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Interruption of DNA injection by X-irradiation of phage λ
VIIIOLOGY
43, 16(i-175 (1971)
Interruption
of DNA JOHN
Injection
D. SHARP’
Graduate Departrnenl of Biochemistry,
by X-Irradiation DAVID
AND
of Phage
x
FREIFELDER2
BTandeis UniveTsily,
Waltham,
Massachusetts
02164
Accepted September I, 1970
When X phage is X-irradiated, it loses the capacity to inject all of its DNA into Escherichia coli. Autoradiographic measurement of the amount of DNA injected by X-irradiated phages leads to the conclusion that only a single DNA fragment is injected-a piece extending from the end normally injected first to the first double-strand break. This partial injection accounts for about half of the lethal effects of X-irradia. tion in phagex. Loss of adsorptive capacit.y and gross destruction of the phage head are shown to contribute only very slightly to the inactivat,ion process. INTRODUCTION
The process of infection of a sensitive bacterium by a phage consists of the following stages: adsorption of phage to bacterium, injection of phage DNA into the bacterium, macromolecular synthesis, phage assembly, and lysis of the bacterium (Stent’, 1963). All but one of these stages, i.e., injection, have been extensively studied during the past two decades. In this paper we report the results of an investigation of one aspect of the injection process. In particular, we ask the question: Can a phage inject its entire complement of DnTA even if the DNA molecule is fragmented by X-rays? An important problem in understanding the injection process is how a free end of the DNA molecule gets t’hreaded through the tail orifice. One might imagine that t’his problem could best be met during phage assembly if the phage tail were either synthesized around the free end or at’tached t,o the filled head so that the free end was contained in the tail. Caro (1965) has obtained electron micrographs of osmotically shocked X phage which show the DNA passing out of the head and threading through t,he tail, which suggests 1 Present address: Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115. z To whom reprint requests should be addressed. Reprints will not be available within the United States.
that the problem of initiation of injection might be so handled during assembly. If this is t’he case, it might be predicted that if a double-strand break were introduced into the DNA while still in the phage head, the first piece of DNA, i.e., up to the doublestrand break, would be injected but the new end would not be able to find the t,ail opening. Alternatively, if t,he injection mechanism itself were somehow dependent on t,he intactness of the DNA molecule so that DNA damage would prevent injection from even being initiated, or if for example some X-ray damage to the phage tail completely blocked injection, X-rays would produce a mixture of noninjecting and totally injecting phage particles. These possibilities can be stated as two models for phage injection: First-piece-only injection model. By this model, an X-ray-induced double-strand break is assumed to allow injection of only one fragment of DNA-that one extending from the end normally injected first to the first double-strand break. All-or-none injection model. By this model, an X-ray damaged phage particle is assumed to inject either its full complement of DNA or no DNA at all, but never an intermediate amount. This investigation made use of the two facts: (1) Double-strand breaks can be introduced into the DNA within the phage
166
INTERRUPTION
OF PHAGE
x INJECTION
167
using a Phillips (Miiller) RT-100 X-ray machine operating at 100 kV and 7 mA or 8 mA, with a beryllium window and a 0.78 mm aluminum filter. The sample, 0.05-0.4 ml, was placed in a small glass vial in a brass holder which was immersed in ice during the irradiation. This holder could be sealed with a Lucite cap for irradiation under N2 or a similar perforated cap for irradiation in air. Dosimetry was performed by t’he FeS04 met’hod of Battaerd and Tregear (1966). The X-irradiation done in the experiments reported in this and the accompanying paper was all done in Mg-T (except as noted) containing 5 mM histidine, under a Nz atmosphere. The Nz was found necessary t,o obtain maximum complementation in the experiMETHODS AND MATERIALS ments of the accompanying paper. This phedIecliu. X broth = 10 g Difco Bactonomenon will be discussed in a future pubtryptone, 10 g NaCl, 1000 ml HzO. Autolication. clave, t’hen add 20 ml sterile 20% (w/v) malDNA strand breakage was measured by tose. X agar = 10 g Difco Bacto-tryptone, 5 analyzing DNA sedimenting boundaries in g NaCl, 12 g Difco agar, 1000 ml HSO, auto- the UltracenOrifuge as described by Freifelder clave. NBSA (Sutrient broth soft agar) = (1965). 5 g Difco nutrient broth, 5 g NaCI, 8 g Difco Test for DNA release by X-rays. X-iragar, 1000 ml HzO. llledium 1.21.4.68 g NaCl, radiated Xhc phage (20 pg/ml) was sedi1.49 g KCl, 1.07 g NH&l, 0.203 g MgCl~. mented in the Beckman Model E ultracen6H20, 2.22 g CaCls, 2.S4 g Na2S04, 14.5 g trifuge at 9945 rpm in 1 M NaCI. The sediTris(hydroxymethyl)aminomethane, H,O to menting boundary was seen in UV photo1000 ml, adjust to pH 7.5 with HCl, autographs taken within the first 10 min. The clave. MB-P. 0.01 M MgSO+ 0.01 M po- speed was then increased to 33,450 rpm and tassium phosphate, pH 6.8. Concentrated more photographs were taken. At the lower stocks are autoclaved separately and mixed speed the only visibly moving boundary was when cool. Mg-!Z’. 0.01 III MgSO+ 0.01 M that of phages, whereas at the higher speed, tris(hydroxymethyl)aminomethane-HCI, pH DNA sedimentation was observed. 7.5. Trichloroacetic acid-precipitable radioacE. coli and lambda strains. C600 (suII+ tivity assays. l\lost radioactivity assays were BI- thr- leu- lac- TIR T5R), and CR34(Xi434) done by precipitation with 5% trichloro( surI+ thv- BI- thr- leu- met- TIR) were acetic acid (TCA). Herring sperm DNA, 3-5 obtained-from A. D. Kaiser. C600(Plkc) pg/ml, was added to the sample, followed by was obt,ained from the collection of l\iI. an equal volume of 10 % TCA and the prel\leselson. Xhc,2004, referred t’o here as cipitate was collected on a J!Iillipore filter simply Xhc, was isolated in the laboratory of (0.45 p pore size) by suction filtration, R. Thomas and obtained from K. Brooks. washed several times with 10% TCA, and Plaque assays were done by standard finally with 95 % ethanol. The filters were methods using x agar and NBSA for bottom dried in a 100°C oven, placed in vials conand top agar, respectively. X-Ray survival taining 10 ml scintillation fluid (toluene, of Xi4a4was measured using C600 as indicacontaining Liquifuor, New England Nutor. Plating bact,eria were grown with shakclear Corp.), and counted in a liquid scint,iling at 37” in X broth to 5 X 108/ml, centrilation counter. fuged and resuspended in Mg-P at log/ml, TCA-soluble radioactivity assays. TCAand stored on ice less than 4 hr before use. was measured in the X-irradiation. All X-irradiation was done soluble radioactivity
head by X-irradiabion of the intact phage (Freifelder, 1966, 1968); and (2) uninject’ed DNA can be removed from a lambda phagebacterium complex by the classical blending procedure of Hershey and Chase (1952). It was thus possible to prepare bacteria containing only injected radioactive phage DNA and free of uninjected DNA, and to examine t,hem autoradiographicallp to determine the dist,ribution of amount of phage DNA injected. This study revealed part,ial injection as a frequent consequence of Xirradiation of phage X, in quantit’ative agreement wit,h t,he model for “first-piece-only” injection.
