VIROLOGY

68,

1-13 (1975)

Effects of Radiophosphorus I. The Mechanism JACK Department

of Genetics,

Uniuersit,v Accepted

Decay in Bacteriophage

T4D

of Phage Inactivation N. LEVY’ of Washington,

Seattle,

Washington

98105

May 14, 1975

Decay of “P incorporated into the DNA of bacteriophage T4D inactivates the phage and produces single- and double-strand breaks in phage DNA, all with single-hit kinetics. The lethal efficiency for 32P-labeled phage stored in buffer at 4’ was O.!O, and double-strand breaks in the DNA are formed at the same rate. When the storage medium is supplemented with 2.8% (w/v) AET (2-aminoethyl isothiouronium bromide hydrogen bromide, a free-radical trap), double-strand breaks and lethal damages occur at the rate of 0.06 per szP decay. This suggests that double-strand breaks are the lethal damages. Single-strand breaks accumulate at the rate of one per 32P decay for S2P-labeled phage stored in buffer at 4’. The lethal efficiency of ssP for phage stored in buffer is about 65% that of 32P. The protective effect of AET is nearly as great for 33P-labeled T4D as it is for 32P-labeled T4D. For 32P, between 35 and 89%) of the lethality is due to recoil. Not more than 10% of the lethality is due to radiation effects, and the remainder (if any) is due to transmutation. For 33P, recoil accounts for less than half (probably no more than 5%) of the lethality. Radiation can account for nearly half of the lethality. Transumutation could account for all of the lethality and probably accounts for over half. INTRODUCTION

Since the initial demonstration of the lethal effects of decay of 32P incorporated into bacteriophage T2 DNA (Hershey et al., 1951) more than a dozen laboratories have reported such measurements in a variety of bacterial viruses. These data have been tabulated elsewhere (Levy, 1972). For most free phage having doublestranded DNA and stored in aqueous buffer at 4”, reported lethal efficiencies (i.e., lethal damages per radioactive decay) vary from 0.07 (Stent, 1953) to 0.19 (Denhardt and Sinsheimer, 1965; Stent and Fuerst, 1955). Damages to the DNA resulting from radiophosphorus decay have also been detected in the form of double- and single-strand breaks. There is general agreement that the efficiency of singlestrand break production is about one per ‘Present address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Mass. 02111. Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.

32P decay. Reported frequencies for double-strand breakage (again in buffer at 4”) vary from 0.056 (Tomazawa and Ogawa, 1967) to 0.20 per decay (Thomas, 1959). These results have also been tabulated in detail elsewhere (Levy, 1972). It has been difficult to draw conclusions about the physical cause of lethal lesions because of the variations in these measurements from one laboratory to the next and because careful measurements of both biological and physical lesions made on the same preparation in the same laboratory on samples prepared similarly, were lacking. This paper presents the results of both kinds of measurement, and it is concluded that all 32P decays cause single-strand breaks and about a tenth of the decays break both strands. Ley and Krisch (1974) have recently published a similar analysis. In order to understand the mechanism by which decay of incorporated radiophosphorus produces lethal lesions, I have studied the effect of 32P decay under various

2

.JA(‘K N. IX\‘\

storage conditions and also compared the effect of 32P decay with the effect of decay of a second phosphorus isotope, “P, similarly incorporated into phage DNA. The decay schemes for these two isotopes are: ‘P + 1”:

f:S + Ed

=p 16 +

ES + e-

The mean P-particle energy for 32P decay is 0.70 MeV, while that for 33P decay is only 0.093 MeV (Strauss, 1958). The generally accepted model for the mechanism of strand breakage by 32P is that of Stent (1953). This model proposes that all 32P decays result in single-strand disruptions due either to (a) instability of the sulfur diester bond that replaces a phosphodiester bond upon transmutation of 32P to 32Sor (b) the ejection of the sulfur nucleus from the backbone of the DNA strand as a result of recoil from the emitted P-particle. The model further proposes that double-strand breaks result when the recoiling sulfur atom has sufficient energy and proper orientation to break the complementary strands as well (Stent and Fuerst, 1955). Several observations are difficult to reconcile with this model. These include: (a) The finding that phage stored in buffer supplemented with AET’ are killed by decay of incorporated 32P only about 60%) as fast as phage not so protected (Matheson and Thomas, 1960); (b) DNA may be less sensitive to double-strand breakage after release from virus particles than when packaged (Tomazawa and Ogawa, 1967; but see also Ley and Krisch, 1974); (c) early infective centers are less sensitive to 32P decay than the corresponding free bacteriophage particles (Stent. 1953; Symonds and McCloy. 1958) and (d) 33P has a lethal efficiency nearly as great as that of 32P, although its recoil energy is much smaller (Krisch, 1970). I confirm here the finding of Krisch (1970) that decay of incorporated 33P kills T4 with nearly as high an efficiency as does decay of incorporated 32P. Further. it is

shown that AET protects against the lethal effects of 33P decay to nearly the same extent as it protects against 32P decay. The mechanism by which lethal damages are produced is discussed in light of these findings. It is also shown that 32P produces double-strand breaks in T3 DNA at the same rate as lethal damages and that both are suppressed to the same extent by AET. MATERIAI,S

