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The arrangement of DNA in lambda phage heads
J. Mol. Biol. (1971) 62, 493-502
The Arrangement of DNA in Lambda Phage Heads I. Biological Consequences of Micrococcal Nuclease Attack on a Portion of the Chromosome Exposed in Tailless Heads VERNON C. BODE? AND FRANCES D. GILLIN$ of Biology, Kansas State University Manhattan, Kansas 66502 ati Department of Biochemistry, University of Maryland Medical School Baltimore, Md 21228, U.X.A. Division
(Received 1 September 1970, and in revised form 14 July 1971) If they lack tails, lambda heads are sensitive to inactivation by micrococcal DNase. Nuclease-treated heads still. join with tails, producing phage which adsorb to host cells and inject their DNA normally. The injected chromosomes are defective since both lytic phage production and lysogeny are decreased 85 to 95%. The intracellular circularization of ADNA is the first step in phage development altered by nuclease damage. When extracted from an infected cell, most parental molecules from nuclease-treated heads are of normal size but linear. The failure to cyclize
in, wivo suggests
the cohesive
ends are damaged.
1. Introduction Little is known about how DNA is arranged in a phage head to permit both very compact packaging and rapid injection. During h morphogenesis, the DNA-filled heads and the tails are synthesized by independent biochemical pathways and then joined (Weigle, 1966,196s). When mutants with a suppressor-sensitive (SW) mutation in a tail gene are grown in non-permissive hosts, the resulting lysates lack tails or completed phage but contain normal amounts of heads. Lambda heads containing mature phage DNA molecules can be purified from such lysates and quantitatively joined to isolated tails to produce infectious phage (Harrison & Bode, unpublished results). During
injection,
X DNA passes from the head through
the tubular
tail and into the
host cell (Caro, 1965). Therefore, either the icosahedral head contains a hole at that vertex where the tail attaches or tail attachment produces a hole. Normally, the protein coat of phage h completely shields the encased DNA molecule from hydrolysis by external nucleases. If h heads contain a preformed opening at the tail attachment site, then a nuclease might be able to penetrate the head membrane through the hole or t Please send reprint requests to Division of Biology, Kansas State University, Manhattan, Kansas 66502, U.S.A. $ Present address: Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Md 20014, U.S.A. 493
494
V. C. BODE
AND
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DNA might protrude from the hole. In either case, a region of the DNA molecule packaged near the opening would be uniquely susceptible to nucleases. Therefore, a study of the extent and site of nuclease attack on the DNA in h heads may yield information on the physical arrangement of packaged phage DNA. This paper demonstrates that micrococcal nuclease can damage the DNA in tailless heads and considers the physiological consequences of micrococcal nuclease treatment. The accompanying paper (Gillin & Bode, 1971) describes the nature of the alteration and its location on the h DNA molecule.
2. Materials and Methods (a) Media
a&
buffers
K medium contains 1 g NH,Cl, 6 g Na,HPO*, 3 g KH,P04 and 1 g NaCl in 650 ml. glass-distilled water. After autoclaving and cooling, the following sterile solutions are added: 2 ml of 1 M-MgSO,, 10 ml. of 2.5 x lo- 2 M-CaCl,, and 150 ml. of 10% Casamino acids which have been treated with Norit A to decolorize and to remove aromatic amino acids. h diluent consists of 10-e M-pOtaSSiLUD phosphate buffer (pH 7), 10m2 m.-Mgcl, and 10 pg bovine serum albumin/ml. TPBE buffer contains 10e2 M-Tris*HCl (pH 7*1), 10T2 M-putrescine+HCl, 10 mg bovine serum albumin/ml., and 2 x 10T4 M-EDTA. TCM buffer contains Tris*HCl (pH 7-l), CaCl, and MgSO, each at a concentration of lo-2
M.
Tris-Mg is 10m2 m-Tris*HCl with 10m2 M-MgSO&, pH 7.1. Tris-EDTA is 10m2 M-Tris.HCl with 10e3 M-EDTA (sodium salt), Tryptone broth contains 10 g Bactotryptone and 5 g NaCl/l. (b) Purification
pH 7.1.
of h heads
Heads were prepared according to the procedure of Harrison & Bode (unpublished information). A liter culture of W3350 pm-( XsusJ27) was grown in K medium at 37°C to 5 x lo* cells/ml. The cells were chiied, induced with ultraviolet light and then returned to lysis was complete. (When preparing heads with the shaker bath until the A,,, indicated 3H-labeled DNA, 1 mCi of [methyZ-3H]thymidine was added per liter of culture at 20 mm after induction.) After chilling, 1 ml. of CHCI, was added and the debris removed by centrifugation. One liter of lysate was placed in a 2-l. separatory funnel and mixed with 30 g NaCI, 10 ml. of 1 M-putrescine*HCl, 10 ml. of 20% (w/v) sodium dextran sulfate and 250 ml. of 30% (w/v) polyethylene glycol. After 16 hr at 4”C, the heads accumulate as part of a gelatinous mass at the polymer interface. The bottom phase (dextran sulfate) and the interphase material were collected and centrifuged at 5000 g for 15 min. The contaminating upper and lower phase material was poured away from the solid middle layer which was then dissolved in 10 ml of TPBE buffer. Dextran sulfate was precipitated by the addition of 1.6 ml. of 3 M-KCl. The precipitate was removed after 20 hr at 4°C. The supernatant was still very viscous and was not processed further until a second precipitate formed and the viscosity dropped (7 to 14 days). After this occurred, the solution (4 ml. per tube) was layered on a gradient of 15 to 35% sucrose in TPBE buffer and centrifuged at 5°C in the Spinoo SW25.2 rotor for 90 min at 25,000 rev./min. The lower light-scattering band was collected and usually contained about 5 x 1011 heads/ml. (0) Preparation
of X tail8
The source of tails in these experiments was W3350 pm- (hszcsA32) grown in K medium and induced as described for heads. After the addition of CHC13 and the removal of cell debris by centrifugation, lysates were stored at 4°C. Crude lysates contained 8 x lOlo to 2 x loll tails/ml. and the tails remain active for months (Harrison & Bode, unpublished results).