168
SHARP
AND
degradation experiment as follows. Fiftymicroliter samples were mixed with 0.2 ml of 1 1M NaOH. Within 30 min, 0.2 ml herring sperm DNA (2.5 mg/ml) was added, followed by 0.55 ml of 10% TCA. The samples were mixed and centrifuged for 10 min at 7000 g; an aliquot of the supernatant was counted in 10 ml of Bray’s scintillation mixture (Bray, 1960). Radioactive phage. Thymine-2-14C or thymine-methyl-3H was used to prepare Xi434 containing radioactive DNA by UV induction of CR34(Xi434). The lysogens were grown with shaking at 37” to 2 to 3 X 108 cells/ml in medium 121 supplemented with 10m3M KH2P04, 0.2 % Bacto-casamino acids, 0.4 % glucose and 5 pg/ml thymine. They were centrifuged and resuspended in the same medium except that 2 pg/ml radioactive thymine replaced the unlabeled thymine. The culture was then induced with UV and grown for 2-3 hours, by which time lysis was complete. For the highly radioactive Xi434-3H used in the autoradiography experiments, 3 pg/ml catalase was added to the thymine-3H (to remove peroxides) before mixing with the culture; this maintained the phage yield at nearly normal levels. Bacterial debris was removed by centrifugation and the phage were separated from most of the nonphage radioactivity either by zone sedimentation in a 5-20% (w/v) sucrose gradient or by sedimentmg into a preformed CsCl step gradient. This latter method gave phages of higher purity and at higher concentration, and was done as follows: Four consecutive 2 ml layers were placed in a tube for the Beckman SW 25.1 rotor: CsCl, p = 1.7; CsCl, p = 1.5; CsCl, p = 1.3; and 20 % sucrose, all containing Mg-T. The phage lysate (20 ml) was then carefully layered onto the sucrose layer and the tube was spun at 24,000 rpm for 90 min at 20”. Fractions were collected from the tube bottom (12-14 fractions, each 0.5 ml) and those containing the phage peak were pooled and dialyzed against Mg-T after adding 10 pg/ml gelatin. Phage adsorption and injection. CR34 (xi434) bacteria were prepared as for plaque assays, at log cells/ml in Mg-P, but with 5 pg/ml thymine added to both the growth
FREIFELDER
medium (X broth) and the Mg-P. Radioactive phage was added, and the mixture was incubated for 20 min at 0” for adsorption to occur. Further incubation for 20 min at, either 37” or O”, respectively, allowed or prevented injection (Bode and Kaiser, 1965). The blended-bacterium complexes were then blended in a Waring Blendor stainless steel micro-at.tachment (3-20 ml capacity) with blades about x-inch long. The complexes were then centrifuged for 10 min at 7000 g, and the supernatants were removed, TCA precipitated, and counted. The precipitates were resuspended in Mg-P, TCA precipitated, and counted. From the amount of radioactivity in the supernatant (S) and pellet (P) the percent adsorbed, A, was calculated: A = 100 P/(S + PI. Control experiments indicated that a blending speed of about 5000 rpm and 10 min blending time were best in terms of producing the maximum difference in adsorbed radioactivity for injected vs. uninjected phage. Under these conditions less than 10% of the radioactivity was usually removable from injected phage-bacterium complexes, whereas at least 90 % was usually removable from uninjected complexes. These controls were always run, as well as an unblended control bo measure initial levels of unadsorbed phage, when adsorption or injection by X-irradiated phage was measured. The “percent adsorbed” of X-irradiated phage was calculated relative to the percent of radioactivity adsorbed to the bacteria in unirradiated phage which was always greater than 70% for the results reported. Adsorption and injection decreased with age in 14Cand 3H phage preparations, and this was compensated for by these controls. For the auhoradiography measurements the Ai434-3Hwas very highly radioactive and lost adsorption and injection ability measurably within a week. Thus in the experiments reported here t,hese phage were always used 1 or 2 days after growth. Autoradiography was carried out essentially according to Caro and van Tubergen (1962). Dichromate-washed glass slides were “subbed” by dipping into a solution of 0.5 % gelatin + 0.05 % chromium potassium sulfate. Drops of bacteria in 1Ig-P
INTERRUPTION
OF PHAGE
were spread gently with the edge of a coverslip and allowed to dry. Kodak AR. 10 stripping film was applied by standard methods. After drying in a stream of air, the slides were placed in a desiccated, lighttight box at 4” for exposure. The slides were developed in Kodak D-19 developer for 2 min, rinsed in 1% acetic acid for lo-15 set, fixed in 30% (w/v) Na&&0~ for 1 min, washed in water for 1 min, dried, and stained. Staining, necessary to visualize the bacteria, was done by dipping a slide for 2-3 min in 0.02 % crystal violet in 30 % ethanol, then 15% ethanol for 10 set, washing in water for 1 min, and then drying in a stream of air. Grains were counted with a microscope at 1600X under oil immersion. RESULTS
Survival Curves for Phage Viability Breakage
and DNA
Figure 1 shows the results of X-irradiation of phage X in a histidine-containing buffer in terms of phage inactivation (loss of plaqueforming ability) and DNA breakage. This agrees with earlier results for phages T7 and X (Freifelder, 1968), in that the rate of DNA breakage is approximately half that of inactivation. Efects of X-Irradiation on Adsorption and Gross Phage Structure The effect of X-irradiation on the adsorption process was measured as follows:
DNA
FIG. 1. Relative rates of X phage killing and DNA breakage by X-rays. Xhc was irradiated at 10.0 kradlmin. The DNA was released and sedimented in the analytical ultracentrifuge to measure the amount of broken DNA.
169
X INJECTION
OIL--J! 0
’ 2
I X-Ray
’ 3 Dose
I
I
I
I
I
4
5
G
7
8
(I Untt=
185
Klloradsl
FIG. 2. Effect of X-rays on adsorption of phage X to Escherichia coli cells. XiQ4-W or 3H were Xirradiated and adsorption was measured as described in Methods and Materials. Solid line is X0 the slope of the phage survival curve, for comparison with the data.
Xi434-14Cphage were X-irradiated and allowed to adsorb at 0” to bacteria. At this temperature adsorption without injection occurs (Bode and Kaiser, 1965; and see below). The bacteria were then separated from unadsorbed phage by centrifugation. Figure 2 shows the results of such an experiment. X-irradiation has a small but measurable effect in agreement with the finding of HradeEn& (1966). To test whether this loss of adsorption is due to breakage of the phage head and release of the DNA rather than to a direct effect on adsorption, the intactness of the phage structure was examined after X-irradiation. Phages were X-irradiated and then sedimented in the analytical ultracentrifuge at low and then high speed in order to see the phage and DNA boundaries, respectively, as described in Methods and Materials. If the phage particle had burst and released its DNA, one would expect to see some DNA sedimenting more slowly than the intact phage. No such material was seen even with a dose yielding 0.01% survival, so that there is little or no breakage of the phage head (<0.003 per lethal hit). Hence the small loss of adsorption reported above is probably due to an effect on the adsorption apparatus. Lack of Degradation of DNA Injected by X-Irradiated Phage Before quantitative studies of injected DNA could be carried out it was necessary
170
SHARP
AND
FREIFELDER
trol. Note that limit’ed nuclease activity on irradiated DKA is not detected by this test, but only degradation sufficient to make the DNA acid-soluble. This is the appropriate control, since acid precipitability is the criterion used to detect DNA in this and the accompanying paper (Sharp et al., 1970). E$ect of X-Irradiation on the Inject&m Capacity of the Phaye Population 60
0 Time
After
Id0
120 InjeCtiOn,
Min.