AND

METHODS

Media. The following media were prepared in accordance with recipes in the literature cited: Hershey broth, referred to below merely as broth (Chase and Doermann, 1958); H medium, a Tris-buffered minimal salts medium (Hershey, 1955); T2 buffer, a phosphate-buffered storage solution (Hershey and Chase, 195%) and top and bottom agar (Chase and Doermann, 1958). H buffer is H medium without glucose, phosphate or sulfate. Amino acid stock solution contained X.0 mg/ml or saturating amounts (whichever is the lesser at refrigerator temperatures) of the following amino acids: alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine. glycine, histidine. isoleucine, leucine, lysine, phenylalanine. proline. serine. threonine, tryptophan. tyrosine, and valine. Phage stored anaerobically were bubbled with nitrogen and sealed in glass ampoules until the time of assay. Special chemical. AET was obtained from Sigma Chemical Company and purified by the dialysis method of Litt (1958). Samples of AET from Mann Research Laboratories gave similar results in my hands. The AET solutions were always neutralized before use by addition of NaOH, since the buffering capacity of I“2 and H buffers alone was insufficient to neutralize the stronglv acid AET. Bacteria. Escher;chia coli strains CR63(Xh) (restrictive for rII but permissive for amber mutants) and S/6 (permissive for rI1 but restrictive for amber mutants) were used as plating bacteria in assaying phage stocks and came from the collection of A. H. Doermann. E. coli BB #552. also from ‘Ahhreviations used: AET. 2.8’, (w/v) Z-aminoethyl isothiouronium bromide hydrogen bromide; CY,, the Doermann collection. was used in preparing stocks of T3D ac41 rh45. On this lethal efficiency of radiophosphorus decay (in lethal e:Jents per decay). strain. rI1 mutants undergo lysis inhibi-

3zP INACTIVATION

tion, which results in higher stock titers than obtained with normal E. coli B. Overnight cultures were prepared by innoculating 30 ml of broth with a loop of bacterial culture and growing the culture so prepared at 30” with aeration for at least 8 hr. Exponential-plating bacteria were prepared by diluting overnight cultures 50-fold into fresh broth, growing with aeration at 30” for 2.5 hr, spinning down in the centrifuge and resuspending the cells in one-fourth volume of prechilled broth. Such bacteria were stored on ice or in a refrigerator until discarded. Overnight and plating bacteria used for the experiments of Table 1, Part B, were prepared at 37”. Plating bacteria made at this temperature were harvested after 1.75 hr of growth. Phage strains. All 32P-labeled phage used for the experiments reported in this paper were T4D ac41 rb45, a double mutant obtained by crossing the two single mutants, which in turn are from the collection of A. H. Doermann. Survival of these phage was followed by periodic plating on E. coli S/6. In all experiments, the survival of a parallel 31P-labeled T4D ac41 rb45 preparation was also followed (external control). In many experiments 31P-labeled T4D amN122 was added to the 32P-labeled preparations (internal control) and the survival of these phage followed by periodic assay on E. coli CR63(Xh). Preparation of phage stocks. Phage stocks were prepared from 4-5-hr plaques by the method of Doermann and Hill (1953). 32P-lat)eled phage were prepared as follows: An overnight culture of E. co/i BB was grown at 37” in H medium supplemented with 2 x 10m4M (each) phosphate and sulfate and a 100~ dilution of amino acid stock solution. This culture was diluted lOOO-fold into prewarmed medium of similar composition, but 5 x 1O-5 M in phosphate and 1O.-4 M in sulfate. The diluted culture was then grown at 37” with aeration for 4 hr, at which time “PO, was added to make the total phosphate concentration lo- 4 M, and “‘PO, was added to make the specific activity of 32P equal to the final desired 32P activity. In no case did the 32P exceed 0.001 of the total phosphorus. At 5.5 hr, sulfate and phosphate

OF T4D

3

were added to bring the final concentration of both to 2 x 10e4 M. At the same time [32P]phosphate was added to keep the specific activity of 32P at its previous level, and the bacteria were infected at a multiplicity of two. Adding fresh phosphate at the time of infection resulted in higher phage titers than were obtained if the same total amount of phosphate was present from the time of the initial bacterial inoculation. By adding 32P at its final specific activity two to three bacterial generations before phage infection, uniform labeling of phosphate pools was assured. 33P-labeled phage stocks were prepared similarly. Phage used for inoculating labeled cultures were from a highly concentrated and purified stock stored in T2 buffer. The contribution of phosphate from the inoculum was in all cases less than 1%) of the total phosphate. Isotopically labeled phage were harvested 5.5 hr after infection by first treating infected cells with chloroform and then spinning out cell debris. Labeled stocks were stored between 4 and 6”. (In this text such stocks are referred to as having been stored at 4”.) They were kept at an activity of less than 1 pCi/ml for most experiments. Isotopes and specific activity of labeling:. Both phosphorus isotopes were obtained from New England Nuclear Corporation in the form of H,PO,. The scintillation counter used in preparing all samples except those of Table 1B was calibrated against a 32P standard from the same supplier and used at channel settings which gave 0.90 counts/disintegration for 32P. The specific activity of 32P in the growth medium was taken as the ratio of 32P measured on this counter to 31P (as K,HPO, and KH,PO,) weighed out to make a stock solution and appropriately diluted from this. The number of labeled atoms per phage was determined from this specific activity, assuming that each T4 genome contains 4 x lo5 phosphorus atoms. Analysis of DNA fragment sizes. The molecular size distribution in samples of native or denatured DNA was determined by zone sedimentation in neutral or alkaline sucrose, respectively. Neutral sucrose