THE
ARRANGEMENT
OF DNA (d) In vitro
IN
LAMBDA
PHAGE
HEADS
495
head-tail joining
To assay heads, 0.1 ml. of an appropriate dilution of heads was mixed with 0.1 ml. of undiluted tail-containing lysate and 0.8 ml. of K medium or TPBE buffer. After incubation at 4’C for 20 hr, the mixture was diluted and plated on the sensitive strain, Escherichiu coli CFOO. In this study, a h head was designated as active or infectious if it combined with a tail to yield a particle that formed a plaque on an appropriate lawn of sensitive bacteria. For preparative-scale reactions, h heads were mixed with an excess of tails and incubated overnight at 4°C. The phage produced were then purified. (e) Phage purification
on CsCl step gradients
Phage were purified and concentrated on gradients containing successively layered solutions of CsCl with the following densities: first, 4 ml. of I.7 g/ml., then 9 ml. of 1*5g/ml., then 11 ml. of 1.3 g/ml. and finally 115 ml. of phage sample. All solutions were prepared in Tris-Mg buffer. The gradients were centrifuged at 5°C in the Spinco SW27 rotor for 3 hr at 22,000 rev./min. The phage band was collected and dialyzed against Tris-Mg buffer. (f) Micrococcal
nucleate treatment from Staphylococcus aureus was purchased
Crystalline micrococcal nuclease purified from the Worthington Corporation. Stock solutions of the enzyme (1 mg/ml.) were prepared in 1O-2 M-Tris-HCl (pH S), containing 0.5 mg bovine serum albumin/ml. and stored frozen at -70°C. Assay of MN? by the Worthington procedure showed that the enzyme preparation used contained at least 1.1 x lo4 units/ml. Purified )( susJ27 heads were suspended at a concentration of approximately 3 x lOlo particles/ml. in 0.05 M-Tris.HCl (pH 9), 0.5 mg bovine serum albumin/ml., 10m3 M-CaCl, and 10e2 M-putrescine~HC1. Microcoocal nuclease (10 pg/ml.) was added to one half of the mixture and both halves were incubated at 37°C for 30 min. After chilling, the heads were joined to tails and the phage purified as above. (g) Terminology
MN-phczge are phage particles prepared by joining tails to h heads that have been treated with micrococcal nuclease, as described. ContToZphage have received treatment and purification identical to MN phage, except that micrococcal nuclease was omitted. MN-DNA is DNA extracted from micrococcal nuclease-treated heads or MN-phage. Control DNA is DNA extracted from the control heads or control phage.
3. Results (a) Head inactivation
by micrococ& nuclease
Among a variety of nucleases initially screened, only micrococcal nuclease damaged the DNA packaged in X heads extensively enough to impair subsequent plaque formation (Table 1). Purified XsusJ27 heads were incubated with one of six nucleases and then allowed to join with &us A32 tails to form phage. Under the conditions employed, greater than 90% of untreated heads join with tails (Harrison & Bode, unpublished results). In contrast to the MN-sensitivity of heads, completed phage particles are not afFected by a 50-fold greater concentration of this n&ease (Table 2). The inactivation has the calcium ion requirement of MN (Cuatrecasas, Fuchs & Anfinsen, 1967) and has a greatly reduced rate at 0°C. The kinetics of inactivation at several enzyme concentrations is shown in Figure 1. Reactions were followed for 6 hours but only the first 32 minutes are shown. In all cases where enzyme was present, a maximum inactivation of 95% was observed, This occurs in 8 minutes with 10 units of enzyme. With O-4 unit, 128 minutes are required t Abbreviation
used: MN, micrococcal
nuclease.
496
V. C. BODE
AND
F. D. GILLIN
TABLE 1
Nucleate treatment of heads Nuclease Micrococcal Pancreatic
O/oActive
DNase (2 units) DNase (2 pg)
7.8 80
DNase II, porcine
spleen (350 units)
Phosphodiesterase
II, bovine spleen (0.8 unit)
X Exonuclease Endonuclease
heads
(3 units) I, E. co&i (0.01 unit)
110 80 103
92
Heads were incubated with the various enzymes for 30 min at 37°C. Buffers and divalent metal ions varied according to the requirements of the enzyme but all solutions contained 10-s M-putrescine*HCl and at least 0.5 mg bovine serum albumin/ml. to help stabilize the heads. The total volume was O-2 ml. In every case, controls lacking only enzyme retained at least 80% of their initial titer of active heads. The activities of exonuolease and of endonuclease I were determined by the methods of Little (1967), and Lehman, Roussos & Pratt (1962), respectively. Both enzymes were kindly given and assayed by Dr Marcus Rhoades. The remaining nucleases were purchased from the Worthington Corporation and the activities shown are as supplied by them.
TABLE 2
Sensitivity of heads and resistance of phage to inactivation by micrococcal n&ease Percentage infectious particles remaining
Treatment
A. Phage 119
(a) No enzyme
109
(b) MN (100 units/ml.) B. Heads (a) MN (2 units/ml.)
19
(b) MN (O-5 unit/ml.) (c) Omit MN
57 90
(d) Omit Ca2+ (MN = 2 units/ml.) (e) Incubate
at 0°C (MN
= 2 units/ml.)
Incubation
99 95
was for 30 min at 37’C except in (e).
but the same level is reached. The extent of inactivation varies between 85 and 95% in different experiments. It is unaltered by the addition of more enzyme either initially or after the inactivation plateau is reached. The enzyme is stable during the reaction and will inactivate a second portion of heads, but again only to the 95% level. Later, we will discuss the origin of this residual activity.
497
THEARRANGEMENTOFDNAINLAMBDAPHAGEHEADS
I
I
20
30
Time (mm) FIG. 1. The kinetics of head inactivation at various MN concentrations. Purified X heads were incubated under standard conditions with varying amounts of MN. Samples were removed and diluted lOOO-fold in cold TPBE buffer containing crude tails. Head-tail joining proceeded for 20 hr at 5”C, then titrations were performed.
(b) Properties of MN-heads The possibility that MX-inactivated heads are unable to join with tails was investigated. If tails attach only to the 5 to 15% of the heads that remain active, the phage particles formed would have normal infectivity but the yield would be only 5 to 15% that of the control. If, on the other hand, the treated heads combine normally with tails but only 5 to 15% of these particles give rise to plaques, the yield of completed phage particles would be normal compared to an untreated control but their specific infectivity (in plaque-forming units per particle) would be low. Phage prepared by incubating MN-treated [3H]thymidine-labeled heads with excess tails were purified on CsCl step gradients. Heads that have not joined with a tail lose their DNA during this purification procedure. If any heads survived, they would seek a higher density TABLE
3
SpeciJc infectivity of puri$ed p?mge Total recovery Phage origin Control
(cts/min)
heads
MN-treated
heads
(P.f.U.)T
(p.f.u./cts/min)
%
87,800
4.6 x 1011
5.1 x 10s
100
88,600
5.6 x 10n’
6.1 x IO5
12
Purified Xsus-J heads, labeledwith [methyl-3H]thymidine, were suspended in 5 x IO-” M-TrhHC1 (pH 9.0), 10V3 &r-CaCl,, 10m2 M-putrescineHC1, and 5 mg bovine serum albumin/ml. The mixture was divided in half and 22 pg MN/ml. was added to one half. Both the treated and control head suspensions were incubated at 37’C for 30min, then chilled and mixed with lysate oontainingexcess, X sus-A tails. The phage formed after 4 days incubation at 5”C, were purified by centrifugation on CsCl step gradients. 72% of the original tritium counts were recovered. t p.f.u., Plaque-forming units.