FIG. 3. Stability of DNA injected by X-irradiated Xi434-14C. Phage were irradiated and allowed to adsorb to CR34(Xi434) and inject their DNA (20 minutes at 37”) as for the blending experiments (see Methods and Materials). The infected complexes were kept at 0” for various periods of time after the 20-min adsorption incubation. The incubation was terminated by lysing the cells with NaOH and precipitation with trichloroacetic acid. -U--O-, phage not X-irradiated; -O-O-, phage X-irradiated to 1OW survival; -O-O--, phage adsorbed to C6OO(Pl), and incubation continued at 37”, for maximum degradation.
to ascertain that injected, irradiated DNA was not degraded under the conditions used. This point is raised by the recent finding of Huston and Pollard (1967) that E. coli cells treated with ionizing radiation degrade their own DNA. Xi434-14Cwas X-irradiated to a survival of 1O-3 and mixed with E. coli cells for adsorption and injection. As in the experiments reported in this and the accompanying paper, bacteria lysogenic for Xi4s4were used so that phage growth and cell lysis, but not adsorption or injection, were blocked. TCA-soluble radioactivity was measured after lysing the cells at various times with NaOH (see Methods and Materials). As a test of the method to show that degradation could be seen, cells lysogenic for phage Pl were also infected with hi4s4-14C.Arber and Dussoix (1962) have shown t’hat the presence of the Pl prophage causes the X DNA to be degraded. The results of this experiment are given in Fig. 3, in which it is shown that there is negligible degradation of the x DNA with or without X-irradiation, but 60 % degradation in the Pl lysogen con-
Xi434-14Cphage were X-irradiated with various doses and allowed to adsorb to bacteria and inject their DNA. The injected phage-bact.erium complexes were then blended to remove uninjected DNA. The bacteria containing the injected DNA were separated from the uninjected DNA by sedimentation and the amount injected was determined as a function of X-ray dose. The results of this experiment are shown in Fig. 4. The points agree with the curve labeled “Partial Injection Prediction, Eq. 2” which represents the first-piece-only model (see Appendix 1). It is clear from Fig. 4 that more DNA than is represented by unbroken molecules must be injected
:
kOO6 0.04 0
I 0.5 X-Rrv
1.5
Dose
I I Unit
2.0
2.5
,\ 3.0
1 3.5
= 185 Kilorodsl
of X-rays on injection of DNA by phage X. 14C- or 3H-labeled Xi4$4were X-irradiated and injection was measured by adsorption and blending as described in Methods. The unit X-ray dose chosen is the DZT for DNA double-strand breaks, so that the abscissa represents z, the average number of double-strand breaks per DNA molecule. The prediction for all-or-none injection is represented by the line labeled “Fraction of Unbroken DNA.” FIG.
4. Effect
I IO
INTERRUPTION
OF PHAGE
by the X-irradiated phage. The question remains whether this broken DNA is injected according to the first-piece-only model, or independently of DNA breakage. To answer this, the amount of DNA injected by individual phages after X-irradiation was measured autoradiographically.
x INJECTION
lil
6
Autoradiographic Measurement of the Effect of X-Irradiation on the Injection Capacity of Single Phages Injected complexes of Xi434-3H phage X-irradiated with various doses were prepared as for the blending experiment in the preceding section, using a low (0.1-0.2) multiplicity of infection to avoid multiple infection. After three cycles of centrifugation and resuspension, the bacteria, free of unadsorbed phage and DNA removed by the blending, were dried onto glass slides which were then covered with autoradiographic stripping film, dried, and exposed. After suitable periods of time the slides were developed and stained. Silver grains were counted microscopically and the grain distribution over bacterial cells was plotted. Since the effect of blending on leakage of intracellular components is not well known, it is important that blending of the infected complexes with unirradiated phage 7
P
d
6 t
-2 1234567 m = No Groins Over Cells FIG. 5. Autoradiographic grain distributions for unirradiated phage. CR34@?) cells infected with Xi434-3H were placed on slides for autoradiography before (0) and after (X) removing uninjetted DNA by shearing and centrifugation. Exposure was for 27 days. sm is the fraction of cells having m grains, and gm is a straight line when s,,, is a Poisson distribution (see Appendix 2). Average grain number per infected cell was z = 2.9.
o 2 4 6 '3 m= No Grains Over Cells
grain distributions FIG. 6. Autoradiographic for X-irradiated phage. Xi434-3H were X-irradiated to a survival of 5 X 1O-3, mixed with CR34(Xi434) bacteria and allowed to adsorb and inject. The infected cells were placed on slides for autoradiography before (0) and after (0) being sheared in a blendor and washed by centrifuging. Exposure was for 60 days. sm is the fraction of cells having m grains, and gm is a straight line when sm is a Poisson distribution (see Appendix 2). The straight line was drawn to fit the distribution for unsheared cells, giving an average of 4.4 grains per infected cell. Equation 13 of Appendix 2 was used to calculate the curved line, based on this average number (z = 4.4) and the phage survival, from which the average number of double-strand breaks was calculated: x = -x log, (5 X 1OW) = 2.65.
does not alter the shape of the grain distribution from that obtained with unblended samples. As explained in Appendix 2, the way used here for plotting the grain distributions gives no change from the straight line (Poisson distribution) when fewer cells contain radioactive phage DNA, as long as the distribution remains the same among cells with grains. Thus although a small fraction of the phages do not inject and are removed by blending, this should not change the distribution. This is shown to be the case in Fig. 5. The results of this experiment when done with X-irradiated phage are shown in Figs. 6 and 7. The theoretical grain distributions (calculated in Appendix 2) for the firstpiece-only model are shown in the figures (curved lines). The all-or-none injection model predicts no change on shearing, as was the case for unirradiated phage. It is important to note that the vertical separation of the curves of approximately one
172
SHARP AND FREIFELDER 6 5 E -.““; .‘;
0
Y z J II E CD
4
2 0 I
Sheared
0 -I
yzIff?r
Unsheared
3
[I
-2 234567 m = No. Groins
Over
Cells
FIG. 7. Autoradiographic grain distributions for X-irradiated phage. Same experiment as Fig. 6, except exposure was for 27 days. The slope of the “unsheared” line gives z = 2.64 grains per cell. The curved line is calculated from Eq. 13 of Appendix 2, using x = 2.65 and z = 2.64.
unit represents a factor of e (natural log base) difference in s,, the relative frequency of cells with m grains. Hence it is clear that the data agree well only with the first-pieceonly model. DISCUSSION
It has been shown that if one or more double-strand breaks are introduced into a x DNA molecule in an intact phage, only the first piece, i.e., up to the first doublestrand break, is injected. Hence the phage probably does not have a mechanism for threading the newly generated free end through the tail. The fact that first-pieceonly injection occurs makes possible a determination of the direction of injection of X DNA since one may ask, by genetic analysis, which genes have been injected, or by physical analysis, which end has been injected. The results of these studies are presented in the accompanying publication (Sharp et al., 1970). The phenomenon of first-piece-only injection answers an important question in phage radiobiology concerning the relation between lethal hits and double-strand breaks. It has been shown that the rate of double-strand breakage is half the X-ray inactivation rate and that if the irradiation is done in the absence of oxygen (in which case the inactivation rate decreases2-fold), the inactivation rate equals the rate of double-strand breakage (Freifelder, 1965,
1966). Hence it was tentatively concluded that double-strand breakage is a major cause of X-ray inactivation. However, unless al2 the chemical effects of X-irradiation are known, agreement of rates can be fortuitous, as has been pointed out previously (Freifelder, 1966). For example, it is possible that if all fragments were injected, the injected fragments might somehow be reassembled in viva-lethality then being due to other, unknown effects. However, the results presented here show that firstpiece-only injection is a frequent consequence of X-irradiation so that a significant fraction of the lethal hits are chemical changes that lead to incomplete injection. This is apparently the first example of an unambiguous relation between a biological change produced by X-rays and lethality. Rigorously, however, double-strand breakage is only strongly implicated as the cause of first-piece-only injection; proof that this is the case still rests upon agreement between the rate of production of doublestrand breaks and of the lesion producing first-piece-only injection. ACKNOWLEDGMENT This work was supported by contract AT(301)3797 from the Atomic Energy Commission, Grant GM-14358 from the National Institutes of Health, and E-509 from the American Cancer Society. JDS was supported by a predoctoral fellowship (GM-31,266) from the National Institutes of Health; DF by a Career Development Award (GM-7617) from the National Institute of General Medical Science. This is publication No. 745 of the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154. REFERENCES ARBER, W., and DUSSOIX, D. (1962). Host specificit,y of DNA produced by E. coli: I. Host controlled modification of bacteriophage X. J. Mol. Biol. 5, 1836. BATTAERD, H. A. J. B., and TREGEAR, G. W.
(1966). Radiation ferrous sulphate
dosimetry-the Fricke-Miller dosimeter. Rev. Pure Appl.