4

JACK N. LEVI

was prepared in H buffer, adjusted to 1 N in NaCl and pH 7.3. Linear gradients containing 12 ml of 5-20% (w/v) sucrose were prepared in nitrocellulose tubes for a Beckman SW 41 rotor. Native DNA was extracted from phage by using freshlv distilled phenol that was saturated with H buffer (pH 7.3) and was 10m3M in EDTA. Two volumes of phenol were gently pipetted into a centrifuge tube containing 0.5-1.0 ml of phage solution at a concentration of no more than 4 x 10” (live plus 32P-killed) phage/ml (about 10 pg of DNA/ ml). This was carefully rolled by hand to allow maximal contact between phases and allowed to stand 30 min at room temperature. The tubes were then chilled on ice, centrifuged briefly at low speed to separate phases, and the phenol layer removed with a Pasteur pipet. This was followed by two similar phenol extractions with lo-min incubations. Finally, 0.3 ml of aqueous (DNA) phase was layered onto the gradient using the large end of a disposable l.O-ml plastic pipet. The gradients were spun in a Beckman L2-65B ultracentrifuge for 5.5 hr at 33,000 rpm, after which fractions of 10 or 12 drops were collected (from the bottom of the gradient) directly onto Whatman GF/C glass-fiber filters. The filters were batch washed in three changes of 5% cold trichloroacetic acid and one change of 95q’ ethanol, dried, placed in scintillation counting vials with 10 ml of toluene-based scintillation fluid, and counted in a Model No. 547 Packard Tri-Carb liquid scintillation spectrometer. Alkaline sucrose contained 4 x lo-’ M phosphate and was also 1 N in NaCl. After addition of other ingredients, it was adjusted to pH 12.2 with 4 M KOH. DNA was released from phage directly on top of the gradients in pH 11.8 buffer according to the method of Yamagishi (1968). This minimized the breakage of single-stranded DNA due to shearing during handling. Alkaline gradients also contained 12 ml of 5-20%) (w/v) sucrose and were spun in an SW 41 rotor of an L2-65B ultracentrifuge for 5.75 hr at 33,000 rpm. Collection of fractions and preparation of filters for counting were as for neutral gradients.

In order to determine the average number of breaks per molecule in native or denatured DNA samples. a computer program was written which generated theoretical gradient profiles to which the experimental profiles were compared. The computer-generated profiles were based upon (i) the sedimentation profile of a homogeneous DNA population, as determined empirically by using (unbroken) DNA from “C-labeled T4D (this allows correction for the spreading of a homogeneous sample during sedimentation which occurs as a result of diffusion, interaction with the walls of the centrifuge tube. etc.); (ii) the size distribution of fragments obtained when an initially uniform population of polymers is subjected to a given average number of breaks per molecule (the breaks being inflicted at random among the molecules and at random positions within molecules) as derived by Montroll and Simha (1940); (iii) the relations between sedimentation rate and molecular weight for native DNA in neutral sucrose and for denatured DNA in alkaline sucrose, as determined by Studier (1965); and (iv) the position in the gradient of unbroken molecules (determined for each centrifuge run by appropriate [l%]DNA control). Given the position of whole molecules and Studier’s relations the computer uses the program to determine the size of the largest and smallest fragments which would be found in each gradient fraction from the top of the gradient to the whole molecule position. Given the Montroll-Simha relationship and the average number of breaks per molecule, the computer determines the fraction of nucleotides occurring in each size fragment and then sums over the size range of fragments to be found in each of the gradient fractions. Finally, the whole molecule “peak” is redistributed according to the specific profile observed in the 14C control. Theoretical profiles were fitted to the experimental profiles by eye. The fits were close enough to allow unambiguous measurement of the average number of breaks per molecule to within 20? for low amounts of breakage and to within 10% for samples receiving an average of more than

=P INACTIVATION

two breaks per molecule. Copies of the programs, sample printouts, and a series of profiles for varying numbers of breaks are given in Levy (1972), along with a more detailed explanation of the program itself. RESULTS

AND DISCUSSION

Survival of Radiophosphorus-Labeled T4. Any of three effects might contribute to the lethality of radiophosphorus decay. These effects are: (i) nuclear recoil, sustained by the newly produced sulfur nucleus when the P-particle is emitted; (ii) transmutation effects, due to the substitution of a sulfur atom for a phosphorus atom in the DNA backbone (these effects would include bond breakage due to substitution of sulfur for phosphorus or through dissipation of the excitation energy of the sulfur atom), and (iii) radiation effects, that is, loss of energy by the exiting P-particle. Results of representative phage survival experiments are presented in Table 1. (Survival data on phage samples which contained less than 60 radiophosphorus atoms per phage particle have not been included, but were in close agreement with those in the table.) These experiments have been carried out in two different laboratories, and data coming from the two laboratories has been segregated in Table 1 since lethal efficiencies for 32Pwere slightly but consistently higher as measured at Tufts University than they were at the University of Washington. The cause of this variation is not known. All experiments to be reported in other sections of this paper and in the companion article (Levy, 1975) were carried out at the University of Washington. Because of this variability, I have based my conclusions upon comparisons of the survival of portions of a single phage preparation stored under a variety of conditions or upon comparison of 32P- and 33P-labeled phage samples prepared and stored in parallel (i.e., samples 8 and 9 of Table 1). The results do not permit exact calculation of the fraction of lethality due to each of the three sources listed above, but do range of greatly restrict the “allowable” each of these variables. To facilitate visual-

5

OF T4D TABLE

1

LETHAL EFFECTS OF RADIOPHOSPHORUSDECAY” Phage SamPie

storage Medium

Isotope

Atoms Per phw

a,.

