FRACTION FIG. 2. The endonucleolytic activity of cell fraction. 13*PlT4 DNA (2 pg/ml) was incubated with cell fraction (4.5 x 10” phage equivalents of protein per milliliter and centrifuged after addition of [3H]T4 DNA through alkaline sucrose gradients at 40,000 rpm for 1.5 hr. (a) 13*P]T4 DNA after incubation with cell fraction of uninfected E. coli 1100; (b) 13*PlT4 DNA after incubation with cell fraction of uninfected E. coli B; (cl 13*P3T4 DNA after incubation with cell fraction of E. coli 1100 infected with phage and aerated for 25 min; (d) [3H]T4 DNA.
lytic activity is not due to host enzyme. In addition, the responsible substance is associated with phage structures but is not active unless the structure of the virion is disrupted. To confirm the latter hypothesis the following experiments were carried out. Ghosts were sedimented through a neutral glycerol gradient, and fractions were collected from the bottom of the tube. Evennumbered fractions were used for titration of infectious phages which still remained after osmotic shock and for assay of protein concentrations. As seen in Fig. 3, protein distributed in two peaks. The rapidly sedimenting fractions contained infectious phages, and such phages constitute approximately 20% of the amount of protein in these fractions, indicating that they were composed of DNA-containing ghosts and aggregation of ghosts besides phage particles. Odd-numbered fractions were used for assay of the endonucleolytic activity. They were incubated with fd DNA and treated by heat as described in Materials and Methods, and the infectivity of fd DNA was assayed with spheroplasts. The nucleolytic activity was expressed as a per-
FIG. 3. Sedimentation of ghosts and the nucleolytic activity. A ghost preparation was sedimented through a 4-ml glycerol gradient (10 to 30% in 0.1 M NaCl-0.02 M phosphate buffer at pH 6.0) by centrifugation at 15,000 rpm for 1 hr. Ten-drop fractions were collected from the bottom. Infectious phages (a) and protein (b) were titrated in the even-numbered fractions. The nucleolytic activity (c) was measured in the odd-numbered fractions by spheroplast assay of fd DNA as described in Materials and Methods and was expressed as a percentage of decrease of infectivity of the DNA.
centage of the decrease of infectivity of fd DNA incubated with the fraction to that of infectivity of fd DNA incubated without the fraction. As seen in the figure, its distribution was grossly parallel to that of protein. The activity would be underestimated in this method, since contaminating double-stranded DNA would interfere with the reaction. This may be a possible explanation for the low activity in the rapidly sedimenting peak fractions. On the other hand, the activity would be overestimated when a spheroplast was coinfected with fd DNA and abortive T4 phages, since fd is excluded by T4 (Rye, personal communication). The above experiment showed that the endonucleolytic substance is associated with ghost structures. The next experiment was carried out to fjnd if unbroken phages contain such an activity. To prevent plaque formation in the spheroplast assay, uv-irradiated phages or an amber mutant of early genes, am X5, was used. From the same preparative batch, an aliquot of phages was disassembled with
guanidine and another aliquot was irradiated by uv (1 x lop4 survival). Fd DNA was incubated with various concentrations of those phages or of am X5. Infective phages still remaining after uv-irradiation were inactivated by the heat treatment after incubation. Survival of fd DNA infectivity was titrated by spheroplast assay (Fig. 4). Infectivity of fd DNA was rapidly lost with increasing amounts of disassembled phages but only slightly with uvphages or am X5. The decrease of the latter at higher concentrations may be overestimated as described above, if we consider that the concentration of spheroplasts in the incubation mixture was derived from approximately 1 x 10” cells/ml. These experiments indicate that intact phages have no nucleolytic activity but when phages are broken the activity becomes distinct, and that the responsible substance is associated with ghosts. Effect of pH on the Endonucleolytic Activity Effect of pH on the endonucleolytic activity was examined by using disassem-
PROTEIN(X?d’PHGE EQIML) endonucleolytic activity at various concentrations of phages and disassembled phages. fd DNA was incubated with disassembled phages (a); uv-irradiated phages (b) (1 x IO-“ survival); or am x 5 cc). Infectivity of fd DNA was assayed with spheroplasts. Infective particles remaining in uvphages were inactivated by heat treatment at the termination of incubation. FIG.
