Proc. Nati. Acad. Sci. USA Vol. 76, No. 7, pp. 3228-3232, July 1979
Biochemistry
Inactivation of phage T7 by near-ultraviolet radiation plus hydrogen peroxide: DNA-protein crosslinks prevent DNA injection (photobiology/double-strand DNA breaks/17 proteins)
P. S. HARTMAN, A. EISENSTARK, AND P. G. PAUW Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211
Communicated by Ernest R. Sears, April 19, 1979
ABSTRACT A nonlethal concentration of H202 (0.05%) greatly enhances near-ultraviolet (NUV) inactivation of phage T7. Simultaneous treatment with H202 and NUV reduces the amount of DNA injected into the bacterial host, but not- the number of phage adsorbed. Not only were recombination and gene expression of late markers reduced upon treatment of phage '11 with NUV plus H202, but also a gradient of recombination resulted, with markers injected first reduced to a lesser extent than those injected last. Double-strand DNA breaks were not detected; however, DNA-protein crosslinks were observed upon NUV plus H202 treatment of double-labeled T7. Previous studies demonstrated that single-strand DNA breaks did not account for phage death by NUV plus H20%. It is concluded that the DNA-protein crosslinks prevent normal injection of T7 phage DNA; such crosslinks may be important lesions in NUV cellular damage.
Labeling Techniques. Phage used for adsorption experiments were labeled as reported (9). The method of Hawkins (11) was used in double labeling. Both procedures yielded from 10-6 to 10-7 cpm per phage. Adsorption and Injection Studies. Phage were separated by the polyethylene glycol/dextran sulfate method (12) and further purified by differential centrifugation (13) prior to gradient centrifugal studies. Titers of ca. 5 X 1012 plaqueforming units per ml were routinely obtained. For phage adsorption and injection assays (14) E. coli B was infected with treated phage; 8 min after infection of cells, chloramphenicol was added, a sample was removed to assay radioactivity, and infected bacteria were removed by centrifugation. The pellet was resuspended and an aliquot was withdrawn to determine percentage of phage adsorbed. To determine the fraction of DNA injected into the host, cells were lysed, treated with DNase I, and centrifuged to remove bacterial debris as well as whole phage particles, and the supernatant was then assayed for radioactivity. This procedure is based on the assumption that the solubilized radioactivity represents the amount of DNA that is injected into the bacterial host, because only it is available for
Current interest in 300- to 400-nm near-ultraviolet radiation (NUV) is due in part to an increasing concern over potential environmental hazards (1). Induction of pyrimidine dimers and single-strand (SS) DNA breaks, and interference with DNA repair and transport systems, have been documented as NUV effects (1). Lethal and mutagenic effects of NUV are distinct from those of far-UV (254-nm) radiation (1, 2). NUV photolysis of tryptophan also generates photoproducts that are lethal to rec mutants of Salmonella typhimurium and Escherichia coil (3), as well as to mammalian cells in culture (4), H202 has been demonstrated as the biologically active photoproduct (5-7). H202 generation from tryptophan can enhance other biological actions by the same NUV wavelengths necessary for its initial generation. Simultaneous treatment with NUV plus H202 results in SS DNA breaks in bacteria (8). H202 also enhances NUV lethality in phage T7 (6, 9), thus providing a simple model system for the study of these synergistic interactions. While action spectrum analysis indicates synergism is maximal at 340 nm, the frequency of SS DNA break induction could not be correlated with peak of cell killing (9); i.e., there is no apparent direct relationship between SS
digestion by deoxyribonuclease. Genetic Recombination. For genetic crosses between amber mutants (15, 16) E. colh 011' (Su+) was coinfected with either T7 amli (gene 8) or am28 (gene 5), and one of a series of different amber mutants (helper phage) located throughout the T7 geqome. Lysates were assayed on 011' (Su+) and B (Su-) for total progeny and wild-type recombinants, respectively. Results are expressed as percentage of normal recombination (16). Average frequencies of wild-type recombinants for the control crosses were compatible with those in previous reports (15, 16). Gel Electrophoresis Studies. E. coli B was grown at 37°C to 4 X 108 cells per ml in M9 medium plus 2 mg of casamino acids per ml, irradiated with 72 J-M2 of far-UV radiation to suppress host cell protein synthesis, and incubated for 30 min at 30°C in the dark. Aliquots (2 ml) were then infected with appropriately treated T7 at a multiplicity of infection of 10 and incubated with 10 ,tCi (1 Ci = 3.7 X 1010 becquerels) of [-5S]methionine (New England Nuclear) per ml for 24 min at 30°C. Samples were washed and concentrated 10-fold by centrifugation and applied to polyacrylamide gels. Sodium dodecyl sulfate/polyacrylamide gradient slab gels were prepared 1.5 mm thick with a linear gradient of 8-13% acrylamide and a 5% acrylamide stacking gel (17). Gels were prepared for fluorography (18), dried, and placed in direct contact with Kodak Royal X-O-Mat film and incubated at -70'C for up to 12 hr. Proteins corresponding to specific T7 genes were identified by comparison with results of others (19, 20) and only those proteins that could be unequivocally assigned were compared.
breaks and cell death. In this report, we demonstrate that NUV plus H202 inactivates phage T7 by producing DNA-protein crosslinks, preventing injection of phage DNA into the host. MATERIALS AND METHODS Bacteria. E. coli B, the nonpermissive (no suppression of amber mutants, Su-) T7 host, and E. coli 011' (thy-), the permissive (Su+) host, were obtained from M. Center. Phage. T7 wild type and amber mutants am 193 (gene 1), am28 (gene 5), amIl (gene 8), am3 (gene 12), and am3l (gene 15) were provided by F. W. Studier (10). Phage stocks were grown, maintained, and assayed according to Studier (10). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
Abbreviations: NUV, near-ultraviolet radiation; DS, double strand; SS, single strand; Su+ and Su, suppressor and nonsuppressor of amber mutations, respectively.
this fact.