A. Experiments done at University of Washington 32P H buffer 89 0.108 1 32P 0.104 H buffer 60 2 32P 80 0.112 3 H buffer 32P 80 0.107 4 T2 buffer 32P 120 0.090 5 T2 buffer 32P 0.079 T2 buffer 160 6 32P 123 0.097 7 T2 buffer H buffer + His 32P 60 0.097 2 H buffer + His 32P 80 0.104 3 32P 0.101 2 Broth 60 H buffer + AET 32P 0.047 2 60 32P T2 buffer + AET 80 0.059 4 T2 buffer + AET 32P 120 0.060 5 =P 0.057 T2 buffer + AET 160 6 32P 0.058 T2 buffer + AET 123 7

8 8 8 9 9 9

B. Experiments done at Tufts University T2 buffer =P 89 =P Broth 89 T2 buffer + AET =P 89 T2 buffer =P 93 33P Broth 93 33P T2 buffer + AET 93

0.142 0.122 0.100 0.084 0.082 0.063

a Stocks of T4D ac41 rb45 with incorporated ‘*P or 33P were prepared, and the number of labeled atoms per phage determined, as described in Materials and Methods. Stocks were stored at 4” in the media listed in the table. Survival was assayed periodically on E. coli S/6 and normalized to the titer of a 3’P-labeled T4D ac41 rb45 stock prepared in parallel (external control) or to the titer of a 31P-labeled T4D amN122 stock added to the same tube and assayed on E. co/i CR63 (hh) (internal control). Results for stocks having at least 60 radiophosphorus atoms per phage are presented and are in close agreement with results from stocks with lesser amounts of label. Values for u, were determined by extrapolating the survival curve to OY and to lOO? isotope decay and computing the final fraction of survivors. This was taken as the zero class of a Poisson distribution and the number of lethal damages required to yield a zero class of this size read from a table of exponential functions. The errors included with mean values in the text were calculated as the sum of the standard deviations of individual determinations and 3%#of the mean, which allows for the (possible) error in instrument calibration. The following abbreviations are used here in addition to those defined above: His, 10m3 M histidine; Cys, lo-’ M cysteine.

6

JACK

ization of the discussion to follow. the reader is referred to Fig. 1, where these three variables, which I wish to evaluate for each of the two isotopes, have been plotted on triangular coordinates. The contribution of each of the three types of damage corresponding to any point within the triangle is taken as the distance from the point to the corresponding side of the triangle. The sum of these distances is always equal to the altitude of the triangle. In Fig. 1 I have taken the lethal efficiency for 32P to be 0.100 and I have adjusted the lethal efficiency for 33P to 65% of this value, or 0.065. In evaluating the data of Table 1, I will reach “hard” and “soft” conclusions regarding the range of contribution to lethality that may come from each of the three sources. Hard conclusions, or restrictions, are those necessitated by theoretical considerations and the data obtained. Soft conclusions are those that seem very likely, but for which less likely alternatives exist. With respect to Fig. 1, the hard conclusions reached below restrict the permissible range of each source of lethality to the shaded area within each triangle; values corresponding to points within the unshaded area are thus eliminated. The soft conclusions further limit the range likely for each source to the dark shaded area, thus the values corresponding to points within the lightly shaded area are possible but unlikely. The lethal efficiency of 32P for phage stored in buffer is (from Table 1A) 0.100 + 0.007. This agrees well with measurements reported previously (see Levy (1972) for a review). It can also be seen from this table that storage of 32P-labeled phage in buffer supplemented with 10m3 histidine or storage in broth results in lethal efficiencies of 32P decay similar to that for phage stored in unsupplemented buffer. These conditions are known to partially protect phage from the lethal effects of X irradiation due to suppression of free-radical mediated damages (Freifelder, 1965). In contrast, AET, which also has been assumed to protect as a result of its action as a free-radical trap (Matheson and Thomas, 1960), is able to prevent about 40% of the lethal damages resulting from

N. LEVY

SOURCESOF 33P LETHALITY

SOURCESOF 32P LETHALITY

B

i‘\

FIG. 1. Mechanism of radiophosphorus lethality. In the form of graphs on triangular coordinates are the contributions to the lethality of T4 phage from each of the three possible mechanisms by which radiophosphorus decays can produce lethal lesions. The contribution of each mechanism corresponding to a point within one of the two graphs is equal to the distance from the point to the side representing that mechanism, i.e., transmutation damages, radiation damages, or recoil damages. Since the triangles are equilateral, the sum of these three distances will always equal the altitude of the triangle. The triangles have therefore been drawn with relative altitudes of 0.065 (A, representing 33P lethality) and 0.100 (B, representing 32P lethality). With each triangle, the unshaded area represents values for the three sources of lethality that are not possible. The lightly shaded area includes the points corresponding to values that are possible but not likely, while the dark portion of the triangle includes the combinations of values that are considered to be likely.