bled ghosts, which were obtained from phage grown on E. coli 1100 (endo I-). The optimum pH for single- and double-strand breaks was the same, 6.0, which is clearly different from that of cell fraction, presumably of endonuclease I, prepared from E. coli (Fig. 5). Since the enzyme activity of disassembled ghosts from phage grown on E. coli B had the same optimum pH, it is likely that endonuclease I of host cells is completely removed from phage particles by the present purification procedure. Heat Sensitivity The endonucleolytic activity of disassembled ghosts was not inhibited by heating at 56” for 15 min. Heating at 65” for 5 min completely inhibited double-strand breaks, but slight activity for singlestrand breaks still remained after 15 min of treatment.
To compare the effect of glucosylation, nonglucosylated 132PlDNA was extracted from 32P-labeled T4* which had been grown on E. coli W4597 (Fukasawa and Saito, 1964). A mixture of L3HlT4 DNA and L3’PlT4* DNA (5 pg/ml each) was incubated with disassembled phages and sedimented through alkaline sucrose gradients (Fig. 6). Nonglucosylated DNA was cleaved better than glucosylated DNA, the difference being small but reproducible. Disassembled phages cleaved singlestranded T4 DNA, but less actively than double-stranded T4 DNA. Cleavage of double-stranded DNA of T3 and of singlestranded DNA of fd was also noted. In this case again, double-stranded DNA seemed to be a better substrate than singlestranded DNA (Table 1). Effect of Other Substances Addition of S-adenosyl methionine and ATP, or yeast tRNA (a gift from Dr. M. Imai) to the complete system had no effect on the endonucleolytic activity of disassembled ghosts (Table 2). Omission of
P H FIG. 5. Effect of pH on the endonucleolytic activity. Acetate buffer was used for pH 4.2 and 5.0, cacodylate buffer for pH 6.0, and Tris buffer for pH 7.4, 8.0, and 9.0. [32P]T4 DNA (1 pg/ml) was incubated with 8.4 x 1O1’ phage equivalents of protein per milliliter of disassembled phages, or cell fraction of E. coli B and centrifuged with 13HlT4 DNA (as a reference) through neutral (curves a and c) and alkaline (curve b) sucrose gradients. The endonucleolytic activity was expressed as a ratio of the difference between the running distance of 13*P1- and 13HlT4 DNA to that of 13HlT4 DNA. (a) and (b) Disassembled phages; and (c) cell fraction of uninfected E. coli B
FRACTION FIG. 6. Comparison of glucosylated and nonglucosylated T4 DNA as substrate. A mixture of 13H]T4 DNA and [“*PlT4* DNA (5 pg/ml each) was incubated with 6.2 x 10” phage equivalents per milliliter of disassembled phages, and centrifuged through an alkaline sucrose gradient at 40,000 rpm for 1.5 hr.
ENDONLJCLEASE TABLE THE
T4 (3.81, T3 (0.21, fd (0.06),
Concn of protein (Phage equivalentslml)
Native Denatured Native Native
4.2 4.2 4.2 1.2 2.4
x 10” x IO” x 10” x 10” x 10”
Incubation periods (min) 30 30 30 30 180
Endonucleolytic activitya 0.31 0.20 0.57 0.10 0.43
fl After [“*P]DNA was incubated with disassembled phages, 1:‘HlDNA was added and centrifuged through alkaline sucrose gradients (5 to 20%). The endonucleolytic activity was expressed as a ratio of the difference between the running distance of lD2PlDNA and [“HIDNA to that of VHIDNA. TABLE EFFECT
OF VARIOUS ENWNUCLEOLYTIC
Complete” +SAM (0.1 md4) -Mg’t
0.12 0.19 0.17 0.20 0.00 0.00 0.00
0.50 0.53 0.53 0.50 0.20 0.00 0.20
+tRNA (500 pg/ml) +EDTA (20 mM) +SDS (0.2%) +Trypsin (50 pg/ml)’ I) After incubation [“HIT4 DNA was added and centrifuged through neutral and alkaline sucrose gradients (5 to 20%). The endonucleolytic activity is expressed as in Table 1. * The complete reaction mixture was composed of 2 pg/ml of [“ZP]T4 DNA, 7 x 10” phage equivalents per ml of disassembled ghosts, 0.005 M MgSO+ and 0.04 M cacodylate buffer at pH 6.0 in 0.2 ml of total volume. V Trypsin (3x crystallized, Worthington) was added 30 min before addition of [52PlT4 DNA.