Chem. 16, 83-90. BODE, V. C., and KAISER, A. D. (1965). Changes in
the structure and activity of lambda DNA in a superinfected immune bacterium. J. Mol. Biol. 14, 399-417. BRAY, G. A. (1960). A simple liquid scintillator
for
INTERRUPTION
OF PHAGE
counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, 279-285. CARO, L. G. (1965). The molecular weight of lambda DNA. vi’irology 25, 226-236. CARO, L. G., and VAN TUBERGEN, R. P. (1962). High-resolution autoradiography. J. Cell. Biol. 15, 173-188. Davmow, P. F., HOLLOWAY, B., and FREIFELDER,
D. (1964). Interruptions strands in bacteriophage
in the polynucleotide DNA. J. Mol. Biol. 8,
l-10. FREIFELDER, D. (1965). Mechanism of inactivation
of coliphage T7 by X-rays. Proc. Nat. Acad. Sci. U. S. 54, 128-134. FREIFELDER, 1). (1966). Lethal changes in bacteriophage DNA produced by X-rays. Radiat. Res. 6, Suppl., 80-96. FREIFELDER, D. (1968). Physicochemical studies on X-ray inactivation of bacteriophage. Virology 36, 613-619. FREIFELDER, D., and DAVISON, P. F. (1962). Studies on the sonic degradation of deoxyribonucleic acid. Biophys. J. 2, 23b247. HERSHEY, A. D., and CHASE, M. (1952). Independent frunctions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36, 39-56. HRADEENA, Z. (1966). X-ray inactivation of lambda phages. I. Damage on adsorption and injection. Znt. J. Radiat. Biol. 10, 443-449. HUSTON, D. C., and POLLARD, E. C. (1967). DNA degradation in E. coli 15 T-L- induced by fast proton bombardment. Biophys. J. 7,555-565. SHARP, J. D., DONTA, S., and FREIFELDER, D. (1971). Lack of polarity of DNA injection by E. coli phage h. Virology 43, 176-184. STENT, G. S. (1963). “Molecular Biology of Bacterial Viruses.” W. H. Freeman, San Francisco, California. Appendix 1: CALCULATION OF THE EFFECT OF DNA DOUBLE-STRAND BREAKS ON THE AMOUNT OF DNA INJECTED BY PHAGE x: FIRST-PIECE-ONLY HYPOTHESIS
It is assumed here that double-strand breaks allow injection of DNA only up to the first break. For this calculation, two specific assumptions, verified by Freifelder and Davison (1962), are made: (1) The breaks
are assumed
to be distributed
received
an average
of J: breaks
molecule, is assumed to be given by the Poisson distribution: F,, = em5 $ 0
Among molecules with exactly n breaks, the average proportion fn of DNA between the injected end and the first break can be expressed as fn = 1’ .z ClP
where x is the length from the injected end, in units of the total length of a lambda DNA molecule. cZP is the differential probability that a given DNA molecule has its breaks arranged so that (a) the first break lies between z and z + dz, and (b) the other n - 1 breaks lie between z and 1, the position of the last-injected end. dP is the product of the probabilities of these two requirements being met, or n dz for requirement (a) and (1 - x)+-l for (b). Thus dP = (1 - ~)~--lndz, from which fn = &zlP 1
1 x(1 - z)“-52 czz= n+1 Although this treatment is valid only for n 2 1, the correct result, fn = 1, is obtained when n = 0 is substituted. The above equation expressesthe intuitive result that a molecule population having n randomly spaced breaks per molecule can only inject a fraction l/(n + 1) of the DNA. Those molecules with n breaks inject a fraction Fnfn of the total DNA of the phage population, and thus, using Eq. (l), the fraction P of DNA injected by all the particles is =
s0
ran-
domly along the length of the DNA molecule; and (2) they are also assumed to be distributed randomly among the molecule population. Thus the fraction F, of molecules suffering n breaks, in a population having
173
X INJECTION
per
P = k (1 -
e-“)
(2)
174
SHARP
AND
Appendix 2: GRAIN DISTRIBUTION FOR FIRST-INJECTED PIECE THEORY: DERIVATION
Assuming the first-piece-only injection hypothesis to be true, it is the purpose here to calculate the autoradiographic grain distribution expected over bacterial cells containing only that radioactive DNA which can be injected by X-rayed, partially-injecting phage, the uninjected DNA having been removed by blending. Consider a population of phage particles with x average randomly spaced doublestrand breaks per particle. A certain fraction dq(x, y) of the phages will inject a fraction between y and y + dy of their DNA, y = 1 representing total injection. The probability of injection of y to y + dy is given by the sum of the probabilities dpn(y) for the class of molecules with exactly n DNA breaks, each multiplied by the probability e-“(xn/n!) of occurrence of that class :
ddx,
Y> =go
b(y)
dpnh)
=
dy
: --6(1 - y) dy
(n 2 1)
(4a)
(n = 0)
(4b)
where 6 is the delta function, Eq. (5):
defined by
dq(x, y) = --6(1 - y>e-” dy + gl n(1 - y)“-le-z
+ xe-’
ccax-y1- l)!yy-’
dy
n=l
(n
-
= --6(1 - y>e-” dy +xe -’ e2(1--y)dy dq(x, y) = --6(1 - y)e-” dy + xe-Xv dy
(6)
Phages injecting y to y + dy of their DNA will give yz grains on the average, where 2 is the average number of grains over cells containing fully injected (unirradiated) phage and exposed for the same time interval. Thus for cells infected by such phage the probability of obtaining exactly m grains is Pm(y, 2) = e-‘” T(yzyz)”
(7)
The probability s,(z, x) of obtaining exactly m grains among the whole phage population is the sum of the probabilities P, for each of the partial-injection classes, multiplied by dq(x, y), the frequency of that class occurring:
Y) s P~(Y, 1
&%(x,
2)
=
!I=0
2)
ddx,
We thus combine the expressions for Pm(y, z) and dq(x, y) from Eq. (7) and Eq. (6), respectively : =
s (YZ)” eAffzz
0
b) dx =
dy
= --6(1 - y)e-” dy
s,(x,z)
6(x -
$ 0
1
a’ sa
Combining Eqs. (3), (4a), and (4b) gives
(3)
e-’
dpn(y) can be calculated as the differential of p,(y), the probability that at least a fraction y of the DNA is injected. p,(y) = (1 - y)“, the probability that all of the n breaks lie anywhere beyond the first fraction y of the DNA, i.e., within the remaining fraction (1 - y). Thus n(1 - y)“-’
FREIFELDER
(5)
1, a6b6a’ ! 0, for all other
b
Equation (4b), giving dpn(y) for n = 0, simply states that for unbroken molecules the probability of injection of y of the DNA is zero for 0 5 y < 1, and 1 for y = 1.
*r--6(1 - y>e-” + xe-“]
dy
Using the property of the delta function a’ 6(x - b)f(x) dx sa = p, I 0
(8)
(a 6 lJ s a’) for all other
b
INTERRUPTION
OF PHAGE
molecule, sm(x, Z) = fraction
leads bo the result ?n ~~(2, x) = eC ?- e-” + 2 31, m! .
I, =
I0
(9)
This distribution
is normalized: m
1 yme-(r+z)”@
IO = f (1 - e-“)
of cells with
m grains, b = x + x.
and has the required property that the average number of grains per cell is decreased by X-rays by the factor calculated in Appendix 1:
Letting b = 2 + X, (10)
2
Integration
175
X INJECTION
mh(5,
2) = z [;1 (1 -
eO]
by parts yields I,
= -ie-’
+gImBl
(11)
In order to compare these theoretical curves directly with experimental data the function
The general form for this integral, satisfying Eqs. (10) and (II), can be shown to be (12) Combining
Eqs. (9) and (12) yields m sm(x, 2) = 5 e-’ (13) 1 where J: = average DNA breaks per molecule; z = average grains per phage DNA
will be plotted against m. This function has t wo useful features: (1) it is a linear function of m if s, is the Poisson distribution, the slope being log,(z), the log of the average number of grains per infected cell; and (2) it is independent of changes in “zero” fraction SO but sensitive to changes in the grain distribution among cells having grains. Thus g, is expected not to change with X-rays for the “all-or-none” model, since the grain distribution will still be Poisson with the same mean, x.