=P INACTIVATION

decay of incorporated 3zP and protects phage from the lethal effects of 33P to a nearly equal extent (Table 1). The difference in the abilities of these compounds to protect against the two types of radiation damage may be due to the fact that free radicals created during radiophosphorus decay arise in the DNA itself while many of the free radicals that are important in Xray-induced lethality arise in the medium (Szybalski, 1967). Thus the protective effects for radiophosphorus decay may depend upon specific associations of AET with the phage DNA (see below). The lethal efficiencies reported in Table 1B are slightly higher than those in Table 1A but agree well with values published by Krisch (1970). The lethal efficiency of 33P for phage stored in buffer or broth in 0.083 * 0.004 or about 65% of the lethal efficiency of 32P in a parallel sample. If nuclear recoil contributes to this lethality, one would expect the effect to be greater for 32P than for 33P since 87% of 32P decays produce nuclear recoils of greater energy than the maximum recoil energy obtainable from a 33Pdecay (Persson, 1964; Strauss, 1955). Indeed, nuclear recoil is the only one of the three possible sources of lethality which would be greater for a 32P decay than for a 33P decay. Therefore the greater lethal efficiency of 32P decay must be a result of nuclear recoil. Thus for 32P, at least 0.035 lethal events per decay must be due to nuclear recoil. For 33P, the mean recoil energy is only 1.7 eV (Krisch and Zelle, 1969). Since the types of chemical bonds found in DNA have energies of 2-4 eV (Pauling, 1948), we must conlcude that for the majority of 33P decays the energy of recoil is insufficient to break such bonds, even if all of it could be directed toward bond breaking. I thus reach the hard conclusions that at least 0.035 lethal events per 32Pdecay are due to recoil and less than 0.032 lethal events per 33P decay, i.e., less than half of the total lethality, result from this source. Furthermore, if the likelihood of a recoil causing a lethal damage increases as the recoil energy increases, then a comparison of the recoil energies for 32P and 33P leads us to the soft conclusion that only a very small fraction of 33P decays can

OF T4D

7

produce lethal damages by this mechanism. This fraction is taken as 5%, i.e., 0.003 lethal events per 33P decay, for purposes of illustration in Fig. 1. If radiation effects contribute to lethality, one can expect about three times as much of an effect from 33P as from 32P (Levy, 1972). The greater effect of 33P @-particles is a consequence of the fact that the lower energy decays of this isotope produce slower electrons than those of 32P, and a slow electron deposits more energy per unit track length than does a fast one, since it has more time to interact with the atoms it passes. It has been shown (Hershey, 1951) that very little if any lethality due to 32P decay can be simply the result of irradiation of one segment of a T4 phage’s DNA by a decay occurring elsewhere within the phage particle. Insight into the lethal effects of P-particles is provided by the work of Funk et al. (1968). These authors have found that the lethal efficiency for tritium decay (for T4) is 0.12 for decays originating in the phage DNA and 0.046 for decays occurring in the phage protein. They also calculate that the path length of the exiting P-particle through the (packaged) phage DNA would be 2.6 times as great for a decay occurring within the DNA as for a decay occurring within the phage protein. Since the lethal efficiencies are proportional to the track lengths they conclude that all lethality due to such tritium decays results from P-particle effects. It seems reasonable that radiation effects would indeed be the major source of lethality from this isotope since the maximum recoil energy for tritium is only 3.2 eV (Strauss, 1958) and since even when the decays occur in DNA it is the (thymine) base rather than the backbone which is disrupted by the transmutation. The linear energy transfer for a tritium P-particle is about 1 eV/A (Funk et al., 1968) while that of a 32P P-particle is only about 0.019 eV/A (Levy, 1972). Thus the lethal efficiency of ST from this source would be only (0.019) (0.12) = 0.0023 per decay. One might imagine that a certain fraction of the lethal events result from the combination of two damages, one occurring at the site of decay, due either to nuclear

8

JACK

N. L,EV\I

recoil or transmutation, and the other resulting from energy loss from the /3-particle, i.e., producing a second lesion in the associated strand. Since phage inactivation by incorporated 32P follows single-hit kinetics (Hershey et al., 1951; and many others), I conclude that such combined damages can only be important when the two lesions result from the same decay event, but perhaps not even then. A quantitative theoretical treatment of this type of damage has set 0.007 lethal events per decay as the upper limit for 32P decay (Levy, 1972). This number is the fraction of 32P decays in which there would be an energy loss event by the exiting P-particle within 20 base pairs of the primary decay. Thus radiation effects are involved in no more than 0.01 lethal events per decay for 32P (10% of the total lethality) and no more than three times this or 0.03 (about 46% of the total) for 33P. These are hard restrictions. We know that if transmutation damages contribute to the lethality of radiophosphorus decay, the contribution must be equal for 32P and 33Psince the chemistry of the two transmutations is identical. We therefore can be sure that no more than about 65% of the total lethality for 32P can result from this sort of damage. This is a hard conclusion which has in essence already been reached, since it was shown above that at least 35% of the 32P lethality is due to recoil effects. Furthermore, if we assume that AET does not protect against recoil damages, then at least 40% of the 32P lethality must be due to transmutation or radiation effects, since this is the amount of protection conferred by AET (see Table 1). Since no more than 10% of the lethality is due to radiation effects, I reach the soft conclusion that at least 30% of the 32P lethality, or 0.03 lethal damages per 32P decay, must result from chemical (transmutation) effects. An additional soft conclusion is that an equal absolute amount of killing (that is, again at least 0.03 lethal damages per decay) must result from transmutation damages for 33P decay. This would be at least 46% of the total lethality due to 33P.