3228
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Hartman et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
3229
Analysis for Double-Strand (DS) DNA Breakage. T7 phage,
after purification, was extracted with phenol (13), layered on preformed 5-20% neutral (pH 7) sucrose gradients, and centrifuged, fractionated, and assayed for radioactivity as described (7). Analysis for DNA-Protein Crosslinks. Protein putatively crosslinked to DNA was separated from remaining protein by sedimentation through alkaline sucrose gradients; 0.4-ml fractions were collected and assayed for radioactivity (7). Elimination of the alkaline sucrose gradient step gave the same results (data not shown). Fractions showing high 3H (DNA) counts were pooled and dialyzed overnight against 0.05 M KHPO4 (pH 7.4). One milliliter of this dialysate was layered on a step gradient of CsCl consisting of 1.4 ml each of 1.75, 1.55, and 1.35 g/cm3, centrifuged at 40,000 rpm for 24 hr at 4VC (SW 50.1 rotor, Beckman L5-50 ultracentrifuge), followed by fractionation and detection of radioactivity (7). A small amount of 14C radioactivity cosedimented with the 3H in the CsCl gradients, even with the control or untreated phage. This material probably represents the fraction of 14C metabolized into DNA during phage labeling. Hawkins (11) noted similar conversions under identical labeling conditions. The following formula was used to calculate the percentage of '4C in a given fraction that was actually protein:
14Ccontrol/% 3Hcontrol) X 3Hi, in which % proteinj equals the percentage of 14C in fraction i %
protein,
= 14Ci - (%
%
that is actually in protein; % "4Ce equals the percentage of 14C radioactivity in fraction i; % 14Ccontrol equals the total percentage of 14C in fractions 5-9 of the control; and 3Hcontrol equals the percentage of 3H in fractions 5-9 of the control. The validity of the calculation was confirmed by performing similar experiments with [MS]methionine. There was no 35S radioactivity that cosedimented with the DNA peak in the control. Irradiation. Radiation was with four General Electric F15T8 BLB integral-filter black-light bulbs, as reported (6). After NUV and/or H202 treatment, catalase was added to destroy H202.
RESULTS Effects of NUV plus H202 on Adsorption and Injection. To test whether adsorption or injection might be impaired by NUV plus H202 treatment, the fate of 3H-labeled DNA of untreated and treated T7 phage was measured by a modified Hershey-Chase experiment (14, 21). Separate exposure to NUV radiation or H202 resulted in little or no decrease in viability, adsorption, or injection (Table 1). Phage adsorption was not significantly diminished by synergistic treatment; in contrast,
c 0
*m,
11
.0
E 0
'EE 0
c
0 OI
T7 genetic
map
FIG. 1. Frequency of recombination between amber mutants of T7 (identified by gene number on genetic map). (Left) T7 am28 (gene 5) was irradiated with 600 (o), 1200 (-), or 1800 (0) J-m-2, each in the presence of 0.05% H202. (Right) T7 amll (gene 8) was irradiated with 300 (o), 900 (M), or 1500 (0) J-m-2, each in the presence of 0.05% H202-
both viability and DNA injection were greatly reduced. The drop in viability was more drastic than the drop in DNA injection (Table 1). This discrepancy could have one of several explanations: (i) All-or-nothing injection: a fraction of the treated phage inject no DNA, but the remainder inject all of their DNA, albeit damaged. (ii) Partial injection: despite inactivation, phages are still able to inject a portion of their genome. (iii) A combination of i and ii could occur. Possibility ii may be distinguished from i and iii by comparing the amounts of each of several genes that are injected and transcribed. NUV plus H202 Effects of Genetic Recombination. To distinguish among the above possibilities, genetic crosses were conducted in which partially injected DNA could be "rescued." A particular amber mutant was treated and crossed with other amber mutants (helper phage) defective in other T7 genes. Because T7 injects its DNA linearly in a single direction with a unique starting point (15, 16, 22), the genes coding for the early proteins are injected first. If NUV plus H202 inactivates by all-or-nothing injection (i), recombination between the treated amber mutant and all helper phage would decrease uniformly, irrespective of map position. With partial injection
Table 1. Effects of NUV, H202, and NUV plus H202 treatments on adsorption and injection of T7* NUV dose, Injection,I Adsorption,T Survival, H202 time,t J.m-2 min Treatment % % % NUV 0.05% H202
NUV plus 0.05% H202
0 54,000
0 0
100.0 96.0
49 (100) 5:3 (108)
35.0 (100) 33.0 (94)
0 0
0 1.5
100.0 98.0
51 (100) 50 (98)
31.0 (100) 30.0 (97)
0 0 32.0 (100) 40 (100) 100.0 0.75 40 (100) 24.0 (75) 50.0 27,000 6.3 (20) 4.5 36 (93) 1.5 54,000 * Experimental procedure and calculations were as described by Karska-Wysocki and coworkers (14). Each value is the average of at least two independent experiments. t The NUV radiation dose of 54,000 J.m-2 is obtained by 1.5 min of exposure. 4 Numbers outside parentheses are absolute values; relative amounts of adsorption and injection are indicated inside parentheses.
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Biochemistry: Hartman et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
(ii), early genes should recombine to a greater extent than those injected last.am28 (gene 5) phage were exposed to several doses of NUV in the presence of 0.05% H202 and crossed with amber mutants defective in genes 1, 8, 12, or 15 (Fig. 1 left). With increasing exposure, there was a gradient of recombination inversely proportional to the order of injection, with genes injected late affected to a greater extent than those injected early. Identical experiments were performed with the later marker amli (gene 8), with confirmatory results (Fig. 1 right). As was the case with injection and adsorption (Table 1), separate NUV or H202 treatment had little effect on viability or recombination frequencies (data not shown). In addition to precluding the possibility of all-or-nothing (i) injection, a gradient of recombination allows a comparison of the decreases in recombination and viability. The degree of correlation between these two values permits assignment of the relative role that the injection defect plays in the NUV plus H202 inactivation of T7. Because a single phage must inject its entire DNA molecule to produce a burst of progeny phage, the percent decrease in distal recombination or maximal decrease in recombination that is possible with a given dose of NUV plus H202 can be extrapolated (Fig. 1). When the percent decrease in distal recombination for a given dose is plotted with the percentage of survivors for the same dose, the values are superimposable (Fig. 2), demonstrating that the injection defect is the primary, and perhaps the sole, cause of the synergistic inactivation of phage T7 by NUV plus H202.