43, 16(i-175 (1971)
Interruption
of DNA JOHN
Injection
D. SHARP’
Graduate Departrnenl of Biochemistry,
by X-Irradiation DAVID
AND
of Phage
x
FREIFELDER2
BTandeis UniveTsily,
Waltham,
Massachusetts
02164
Accepted September I, 1970
When X phage is X-irradiated, it loses the capacity to inject all of its DNA into Escherichia coli. Autoradiographic measurement of the amount of DNA injected by X-irradiated phages leads to the conclusion that only a single DNA fragment is injected-a piece extending from the end normally injected first to the first double-strand break. This partial injection accounts for about half of the lethal effects of X-irradia. tion in phagex. Loss of adsorptive capacit.y and gross destruction of the phage head are shown to contribute only very slightly to the inactivat,ion process. INTRODUCTION
The process of infection of a sensitive bacterium by a phage consists of the following stages: adsorption of phage to bacterium, injection of phage DNA into the bacterium, macromolecular synthesis, phage assembly, and lysis of the bacterium (Stent’, 1963). All but one of these stages, i.e., injection, have been extensively studied during the past two decades. In this paper we report the results of an investigation of one aspect of the injection process. In particular, we ask the question: Can a phage inject its entire complement of DnTA even if the DNA molecule is fragmented by X-rays? An important problem in understanding the injection process is how a free end of the DNA molecule gets t’hreaded through the tail orifice. One might imagine that t’his problem could best be met during phage assembly if the phage tail were either synthesized around the free end or at’tached t,o the filled head so that the free end was contained in the tail. Caro (1965) has obtained electron micrographs of osmotically shocked X phage which show the DNA passing out of the head and threading through t,he tail, which suggests 1 Present address: Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115. z To whom reprint requests should be addressed. Reprints will not be available within the United States.
that the problem of initiation of injection might be so handled during assembly. If this is t’he case, it might be predicted that if a double-strand break were introduced into the DNA while still in the phage head, the first piece of DNA, i.e., up to the doublestrand break, would be injected but the new end would not be able to find the t,ail opening. Alternatively, if t,he injection mechanism itself were somehow dependent on t,he intactness of the DNA molecule so that DNA damage would prevent injection from even being initiated, or if for example some X-ray damage to the phage tail completely blocked injection, X-rays would produce a mixture of noninjecting and totally injecting phage particles. These possibilities can be stated as two models for phage injection: First-piece-only injection model. By this model, an X-ray-induced double-strand break is assumed to allow injection of only one fragment of DNA-that one extending from the end normally injected first to the first double-strand break. All-or-none injection model. By this model, an X-ray damaged phage particle is assumed to inject either its full complement of DNA or no DNA at all, but never an intermediate amount. This investigation made use of the two facts: (1) Double-strand breaks can be introduced into the DNA within the phage
166
INTERRUPTION
OF PHAGE
x INJECTION
167
using a Phillips (Miiller) RT-100 X-ray machine operating at 100 kV and 7 mA or 8 mA, with a beryllium window and a 0.78 mm aluminum filter. The sample, 0.05-0.4 ml, was placed in a small glass vial in a brass holder which was immersed in ice during the irradiation. This holder could be sealed with a Lucite cap for irradiation under N2 or a similar perforated cap for irradiation in air. Dosimetry was performed by t’he FeS04 met’hod of Battaerd and Tregear (1966). The X-irradiation done in the experiments reported in this and the accompanying paper was all done in Mg-T (except as noted) containing 5 mM histidine, under a Nz atmosphere. The Nz was found necessary t,o obtain maximum complementation in the experiMETHODS AND MATERIALS ments of the accompanying paper. This phedIecliu. X broth = 10 g Difco Bactonomenon will be discussed in a future pubtryptone, 10 g NaCl, 1000 ml HzO. Autolication. clave, t’hen add 20 ml sterile 20% (w/v) malDNA strand breakage was measured by tose. X agar = 10 g Difco Bacto-tryptone, 5 analyzing DNA sedimenting boundaries in g NaCl, 12 g Difco agar, 1000 ml HSO, auto- the UltracenOrifuge as described by Freifelder clave. NBSA (Sutrient broth soft agar) = (1965). 5 g Difco nutrient broth, 5 g NaCI, 8 g Difco Test for DNA release by X-rays. X-iragar, 1000 ml HzO. llledium 1.21.4.68 g NaCl, radiated Xhc phage (20 pg/ml) was sedi1.49 g KCl, 1.07 g NH&l, 0.203 g MgCl~. mented in the Beckman Model E ultracen6H20, 2.22 g CaCls, 2.S4 g Na2S04, 14.5 g trifuge at 9945 rpm in 1 M NaCI. The sediTris(hydroxymethyl)aminomethane, H,O to menting boundary was seen in UV photo1000 ml, adjust to pH 7.5 with HCl, autographs taken within the first 10 min. The clave. MB-P. 0.01 M MgSO+ 0.01 M po- speed was then increased to 33,450 rpm and tassium phosphate, pH 6.8. Concentrated more photographs were taken. At the lower stocks are autoclaved separately and mixed speed the only visibly moving boundary was when cool. Mg-!Z’. 0.01 III MgSO+ 0.01 M that of phages, whereas at the higher speed, tris(hydroxymethyl)aminomethane-HCI, pH DNA sedimentation was observed. 7.5. Trichloroacetic acid-precipitable radioacE. coli and lambda strains. C600 (suII+ tivity assays. l\lost radioactivity assays were BI- thr- leu- lac- TIR T5R), and CR34(Xi434) done by precipitation with 5% trichloro( surI+ thv- BI- thr- leu- met- TIR) were acetic acid (TCA). Herring sperm DNA, 3-5 obtained-from A. D. Kaiser. C600(Plkc) pg/ml, was added to the sample, followed by was obt,ained from the collection of l\iI. an equal volume of 10 % TCA and the prel\leselson. Xhc,2004, referred t’o here as cipitate was collected on a J!Iillipore filter simply Xhc, was isolated in the laboratory of (0.45 p pore size) by suction filtration, R. Thomas and obtained from K. Brooks. washed several times with 10% TCA, and Plaque assays were done by standard finally with 95 % ethanol. The filters were methods using x agar and NBSA for bottom dried in a 100°C oven, placed in vials conand top agar, respectively. X-Ray survival taining 10 ml scintillation fluid (toluene, of Xi4a4was measured using C600 as indicacontaining Liquifuor, New England Nutor. Plating bact,eria were grown with shakclear Corp.), and counted in a liquid scint,iling at 37” in X broth to 5 X 108/ml, centrilation counter. fuged and resuspended in Mg-P at log/ml, TCA-soluble radioactivity assays. TCAand stored on ice less than 4 hr before use. was measured in the X-irradiation. All X-irradiation was done soluble radioactivity
head by X-irradiabion of the intact phage (Freifelder, 1966, 1968); and (2) uninject’ed DNA can be removed from a lambda phagebacterium complex by the classical blending procedure of Hershey and Chase (1952). It was thus possible to prepare bacteria containing only injected radioactive phage DNA and free of uninjected DNA, and to examine t,hem autoradiographicallp to determine the dist,ribution of amount of phage DNA injected. This study revealed part,ial injection as a frequent consequence of Xirradiation of phage X, in quantit’ative agreement wit,h t,he model for “first-piece-only” injection.