Strauss (1958) has given the excitation energy of the sulfur nucleus resulting from 32P decay as about 60 eV. An equally excited sulfur atom would result from 33P decay. This energy must be dissipated to surrounding atoms during return of the excited atom to the ground state, and such energy transfers might result in production of free radicals. Although it is not known how the excitation energy is dissipated, it is apparent that the energy is sufficient to break chemical bonds with strengths of 2-4 eV. It thus seems quite possible that a major fraction of the 32P and 33P lethality could result from this mechanism. The fact that AET shows nearly as much protection for 33P-labeled phage as for 32P-labeled phage (see Table 1) supports the contention that the damages which it prevents do not result from recoil effects. However I have considered these to be soft conclusions since the possibility remains that AET can provide some protection against recoil damages. This might be the case if some recoil damages are produced via free-radical intermediates or if recoil damages can be prevented by physical stabilization of the double helix. It is known that the binding of bis(2-guanidoethyl)disulfide (GED. a dimer of AET which forms spontaneously in aqueous solutions, though at a slow rate) to calf thymus DNA can raise the melting temperature by 15” (Jellum, 1965). Finally, I reach the hard conclusion that no more than 89% of the 32P lethality can result from recoil. At least half of the 33P lethality, i.e., 0.032 lethal events per decay, is due to transmutation and/or radiation effects. If all of this lethality were due to transmutation, then no more than 68%’ of the 32P lethality (0.068 lethal events per decay) could result from recoil, since there is an equal transmutation effect for both isotopes. We also know that the radiation effect is one-third as great for 32Pas for 33P, so that if this half of the 33P lethality results from radiation (P-particle) effects, then at least 11% (0.011 lethal events per decay) of the 32P lethality must be due to radiation effects. In this case no more than 89% of the 32P lethality can be due to

=P INACTIVATION

recoil. If both transmutation and radiation contribute to 33P lethality, the upper limit on the recoil effect for 32P would be between 68 and 89%. This limit is also indicated in Fig. 1. Breakage of LINA by 32P Decay

9

OF T4D

and the corresponding theoretical profile. The DNA sample used for this gradient came from phage which had received 30.2 32P decays per strand, i.e., 60.4 decays per phage. The theoretical profile which best fits this data is that for 26 single-strand breaks. Figure 3 shows the results of neu-

The extent to which DNA of T4 phage was broken by decay of incorporated 32P was determined by zonal sedimentation in alkaline or neutral sucrose gradients to measure single-strand and double-strand breaks, respectively. The measured distribution of 32P counts in each gradient was compared with a computer-generated gradient profile in order to determine the corresponding number of breaks. Figure 2 shows an example of an alkaline gradient

n

buffer

buffer + AET

*-r -..,....... j ” 100

80

distance

60

A

B

-1

distance

y

/

40

sedimented

FIG. 2. Alkaline sucrose gradient profile from phage stored at 4” in buffer. A small amount (10 ~1) of azP-T4D ac41 rb45 which had sustained an average of 30.2 %*P decays per DNA strand during storage in buffer at 4” was added to 0.25 ml of pH 11.8 Yamagishi buffer, which had been previously layered at the top of a 12-ml 520% alkaline (pH 12.4) sucrose gradient. After 30-min incubation at 4” the gradient was placed in an L2-65B SW 41 rotor and was spun for 5.75 hr at 33,000 rpm. A “C-labeled T4D ac41 rb45 control (not shown) was spun at the same time in a separate tube and was used to determine the position to which unbroken molecules would have sedimented. The relative recovery of radioactivity is plotted as a function of the percentage of the distance between the top of the gradient and the whole molecule peak. The solid line gives the computer-generated profile for randomly broken single strands which best fits this data and corresponds to 26 single-strand breaks per DNA strand.

sedimented

Fro. 3. Neutral sucrose gradient profiles of DNA from phage stored at 4” in (A) H buffer and (B) H buffer supplemented with 2.8’; (w/v) AET. The two samples are from the same phage lysate. Each 32Plabeled sample was mixed with “C-labeled T4D ac41 rb45 and the DNA extracted three times with buffered phenol (pH 7.3), as indicated in Materials and Methods. A small amount (0.3 ml) of the DNA solution was layered on top of a 12.ml 5-20% neutral sucrose gradient and spun in an L2-65B centrifuge, SW 41 rotor for 5.5 hr at 33,000 rpm. The relative recovery of ‘*P radioactivity in the various gradient fractions is plotted as a function of the percentage of the distance between the top of the gradient and the whole molecule peak. The “C control (not shown) was used to determine the position and shape of the whole molecule peak. The solid line gives the computergenerated sedimentation profile for randomly broken DNA which best fits the experimental data. The phage used for (A) had incurred an average of 2.7 phage lethal hits per particle of the time of DNA extraction and the DNA had received an average of 2.6 double-strand breaks per molecule: the sample in (B) had received 1.5 phage lethal hits and 1.5 doublestrand breaks.

10

*JACK N. LEVE

tral gradients run on DNA from two portions of a phage preparation, one of which was stored during 32P decay in unsupplemented buffer and the other in buffer supplemented with 2.8%’ AET. The sample stored in buffer had received 2.7 phage lethal hits at the time of DNA extraction, and the experimental points best fit the theoretical curve corresponding to 2.6 double-strand breaks per molecule. The same phage stored in buffer + 2.8% AET had by this time received only 1.5 phage lethal hits and only 1.5 double-strand breaks per molecule. The theoretical profiles have been generated on the assumption that breaks are introduced at random sites, and the close agreement between the theoretical and experimental gradient profiles indicates that breakage is indeed at random, or very nearly so. The results of a number of such experiments are summarized in Fig. 4 and 5. It is clear from Fig. 4 that single-strand breaks accumulate at the rate of about one per 32P decay. Double-strand breaks occur at the rate of one per lethal event, or about 0.1 per decay. I conclude that each 32P decay creates a single-strand interruption at the site of the decay and that the lethal damages are probably double-strand breaks. The latter conclusion is strengthened by the observation that, when phage are stored in buffer supplemented with 2.8% AET during 32P decay, not only is lethality reduced by about 40% but doublestrand breaks in the DNA are decreased by a similar amount (Fig. 3). The fact that both double- and single-strand breaks accumulate in strict proportionality to dose indicates that, in the dose range covered, the observed damages result from single events and not from the accumulation of other damages, e.g., double-strand breaks do not result from the accumulation of single-strand breaks. Since single-strand breaks do not appear to contribute to the lethality of 32P decay, I conclude that they do not prevent injection and that they must be efficiently repaired in the host cell. Note that if every decay produces a singlestrand break and a tenth of the decays also produces a break in the associated strand,