498
V. C. BODE
AND
F. D. GILLIN
stratum than phage. The yield of completed MN phage particles, as estimated by the tritium in labeled DNA, is the same as that obtained from an equal number of heads subjected to identical treatment except for the omission of nuolease, but the specific infectivity is only 12% of the control (Table 3). Thus, MN-inactivated h heads join with tails as efficiently as untreated heads, but they yield defective phage particles. (c) Properties of MN-phage (i) Adsorption and DNA injection In order to measure the efliciency with which these particles adsorb to sensitive bacteria and inject their DNA, [3H]thymidine-labeled MN-phage and control phage were mixed with sensitive bacteria. Approximately 90% of both the control and MNphage adsorb to the bacteria (Table 4). After shearing these complexes to remove phage TABLE
4
Adsorption and injection of micrococcal nuclease-treated and control phage Control
MN-phage
(a) (b) (c) d)
Infecting phage Unadsorbed phage Cts/min removed by blendor shearing Injected phage: (a)-((b) + (c))
(&s/mm)
%
15,275 1597 2720 10,958
100 10 1s 72
(ots/min) 15,000 1334 2330 11,336
phage % 100 S 16 76
Sensitive E. coli W3104 bacteria grown in Tryptone broth to 7 x lo* cells/ml., were sedimented and resuspended in l/5 vol. of h diluent. Half of the cells were mixed with an equal volume of [3H]thymidine-labeled control phage (multiplicity of infection = 4). The other half was infected similarly with MN-phage. Both were incubated at 37°C for 15 min to allow the phage to adsorb and to inject their DNA. The cells were then pelleted and samples of the supernatant solution, contaming unadsorbed phage, were dried and counted to determine adsorption (line (a) minus line (b)). The infected cells were resuspended in X diluent and sheared in a chilled Waring blendor for 6 mm. Again the cells were sedimented, and samples of both the supernatant and the pellet counted; the supernatant values are listed on line (c) above.
with uninjected DNA (Bode & Kaiser, 1965), 76% of the original DNA from control phage and 72% of that from MN-treated phage still sediments with the cells. Thus, the low plating efficiency of MN-phage cannot be attributed either to poor adsorption or to impaired injection. (ii) Infectivity
in a heteroimmune lysogen
If the MN phage are defective because they inject damaged DNA, it might be possible to increase their plaque forming ability by marker rescue or complementation. Table 5 shows that the plating efficiency of MN-phage on a heteroimmune lysogen is increased fivefold over the etllciency on a sensitive host. In other experiments, not shown in the Table, where the average burst ranged from 20 to 30 phage per cell, the burst size of MN-phage was at least 80% that of control phage both in a sensitive and in a heteroimmune lysogenic host. Furthermore, singleburst experiments using all four combinations of MN and control phage with sensitive and lysogenic host cells failed to reveal a significant difference in the distribution of burst sizes. Single-step growth curves indicated that those MN-phage which grow, exhibit the same latent period as control phage.
499
THEARRANGEMENTOFDNAINLAMBDAPHAGEHEADS
TABLE 5 Relative plating
C600 C600 (21 hy) C600 C600 (21 hy)
heads
MN-treated
Plaque forming
Lawn
Phage origin Control
on sensitive and lysogenic
eficiency
heads
1.4 1.6 8.9 4.5
x x x x
bacteria
units/ml.
1012 1O1a lOlo 1011
y0 100 114 6.4 32.1
Purified MN-treated and control phage suspensions with an equal number of particles, as indicated by their absorption at 260 nm, were titrated using the indicated lawn bacterial at 37°C. The plating efficiency of control phage on the C600 lawn was taken as 100%.
(iii) The fate of MN-phage
infected
cells
Since lambda is a temperate phage, one possible explanation for the low plaqueforming efficiency of MN-phage is a greatly increased lysogenization frequency. Table 6 indicates, however, that both the lytic and lysogenic responses are inhibited more than 80%. It also shows that most of the MN-infected cells survive and produce lambda-sensitive clones.
TABLET Response of MN-phage-infected
cells
Control (no./1000 cells) Infecting phage Infected cells? Infectious centers Lysogenio cells Surviving cells (1) expected5 (2) found
MN (no./1000 cells)
320 270 200
640 470 380
450 360 26
900 590 49
(&
(&
(O&
(Oi61)j:
740 700
550 600
640 970
410 850
Strain C600, 6 x lo* cells/ml and MN or control phage at two multiplicities were mixed at 0°C in h dil. After 15 min for adsorption, the cells were warmed to 37°C for 10 mm then diluted tenfold into X dil with anti-X serum (K = 1). After 10 min at 37’C to neutralize any unadsorbed phage, the cells were diluted further then (a) plated for infectious centers on a C600 lawn, (b) plated without the lawn bacteria to measure total viable surviving cells and (c) spread with antiserum on the surface of an agar plate and grown overnight. The colonies on the latter plates were replica-plated on a C600 lawn then exposed to an inducing dose of ultraviolet light. Those colonies which produced phage were scored as lysogenio. Similar frequencies of lysogenization were obtained when individual colonies were tested for immunity as an indication of lysogeny. t Calculated assuming a poison distribution and 100% absorption. t Lysogenic cells per 100 infecting phage. 0 Total cells minus infected cells plus lysogenic cells.
(d) Covalently-closed
circle formation
after
infection
Soon after X infection at least 50% of the infecting DNA can be recovered in extracts as a covalently-closed twisted circular form, species I (Bode & Kaiser, 1965). Since MN-DNA is injected into bacteria normally and, at least under certain conditions, forms hydrogen-boncled circles in vitro (Gillin & Bode, 197 l), it was of interest to know if MN-DNA is converted to species I in vivo. DNA was extracted from sensitive
V. C. BODE
500
AND
I?. D. GILLIN
bacteria 15 minutes after injection with [3H]thymidine-labeled MN or control phage. Although sedimentation reveals a normal conversion of injected control DNA to the rapidly sedimenting closed circular form, very little of this species is detected in extracts containing injected MN-DNA (Fig. 2). A similar result is obtained when any complications of vegetative growth are eliminated by using an immune lysogen as the host. The recovery of intracellular 3H labeled MN-DNA was frequently 20% less than that of control DNA, but the gradients show no low molecular weight fragments as would be expected if MN-DNA were subjected to extensive degradation after injection.