Mg”+ from the complete system or an increase of Mg’+ to 0.01 M also had no effect, though 0.005 M MgSO, was added to the reaction mixture in the present experiments. Addition of EDTA or SDS inhibited the enzyme activity. The inhibitory effect of EDTA varied from preparation to preparation of ghosts, possibly due to contamination of divalent cations which are re-
quired for the reaction. Prior incubation of disassembled ghosts with trypsin (3 x crystallized, Worthington) inhibited the nucleolytic activity. It is worth noting that when intact phage was treated with trypsin before disassembly, no inhibitory effect was observed. The Enzyme Activity
of Defective Capsids
It is well known that upon lethal infection with am and ts mutants, defective capsids are formed. DNA maturation occurs with some classes of mutants but not with others. Therefore, it is of interest to know if defective capsids have the the enzyme activity. All defective capsids were obtained by infection of E. coli 1100 with am mutants of 37”, and capsids of tsL51 (gene 17) were obtained at 30 and 42”. DNA of the wild type and ts L51 phages grown at 30” were removed by osmotic shock and ghosts were sonicated. Other capsids were prepared as described in Materials and Methods, and sonicated. The experimental results are summarized in Table 3. Capsids of mutants of genes 4, 10, 14, 16, and 49 had as high an activity of enzyme which produces single- and double-strand breaks as ghosts of the wild-type, whereas capsids of three am mutants of gene 17 had no or a slight activity. Capsids of mutant am NG178 of the same gene had comparable activity to those of the wild type. This mutant am NGl78, distal to gene 16, could grow well for unknown reasons on the host bacteria 1100; the efficiency of plating on this strain and the burst size were close to those on CR63, whereas this was not the case for the other am mutants. This result would explain why the enzyme activity is relatively high with capsids of am NG178. Therefore, we can conclude that capsids formed in the absence of gene l7-product are not associated with the endonuclease. This conclusion is further supported by another experiment using a ts mutant of gene 17; the enzyme activity of the capsids formed with this mutant at 30” was comparable to that of wild capsids, but the activity of capsids formed at 42” was much reduced. Temperature sensitivity of the en-
W am N112 (4) am B255 (10) am B 20 (14) am N 66 (16) am NG 507 (16) am NG 10 (17) am N 56 (17) am NG60 (17) am NG178 (17) ts L 51 (17)’ ts L 51 (17F am E727 (49)
0.35 0.29 0.37 0.33 0.29 0.41
0.00 0.05 0.37
0.06 0.33 0.08
n Capsids were prepared by infection at 37”, except in c (30”) and d (42”). b The endonucleolytic activity is expressed as in Table 1.