; IO 1 :/‘/ 0

0

IO

/ ..._1-

20

30

32P decays FIG. 4. Rate of formation of single-strand breaks in DNA of 32P-Jabeled phage T4D stored in buffer at 4”. The number of single strand breaks was determined as described in the legend for Fig. 2, and the number of “P decays per single strand (N&2) at the time of DNA extraction was determined from the relation N&2 O.iO(S.A.) (1 co nrs5’), where S.A. is the specific activity of the medium in which the phage were prepared (in millicuries per milligram of P) and t is the elapsed time (in days) between the preparation of the 32P-labeled phage and sedimentation of the extracted DNA. The number of singlestrand breaks per single strand is plotted as a function of the average number of3*P decays per single strand. Error flags indicate uncertainties in breakage measurements. The straight line best fitting the experimental points was determined by linear regression analysis. The line has a slope indicating 0.95 singlestrand breaks per 32P decay.

the actual rate of accumulation of singlestrand breaks should be 1.1 per 32P decay. Thus the accuracy with which I am able to measure breaks is probably no better than 10%. CONCLUSIONS

The main conclusions to be drawn from the work reported here are: (i) The lethal lesions for T4 damaged by decay of incorporated 32P are double-strand breaks, which occur at the rate of about 0.10 per 32P decay for phage stored at 4” in buffer and 0.065 per 32P decay for phage stored in buffer supplemented with 2.8% (w/v) AET. (ii) Single-strand breaks accumulate in 32P-labeled T4 stored in buffer at 4” at the rate of one per decay and are nonlethal and

32P INACTIVATION

lethal

damages

Fro. 5. Hate of formation of double-strand hreaks in DNA of 32P-lat)eled T4D stored in buffer at 4”. The survival of 32P-labeled T4D ac41 rh45 was followed during anP decay by assay on E. co/i S/6. At intervals. samples were analyzed for double-strand hrcaks by zone sedimentation, as described in the lep;end for Fig. 3. As described in the text. the theoretical gradient profile which best fits the experimental data was selected by eye. Error flags on the experimental points indicate a visually determined range which the author felt must certainly include the theoretical profile giving the best fit. The number of double-strand breaks is plotted as a function of the average number of lethal damages sustained hy the phage at the time of DNA extraction. The straight line best fitting the experimental points was determined hy linear regression analysis and has a slope indicating 0.94 douhlestrand hreaks per lethal damage.

are thus presumably repaired during infection. (iii) The physical breaks produced in DNA by decay of incorporated 32P appear to be randomly located. (iv) The lethal efficiency of 33P is about 65% that of 32Pfor T4 stored in buffer at 4 ‘. (v) The protective effect of AET is similar for the two radiophosphorus isotopes. (vi) These results and theoretical considerations, taken together, restrict the amount of lethality for each isotope which can be attributed to each of the three possible mechanisms of killing, recoil, transmutation, and irradiation, to within narrow limits, as indicated in figure 1. For 32P, between 35 and 89% of the lethality is due to recoil effects. No more than 10% of the lethality is due to radiation

OF T4D

11

effects. For 33P, not more than 50% of the lethality is due to recoil and not more than 50% is due to radiation. Since it is unlikely that recoil damages do contribute as much as 50% to the 33P lethality, it is likely that transmutation damages make some contribution towards 33P lethality. Any involvement of transmutation in 33P lethality requires an equal involvement of The simtransmutation in 32P lethality. plest, though not necessarily the correct model consistent with these restrictions would be that transmutation effects account for all of the 33P lethality and contribute equally toward 32P lethality; recoil effects account for the greater lethal efficiency of 32P. The participation of transmutation and/or radiation effects (in addition to recoil) in creating the lethal damages explains why 33P also kills T4 phage with high efficiency. The release of DNA from a phage head, either into a host cell or into storage medium, would increase the chance of energy transfers occurring between the DNA and other molecules rather than between two portions of the DNA molecule itself. This might explain the slightly reduced killing and breakage ability of incorporated 32P after release of the DNA from phage. Ley and Krisch (1974) have made similar measurements of the effects of the two radiophosphorus isotopes on phage survival and DNA breakage. The results of these workers are generally in agreement with results presented here, the major exception being that they find that only can be about 75% of the 32P lethality accounted for by double-strand breaks, when 3”P-labeled T4 are stored in buffer at 4”. The reason for the discrepancy between my own result and that of Ley and Krisch is not clear. Kirsch (personal communication) has pointed out that in their experiments phage were stored in a glycerol casamino acids (GCA) medium rather than buffer and that the constituents of this medium may offer some protection. If the storage medium is responsible for the observed difference then apparently decays which would produce lethal double-strand