i
(a)
BOC
60C
(b)
60C)
4oc 1 i
2oc
Fraction number
FIG. 2. Sedimentation of 3H-labeled DNA from MN-treated and control phage extracted after injection into a sensitive or into a lysogenic host. (a) Tritiated MN- or control phage (6 x lOa cts/ min) were suspended in h diluent and mixed with the sensitive strain, W3104, at a multiplicity of 12 particles per bacterium. After incubation at 37°C for 15 min, 70% of the tritium sedimented with the bacteria. The infected-cell pellet was suspended in Tryptone broth, shaken at 37°C for 15 min, and then sheared in a Waring Blendor to remove uninjected DNA. Only 46% (MN) and 56% (control) of the counts originally associated with the attached phage sedimented with the cells after growth and blending. The DNA was extracted and sedimented in sucrose gradients as described by Bode t Kaiser (1965). Since the relative position of a marker X DNA (3ZP-labeled) was identical in the two separate gradient tubes, the tritium profiles of MN and control DNA are superimposed and the position of the marker DNA indicated by an arrow. -e-e-, MN-phage; -- x -- x --, control phage. (b) The experiment was performed as described above except the host strain was an immune lysogen, CSOO(A), and the multiplicity was 8 particles per bacterium with 45% of the input tritium injected as measured by insensitivity to removal by shear forces.
THEARRANGEMENTOFDNAINLAMBDA
PHAGE
HEADS
501
(e) .MN-phage infectivity as a function of temperature As reported in the accompanying paper (Gillin & Bode, 1971), MN-DNA forms hydrogen-bonded circles in vitro. In TCM buffer, the T, for melting these circles is 36°C about 25 degrees lower than the T, of normal X DNA circles., Therefore, MN treatment probably removes severs1 bases from a single-stranded cohesive end of the DNA. If their replacement is essential for MN-phage infectivity, then the formation of hydrogen-bonded circles in viva would be necessary, as a first step, to provide the template for cellular repair enzymes. The in viva conditions for circle formation and opening are not known. However, the experiment shown in Figure 2 suggests that, if MN-DNA circles form, they are not repaired and covalently closed. If cyolization is an obligatory step for plaque formation and circles are meltmg in the cell at normal TABLE
7
Phage infectivity at different temperatures Temperature (“C) 32 37 40
Control
MN-treated (p.f.u./ml.)t 9.8 x lo9 6.2 x lo9 2.9 x 109
(%I 19-l 11.9 5.8
(p.f.u./ml.) 5.14 x 1010 5.21 x 10’0 4.96 x lOi0
(%) 100 100 100
Suspensions of purified MN-treated and control phttge with the s&me absorption at 260 nm were diluted in X diluent, mixed with 0.2 ml. of strain C600 lawn culture, and inoubsted for 15 min at 32 or 37 or at 40°C for 10 min. After plating, the Petri dishes remained at 25°C for 15 min while the soft agar layer hardened. Then the plates were incubated at the temperature of the previous incubation. t p.f.u., Plaque forming
units.
growth temperatures, the plating eficiency of MN-phage might change with temperature. Control phage plate with equal efficiences at 40,37 and 32°C (Table 7). In contrast, the efficiency of plaque formation by MN-phage decreases threefold between 32 and 40°C. This difference is consistent with the idea that lower temperatures enhance the infectivity of MN-phage by increasing circle formation which is necessary for repair of the MN damage.
4. Discussion Since joining with a tail renders &us-J heads completely resistant to inactivation by MN, the head’s tail attachment site is also the most likely site for nuclease attack on the DNA in the head. That MN treatment does alter the DNA is documented by genetic and biochemical observations. The increased plating efficiency of MN-phage on a lysogenic host compared to a sensitive host suggests they are damaged genetically. The absence of normal amounts of covalently-closed circles in vivo indicates that nuclease treatment changes the biochemical properties of the h DNA packaged in heads. The following paper (Gillin & Bode, 1971) will further document changes in the physical and biochemical properties of MN-DNA. It was shown here that MN-treated heads join normally to tails, yielding phage, most of which do not produce plaques even though adsorption and injection are normal. Several observations argue that the residual plaque-forming ability of MN-phage is a
502
V. C. BODE
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property of all the particles, rather than of a separate nuclease-resistant population. The plating efficiency of a given MN-phage preparation can vary from 5 to 30% depending upon physiological conditions that do not significantly effect the plaque-forming efficiency of control phage, e.g. plating on a heteroimmune lysogen or varying the plating temperature between 32 and 40°C. Since there is variation in the residual infectivity, it cannot be due to a fixed number of undamaged normal particles. A reduced amount of twisted circular DNA (l?ig. 2) is the earliest known difference in the life cycles of MN and control phage. To become species I, it is necessary for MN-DNA to form hydrogen-bonded circles in vivo, to undergo repair replication, and to be covalently sealed. Attributing the defectiveness of MN-phage to a decreased ability to form circles in vivo is consistent with the increased infectivity of MN-phage at lower temperatures where MN-DNA circle formation may be promoted. The residual infectivity, then, may represent the probability that an MN-DNA molecule can circularize and undergo repair. Although our primary purpose in characterizing MN-phage and DNA is to learn more about how h DNA is arranged in the head, it appears that they will be useful in other problems, particularly when the exact alteration in MN-DNA is known. Since the DNA can be injected into the cell normally, it provides a way to study the effect of this specific DNA lesion on in vivo repair, replication and recombination as well as gene function. It is not known whether heads are uniquely sensitive to MN because of the enzyme’s small size or because of some other property. Similarly, it is not known whether a small amount of DNA protrudes from the head and is attacked by MN or whether the nuclease partially enters the head. It is clear that even at high enzyme concentrations, or extended times, only a few nucleotides can be removed from the head DNA by this enzyme and that these are not available to several other n&eases. For example, pancreatic DNase-treated heads have been studied extensively (Wong & Bode, unpublished results) and contain DNA whose cohesive ends are completely intact. The authors wish to acknowledge the expert assistance given by Mm Christina Wong, Mrs Sarah Brown and Mr John Blotzer. This work was supported by grants from the National Science Foundation (GB6961 and GB25153) and the Public Health Service (Al06493 and GM18182). REFERENCES Bode, V. C. & Kaiser, A. D. (1965). J. MoZ. Biol. 14, 399. Cmo, L. (1965). l’i7iroZogy, 25, 226. Cuatrescasas, P., Fuchs, S. & Anlinsen, C. B. (1967). J. Biol. Chem. 242, 1541. Gillin, F. D. & Bode, V. C. (1971). J. Mol. BioZ. 62, 503. Lehman, I. R., Roussos, G. G. & Pratt, E. A. (1962). J. Biol. Chem. 237, 819. Little, J. (1967). In Methods in Enzymology, ed. by L. Grossman & K. Moldave, part A, p. 263. New York: Academic Press. Weigle, J. (1966). Proc. Nat. Acad. Sci., Wash. 55, 1462. Weigle, J. (1968). J. Mol. Biol. 33, 483.