zyme activity of ts L51 capsids formed at 30” was compared to that of the wild type: There was no significant difference. Preparations of defective capsids were inspected before sonication by electron microscopy (by Fujisawa and Matsuo). More than 80% were empty capsids without tails, but preparations were contaminated with empty capsids with tails and a small fraction of DNA-containing capsids. The least contaminated was the preparation of am B255 (gene 10); approximately 95% were empty capsids without tails and 5% were those with tails. Since the enzyme activity of these capsids was as active as that of ghosts of the wild-type, the enzyme is assumed to be associated with the head capsids and not with tails. DISCUSSION
Several T4-specific endonucleases have been found, and some of them have been purified and characterized. The endonuclease of T4 capsids described here is appar-
ently a new type. Since it cleaves native T4 DNA, it is different from endonucleases II (Sadowski and Hurwitz, 1969a), IV (Sadowski and Hurwitz, 1969b), and V (Yasuda and Sekiguchi, 1970). The enzyme also cleaves denatured T4 DNA and fd DNA in contrast to endonuclease III (Altman and Meselson, 1970) which can attack native T4 DNA but neither denatured T4 DNA nor $X-174 DNA. Frankel et al. (1971) found an endonuclease in T4-infected cells, the synthesis of which is controlled by gene 49. The capsid endonuclease was found in gene 49- capsids but not in gene 17- capsids (Table 3). Furthermore, these enzymes have different optimum pH and substrate specificities, and the activity of the gene 49-controlled enzyme was not detectable in capsids (manuscript in preparation). When doublestranded T4 DNA, glucosylated or nonglucosylated, was used as a substrate, the ratio of single- to double-strand breaks was 10 to 15. This implies that doublestrand breaks do not result from random accumulation of single-strand breaks. Since the optimum pH for both kinds of breaks was the same, double-strand breaks may be caused by a single enzyme. But the possibility is not ruled out that more than a single enzyme is involved. The present experiments have shown that the endonuclease activity was very weak with purified intact phages, but became distinct when ghosts were disrupted or disassembled, and cosedimented with ghosts. These facts clearly indicate that the enzyme is associated with the phage structure. The enzyme activity was evident with tail-less capsids of a gene 10 mutant, indicating possible localization of the enzyme on the phage head structure. On the other hand, the enzyme activity was very low with capsids of four mutants of gene 17, when grown under the restrictive condition. This indicates that gene 17 controls synthesis of the enzyme which is a component of capsids or synthesis of a structural component which enables the enzyme to associate with capsids. That the enzyme is not simply adsorbed to the capsid surface is supported by the finding that trypsin digestion of intact phages had no
effect on the enzyme activity. The enzyme activity of disassembled ghost, however, was inhibited by trypsin digestion. According to Snustad’s criterion (19681, the product of gene 17 is stoichiometric in its function, suggesting that it is a structural component, though no one has proved this yet. A similar situation, where a protein has a dual function as a structural component and an enzyme, has been described for an endonuclease of adenovirus (Burlingham et al., 19711, which constitutes pentons of the virus particles. Kozloff et al. (1970) found that dihydrofolate reductase was a structural component of tail plates of T4 and its enzymological property was correlated with phage viability. Conversion of precursor DNA of T4 to mature DNA correlates with head formation; it does not occur when assembly of capsid components is defective and also when assembly of capsids and DNA is abortive. For the latter step, the products of genes 16, 17, and 49 are required (Frankel, 1968; Minagawa and Fujisawa, 1968a,b; and Fujisawa and Minagawa, 1971). Assuming that the length of mature T4 DNA is determined by the size of capsids, these gene products should be pertinent to packaging a portion of precursor DNA in capsids or to cleaving the DNA strand in two portions, the packaged and the outer remaining. Capsids filled with DNA were observed in thin sections of cells infected with a mutant of gene 49 (Granboulan et al., 1971), and a significant amount of DNA associated with capsids was detectable by sedimentation and density analysis of gene 49- capsids which had been treated with DNase (Luftig and Ganz, 1972a,b; Laemmli and Favre, 19731, indicating that gene 49-controlled protein is not a packaging factor. DNA-filled capsids were not observed in thin-sectioned specimens of cells infected with mutants of genes 16 and 17 (Granboulan et al., 1971; Simon, 1972; Luftig and Ganz, 1972b), but those capsids sedimented with a small amount of DNase-resistant DNA (Luftig and Ganz, 1972). This may imply the following possibilities: Either DNA is not encapsidated in the absence of products of those genes, or alternately, if DNA is en-