168
SHARP
AND
degradation experiment as follows. Fiftymicroliter samples were mixed with 0.2 ml of 1 1M NaOH. Within 30 min, 0.2 ml herring sperm DNA (2.5 mg/ml) was added, followed by 0.55 ml of 10% TCA. The samples were mixed and centrifuged for 10 min at 7000 g; an aliquot of the supernatant was counted in 10 ml of Bray’s scintillation mixture (Bray, 1960). Radioactive phage. Thymine-2-14C or thymine-methyl-3H was used to prepare Xi434 containing radioactive DNA by UV induction of CR34(Xi434). The lysogens were grown with shaking at 37” to 2 to 3 X 108 cells/ml in medium 121 supplemented with 10m3M KH2P04, 0.2 % Bacto-casamino acids, 0.4 % glucose and 5 pg/ml thymine. They were centrifuged and resuspended in the same medium except that 2 pg/ml radioactive thymine replaced the unlabeled thymine. The culture was then induced with UV and grown for 2-3 hours, by which time lysis was complete. For the highly radioactive Xi434-3H used in the autoradiography experiments, 3 pg/ml catalase was added to the thymine-3H (to remove peroxides) before mixing with the culture; this maintained the phage yield at nearly normal levels. Bacterial debris was removed by centrifugation and the phage were separated from most of the nonphage radioactivity either by zone sedimentation in a 5-20% (w/v) sucrose gradient or by sedimentmg into a preformed CsCl step gradient. This latter method gave phages of higher purity and at higher concentration, and was done as follows: Four consecutive 2 ml layers were placed in a tube for the Beckman SW 25.1 rotor: CsCl, p = 1.7; CsCl, p = 1.5; CsCl, p = 1.3; and 20 % sucrose, all containing Mg-T. The phage lysate (20 ml) was then carefully layered onto the sucrose layer and the tube was spun at 24,000 rpm for 90 min at 20”. Fractions were collected from the tube bottom (12-14 fractions, each 0.5 ml) and those containing the phage peak were pooled and dialyzed against Mg-T after adding 10 pg/ml gelatin. Phage adsorption and injection. CR34 (xi434) bacteria were prepared as for plaque assays, at log cells/ml in Mg-P, but with 5 pg/ml thymine added to both the growth
FREIFELDER
medium (X broth) and the Mg-P. Radioactive phage was added, and the mixture was incubated for 20 min at 0” for adsorption to occur. Further incubation for 20 min at, either 37” or O”, respectively, allowed or prevented injection (Bode and Kaiser, 1965). The blended-bacterium complexes were then blended in a Waring Blendor stainless steel micro-at.tachment (3-20 ml capacity) with blades about x-inch long. The complexes were then centrifuged for 10 min at 7000 g, and the supernatants were removed, TCA precipitated, and counted. The precipitates were resuspended in Mg-P, TCA precipitated, and counted. From the amount of radioactivity in the supernatant (S) and pellet (P) the percent adsorbed, A, was calculated: A = 100 P/(S + PI. Control experiments indicated that a blending speed of about 5000 rpm and 10 min blending time were best in terms of producing the maximum difference in adsorbed radioactivity for injected vs. uninjected phage. Under these conditions less than 10% of the radioactivity was usually removable from injected phage-bacterium complexes, whereas at least 90 % was usually removable from uninjected complexes. These controls were always run, as well as an unblended control bo measure initial levels of unadsorbed phage, when adsorption or injection by X-irradiated phage was measured. The “percent adsorbed” of X-irradiated phage was calculated relative to the percent of radioactivity adsorbed to the bacteria in unirradiated phage which was always greater than 70% for the results reported. Adsorption and injection decreased with age in 14Cand 3H phage preparations, and this was compensated for by these controls. For the auhoradiography measurements the Ai434-3Hwas very highly radioactive and lost adsorption and injection ability measurably within a week. Thus in the experiments reported here t,hese phage were always used 1 or 2 days after growth. Autoradiography was carried out essentially according to Caro and van Tubergen (1962). Dichromate-washed glass slides were “subbed” by dipping into a solution of 0.5 % gelatin + 0.05 % chromium potassium sulfate. Drops of bacteria in 1Ig-P
INTERRUPTION
OF PHAGE
were spread gently with the edge of a coverslip and allowed to dry. Kodak AR. 10 stripping film was applied by standard methods. After drying in a stream of air, the slides were placed in a desiccated, lighttight box at 4” for exposure. The slides were developed in Kodak D-19 developer for 2 min, rinsed in 1% acetic acid for lo-15 set, fixed in 30% (w/v) Na&&0~ for 1 min, washed in water for 1 min, dried, and stained. Staining, necessary to visualize the bacteria, was done by dipping a slide for 2-3 min in 0.02 % crystal violet in 30 % ethanol, then 15% ethanol for 10 set, washing in water for 1 min, and then drying in a stream of air. Grains were counted with a microscope at 1600X under oil immersion. RESULTS
Survival Curves for Phage Viability Breakage
and DNA
Figure 1 shows the results of X-irradiation of phage X in a histidine-containing buffer in terms of phage inactivation (loss of plaqueforming ability) and DNA breakage. This agrees with earlier results for phages T7 and X (Freifelder, 1968), in that the rate of DNA breakage is approximately half that of inactivation. Efects of X-Irradiation on Adsorption and Gross Phage Structure The effect of X-irradiation on the adsorption process was measured as follows:
DNA
FIG. 1. Relative rates of X phage killing and DNA breakage by X-rays. Xhc was irradiated at 10.0 kradlmin. The DNA was released and sedimented in the analytical ultracentrifuge to measure the amount of broken DNA.
169
X INJECTION
OIL--J! 0
’ 2
I X-Ray
’ 3 Dose
I
I
I
I
I
4
5
G
7
8
(I Untt=
185
Klloradsl
FIG. 2. Effect of X-rays on adsorption of phage X to Escherichia coli cells. XiQ4-W or 3H were Xirradiated and adsorption was measured as described in Methods and Materials. Solid line is X0 the slope of the phage survival curve, for comparison with the data.
Xi434-14Cphage were X-irradiated and allowed to adsorb at 0” to bacteria. At this temperature adsorption without injection occurs (Bode and Kaiser, 1965; and see below). The bacteria were then separated from unadsorbed phage by centrifugation. Figure 2 shows the results of such an experiment. X-irradiation has a small but measurable effect in agreement with the finding of HradeEn& (1966). To test whether this loss of adsorption is due to breakage of the phage head and release of the DNA rather than to a direct effect on adsorption, the intactness of the phage structure was examined after X-irradiation. Phages were X-irradiated and then sedimented in the analytical ultracentrifuge at low and then high speed in order to see the phage and DNA boundaries, respectively, as described in Methods and Materials. If the phage particle had burst and released its DNA, one would expect to see some DNA sedimenting more slowly than the intact phage. No such material was seen even with a dose yielding 0.01% survival, so that there is little or no breakage of the phage head (<0.003 per lethal hit). Hence the small loss of adsorption reported above is probably due to an effect on the adsorption apparatus. Lack of Degradation of DNA Injected by X-Irradiated Phage Before quantitative studies of injected DNA could be carried out it was necessary
170
SHARP
AND
FREIFELDER
trol. Note that limit’ed nuclease activity on irradiated DKA is not detected by this test, but only degradation sufficient to make the DNA acid-soluble. This is the appropriate control, since acid precipitability is the criterion used to detect DNA in this and the accompanying paper (Sharp et al., 1970). E$ect of X-Irradiation on the Inject&m Capacity of the Phaye Population 60
0 Time
After
Id0
120 InjeCtiOn,
Min.