vol. 12,
The Arrangement of DNA in Lambda Phage Heads I. Biological Consequences of Micrococcal Nuclease Attack on a Portion of the Chromosome Exposed in Tailless Heads VERNON C. BODE? AND FRANCES D. GILLIN$ of Biology, Kansas State University Manhattan, Kansas 66502 ati Department of Biochemistry, University of Maryland Medical School Baltimore, Md 21228, U.X.A. Division
(Received 1 September 1970, and in revised form 14 July 1971) If they lack tails, lambda heads are sensitive to inactivation by micrococcal DNase. Nuclease-treated heads still. join with tails, producing phage which adsorb to host cells and inject their DNA normally. The injected chromosomes are defective since both lytic phage production and lysogeny are decreased 85 to 95%. The intracellular circularization of ADNA is the first step in phage development altered by nuclease damage. When extracted from an infected cell, most parental molecules from nuclease-treated heads are of normal size but linear. The failure to cyclize
in, wivo suggests
the cohesive
ends are damaged.
1. Introduction Little is known about how DNA is arranged in a phage head to permit both very compact packaging and rapid injection. During h morphogenesis, the DNA-filled heads and the tails are synthesized by independent biochemical pathways and then joined (Weigle, 1966,196s). When mutants with a suppressor-sensitive (SW) mutation in a tail gene are grown in non-permissive hosts, the resulting lysates lack tails or completed phage but contain normal amounts of heads. Lambda heads containing mature phage DNA molecules can be purified from such lysates and quantitatively joined to isolated tails to produce infectious phage (Harrison & Bode, unpublished results). During
injection,
X DNA passes from the head through
the tubular
tail and into the
host cell (Caro, 1965). Therefore, either the icosahedral head contains a hole at that vertex where the tail attaches or tail attachment produces a hole. Normally, the protein coat of phage h completely shields the encased DNA molecule from hydrolysis by external nucleases. If h heads contain a preformed opening at the tail attachment site, then a nuclease might be able to penetrate the head membrane through the hole or t Please send reprint requests to Division of Biology, Kansas State University, Manhattan, Kansas 66502, U.S.A. $ Present address: Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Md 20014, U.S.A. 493
494
V. C. BODE
AND
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DNA might protrude from the hole. In either case, a region of the DNA molecule packaged near the opening would be uniquely susceptible to nucleases. Therefore, a study of the extent and site of nuclease attack on the DNA in h heads may yield information on the physical arrangement of packaged phage DNA. This paper demonstrates that micrococcal nuclease can damage the DNA in tailless heads and considers the physiological consequences of micrococcal nuclease treatment. The accompanying paper (Gillin & Bode, 1971) describes the nature of the alteration and its location on the h DNA molecule.
2. Materials and Methods (a) Media
a&
buffers
K medium contains 1 g NH,Cl, 6 g Na,HPO*, 3 g KH,P04 and 1 g NaCl in 650 ml. glass-distilled water. After autoclaving and cooling, the following sterile solutions are added: 2 ml of 1 M-MgSO,, 10 ml. of 2.5 x lo- 2 M-CaCl,, and 150 ml. of 10% Casamino acids which have been treated with Norit A to decolorize and to remove aromatic amino acids. h diluent consists of 10-e M-pOtaSSiLUD phosphate buffer (pH 7), 10m2 m.-Mgcl, and 10 pg bovine serum albumin/ml. TPBE buffer contains 10e2 M-Tris*HCl (pH 7*1), 10T2 M-putrescine+HCl, 10 mg bovine serum albumin/ml., and 2 x 10T4 M-EDTA. TCM buffer contains Tris*HCl (pH 7-l), CaCl, and MgSO, each at a concentration of lo-2
M.
Tris-Mg is 10m2 m-Tris*HCl with 10m2 M-MgSO&, pH 7.1. Tris-EDTA is 10m2 M-Tris.HCl with 10e3 M-EDTA (sodium salt), Tryptone broth contains 10 g Bactotryptone and 5 g NaCl/l. (b) Purification
pH 7.1.
of h heads
Heads were prepared according to the procedure of Harrison & Bode (unpublished information). A liter culture of W3350 pm-( XsusJ27) was grown in K medium at 37°C to 5 x lo* cells/ml. The cells were chiied, induced with ultraviolet light and then returned to lysis was complete. (When preparing heads with the shaker bath until the A,,, indicated 3H-labeled DNA, 1 mCi of [methyZ-3H]thymidine was added per liter of culture at 20 mm after induction.) After chilling, 1 ml. of CHCI, was added and the debris removed by centrifugation. One liter of lysate was placed in a 2-l. separatory funnel and mixed with 30 g NaCI, 10 ml. of 1 M-putrescine*HCl, 10 ml. of 20% (w/v) sodium dextran sulfate and 250 ml. of 30% (w/v) polyethylene glycol. After 16 hr at 4”C, the heads accumulate as part of a gelatinous mass at the polymer interface. The bottom phase (dextran sulfate) and the interphase material were collected and centrifuged at 5000 g for 15 min. The contaminating upper and lower phase material was poured away from the solid middle layer which was then dissolved in 10 ml of TPBE buffer. Dextran sulfate was precipitated by the addition of 1.6 ml. of 3 M-KCl. The precipitate was removed after 20 hr at 4°C. The supernatant was still very viscous and was not processed further until a second precipitate formed and the viscosity dropped (7 to 14 days). After this occurred, the solution (4 ml. per tube) was layered on a gradient of 15 to 35% sucrose in TPBE buffer and centrifuged at 5°C in the Spinoo SW25.2 rotor for 90 min at 25,000 rev./min. The lower light-scattering band was collected and usually contained about 5 x 1011 heads/ml. (0) Preparation
of X tail8
The source of tails in these experiments was W3350 pm- (hszcsA32) grown in K medium and induced as described for heads. After the addition of CHC13 and the removal of cell debris by centrifugation, lysates were stored at 4°C. Crude lysates contained 8 x lOlo to 2 x loll tails/ml. and the tails remain active for months (Harrison & Bode, unpublished results).