Effects of NUV plus H202 on Protein Synthesis. An injection defect should also result in a "map-position-dependent" decrease in protein synthesis, because late genes would be transcribed to a lesser extent than early genes. Accordingly, wild-type T7 phage was exposed to three dosages of NUV and 0.05% H202 and used to infect E. coli B in the presence of [l5S]methionine, and the resulting proteins were electrophoresed through polyacrylamide gels. As with the genetic recombination experiments, a gradient resulted, with gene expression greater in early genes than in late genes (Fig. 3). Densitometric scans of the fluorogiams revealed that, as with the correlation between decreases in recombination and viability (Fig. 2), the values for maximal decrease in protein synthesis (34%, 8%, and 0.52%) correspond favorably with the survival at each dose (60%, 10%, and 0.17%, respectively) (data not shown). Insignificant Production of DS DNA Breaks by NUV plus H202. As noted previously (6, 9), the contribution of SS DNA breaks to death in T7 is insignificant. However, DS breakage could account for the striking enhancement of NUV lethality by H202, because x-ray inactivation of DNA-containing phages, including T7, is known to be due mainly to DS DNA breaks (23), which can impede injection (15, 24). The possibility of death by DS breaks was examined by the sedimentation of 3H-labeled T7 DNA through neutral sucrose gradients. T7 was either irradiated with 20,000 J.m-2 of NUV in the presence of 0.05% H202 or held in the dark. Even after a dose that resulted
_
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Dose, J-m-2 x 10-2 FIG. 2. Effects of NUV plus 0.05% H202 on survival (open symbols) and decrease in distal recombination (closed symbols) of phage T7. Recombination values were extrapolated from Fig. 1 as explained in the text. 0, Survival of am28; 0, distal recombination of am28; 0, survival of am 11; *, distal recombination of amlI.
-0~
-PO03
c d FIG. 3. Fluorogram of 35S-labeled proteins from T7-infected E. coli B after electrophoresis. Phage T7 was exposed to 0 (lane a), 830 (b), 6600 (c), or 13,000 (d) J.m-2 of NUV, each in the presence of 0.05% H202. Protein identifications are as in refs. 19 and 20. a
b
Biochemistry:
Hartman et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
3231
The sodium dodecyl sulfate lysis step also preceded alkaline sucrose gradient centrifugation to facilitate preliminary separation of the DNA from the bulk of the protein. While the overall pattern of protein separation was relatively unchanged (Fig. 5), the DNA experienced a slight shift with H202 treatment and a considerable shift with NUV plus H202 treatment. The fractions showing maximal 3H radioactivity were pooled, dialyzed, layered on CsCl step gradients, and centrifuged. Fractions were collected and radioactivities were measured. The peak of 14C radioactivity (protein) cosedimenting with 3H radioactivity (DNA) is of key importance (Fig. 6). There was a substantial increase in the cosedimentation complex only when phage were treated with both NUV and H202. These results may be explained by DNA-protein crosslinks induced by NUV plus H202 treatment of phage T7. This argument is strengthened by the small, but reproducible, decrease in buoyant density of the DNA with NUV plus H202 treatment (1.687 g/cm3) versus that of the control (1.704 g/cm3), NUV (1.702 g/cm3), or H202 (1.701 g/cm3) treatments. Further, Fraction
FIG. 4. NUV plus H202 does not induce DS breaks in phage T7. T7 containing 3H-labeled DNA was irradiated with 20,000 Jm-2 of NUV in the presence of 0.05% H202 (0), or incubated in the dark (0). After phenol extraction, the DNA was layered on 5-20% neutral sucrose gradients, centrifuged, fractionated, and assayed for radioactivity.
in a surviving fraction of about 1 x 10-5 the sedimentation patterns were not substantially changed (Fig. 4). Because a minimum of one DS break per lethal event would be required, it is unlikely that DS breaks play a significant role in inactivation. DNA-Protein Crosslinks Induced by NUV plus H202.
DNA-protein crosslinkage would explain both the impairment of injection and the degree of synergism. Double-labeled T7 ([3H]DNA and [14C]protein) was appropriately treated and sedimented through CsCl step gradients. To avoid artifacts produced by phenol extraction (obtained in preliminary experiments), the phage were lysed with sodium dodecyl sulfate.
0
-( C.
4.
mq
Control
NUV
H202
I
NUV 40
-
H202
? 30
.25
1
30
C.t
0
.t 0
20
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00 51 00
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10
FIG. 5. Alkaline sucrose gradient profiles of treated and lysed phage T7. Double-labeled T7 ([3H]DNA and [14C]protein) was (i) incubated in the dark for 7 min (control) or exposed to (ii) 25,000 J-m-2 of NUV, (iii) 0.05% H202 for 7 min, or (iv) 25,000 Jm-2 of NUV in the presence of 0.05% H202. Catalase (365 units, Sigma) was added to destroy H202 and the samples were gently layered on 5-20% alkaline sucrose gradients. After centrifugation and fractionation, 10-,ul samples were removed and assayed for :3H (0) and 14C (0) radioactivities.
40"*"" 30 20 Fraction FIG. 6. Detection of DNA-protein crosslinkage by CsCl step gradients. Fractions showing peak ;3H radioactivity from alkaline sucrose gradients (Fig. 5) were pooled, dialyzed, and layered on CsCl step gradients. After centrifugation and fractionation, samples were assayed for :H (0) and 14C (0) radioactivities. "0
10
3232
Biochemistry: Hartman et al.
