Incorporation of 5-fluorodeoxyuridine into the DNA of Bacillus subtilis phage PBS2 and its radiobiological consequences

J. Mol. Biol. (1967) 80, 277-200

Incorporation of S-Fluorodeoxyuridine into the DNA of Bacillus subtilis Phage PBS2 and its Radiobiological Consequences HOMER

A.

LoZERON AND WACLAW SZYBALSKI

McArdle LaboraJory, University of Wi8consin Madison, Wi8consin 53706, U.S.A. (Received 23 May 1967, and in revised form 10 August 1967) BaciUus subtili8 cells, infected with phage PBS2 and grown in synthetic mediwn supplemented with 50 p.g [2_14C]5-~uorodeoxyuridinejm1., yield progeny phage in which their normal DNA constItuent, deoxyuridine, has been replaced by 5-fluorodeoxyuridine as evidenced by (1) a buoyant density increase of the phage DNA in CsCI from 1·722 (normal DNA) to 1'737 to 1·745 gjcm3 , and (2) HC incorporation from [2_14C]5-fluorodeoxyuridine specifically into this "heavy" DNA fraction with quantitative recovery of [2-14C]5-fluorouracil from perchloric acid digests ofthe DNA. [2-14C]5-Fluorodeoxyuridine incorporation data indicate that the replacement of 1 mole % of uracil by 5-fluorouracil is accompanied by an approximately 0·6 mgfcm3 increase in the buoyant density of the DNA. Similar replacement of 1 mole % of uracil in PBS2 DNA by thymine (i.e., methylation of DNA) should result in a density decrease of 0·34 mg/cm3 • Although its DNA contains no thymine, the PBS2 phage can undergo host-cell reactivation, since the ultraviolet sensitivity of PBS2 phage plated on non-host reactivating strains of BaciUU8 aubtilis is greater than that for the same phage plated on wild-type strains. 5-Fluorodeoxyuridine incorporation into the PBS2 DNA enchances the ultraviolet sensitivity of the phage.

1. Introduction The halogenated pyrimidine analog, 5-bromouracil (also the 5-chloro and iodo derivatives), is readily incorporated in place of the natural base, thymine, into the DNA of bacteria (Weygand. Wacker & Dellweg, 1952; Zamenhof & Griboff, 1954; Dunn & Smith, 1957), bacteriophage (Litman & Pardee, 1956) and mammalian cells (Hakala, 1959) (representative papers only). 5-Fluorouracil, in contrast, is substituted in place of uracil into many RNA's including bacterial (Horowitz & Chargaff, 1959), mammalian (Chaudhuri, Montag & Heidelberger, 1958), tobacco mosaic virus (Gordon & Staehelin, 1959), polio virus (Munyon & Saltzman, 1962; Tershak, 1964) and coliphage MS2 RNA (Shimura, Moses & Nathans, 1965), but hitherto has never been shown to be incorporated into the DNA of any organism. The specificity of 5-bromo-analog substitution into DNA and of 5-fluoro-analog substitution into RNA is probably a reflection of the close similarity of the Van der Waals radius (quoted by Szybalski, 1962) of the bromine atom (1·95 A) to that of the methyl group (2.0 A), and the fluorine atom (1'35 A) to that of the hydrogen atom (1·2 A), respectively. The in vitro incorporation, catalyzed by DNA polymerase, of FUdRt triphosphate

t Abbreviations used: u.v.~ ultra~iolet light (predom~antly 253·7 mIL); m.o.i., multiplicity of infection; p.f.u., plaque-formmg urut; FU, 5-fiuorouraoil; UdR, deoxyuridine; FUdR, 5.f1uorodeoxyuridine; PBS2.FUdR·DNA, B. aubtilis phage PBS2 DNA containing FUdR in place ofUdR' PBS2-FUdR phage, phage PBS2 containing FUdR'DNA; CAA, casamino acids (Difco). ' 277

278

H. A. LOZERON AND W. SZYBALSKI

into DNA (Richardson, Schildkraut, & Kornberg, 1963) would indicate that the ita. vivo exclusion ofFUdR from DNA must reside at the level of enzymic phosphorylation ofFUdR. Since the DNA of B. subtilis phage PBS2 has been shown to contain uracil in place of thymine (Takahashi & Marmur, 1963), it appeared probable that in this special case uracil could be replaced by its analog, 5-fluorouracil. This communication describes in detail the preparation of PBS2 DNA containing 5-fluorouracil, and some preliminary observations on the photochemical properties of the analog-containing

virus.