THE
ARRANGEMENT
OF DNA (d) In vitro
IN
LAMBDA
PHAGE
HEADS
495
head-tail joining
To assay heads, 0.1 ml. of an appropriate dilution of heads was mixed with 0.1 ml. of undiluted tail-containing lysate and 0.8 ml. of K medium or TPBE buffer. After incubation at 4’C for 20 hr, the mixture was diluted and plated on the sensitive strain, Escherichiu coli CFOO. In this study, a h head was designated as active or infectious if it combined with a tail to yield a particle that formed a plaque on an appropriate lawn of sensitive bacteria. For preparative-scale reactions, h heads were mixed with an excess of tails and incubated overnight at 4°C. The phage produced were then purified. (e) Phage purification
on CsCl step gradients
Phage were purified and concentrated on gradients containing successively layered solutions of CsCl with the following densities: first, 4 ml. of I.7 g/ml., then 9 ml. of 1*5g/ml., then 11 ml. of 1.3 g/ml. and finally 115 ml. of phage sample. All solutions were prepared in Tris-Mg buffer. The gradients were centrifuged at 5°C in the Spinco SW27 rotor for 3 hr at 22,000 rev./min. The phage band was collected and dialyzed against Tris-Mg buffer. (f) Micrococcal
nucleate treatment from Staphylococcus aureus was purchased
Crystalline micrococcal nuclease purified from the Worthington Corporation. Stock solutions of the enzyme (1 mg/ml.) were prepared in 1O-2 M-Tris-HCl (pH S), containing 0.5 mg bovine serum albumin/ml. and stored frozen at -70°C. Assay of MN? by the Worthington procedure showed that the enzyme preparation used contained at least 1.1 x lo4 units/ml. Purified )( susJ27 heads were suspended at a concentration of approximately 3 x lOlo particles/ml. in 0.05 M-Tris.HCl (pH 9), 0.5 mg bovine serum albumin/ml., 10m3 M-CaCl, and 10e2 M-putrescine~HC1. Microcoocal nuclease (10 pg/ml.) was added to one half of the mixture and both halves were incubated at 37°C for 30 min. After chilling, the heads were joined to tails and the phage purified as above. (g) Terminology
MN-phczge are phage particles prepared by joining tails to h heads that have been treated with micrococcal nuclease, as described. ContToZphage have received treatment and purification identical to MN phage, except that micrococcal nuclease was omitted. MN-DNA is DNA extracted from micrococcal nuclease-treated heads or MN-phage. Control DNA is DNA extracted from the control heads or control phage.
3. Results (a) Head inactivation
by micrococ& nuclease
Among a variety of nucleases initially screened, only micrococcal nuclease damaged the DNA packaged in X heads extensively enough to impair subsequent plaque formation (Table 1). Purified XsusJ27 heads were incubated with one of six nucleases and then allowed to join with &us A32 tails to form phage. Under the conditions employed, greater than 90% of untreated heads join with tails (Harrison & Bode, unpublished results). In contrast to the MN-sensitivity of heads, completed phage particles are not afFected by a 50-fold greater concentration of this n&ease (Table 2). The inactivation has the calcium ion requirement of MN (Cuatrecasas, Fuchs & Anfinsen, 1967) and has a greatly reduced rate at 0°C. The kinetics of inactivation at several enzyme concentrations is shown in Figure 1. Reactions were followed for 6 hours but only the first 32 minutes are shown. In all cases where enzyme was present, a maximum inactivation of 95% was observed, This occurs in 8 minutes with 10 units of enzyme. With O-4 unit, 128 minutes are required t Abbreviation
used: MN, micrococcal
nuclease.
496
V. C. BODE
AND
F. D. GILLIN
TABLE 1
Nucleate treatment of heads Nuclease Micrococcal Pancreatic
O/oActive
DNase (2 units) DNase (2 pg)
7.8 80
DNase II, porcine
spleen (350 units)
Phosphodiesterase
II, bovine spleen (0.8 unit)
X Exonuclease Endonuclease
heads
(3 units) I, E. co&i (0.01 unit)
110 80 103
92
Heads were incubated with the various enzymes for 30 min at 37°C. Buffers and divalent metal ions varied according to the requirements of the enzyme but all solutions contained 10-s M-putrescine*HCl and at least 0.5 mg bovine serum albumin/ml. to help stabilize the heads. The total volume was O-2 ml. In every case, controls lacking only enzyme retained at least 80% of their initial titer of active heads. The activities of exonuolease and of endonuclease I were determined by the methods of Little (1967), and Lehman, Roussos & Pratt (1962), respectively. Both enzymes were kindly given and assayed by Dr Marcus Rhoades. The remaining nucleases were purchased from the Worthington Corporation and the activities shown are as supplied by them.
TABLE 2
Sensitivity of heads and resistance of phage to inactivation by micrococcal n&ease Percentage infectious particles remaining
Treatment
A. Phage 119
(a) No enzyme
109
(b) MN (100 units/ml.) B. Heads (a) MN (2 units/ml.)
19
(b) MN (O-5 unit/ml.) (c) Omit MN
57 90
(d) Omit Ca2+ (MN = 2 units/ml.) (e) Incubate
at 0°C (MN
= 2 units/ml.)
Incubation
99 95
was for 30 min at 37’C except in (e).
but the same level is reached. The extent of inactivation varies between 85 and 95% in different experiments. It is unaltered by the addition of more enzyme either initially or after the inactivation plateau is reached. The enzyme is stable during the reaction and will inactivate a second portion of heads, but again only to the 95% level. Later, we will discuss the origin of this residual activity.
497
THEARRANGEMENTOFDNAINLAMBDAPHAGEHEADS
I
I
20
30
Time (mm) FIG. 1. The kinetics of head inactivation at various MN concentrations. Purified X heads were incubated under standard conditions with varying amounts of MN. Samples were removed and diluted lOOO-fold in cold TPBE buffer containing crude tails. Head-tail joining proceeded for 20 hr at 5”C, then titrations were performed.
(b) Properties of MN-heads The possibility that MX-inactivated heads are unable to join with tails was investigated. If tails attach only to the 5 to 15% of the heads that remain active, the phage particles formed would have normal infectivity but the yield would be only 5 to 15% that of the control. If, on the other hand, the treated heads combine normally with tails but only 5 to 15% of these particles give rise to plaques, the yield of completed phage particles would be normal compared to an untreated control but their specific infectivity (in plaque-forming units per particle) would be low. Phage prepared by incubating MN-treated [3H]thymidine-labeled heads with excess tails were purified on CsCl step gradients. Heads that have not joined with a tail lose their DNA during this purification procedure. If any heads survived, they would seek a higher density TABLE
3
SpeciJc infectivity of puri$ed p?mge Total recovery Phage origin Control
(cts/min)
heads
MN-treated
heads
(P.f.U.)T
(p.f.u./cts/min)
%
87,800
4.6 x 1011
5.1 x 10s
100
88,600
5.6 x 10n’
6.1 x IO5
12
Purified Xsus-J heads, labeledwith [methyl-3H]thymidine, were suspended in 5 x IO-” M-TrhHC1 (pH 9.0), 10V3 &r-CaCl,, 10m2 M-putrescineHC1, and 5 mg bovine serum albumin/ml. The mixture was divided in half and 22 pg MN/ml. was added to one half. Both the treated and control head suspensions were incubated at 37’C for 30min, then chilled and mixed with lysate oontainingexcess, X sus-A tails. The phage formed after 4 days incubation at 5”C, were purified by centrifugation on CsCl step gradients. 72% of the original tritium counts were recovered. t p.f.u., Plaque-forming units.