when these fractions were pooled, dialyzed, digested with DNase I, and subjected to a second round of CsCl centrifugation, the liberated '4C banded at ca. 1.3 g/cm3, a position characteristic of protein. Identical treatment of the control, H202, and NUV samples failed to produce any radioactivity in the 1.3 g/cm3 range (data not shown). The most obvious difference between the treatments is the increase in 14C radioactivity in fractions 239 (Fig. 6). Because this material has a buoyant density of about 1.3 g/cm3 and contains no appreciable 3H radioactivity, it represents the increased carryover of protein that is not associated with DNA. Variance in the size of this peak results from the increased overlap of the DNA and protein on the alkaline sucrose gradients, caused by differential SS DNA break induction (Fig. 5). This particular NUV plus H202 treatment resulted in a surviving fraction of 5.9 X 10-5, which corresponds to 9.7 lethal hits per phage (one lethal hit is the dose that yields 37% survival). Under these conditions, 1.48% of phage protein was crosslinked to DNA (taken from Fig. 6) or 0.153% per lethal hit. Because the total molecular weight of the protein per T7 particle is 2.4 X 107 (10), the contribution of one molecule of the product (38,000 daltons) of gene 10, the major head protein (19) is 38,000/(2.4 X 107), or 0.158%. The close agreement of these two values suggests that one molecule of gene 10 protein could crosslink with DNA per lethal event. Because this protein represents over 60% of the total protein in T7, it would be present in sufficient quantities for the juxtaposition necessary for crosslinkage to occur between DNA and protein. DNAprotein association was observed when phage were lysed by either phenol or sodium dodecyl sulfate at high pH (data not shown). Both treatments should have disrupted any noncovalent interactions. DISCUSSION Several independent lines of evidence suggest that H202 plus NUV synergism results from interference with injection of T7 DNA into host. First, far-UV- (but not NUV plus H202)-irradiated T7 exhibit different rates of survival when plated on bacterial hosts that vary in DNA repair capacities (9, 25). Second, the shoulder in the inactivation curve is far less for synergistic H202 plus NUV than for NUV alone (6). The shoulder reflects phage-encoded repair capacity (26); its reduction implicates a defect in either adsorption or injection without opportunity for DNA repair. Direct evidence of an injection defect stems from a modified Hershey-Chase experiment which showed that NUV plus H202 treatment has little or no effect on adsorption, but greatly reduces the amount of DNA injected into the bacterial host (Table 1). Because T7 injects its DNA linearly in a specific order (15, 16, 22), genetic recombination using marker rescue techniques demonstrated partial injection (16). Crosses of amber mutants in genes 5 and 8 not only resulted in a gradient of recombination after NUV plus H202 treatment (Fig. 1) but also provided a one-to-one correlation between decrease in distal recombination and decrease in survival (Fig. 2). Also, differential gene expression in phages inactivated by NUV plus H202 (Fig. 3) correlated with loss of survival. There are several lesions that could cause injection defects. While DS breakage of DNA in x-rayed phage interferes with injection (15, 24), NUV plus H202 yielded a negligible number of DS breaks (Fig. 4). However, DNA-protein complex was observed (Fig. 6) when double-labeled T7 was irradiated with NUV in the presence of 0.05% H202. A straightforward explanation is that NUV plus H202 mediates induction of
Proc. Natl. Acad. Sci. USA 76 (1979)
DNA-protein crosslinks that prevent injection of phage DNA into host. In vivo DNA-protein crosslinkage represents a widespread and diverse biological phenomenon (27). There are normal linkages (e.g., DNA-membrane attachment sites, protein linkers in chromosomes, and gene regulation by noncovalently bound proteins), and, in addition, theories of radiation damage and aging include crosslink lesions to impair DNA template activity. DNA-protein crosslinkage induced by NUV plus H202 treatment of phage T7 represents a specific example in which such a covalent association can be directly correlated with inactivation. These observations also emphasize the important contribution that naturally occurring cellular substances (such as H202) play in accentuating the harmful effects of NUV, from both solar and manufactured sources. The authors gratefully acknowledge the assistance of Cathy Huckins in preparation of figures and F. W. Studier and M. Center in providing phage mutants and bacterial hosts, respectively. This work was supported by U.S. Public Health Service (Food and Drug Administration) Grant 2R01 FD00658-06 and National Institutes of Health Predoctoral Training Grant 5-T32-GM07474 (to P.S.H. and P.G.P.). 1. Webb, R. B. (1977) in Photochemical and Photobiological Redews, ed. Smith, K. C. (Plenum, New York), Vol. 2, pp. 169199. 2. Ferron, W. L., Eisenstark, A. & Mackay, D. (1972) Biochim. Biophys. Acta 277,651-658. 3. Yoakum, G. & Eisenstark, A. (1972) J. Bacteriol. 112, 653655. 4. Stoien, J. D. & Wang, R. J. (1974) Proc. Natl. Acad. Sci. USA 71, 3961-3965. 5. McCormick, J. P., Fisher, R. J., Pachlatko, J. P. & Eisenstark, A. (1976) Science 191, 468-469. 6. Ananthaswamy, H. N. & Eisenstark, A. (1976) Photochem. Photobiol. 24, 439-442. 7. Ananthaswamy, H. N. & Eisenstark, A. (1977) J. Bacteriol. 130, 187-191. 8. Hartman, P. S. & Eisenstark, A. (1978) J. Bacteriol. 133, 769774. 9. Ananthaswamy, H. N., Hartman, P. S. & Eisenstark, A. (1979)
Photochem. Photobiol. 29,53-56. 10. Studier, F. W. (1969) Virology 39,562-574. 11. Hawkins, R. B. (1976) Radiat. Res. 68,300-307. 12. Albertson, P. A. (1967) in Methods in Virology, eds. Maramorosch, K. & Koprowski, H. (Academic, New York), Vol. 2, pp. 303-321. 13. Thomas, C. A. & Abelson, J. (1966) in Procedures in Nucleic Acid Research, eds. Cantoni, G. L. & Davies, D. R. (Harper and Rowe, New York), pp. 553-561. 14. Karska-Wysocki, B., Thibodeau, L. & Verly, W. G. (1976) Biochim. Biophys. Acta 435, 184-191. 15. Pao, C.-C. & Speyer, J. F. (1973) J. Virol. 11, 1024-1026. 16. Karska-Wysocki, B., Mamet-Brately, M. & Verly, G. (1976) J. Virol. 19, 318-324. 17. Laemmli, U. K. (1970) Nature (London) 227,680-685. 18. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 19. Studier, F. W. & Maizel, J. V., Jr. (1969) Virology 39, 575586. 20. Strome, S. & Young, E. T. (1978) J. Mol. Biol. 125,75-94. 21. Hershey, A. D. & Chase, M. (1952) J. Gen. Physiol. 36,39-45. 22. Krisch, R. E. (1974) Int. J. Radiat. Biol. 25,261-276. 23. Freifelder, D. (1968) Virology 36,613-619. 24. Sharp, J. D. & Freifelder, D. (1971) Virology 43, 176-184. 25. Rupert, C. S. & Harm, W. (1966) in Advances in Radiation Biology, eds. Augenstein, L. G., Mason, R. & Zelle, M. R. (Academic, New York), Vol. 2, pp. 1-81. 26. Friedberg, E. C. & King, J. J. (1971) J. Bacteriol. 106, 105. 27. Smith, K. C. (1976) Aging, Carcinogenesis, and Radiation Biology (Plenum, New York).