FIG. 3. Stability of DNA injected by X-irradiated Xi434-14C. Phage were irradiated and allowed to adsorb to CR34(Xi434) and inject their DNA (20 minutes at 37”) as for the blending experiments (see Methods and Materials). The infected complexes were kept at 0” for various periods of time after the 20-min adsorption incubation. The incubation was terminated by lysing the cells with NaOH and precipitation with trichloroacetic acid. -U--O-, phage not X-irradiated; -O-O-, phage X-irradiated to 1OW survival; -O-O--, phage adsorbed to C6OO(Pl), and incubation continued at 37”, for maximum degradation.
to ascertain that injected, irradiated DNA was not degraded under the conditions used. This point is raised by the recent finding of Huston and Pollard (1967) that E. coli cells treated with ionizing radiation degrade their own DNA. Xi434-14Cwas X-irradiated to a survival of 1O-3 and mixed with E. coli cells for adsorption and injection. As in the experiments reported in this and the accompanying paper, bacteria lysogenic for Xi4s4were used so that phage growth and cell lysis, but not adsorption or injection, were blocked. TCA-soluble radioactivity was measured after lysing the cells at various times with NaOH (see Methods and Materials). As a test of the method to show that degradation could be seen, cells lysogenic for phage Pl were also infected with hi4s4-14C.Arber and Dussoix (1962) have shown t’hat the presence of the Pl prophage causes the X DNA to be degraded. The results of this experiment are given in Fig. 3, in which it is shown that there is negligible degradation of the x DNA with or without X-irradiation, but 60 % degradation in the Pl lysogen con-
Xi434-14Cphage were X-irradiated with various doses and allowed to adsorb to bacteria and inject their DNA. The injected phage-bact.erium complexes were then blended to remove uninjected DNA. The bacteria containing the injected DNA were separated from the uninjected DNA by sedimentation and the amount injected was determined as a function of X-ray dose. The results of this experiment are shown in Fig. 4. The points agree with the curve labeled “Partial Injection Prediction, Eq. 2” which represents the first-piece-only model (see Appendix 1). It is clear from Fig. 4 that more DNA than is represented by unbroken molecules must be injected
:
kOO6 0.04 0
I 0.5 X-Rrv
1.5
Dose
I I Unit
2.0
2.5
,\ 3.0
1 3.5
= 185 Kilorodsl
of X-rays on injection of DNA by phage X. 14C- or 3H-labeled Xi4$4were X-irradiated and injection was measured by adsorption and blending as described in Methods. The unit X-ray dose chosen is the DZT for DNA double-strand breaks, so that the abscissa represents z, the average number of double-strand breaks per DNA molecule. The prediction for all-or-none injection is represented by the line labeled “Fraction of Unbroken DNA.” FIG.
4. Effect
I IO
INTERRUPTION
OF PHAGE
by the X-irradiated phage. The question remains whether this broken DNA is injected according to the first-piece-only model, or independently of DNA breakage. To answer this, the amount of DNA injected by individual phages after X-irradiation was measured autoradiographically.
x INJECTION
lil
6
Autoradiographic Measurement of the Effect of X-Irradiation on the Injection Capacity of Single Phages Injected complexes of Xi434-3H phage X-irradiated with various doses were prepared as for the blending experiment in the preceding section, using a low (0.1-0.2) multiplicity of infection to avoid multiple infection. After three cycles of centrifugation and resuspension, the bacteria, free of unadsorbed phage and DNA removed by the blending, were dried onto glass slides which were then covered with autoradiographic stripping film, dried, and exposed. After suitable periods of time the slides were developed and stained. Silver grains were counted microscopically and the grain distribution over bacterial cells was plotted. Since the effect of blending on leakage of intracellular components is not well known, it is important that blending of the infected complexes with unirradiated phage 7
P
d
6 t
-2 1234567 m = No Groins Over Cells FIG. 5. Autoradiographic grain distributions for unirradiated phage. CR34@?) cells infected with Xi434-3H were placed on slides for autoradiography before (0) and after (X) removing uninjetted DNA by shearing and centrifugation. Exposure was for 27 days. sm is the fraction of cells having m grains, and gm is a straight line when s,,, is a Poisson distribution (see Appendix 2). Average grain number per infected cell was z = 2.9.
o 2 4 6 '3 m= No Grains Over Cells
grain distributions FIG. 6. Autoradiographic for X-irradiated phage. Xi434-3H were X-irradiated to a survival of 5 X 1O-3, mixed with CR34(Xi434) bacteria and allowed to adsorb and inject. The infected cells were placed on slides for autoradiography before (0) and after (0) being sheared in a blendor and washed by centrifuging. Exposure was for 60 days. sm is the fraction of cells having m grains, and gm is a straight line when sm is a Poisson distribution (see Appendix 2). The straight line was drawn to fit the distribution for unsheared cells, giving an average of 4.4 grains per infected cell. Equation 13 of Appendix 2 was used to calculate the curved line, based on this average number (z = 4.4) and the phage survival, from which the average number of double-strand breaks was calculated: x = -x log, (5 X 1OW) = 2.65.
does not alter the shape of the grain distribution from that obtained with unblended samples. As explained in Appendix 2, the way used here for plotting the grain distributions gives no change from the straight line (Poisson distribution) when fewer cells contain radioactive phage DNA, as long as the distribution remains the same among cells with grains. Thus although a small fraction of the phages do not inject and are removed by blending, this should not change the distribution. This is shown to be the case in Fig. 5. The results of this experiment when done with X-irradiated phage are shown in Figs. 6 and 7. The theoretical grain distributions (calculated in Appendix 2) for the firstpiece-only model are shown in the figures (curved lines). The all-or-none injection model predicts no change on shearing, as was the case for unirradiated phage. It is important to note that the vertical separation of the curves of approximately one
172
SHARP AND FREIFELDER 6 5 E -.““; .‘;
0
Y z J II E CD
4
2 0 I
Sheared
0 -I
yzIff?r
Unsheared
3
[I
-2 234567 m = No. Groins
Over
Cells
FIG. 7. Autoradiographic grain distributions for X-irradiated phage. Same experiment as Fig. 6, except exposure was for 27 days. The slope of the “unsheared” line gives z = 2.64 grains per cell. The curved line is calculated from Eq. 13 of Appendix 2, using x = 2.65 and z = 2.64.
unit represents a factor of e (natural log base) difference in s,, the relative frequency of cells with m grains. Hence it is clear that the data agree well only with the first-pieceonly model. DISCUSSION
It has been shown that if one or more double-strand breaks are introduced into a x DNA molecule in an intact phage, only the first piece, i.e., up to the first doublestrand break, is injected. Hence the phage probably does not have a mechanism for threading the newly generated free end through the tail. The fact that first-pieceonly injection occurs makes possible a determination of the direction of injection of X DNA since one may ask, by genetic analysis, which genes have been injected, or by physical analysis, which end has been injected. The results of these studies are presented in the accompanying publication (Sharp et al., 1970). The phenomenon of first-piece-only injection answers an important question in phage radiobiology concerning the relation between lethal hits and double-strand breaks. It has been shown that the rate of double-strand breakage is half the X-ray inactivation rate and that if the irradiation is done in the absence of oxygen (in which case the inactivation rate decreases2-fold), the inactivation rate equals the rate of double-strand breakage (Freifelder, 1965,
1966). Hence it was tentatively concluded that double-strand breakage is a major cause of X-ray inactivation. However, unless al2 the chemical effects of X-irradiation are known, agreement of rates can be fortuitous, as has been pointed out previously (Freifelder, 1966). For example, it is possible that if all fragments were injected, the injected fragments might somehow be reassembled in viva-lethality then being due to other, unknown effects. However, the results presented here show that firstpiece-only injection is a frequent consequence of X-irradiation so that a significant fraction of the lethal hits are chemical changes that lead to incomplete injection. This is apparently the first example of an unambiguous relation between a biological change produced by X-rays and lethality. Rigorously, however, double-strand breakage is only strongly implicated as the cause of first-piece-only injection; proof that this is the case still rests upon agreement between the rate of production of doublestrand breaks and of the lesion producing first-piece-only injection. ACKNOWLEDGMENT This work was supported by contract AT(301)3797 from the Atomic Energy Commission, Grant GM-14358 from the National Institutes of Health, and E-509 from the American Cancer Society. JDS was supported by a predoctoral fellowship (GM-31,266) from the National Institutes of Health; DF by a Career Development Award (GM-7617) from the National Institute of General Medical Science. This is publication No. 745 of the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154. REFERENCES ARBER, W., and DUSSOIX, D. (1962). Host specificit,y of DNA produced by E. coli: I. Host controlled modification of bacteriophage X. J. Mol. Biol. 5, 1836. BATTAERD, H. A. J. B., and TREGEAR, G. W.
(1966). Radiation ferrous sulphate
dosimetry-the Fricke-Miller dosimeter. Rev. Pure Appl.