12

JACK

breaks in buffer are producing other types of lesions which are nevertheless lethal in GCA medium. Freifelder (personal communication) called to my attention his revised value for the exponent relating sedimentation rate of native DNA to molecular weight (0.38 rather than 0.35; see Freifelder, (1970)). By using this value in place of Studier’s, a breakage rate of 0.85 double-strand breaks per lethal decay was calculated from the sedimentation data presented here. Although this raises the possibility that not all lethal damages are double-strand breaks, it does not explain the discrepancy with the results of Ley and Krisch, since these workers also used the Studier figure in their analysis. It is interesting that Krisch has also found somewhat different efficiencies of 32P lethality in series of experiments done at different times; compare Krisch (1970) and Ley and Krisch (1974). The observation that some lethal damages are due to transmutation and/or radiation effects also emphasizes the importance of careful control of the conditions of preparation and storage of phage preparations for these types of experiments. Differences in small molecules or proteins packaged with the DNA and/or differences in storage media may acount for the variation in lethal efficiencies measured in different laboratories. ACKNOWLEDGMENTS The colleagues whose advice and encouragement sustained me throughout this work are too numerous to list here, but special thanks are due to A. H. Doermann and E. B. Goldberg for their suggestions and patience. Most of the work was carried out at the University of Washington and was supported by Grant No. GM-13280 from the National Institute of General Medical Sciences to A. H. Doermann and by Training Grant No. 5TOl GM-00182 to the author. A minority of the work done at Tufts University was supported by Grant No. GM-13511 to E. Goldberg from the National Institutes of Health. REFERENCES CHASE, M., and DOERMANN, A. H. (1958). High negative interference over short segments of the genetic structure of bacteriophage T4. Genetics 43, 332-353. DENHARD.~. D. T.. and SINSHEIMER, R. L. (1965). The process of infection with bacteriophage X174. V.

N. LEVY Inactivation of the phageebacterium complex by decay of “P incorporated in the infecting particle. J. Mol. Biol. 12, 663-673. DOERMANN. A. H., and HILL M. D. (1953). Genetic structure of bacteriophage T4 as described by recombination studies of factors influencing plaque morphology. Genetics 38, 79-90. FREIFEI.DEH, D., (1965). Mechanism of inactivation of coliphage T’l by X rays. Proc. Nat. Acad. Sci. USA 54, 128-134. FREIFELDER, D., (1970). Molecular weights of coliphages and coliphage DNA. IV. Molecular weights of DNA from bacteriophages T4, T5 and T7 and the general problem of determination of M. J. Mol. Biol. 54, 567-578. FUNK, F., PEHSON. S., and BOCKHATH. R. C., JR. (1968). The mechanism of inactivation of T4 bacteriophage by tritium decay. Bioph,ss. J. 8, 1037-1050. HERSHEY, A. D. (1955). An upper limit to the protein content of the germinal substance of bacteriophage T2. Virology 1, 108-127. HERSHEY, A. D., and CHASE, M. (1952). Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36, 39-56. HERSHEY, A. D., KAMEN, M. D., KENNEDY, J. W.. and GEST, H. (1951). The mortality of bacteriophage containing assimilated radioactive phosphorus. J. Gen. Physiol. 34, 305-319. JEI,LUM, E. (1965). Interaction of cystamine and cystamine derivatives with nucleic acids and nucleoproteins. Irzt. J. Radiat. Biol. 9, 185-200. KRISCH, R. E. (1970). Comparison of the lethal effects of “P and 33P decay in E. coli. and in bacteriophages. Int. J. Radiat. Biol. 18, 259-266. LEVY, J. N. (1972). Biological and Physical Effects of Radiophosphorus Decay, in Bacteriophage T4D. Dissertation, University of Washington, Seattle, Wash. LEVY, J. N. (1975). Effects of radiophosphorus decay in bacteriophage T4D. II. The mechanism of marker rescue. Virology 68, 14-25. LEVY, R. D., and KRISCH, R. E. (1974) Lethality and DNA breakage from 32P decay in bacteriophage T4. Int. J. Radiat. Biol. 25, 531-537. LITT, M. (1958). “The Critical Size of Transforming DNA.” Dissertation, Part II, Harvard University Cambridge, Mass. MATHESON, A. T., and THOMAS, C. A., JR. (1960). The indirect effects accompanying 32P suicide of bacteriophage. Virology 11, 289-291. MONTROL, E. W., and SIMHA, R. (1940). Theory of depolymerization of long chain molecules. J. Chem. Phvs. 8, 721-727. PAULING. L. (1948). “The Nature of the Chemical Bond”, 2nd ed. Cornell University Press, Ithaca, N.Y.

=P INACTIVATION PRESSON,B. (1964). An electron scintillation spectrometer. Nucl. Instrum. Methods. 27, l-9. STENT, G. S. (1953). Mortality due to radioactive radiophosphorus as an index of bacteriophage development. Cold Spring Harbor Symp. Quant. Biol. 18, 255-259. STENT, G. S., and FUERST, C. R. (1955). Inactivation of bacteriophages by decay of incorporated radioactive phosphorus. J. Gen. Physiol. 38, 441-458. STRAUSS, B. S. (1958). The genetic effect of incorporated radioisotopes: The transmutation problem. Radiat. Res. 9, 234-247. STUDIER, F. W. (1965). Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11, 373-390. SYMONDS, N., and MCCLOY, E. W. (1958). The irradiation of phage-infected bacteria: Its bearing on the

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relationship between functional and genetic radiation damage. Virology 6, 649-668. SZYBALSKI, W. (1967). Molecular events resulting in radiation injury, repair and sensitization of DNA. Radiat. Res. Suppl. 7, 1477189. THOMAS, C. A., JR. (1959). The release and stability of the large subunit of DNA from T2 and T4 bacteriophage. J. Germ. Physiol. 42, 503-523. TOMIZAWA, J.-I., and OGAWA, H. (1967). Breakage of polynucleotide strands by disintegration of radiophosphorus atoms in DNA molecules and their repair. II. Simultaneous breakage of both strands. J. Mol. Biol. 30, 7-15. YAMAGISHI, H. (1968). Release of DNA from phage on top of sucrose gradients. Biochim. Biophys. Acta. 166, 738-740.