Biochemistry
Inactivation of phage T7 by near-ultraviolet radiation plus hydrogen peroxide: DNA-protein crosslinks prevent DNA injection (photobiology/double-strand DNA breaks/17 proteins)
P. S. HARTMAN, A. EISENSTARK, AND P. G. PAUW Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211
Communicated by Ernest R. Sears, April 19, 1979
ABSTRACT A nonlethal concentration of H202 (0.05%) greatly enhances near-ultraviolet (NUV) inactivation of phage T7. Simultaneous treatment with H202 and NUV reduces the amount of DNA injected into the bacterial host, but not- the number of phage adsorbed. Not only were recombination and gene expression of late markers reduced upon treatment of phage '11 with NUV plus H202, but also a gradient of recombination resulted, with markers injected first reduced to a lesser extent than those injected last. Double-strand DNA breaks were not detected; however, DNA-protein crosslinks were observed upon NUV plus H202 treatment of double-labeled T7. Previous studies demonstrated that single-strand DNA breaks did not account for phage death by NUV plus H20%. It is concluded that the DNA-protein crosslinks prevent normal injection of T7 phage DNA; such crosslinks may be important lesions in NUV cellular damage.
Labeling Techniques. Phage used for adsorption experiments were labeled as reported (9). The method of Hawkins (11) was used in double labeling. Both procedures yielded from 10-6 to 10-7 cpm per phage. Adsorption and Injection Studies. Phage were separated by the polyethylene glycol/dextran sulfate method (12) and further purified by differential centrifugation (13) prior to gradient centrifugal studies. Titers of ca. 5 X 1012 plaqueforming units per ml were routinely obtained. For phage adsorption and injection assays (14) E. coli B was infected with treated phage; 8 min after infection of cells, chloramphenicol was added, a sample was removed to assay radioactivity, and infected bacteria were removed by centrifugation. The pellet was resuspended and an aliquot was withdrawn to determine percentage of phage adsorbed. To determine the fraction of DNA injected into the host, cells were lysed, treated with DNase I, and centrifuged to remove bacterial debris as well as whole phage particles, and the supernatant was then assayed for radioactivity. This procedure is based on the assumption that the solubilized radioactivity represents the amount of DNA that is injected into the bacterial host, because only it is available for
Current interest in 300- to 400-nm near-ultraviolet radiation (NUV) is due in part to an increasing concern over potential environmental hazards (1). Induction of pyrimidine dimers and single-strand (SS) DNA breaks, and interference with DNA repair and transport systems, have been documented as NUV effects (1). Lethal and mutagenic effects of NUV are distinct from those of far-UV (254-nm) radiation (1, 2). NUV photolysis of tryptophan also generates photoproducts that are lethal to rec mutants of Salmonella typhimurium and Escherichia coil (3), as well as to mammalian cells in culture (4), H202 has been demonstrated as the biologically active photoproduct (5-7). H202 generation from tryptophan can enhance other biological actions by the same NUV wavelengths necessary for its initial generation. Simultaneous treatment with NUV plus H202 results in SS DNA breaks in bacteria (8). H202 also enhances NUV lethality in phage T7 (6, 9), thus providing a simple model system for the study of these synergistic interactions. While action spectrum analysis indicates synergism is maximal at 340 nm, the frequency of SS DNA break induction could not be correlated with peak of cell killing (9); i.e., there is no apparent direct relationship between SS
digestion by deoxyribonuclease. Genetic Recombination. For genetic crosses between amber mutants (15, 16) E. colh 011' (Su+) was coinfected with either T7 amli (gene 8) or am28 (gene 5), and one of a series of different amber mutants (helper phage) located throughout the T7 geqome. Lysates were assayed on 011' (Su+) and B (Su-) for total progeny and wild-type recombinants, respectively. Results are expressed as percentage of normal recombination (16). Average frequencies of wild-type recombinants for the control crosses were compatible with those in previous reports (15, 16). Gel Electrophoresis Studies. E. coli B was grown at 37°C to 4 X 108 cells per ml in M9 medium plus 2 mg of casamino acids per ml, irradiated with 72 J-M2 of far-UV radiation to suppress host cell protein synthesis, and incubated for 30 min at 30°C in the dark. Aliquots (2 ml) were then infected with appropriately treated T7 at a multiplicity of infection of 10 and incubated with 10 ,tCi (1 Ci = 3.7 X 1010 becquerels) of [-5S]methionine (New England Nuclear) per ml for 24 min at 30°C. Samples were washed and concentrated 10-fold by centrifugation and applied to polyacrylamide gels. Sodium dodecyl sulfate/polyacrylamide gradient slab gels were prepared 1.5 mm thick with a linear gradient of 8-13% acrylamide and a 5% acrylamide stacking gel (17). Gels were prepared for fluorography (18), dried, and placed in direct contact with Kodak Royal X-O-Mat film and incubated at -70'C for up to 12 hr. Proteins corresponding to specific T7 genes were identified by comparison with results of others (19, 20) and only those proteins that could be unequivocally assigned were compared.
breaks and cell death. In this report, we demonstrate that NUV plus H202 inactivates phage T7 by producing DNA-protein crosslinks, preventing injection of phage DNA into the host. MATERIALS AND METHODS Bacteria. E. coli B, the nonpermissive (no suppression of amber mutants, Su-) T7 host, and E. coli 011' (thy-), the permissive (Su+) host, were obtained from M. Center. Phage. T7 wild type and amber mutants am 193 (gene 1), am28 (gene 5), amIl (gene 8), am3 (gene 12), and am3l (gene 15) were provided by F. W. Studier (10). Phage stocks were grown, maintained, and assayed according to Studier (10). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
Abbreviations: NUV, near-ultraviolet radiation; DS, double strand; SS, single strand; Su+ and Su, suppressor and nonsuppressor of amber mutations, respectively.
this fact.