Chem. 16, 83-90. BODE, V. C., and KAISER, A. D. (1965). Changes in
the structure and activity of lambda DNA in a superinfected immune bacterium. J. Mol. Biol. 14, 399-417. BRAY, G. A. (1960). A simple liquid scintillator
for
INTERRUPTION
OF PHAGE
counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, 279-285. CARO, L. G. (1965). The molecular weight of lambda DNA. vi’irology 25, 226-236. CARO, L. G., and VAN TUBERGEN, R. P. (1962). High-resolution autoradiography. J. Cell. Biol. 15, 173-188. Davmow, P. F., HOLLOWAY, B., and FREIFELDER,
D. (1964). Interruptions strands in bacteriophage
in the polynucleotide DNA. J. Mol. Biol. 8,
l-10. FREIFELDER, D. (1965). Mechanism of inactivation
of coliphage T7 by X-rays. Proc. Nat. Acad. Sci. U. S. 54, 128-134. FREIFELDER, 1). (1966). Lethal changes in bacteriophage DNA produced by X-rays. Radiat. Res. 6, Suppl., 80-96. FREIFELDER, D. (1968). Physicochemical studies on X-ray inactivation of bacteriophage. Virology 36, 613-619. FREIFELDER, D., and DAVISON, P. F. (1962). Studies on the sonic degradation of deoxyribonucleic acid. Biophys. J. 2, 23b247. HERSHEY, A. D., and CHASE, M. (1952). Independent frunctions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36, 39-56. HRADEENA, Z. (1966). X-ray inactivation of lambda phages. I. Damage on adsorption and injection. Znt. J. Radiat. Biol. 10, 443-449. HUSTON, D. C., and POLLARD, E. C. (1967). DNA degradation in E. coli 15 T-L- induced by fast proton bombardment. Biophys. J. 7,555-565. SHARP, J. D., DONTA, S., and FREIFELDER, D. (1971). Lack of polarity of DNA injection by E. coli phage h. Virology 43, 176-184. STENT, G. S. (1963). “Molecular Biology of Bacterial Viruses.” W. H. Freeman, San Francisco, California. Appendix 1: CALCULATION OF THE EFFECT OF DNA DOUBLE-STRAND BREAKS ON THE AMOUNT OF DNA INJECTED BY PHAGE x: FIRST-PIECE-ONLY HYPOTHESIS
It is assumed here that double-strand breaks allow injection of DNA only up to the first break. For this calculation, two specific assumptions, verified by Freifelder and Davison (1962), are made: (1) The breaks
are assumed
to be distributed
received
an average
of J: breaks
molecule, is assumed to be given by the Poisson distribution: F,, = em5 $ 0
Among molecules with exactly n breaks, the average proportion fn of DNA between the injected end and the first break can be expressed as fn = 1’ .z ClP
where x is the length from the injected end, in units of the total length of a lambda DNA molecule. cZP is the differential probability that a given DNA molecule has its breaks arranged so that (a) the first break lies between z and z + dz, and (b) the other n - 1 breaks lie between z and 1, the position of the last-injected end. dP is the product of the probabilities of these two requirements being met, or n dz for requirement (a) and (1 - x)+-l for (b). Thus dP = (1 - ~)~--lndz, from which fn = &zlP 1
1 x(1 - z)“-52 czz= n+1 Although this treatment is valid only for n 2 1, the correct result, fn = 1, is obtained when n = 0 is substituted. The above equation expressesthe intuitive result that a molecule population having n randomly spaced breaks per molecule can only inject a fraction l/(n + 1) of the DNA. Those molecules with n breaks inject a fraction Fnfn of the total DNA of the phage population, and thus, using Eq. (l), the fraction P of DNA injected by all the particles is =
s0
ran-
domly along the length of the DNA molecule; and (2) they are also assumed to be distributed randomly among the molecule population. Thus the fraction F, of molecules suffering n breaks, in a population having
173
X INJECTION
per
P = k (1 -
e-“)
(2)
174
SHARP
AND
Appendix 2: GRAIN DISTRIBUTION FOR FIRST-INJECTED PIECE THEORY: DERIVATION
Assuming the first-piece-only injection hypothesis to be true, it is the purpose here to calculate the autoradiographic grain distribution expected over bacterial cells containing only that radioactive DNA which can be injected by X-rayed, partially-injecting phage, the uninjected DNA having been removed by blending. Consider a population of phage particles with x average randomly spaced doublestrand breaks per particle. A certain fraction dq(x, y) of the phages will inject a fraction between y and y + dy of their DNA, y = 1 representing total injection. The probability of injection of y to y + dy is given by the sum of the probabilities dpn(y) for the class of molecules with exactly n DNA breaks, each multiplied by the probability e-“(xn/n!) of occurrence of that class :
ddx,
Y> =go
b(y)
dpnh)
=
dy
: --6(1 - y) dy
(n 2 1)
(4a)
(n = 0)
(4b)
where 6 is the delta function, Eq. (5):
defined by
dq(x, y) = --6(1 - y>e-” dy + gl n(1 - y)“-le-z
+ xe-’
ccax-y1- l)!yy-’
dy
n=l
(n
-
= --6(1 - y>e-” dy +xe -’ e2(1--y)dy dq(x, y) = --6(1 - y)e-” dy + xe-Xv dy
(6)
Phages injecting y to y + dy of their DNA will give yz grains on the average, where 2 is the average number of grains over cells containing fully injected (unirradiated) phage and exposed for the same time interval. Thus for cells infected by such phage the probability of obtaining exactly m grains is Pm(y, 2) = e-‘” T(yzyz)”
(7)
The probability s,(z, x) of obtaining exactly m grains among the whole phage population is the sum of the probabilities P, for each of the partial-injection classes, multiplied by dq(x, y), the frequency of that class occurring:
Y) s P~(Y, 1
&%(x,
2)
=
!I=0
2)
ddx,
We thus combine the expressions for Pm(y, z) and dq(x, y) from Eq. (7) and Eq. (6), respectively : =
s (YZ)” eAffzz
0
b) dx =
dy
= --6(1 - y)e-” dy
s,(x,z)
6(x -
$ 0
1
a’ sa
Combining Eqs. (3), (4a), and (4b) gives
(3)
e-’
dpn(y) can be calculated as the differential of p,(y), the probability that at least a fraction y of the DNA is injected. p,(y) = (1 - y)“, the probability that all of the n breaks lie anywhere beyond the first fraction y of the DNA, i.e., within the remaining fraction (1 - y). Thus n(1 - y)“-’
FREIFELDER
(5)
1, a6b6a’ ! 0, for all other
b
Equation (4b), giving dpn(y) for n = 0, simply states that for unbroken molecules the probability of injection of y of the DNA is zero for 0 5 y < 1, and 1 for y = 1.
*r--6(1 - y>e-” + xe-“]
dy
Using the property of the delta function a’ 6(x - b)f(x) dx sa = p, I 0
(8)
(a 6 lJ s a’) for all other
b
INTERRUPTION
OF PHAGE
molecule, sm(x, Z) = fraction
leads bo the result ?n ~~(2, x) = eC ?- e-” + 2 31, m! .
I, =
I0
(9)
This distribution
is normalized: m
1 yme-(r+z)”@
IO = f (1 - e-“)
of cells with
m grains, b = x + x.
and has the required property that the average number of grains per cell is decreased by X-rays by the factor calculated in Appendix 1:
Letting b = 2 + X, (10)
2
Integration
175
X INJECTION
mh(5,
2) = z [;1 (1 -
eO]
by parts yields I,
= -ie-’
+gImBl
(11)
In order to compare these theoretical curves directly with experimental data the function
The general form for this integral, satisfying Eqs. (10) and (II), can be shown to be (12) Combining
Eqs. (9) and (12) yields m sm(x, 2) = 5 e-’ (13) 1 where J: = average DNA breaks per molecule; z = average grains per phage DNA
will be plotted against m. This function has t wo useful features: (1) it is a linear function of m if s, is the Poisson distribution, the slope being log,(z), the log of the average number of grains per infected cell; and (2) it is independent of changes in “zero” fraction SO but sensitive to changes in the grain distribution among cells having grains. Thus g, is expected not to change with X-rays for the “all-or-none” model, since the grain distribution will still be Poisson with the same mean, x.