498
V. C. BODE
AND
F. D. GILLIN
stratum than phage. The yield of completed MN phage particles, as estimated by the tritium in labeled DNA, is the same as that obtained from an equal number of heads subjected to identical treatment except for the omission of nuolease, but the specific infectivity is only 12% of the control (Table 3). Thus, MN-inactivated h heads join with tails as efficiently as untreated heads, but they yield defective phage particles. (c) Properties of MN-phage (i) Adsorption and DNA injection In order to measure the efliciency with which these particles adsorb to sensitive bacteria and inject their DNA, [3H]thymidine-labeled MN-phage and control phage were mixed with sensitive bacteria. Approximately 90% of both the control and MNphage adsorb to the bacteria (Table 4). After shearing these complexes to remove phage TABLE
4
Adsorption and injection of micrococcal nuclease-treated and control phage Control
MN-phage
(a) (b) (c) d)
Infecting phage Unadsorbed phage Cts/min removed by blendor shearing Injected phage: (a)-((b) + (c))
(&s/mm)
%
15,275 1597 2720 10,958
100 10 1s 72
(ots/min) 15,000 1334 2330 11,336
phage % 100 S 16 76
Sensitive E. coli W3104 bacteria grown in Tryptone broth to 7 x lo* cells/ml., were sedimented and resuspended in l/5 vol. of h diluent. Half of the cells were mixed with an equal volume of [3H]thymidine-labeled control phage (multiplicity of infection = 4). The other half was infected similarly with MN-phage. Both were incubated at 37°C for 15 min to allow the phage to adsorb and to inject their DNA. The cells were then pelleted and samples of the supernatant solution, contaming unadsorbed phage, were dried and counted to determine adsorption (line (a) minus line (b)). The infected cells were resuspended in X diluent and sheared in a chilled Waring blendor for 6 mm. Again the cells were sedimented, and samples of both the supernatant and the pellet counted; the supernatant values are listed on line (c) above.
with uninjected DNA (Bode & Kaiser, 1965), 76% of the original DNA from control phage and 72% of that from MN-treated phage still sediments with the cells. Thus, the low plating efficiency of MN-phage cannot be attributed either to poor adsorption or to impaired injection. (ii) Infectivity
in a heteroimmune lysogen
If the MN phage are defective because they inject damaged DNA, it might be possible to increase their plaque forming ability by marker rescue or complementation. Table 5 shows that the plating efficiency of MN-phage on a heteroimmune lysogen is increased fivefold over the etllciency on a sensitive host. In other experiments, not shown in the Table, where the average burst ranged from 20 to 30 phage per cell, the burst size of MN-phage was at least 80% that of control phage both in a sensitive and in a heteroimmune lysogenic host. Furthermore, singleburst experiments using all four combinations of MN and control phage with sensitive and lysogenic host cells failed to reveal a significant difference in the distribution of burst sizes. Single-step growth curves indicated that those MN-phage which grow, exhibit the same latent period as control phage.
499
THEARRANGEMENTOFDNAINLAMBDAPHAGEHEADS
TABLE 5 Relative plating
C600 C600 (21 hy) C600 C600 (21 hy)
heads
MN-treated
Plaque forming
Lawn
Phage origin Control
on sensitive and lysogenic
eficiency
heads
1.4 1.6 8.9 4.5
x x x x
bacteria
units/ml.
1012 1O1a lOlo 1011
y0 100 114 6.4 32.1
Purified MN-treated and control phage suspensions with an equal number of particles, as indicated by their absorption at 260 nm, were titrated using the indicated lawn bacterial at 37°C. The plating efficiency of control phage on the C600 lawn was taken as 100%.
(iii) The fate of MN-phage
infected
cells
Since lambda is a temperate phage, one possible explanation for the low plaqueforming efficiency of MN-phage is a greatly increased lysogenization frequency. Table 6 indicates, however, that both the lytic and lysogenic responses are inhibited more than 80%. It also shows that most of the MN-infected cells survive and produce lambda-sensitive clones.
TABLET Response of MN-phage-infected
cells
Control (no./1000 cells) Infecting phage Infected cells? Infectious centers Lysogenio cells Surviving cells (1) expected5 (2) found
MN (no./1000 cells)
320 270 200
640 470 380
450 360 26
900 590 49
(&
(&
(O&
(Oi61)j:
740 700
550 600
640 970
410 850
Strain C600, 6 x lo* cells/ml and MN or control phage at two multiplicities were mixed at 0°C in h dil. After 15 min for adsorption, the cells were warmed to 37°C for 10 mm then diluted tenfold into X dil with anti-X serum (K = 1). After 10 min at 37’C to neutralize any unadsorbed phage, the cells were diluted further then (a) plated for infectious centers on a C600 lawn, (b) plated without the lawn bacteria to measure total viable surviving cells and (c) spread with antiserum on the surface of an agar plate and grown overnight. The colonies on the latter plates were replica-plated on a C600 lawn then exposed to an inducing dose of ultraviolet light. Those colonies which produced phage were scored as lysogenio. Similar frequencies of lysogenization were obtained when individual colonies were tested for immunity as an indication of lysogeny. t Calculated assuming a poison distribution and 100% absorption. t Lysogenic cells per 100 infecting phage. 0 Total cells minus infected cells plus lysogenic cells.
(d) Covalently-closed
circle formation
after
infection
Soon after X infection at least 50% of the infecting DNA can be recovered in extracts as a covalently-closed twisted circular form, species I (Bode & Kaiser, 1965). Since MN-DNA is injected into bacteria normally and, at least under certain conditions, forms hydrogen-boncled circles in vitro (Gillin & Bode, 197 l), it was of interest to know if MN-DNA is converted to species I in vivo. DNA was extracted from sensitive
V. C. BODE
500
AND
I?. D. GILLIN
bacteria 15 minutes after injection with [3H]thymidine-labeled MN or control phage. Although sedimentation reveals a normal conversion of injected control DNA to the rapidly sedimenting closed circular form, very little of this species is detected in extracts containing injected MN-DNA (Fig. 2). A similar result is obtained when any complications of vegetative growth are eliminated by using an immune lysogen as the host. The recovery of intracellular 3H labeled MN-DNA was frequently 20% less than that of control DNA, but the gradients show no low molecular weight fragments as would be expected if MN-DNA were subjected to extensive degradation after injection.