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Proc. Natl. Acad. Sci. USA 76 (1979)
3229
Analysis for Double-Strand (DS) DNA Breakage. T7 phage,
after purification, was extracted with phenol (13), layered on preformed 5-20% neutral (pH 7) sucrose gradients, and centrifuged, fractionated, and assayed for radioactivity as described (7). Analysis for DNA-Protein Crosslinks. Protein putatively crosslinked to DNA was separated from remaining protein by sedimentation through alkaline sucrose gradients; 0.4-ml fractions were collected and assayed for radioactivity (7). Elimination of the alkaline sucrose gradient step gave the same results (data not shown). Fractions showing high 3H (DNA) counts were pooled and dialyzed overnight against 0.05 M KHPO4 (pH 7.4). One milliliter of this dialysate was layered on a step gradient of CsCl consisting of 1.4 ml each of 1.75, 1.55, and 1.35 g/cm3, centrifuged at 40,000 rpm for 24 hr at 4VC (SW 50.1 rotor, Beckman L5-50 ultracentrifuge), followed by fractionation and detection of radioactivity (7). A small amount of 14C radioactivity cosedimented with the 3H in the CsCl gradients, even with the control or untreated phage. This material probably represents the fraction of 14C metabolized into DNA during phage labeling. Hawkins (11) noted similar conversions under identical labeling conditions. The following formula was used to calculate the percentage of '4C in a given fraction that was actually protein:
14Ccontrol/% 3Hcontrol) X 3Hi, in which % proteinj equals the percentage of 14C in fraction i %
protein,
= 14Ci - (%
%
that is actually in protein; % "4Ce equals the percentage of 14C radioactivity in fraction i; % 14Ccontrol equals the total percentage of 14C in fractions 5-9 of the control; and 3Hcontrol equals the percentage of 3H in fractions 5-9 of the control. The validity of the calculation was confirmed by performing similar experiments with [MS]methionine. There was no 35S radioactivity that cosedimented with the DNA peak in the control. Irradiation. Radiation was with four General Electric F15T8 BLB integral-filter black-light bulbs, as reported (6). After NUV and/or H202 treatment, catalase was added to destroy H202.
RESULTS Effects of NUV plus H202 on Adsorption and Injection. To test whether adsorption or injection might be impaired by NUV plus H202 treatment, the fate of 3H-labeled DNA of untreated and treated T7 phage was measured by a modified Hershey-Chase experiment (14, 21). Separate exposure to NUV radiation or H202 resulted in little or no decrease in viability, adsorption, or injection (Table 1). Phage adsorption was not significantly diminished by synergistic treatment; in contrast,
c 0
*m,
11
.0
E 0
'EE 0
c
0 OI
T7 genetic
map
FIG. 1. Frequency of recombination between amber mutants of T7 (identified by gene number on genetic map). (Left) T7 am28 (gene 5) was irradiated with 600 (o), 1200 (-), or 1800 (0) J-m-2, each in the presence of 0.05% H202. (Right) T7 amll (gene 8) was irradiated with 300 (o), 900 (M), or 1500 (0) J-m-2, each in the presence of 0.05% H202-
both viability and DNA injection were greatly reduced. The drop in viability was more drastic than the drop in DNA injection (Table 1). This discrepancy could have one of several explanations: (i) All-or-nothing injection: a fraction of the treated phage inject no DNA, but the remainder inject all of their DNA, albeit damaged. (ii) Partial injection: despite inactivation, phages are still able to inject a portion of their genome. (iii) A combination of i and ii could occur. Possibility ii may be distinguished from i and iii by comparing the amounts of each of several genes that are injected and transcribed. NUV plus H202 Effects of Genetic Recombination. To distinguish among the above possibilities, genetic crosses were conducted in which partially injected DNA could be "rescued." A particular amber mutant was treated and crossed with other amber mutants (helper phage) defective in other T7 genes. Because T7 injects its DNA linearly in a single direction with a unique starting point (15, 16, 22), the genes coding for the early proteins are injected first. If NUV plus H202 inactivates by all-or-nothing injection (i), recombination between the treated amber mutant and all helper phage would decrease uniformly, irrespective of map position. With partial injection
Table 1. Effects of NUV, H202, and NUV plus H202 treatments on adsorption and injection of T7* NUV dose, Injection,I Adsorption,T Survival, H202 time,t J.m-2 min Treatment % % % NUV 0.05% H202
NUV plus 0.05% H202
0 54,000
0 0
100.0 96.0
49 (100) 5:3 (108)
35.0 (100) 33.0 (94)
0 0
0 1.5
100.0 98.0
51 (100) 50 (98)
31.0 (100) 30.0 (97)
0 0 32.0 (100) 40 (100) 100.0 0.75 40 (100) 24.0 (75) 50.0 27,000 6.3 (20) 4.5 36 (93) 1.5 54,000 * Experimental procedure and calculations were as described by Karska-Wysocki and coworkers (14). Each value is the average of at least two independent experiments. t The NUV radiation dose of 54,000 J.m-2 is obtained by 1.5 min of exposure. 4 Numbers outside parentheses are absolute values; relative amounts of adsorption and injection are indicated inside parentheses.
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Proc. Nati. Acad. Sci. USA 76 (1979)
(ii), early genes should recombine to a greater extent than those injected last.am28 (gene 5) phage were exposed to several doses of NUV in the presence of 0.05% H202 and crossed with amber mutants defective in genes 1, 8, 12, or 15 (Fig. 1 left). With increasing exposure, there was a gradient of recombination inversely proportional to the order of injection, with genes injected late affected to a greater extent than those injected early. Identical experiments were performed with the later marker amli (gene 8), with confirmatory results (Fig. 1 right). As was the case with injection and adsorption (Table 1), separate NUV or H202 treatment had little effect on viability or recombination frequencies (data not shown). In addition to precluding the possibility of all-or-nothing (i) injection, a gradient of recombination allows a comparison of the decreases in recombination and viability. The degree of correlation between these two values permits assignment of the relative role that the injection defect plays in the NUV plus H202 inactivation of T7. Because a single phage must inject its entire DNA molecule to produce a burst of progeny phage, the percent decrease in distal recombination or maximal decrease in recombination that is possible with a given dose of NUV plus H202 can be extrapolated (Fig. 1). When the percent decrease in distal recombination for a given dose is plotted with the percentage of survivors for the same dose, the values are superimposable (Fig. 2), demonstrating that the injection defect is the primary, and perhaps the sole, cause of the synergistic inactivation of phage T7 by NUV plus H202.