capsidated it leaks out of capsids because of a fragility of the complex (Granboulan et al., 1971). If gene 17- capsids are the remaining structure after precursor DNA leaks out, the capsid nuclease is assumed to be one of the candidates for determining the length of T4 DNA. The present experiments have shown that the enzyme cleaves T4 DNA endonucleolytically by double-strand breaks, and its activity is associated with all capsids but those of mutants of gene 17. The length of the reaction product was less than one-hundredth of that of T4 DNA. This implies that the enzyme does not have a strict requirement for the base sequence and structure of the substrate DNA, being in strong contrast to the product of gene A of h which has a ter function (Wang and Kaiser, 1973). These properties of the capsid nuclease are consistent with the “Headful Model” (Streisinger et al., 1967) for T4 formation. The present experiments do not provide information as to whether the cleavage is catalyzed by a single or more enzymes. One might suspect it is carried out by cooperation of the capsid nuclease and gene 49controlled enzyme, which is another candidate for the cleavage of precursor DNA (Frankel et al., 1971). This is, however, ruled out, since the latter enzyme activity was not detectable in capsid preparations and the “nicked” T4 DNA by the capsid nuclease could not be a substrate for the latter enzyme (manuscript in preparation). If gene 17- capsids are the product which is formed when DNA packaging is blocked, the capsid endonuclease could be assumed to be essential for the initial step of assembly of precursor DNA and capsid. Experimental evidence has accumulated showing that heads of various phages are formed by assembly of precursor capsids and DNA. Laemmli and Favre (1973) have observed that several kinds of particles are formed by labeling with [“Hlleucine in wild-type infected cells, and such labeled particles are converted to phage particles after chase. According to Bijlenga et al. (1973, 1974), 24--tau particles are transformed to phage particles in a conservative mode. Matsuo-Kato and Fujisawa (1975)
have found that in the absence of DNA synthesis of a T3 mutant, a capsid-like structure is synthesized and accumulated. After restoration of DNA synthesis this structure is converted conservatively to T3 particles and another empty capsid-like structure. Furthermore, the particles formed in the absence of DNA synthesis are convertible to infectious T3 by in vitro complementation (Miyazaki and Minagawa, unpublished data). Direct evidence that DNA is packaged into precapsid has been provided by Kaiser and Masuda (1973), who have shown in vitro assembly of petit A and DNA in the presence of some other factors. One of the essential factors is the product of gene A (Kaiser and Masuda, 1973). The gene A product has been proved to have an endonucleolytic activity, cleaving covalently closed circular A-DNA to yield cohesive ends (Wang and Kaiser, 19731, and it is assumed that this product is involved in the initial assembly step of the particles and DNA (Kaiser et al., 1975). This schedule is analogous to the above assumption for T4 capsid endonuclease. In this case, gene 17- capsids would be an abortive side product, which results from nonassembly of precapsid and DNA. We have tried to see if particles of ts mutants of gene 17 formed at 42” are converted to DNA-containing particles after shift-down to 30”. So far all efforts have been unsuccessful. Similar results have been reported by Luftig and Ganz (1972b). Finally, the capsid nuclease may have both of the above functions. It could initiate association of precursor capsid and DNA, and packaging would proceed in a still unknown way. When the capsid was filled with a part of concatemer DNA, the part of the DNA that remains outside would be cleaved by the enzyme. It is of interest to note that capsids might play some role in determining even the length of phage DNA which has cohesive ends. A mutant of A has been isolated which has two functional cohesive end sites (Emmons, 1974; Feiss and Campbell, 19741, suggesting that the length of A DNA is determined not only by ter-function but also by some other factor. The factor may be capsid size (Emmons, 1974). In the in vitro assembly experiment using P2 pro-
tein and P4 DNA, 90% of infectious assembled contained three copies DNA in a P2 capsid, which is three larger than a P4 capsid (Pruss et al.,
phage of P4 times 1974).
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