i
(a)
BOC
60C
(b)
60C)
4oc 1 i
2oc
Fraction number
FIG. 2. Sedimentation of 3H-labeled DNA from MN-treated and control phage extracted after injection into a sensitive or into a lysogenic host. (a) Tritiated MN- or control phage (6 x lOa cts/ min) were suspended in h diluent and mixed with the sensitive strain, W3104, at a multiplicity of 12 particles per bacterium. After incubation at 37°C for 15 min, 70% of the tritium sedimented with the bacteria. The infected-cell pellet was suspended in Tryptone broth, shaken at 37°C for 15 min, and then sheared in a Waring Blendor to remove uninjected DNA. Only 46% (MN) and 56% (control) of the counts originally associated with the attached phage sedimented with the cells after growth and blending. The DNA was extracted and sedimented in sucrose gradients as described by Bode t Kaiser (1965). Since the relative position of a marker X DNA (3ZP-labeled) was identical in the two separate gradient tubes, the tritium profiles of MN and control DNA are superimposed and the position of the marker DNA indicated by an arrow. -e-e-, MN-phage; -- x -- x --, control phage. (b) The experiment was performed as described above except the host strain was an immune lysogen, CSOO(A), and the multiplicity was 8 particles per bacterium with 45% of the input tritium injected as measured by insensitivity to removal by shear forces.
THEARRANGEMENTOFDNAINLAMBDA
PHAGE
HEADS
501
(e) .MN-phage infectivity as a function of temperature As reported in the accompanying paper (Gillin & Bode, 1971), MN-DNA forms hydrogen-bonded circles in vitro. In TCM buffer, the T, for melting these circles is 36°C about 25 degrees lower than the T, of normal X DNA circles., Therefore, MN treatment probably removes severs1 bases from a single-stranded cohesive end of the DNA. If their replacement is essential for MN-phage infectivity, then the formation of hydrogen-bonded circles in viva would be necessary, as a first step, to provide the template for cellular repair enzymes. The in viva conditions for circle formation and opening are not known. However, the experiment shown in Figure 2 suggests that, if MN-DNA circles form, they are not repaired and covalently closed. If cyolization is an obligatory step for plaque formation and circles are meltmg in the cell at normal TABLE
7
Phage infectivity at different temperatures Temperature (“C) 32 37 40
Control
MN-treated (p.f.u./ml.)t 9.8 x lo9 6.2 x lo9 2.9 x 109
(%I 19-l 11.9 5.8
(p.f.u./ml.) 5.14 x 1010 5.21 x 10’0 4.96 x lOi0
(%) 100 100 100
Suspensions of purified MN-treated and control phttge with the s&me absorption at 260 nm were diluted in X diluent, mixed with 0.2 ml. of strain C600 lawn culture, and inoubsted for 15 min at 32 or 37 or at 40°C for 10 min. After plating, the Petri dishes remained at 25°C for 15 min while the soft agar layer hardened. Then the plates were incubated at the temperature of the previous incubation. t p.f.u., Plaque forming
units.
growth temperatures, the plating eficiency of MN-phage might change with temperature. Control phage plate with equal efficiences at 40,37 and 32°C (Table 7). In contrast, the efficiency of plaque formation by MN-phage decreases threefold between 32 and 40°C. This difference is consistent with the idea that lower temperatures enhance the infectivity of MN-phage by increasing circle formation which is necessary for repair of the MN damage.
4. Discussion Since joining with a tail renders &us-J heads completely resistant to inactivation by MN, the head’s tail attachment site is also the most likely site for nuclease attack on the DNA in the head. That MN treatment does alter the DNA is documented by genetic and biochemical observations. The increased plating efficiency of MN-phage on a lysogenic host compared to a sensitive host suggests they are damaged genetically. The absence of normal amounts of covalently-closed circles in vivo indicates that nuclease treatment changes the biochemical properties of the h DNA packaged in heads. The following paper (Gillin & Bode, 1971) will further document changes in the physical and biochemical properties of MN-DNA. It was shown here that MN-treated heads join normally to tails, yielding phage, most of which do not produce plaques even though adsorption and injection are normal. Several observations argue that the residual plaque-forming ability of MN-phage is a
502
V. C. BODE
AND
I?. D. GILLIN
property of all the particles, rather than of a separate nuclease-resistant population. The plating efficiency of a given MN-phage preparation can vary from 5 to 30% depending upon physiological conditions that do not significantly effect the plaque-forming efficiency of control phage, e.g. plating on a heteroimmune lysogen or varying the plating temperature between 32 and 40°C. Since there is variation in the residual infectivity, it cannot be due to a fixed number of undamaged normal particles. A reduced amount of twisted circular DNA (l?ig. 2) is the earliest known difference in the life cycles of MN and control phage. To become species I, it is necessary for MN-DNA to form hydrogen-bonded circles in vivo, to undergo repair replication, and to be covalently sealed. Attributing the defectiveness of MN-phage to a decreased ability to form circles in vivo is consistent with the increased infectivity of MN-phage at lower temperatures where MN-DNA circle formation may be promoted. The residual infectivity, then, may represent the probability that an MN-DNA molecule can circularize and undergo repair. Although our primary purpose in characterizing MN-phage and DNA is to learn more about how h DNA is arranged in the head, it appears that they will be useful in other problems, particularly when the exact alteration in MN-DNA is known. Since the DNA can be injected into the cell normally, it provides a way to study the effect of this specific DNA lesion on in vivo repair, replication and recombination as well as gene function. It is not known whether heads are uniquely sensitive to MN because of the enzyme’s small size or because of some other property. Similarly, it is not known whether a small amount of DNA protrudes from the head and is attacked by MN or whether the nuclease partially enters the head. It is clear that even at high enzyme concentrations, or extended times, only a few nucleotides can be removed from the head DNA by this enzyme and that these are not available to several other n&eases. For example, pancreatic DNase-treated heads have been studied extensively (Wong & Bode, unpublished results) and contain DNA whose cohesive ends are completely intact. The authors wish to acknowledge the expert assistance given by Mm Christina Wong, Mrs Sarah Brown and Mr John Blotzer. This work was supported by grants from the National Science Foundation (GB6961 and GB25153) and the Public Health Service (Al06493 and GM18182). REFERENCES Bode, V. C. & Kaiser, A. D. (1965). J. MoZ. Biol. 14, 399. Cmo, L. (1965). l’i7iroZogy, 25, 226. Cuatrescasas, P., Fuchs, S. & Anlinsen, C. B. (1967). J. Biol. Chem. 242, 1541. Gillin, F. D. & Bode, V. C. (1971). J. Mol. BioZ. 62, 503. Lehman, I. R., Roussos, G. G. & Pratt, E. A. (1962). J. Biol. Chem. 237, 819. Little, J. (1967). In Methods in Enzymology, ed. by L. Grossman & K. Moldave, part A, p. 263. New York: Academic Press. Weigle, J. (1966). Proc. Nat. Acad. Sci., Wash. 55, 1462. Weigle, J. (1968). J. Mol. Biol. 33, 483.
vol. 12,