Effects of NUV plus H202 on Protein Synthesis. An injection defect should also result in a "map-position-dependent" decrease in protein synthesis, because late genes would be transcribed to a lesser extent than early genes. Accordingly, wild-type T7 phage was exposed to three dosages of NUV and 0.05% H202 and used to infect E. coli B in the presence of [l5S]methionine, and the resulting proteins were electrophoresed through polyacrylamide gels. As with the genetic recombination experiments, a gradient resulted, with gene expression greater in early genes than in late genes (Fig. 3). Densitometric scans of the fluorogiams revealed that, as with the correlation between decreases in recombination and viability (Fig. 2), the values for maximal decrease in protein synthesis (34%, 8%, and 0.52%) correspond favorably with the survival at each dose (60%, 10%, and 0.17%, respectively) (data not shown). Insignificant Production of DS DNA Breaks by NUV plus H202. As noted previously (6, 9), the contribution of SS DNA breaks to death in T7 is insignificant. However, DS breakage could account for the striking enhancement of NUV lethality by H202, because x-ray inactivation of DNA-containing phages, including T7, is known to be due mainly to DS DNA breaks (23), which can impede injection (15, 24). The possibility of death by DS breaks was examined by the sedimentation of 3H-labeled T7 DNA through neutral sucrose gradients. T7 was either irradiated with 20,000 J.m-2 of NUV in the presence of 0.05% H202 or held in the dark. Even after a dose that resulted
_
-P 16
-,.....'-
-Pi
C
0 C c
E0.
-P9
6.,
-PO.7. 1.3 C
-P6
C._
-P3.5
-up'l"
Dose, J-m-2 x 10-2 FIG. 2. Effects of NUV plus 0.05% H202 on survival (open symbols) and decrease in distal recombination (closed symbols) of phage T7. Recombination values were extrapolated from Fig. 1 as explained in the text. 0, Survival of am28; 0, distal recombination of am28; 0, survival of am 11; *, distal recombination of amlI.
-0~
-PO03
c d FIG. 3. Fluorogram of 35S-labeled proteins from T7-infected E. coli B after electrophoresis. Phage T7 was exposed to 0 (lane a), 830 (b), 6600 (c), or 13,000 (d) J.m-2 of NUV, each in the presence of 0.05% H202. Protein identifications are as in refs. 19 and 20. a
b
Biochemistry:
Hartman et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
3231
The sodium dodecyl sulfate lysis step also preceded alkaline sucrose gradient centrifugation to facilitate preliminary separation of the DNA from the bulk of the protein. While the overall pattern of protein separation was relatively unchanged (Fig. 5), the DNA experienced a slight shift with H202 treatment and a considerable shift with NUV plus H202 treatment. The fractions showing maximal 3H radioactivity were pooled, dialyzed, layered on CsCl step gradients, and centrifuged. Fractions were collected and radioactivities were measured. The peak of 14C radioactivity (protein) cosedimenting with 3H radioactivity (DNA) is of key importance (Fig. 6). There was a substantial increase in the cosedimentation complex only when phage were treated with both NUV and H202. These results may be explained by DNA-protein crosslinks induced by NUV plus H202 treatment of phage T7. This argument is strengthened by the small, but reproducible, decrease in buoyant density of the DNA with NUV plus H202 treatment (1.687 g/cm3) versus that of the control (1.704 g/cm3), NUV (1.702 g/cm3), or H202 (1.701 g/cm3) treatments. Further, Fraction
FIG. 4. NUV plus H202 does not induce DS breaks in phage T7. T7 containing 3H-labeled DNA was irradiated with 20,000 Jm-2 of NUV in the presence of 0.05% H202 (0), or incubated in the dark (0). After phenol extraction, the DNA was layered on 5-20% neutral sucrose gradients, centrifuged, fractionated, and assayed for radioactivity.
in a surviving fraction of about 1 x 10-5 the sedimentation patterns were not substantially changed (Fig. 4). Because a minimum of one DS break per lethal event would be required, it is unlikely that DS breaks play a significant role in inactivation. DNA-Protein Crosslinks Induced by NUV plus H202.
DNA-protein crosslinkage would explain both the impairment of injection and the degree of synergism. Double-labeled T7 ([3H]DNA and [14C]protein) was appropriately treated and sedimented through CsCl step gradients. To avoid artifacts produced by phenol extraction (obtained in preliminary experiments), the phage were lysed with sodium dodecyl sulfate.
0
-( C.
4.
mq
Control
NUV
H202
I
NUV 40
-
H202
? 30
.25
1
30
C.t
0
.t 0
20
m 0
10!,
0IR
00 51 00
51 00
510 0
Fraction
5
10
FIG. 5. Alkaline sucrose gradient profiles of treated and lysed phage T7. Double-labeled T7 ([3H]DNA and [14C]protein) was (i) incubated in the dark for 7 min (control) or exposed to (ii) 25,000 J-m-2 of NUV, (iii) 0.05% H202 for 7 min, or (iv) 25,000 Jm-2 of NUV in the presence of 0.05% H202. Catalase (365 units, Sigma) was added to destroy H202 and the samples were gently layered on 5-20% alkaline sucrose gradients. After centrifugation and fractionation, 10-,ul samples were removed and assayed for :3H (0) and 14C (0) radioactivities.
40"*"" 30 20 Fraction FIG. 6. Detection of DNA-protein crosslinkage by CsCl step gradients. Fractions showing peak ;3H radioactivity from alkaline sucrose gradients (Fig. 5) were pooled, dialyzed, and layered on CsCl step gradients. After centrifugation and fractionation, samples were assayed for :H (0) and 14C (0) radioactivities. "0
10
3232
Biochemistry: Hartman et al.
when these fractions were pooled, dialyzed, digested with DNase I, and subjected to a second round of CsCl centrifugation, the liberated '4C banded at ca. 1.3 g/cm3, a position characteristic of protein. Identical treatment of the control, H202, and NUV samples failed to produce any radioactivity in the 1.3 g/cm3 range (data not shown). The most obvious difference between the treatments is the increase in 14C radioactivity in fractions 239 (Fig. 6). Because this material has a buoyant density of about 1.3 g/cm3 and contains no appreciable 3H radioactivity, it represents the increased carryover of protein that is not associated with DNA. Variance in the size of this peak results from the increased overlap of the DNA and protein on the alkaline sucrose gradients, caused by differential SS DNA break induction (Fig. 5). This particular NUV plus H202 treatment resulted in a surviving fraction of 5.9 X 10-5, which corresponds to 9.7 lethal hits per phage (one lethal hit is the dose that yields 37% survival). Under these conditions, 1.48% of phage protein was crosslinked to DNA (taken from Fig. 6) or 0.153% per lethal hit. Because the total molecular weight of the protein per T7 particle is 2.4 X 107 (10), the contribution of one molecule of the product (38,000 daltons) of gene 10, the major head protein (19) is 38,000/(2.4 X 107), or 0.158%. The close agreement of these two values suggests that one molecule of gene 10 protein could crosslink with DNA per lethal event. Because this protein represents over 60% of the total protein in T7, it would be present in sufficient quantities for the juxtaposition necessary for crosslinkage to occur between DNA and protein. DNAprotein association was observed when phage were lysed by either phenol or sodium dodecyl sulfate at high pH (data not shown). Both treatments should have disrupted any noncovalent interactions. DISCUSSION Several independent lines of evidence suggest that H202 plus NUV synergism results from interference with injection of T7 DNA into host. First, far-UV- (but not NUV plus H202)-irradiated T7 exhibit different rates of survival when plated on bacterial hosts that vary in DNA repair capacities (9, 25). Second, the shoulder in the inactivation curve is far less for synergistic H202 plus NUV than for NUV alone (6). The shoulder reflects phage-encoded repair capacity (26); its reduction implicates a defect in either adsorption or injection without opportunity for DNA repair. Direct evidence of an injection defect stems from a modified Hershey-Chase experiment which showed that NUV plus H202 treatment has little or no effect on adsorption, but greatly reduces the amount of DNA injected into the bacterial host (Table 1). Because T7 injects its DNA linearly in a specific order (15, 16, 22), genetic recombination using marker rescue techniques demonstrated partial injection (16). Crosses of amber mutants in genes 5 and 8 not only resulted in a gradient of recombination after NUV plus H202 treatment (Fig. 1) but also provided a one-to-one correlation between decrease in distal recombination and decrease in survival (Fig. 2). Also, differential gene expression in phages inactivated by NUV plus H202 (Fig. 3) correlated with loss of survival. There are several lesions that could cause injection defects. While DS breakage of DNA in x-rayed phage interferes with injection (15, 24), NUV plus H202 yielded a negligible number of DS breaks (Fig. 4). However, DNA-protein complex was observed (Fig. 6) when double-labeled T7 was irradiated with NUV in the presence of 0.05% H202. A straightforward explanation is that NUV plus H202 mediates induction of
Proc. Natl. Acad. Sci. USA 76 (1979)
DNA-protein crosslinks that prevent injection of phage DNA into host. In vivo DNA-protein crosslinkage represents a widespread and diverse biological phenomenon (27). There are normal linkages (e.g., DNA-membrane attachment sites, protein linkers in chromosomes, and gene regulation by noncovalently bound proteins), and, in addition, theories of radiation damage and aging include crosslink lesions to impair DNA template activity. DNA-protein crosslinkage induced by NUV plus H202 treatment of phage T7 represents a specific example in which such a covalent association can be directly correlated with inactivation. These observations also emphasize the important contribution that naturally occurring cellular substances (such as H202) play in accentuating the harmful effects of NUV, from both solar and manufactured sources. The authors gratefully acknowledge the assistance of Cathy Huckins in preparation of figures and F. W. Studier and M. Center in providing phage mutants and bacterial hosts, respectively. This work was supported by U.S. Public Health Service (Food and Drug Administration) Grant 2R01 FD00658-06 and National Institutes of Health Predoctoral Training Grant 5-T32-GM07474 (to P.S.H. and P.G.P.). 1. Webb, R. B. (1977) in Photochemical and Photobiological Redews, ed. Smith, K. C. (Plenum, New York), Vol. 2, pp. 169199. 2. Ferron, W. L., Eisenstark, A. & Mackay, D. (1972) Biochim. Biophys. Acta 277,651-658. 3. Yoakum, G. & Eisenstark, A. (1972) J. Bacteriol. 112, 653655. 4. Stoien, J. D. & Wang, R. J. (1974) Proc. Natl. Acad. Sci. USA 71, 3961-3965. 5. McCormick, J. P., Fisher, R. J., Pachlatko, J. P. & Eisenstark, A. (1976) Science 191, 468-469. 6. Ananthaswamy, H. N. & Eisenstark, A. (1976) Photochem. Photobiol. 24, 439-442. 7. Ananthaswamy, H. N. & Eisenstark, A. (1977) J. Bacteriol. 130, 187-191. 8. Hartman, P. S. & Eisenstark, A. (1978) J. Bacteriol. 133, 769774. 9. Ananthaswamy, H. N., Hartman, P. S. & Eisenstark, A. (1979)
Photochem. Photobiol. 29,53-56. 10. Studier, F. W. (1969) Virology 39,562-574. 11. Hawkins, R. B. (1976) Radiat. Res. 68,300-307. 12. Albertson, P. A. (1967) in Methods in Virology, eds. Maramorosch, K. & Koprowski, H. (Academic, New York), Vol. 2, pp. 303-321. 13. Thomas, C. A. & Abelson, J. (1966) in Procedures in Nucleic Acid Research, eds. Cantoni, G. L. & Davies, D. R. (Harper and Rowe, New York), pp. 553-561. 14. Karska-Wysocki, B., Thibodeau, L. & Verly, W. G. (1976) Biochim. Biophys. Acta 435, 184-191. 15. Pao, C.-C. & Speyer, J. F. (1973) J. Virol. 11, 1024-1026. 16. Karska-Wysocki, B., Mamet-Brately, M. & Verly, G. (1976) J. Virol. 19, 318-324. 17. Laemmli, U. K. (1970) Nature (London) 227,680-685. 18. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 19. Studier, F. W. & Maizel, J. V., Jr. (1969) Virology 39, 575586. 20. Strome, S. & Young, E. T. (1978) J. Mol. Biol. 125,75-94. 21. Hershey, A. D. & Chase, M. (1952) J. Gen. Physiol. 36,39-45. 22. Krisch, R. E. (1974) Int. J. Radiat. Biol. 25,261-276. 23. Freifelder, D. (1968) Virology 36,613-619. 24. Sharp, J. D. & Freifelder, D. (1971) Virology 43, 176-184. 25. Rupert, C. S. & Harm, W. (1966) in Advances in Radiation Biology, eds. Augenstein, L. G., Mason, R. & Zelle, M. R. (Academic, New York), Vol. 2, pp. 1-81. 26. Friedberg, E. C. & King, J. J. (1971) J. Bacteriol. 106, 105. 27. Smith, K. C. (1976) Aging, Carcinogenesis, and Radiation Biology (Plenum, New York).