2. Materials and Methods (a) Phage and bacterial strains Bacillus subtilis phage PBS2 and the B. suhtilis strain SB19 were kindly provided by I. Takahashi, McMaster University, Hamilton, Ontario, Canada. The u.v.-sensitive strains 168 uvr- (Reiter & Strauss, 1965) and mms- (Searashi & Strauss, 1965), 168 hcr-9 (Okubo & Romig, 1965), and the u.v.-resistant strain 168M of B. subtilis were obtained from B. StraUSB, University of Chicago, W. R. Romig, University of California and D. M. Green, University of Pittsburgh, respectively.

(b) Growth oj normal phage stock8 B. subtilis SB19 was grown in Penassay broth (Difco Antibiotic Medium 3) at 37°C to a cell concentration of lOB/mI., spun down at 2500 g (5000 rev_/min in the Lourdes VRA rotor) for 10 min at room temperature, and the pellets resuspended in 0·5 vol. of fresh Penassay broth at 37°C (cell concentration 2 X 10B/mL)_ CultureB of 500 mI. contained in 2-1. Erlenmeyer flasks were inoculated immediately with PBS2 phage (m.o.i. = 3) and phage growth was continued with vigorous shaking (model G25 Gyrotory shaker, New Brunswick Scientific 00., Inc., New Brunswick, N.J.) at 37°0 for 1·5 hr, at which time the suspension rapidly began to clear. The lysate was incubated without shaking another 2 hr to allow completion of the lysis. The use of young, exponentially growing cells, resuspended at a relatively high cell density in fresh Penassay medium, gave us somewhat higher phage titers (1 to 2 X 101D/mI.) than that obtained in the procedure previously outlined (Takahashi, 1963), which in our hands yielded titers of 3 to 5 X 109/ml. (c) Growth oj 5-Jluorodeoxyuridine-labeled phage stock8

(i) Penassay broth lysates

The procedures outlined above for the growth of normal PBS2 stocks were followed. FUdR was added at the time of PBS2 inoculation to yield a final concentration of 500 p.g/mI. Phage titers were 2 to 4 X 10 9 /mI. (ii) Synthetic medium lysates Exponential phase SB19 cells at a cell density of lOB/mI. in Penassay broth were diluted 1:9 into VB (minimal medium of Vogel & Bonner (1956) supplemented with 0·50% glucose. 0'10% Casamino acids (Difco), 10 p.g tryptophan/mI., and 10 p.g FeCl3 ,6HaO/ml.) and again grown to a. cell density of lOB/m!. The eells were pelleted, resuspended in 0·5 vol. of fresh VB medium at 37°C, and 20 min after PBS2 phage inoculation (m.o.i. 2 to 3). FUdR was added to yield a. final concentration of 50 p.g/ml. Lysis occurred rapidly after a. further 1·5 hr incubation with vigorous stirring at 37°0. Phage titers were 2 to 5 X 107/ml. For radioactive incorporation studies, [2_l4C]FUdR (a gift of C. Heidelberger) at a specific activity of approximately lOB ets/min/mg, was repurified by preparative highvoltage electrophoresis (Birnie, Kroeger & Heidelberger, 1963) on Whatman 3MM paper to remove contaminating [2-14C]FU from the preparation. Ultraviolet-absorbing material corresponding to [2_l40]FUdR was eluted from the paper with 0·01 M-sodium phosphate buffer (pH 7·0) and mixed with carrier .FUdR (supplied by Hoff~-La.Ro?h.e~ Inc., Nutley, N.J.) to yield stock preparations of [2- 140]FUdR with specific actlVltles of 3'1 X 108 and 3·6 X lOB cu/min/mg.

5·FLUOROURACIL·CONTAINING DNA

279

(d) Purification of PBS2 phage and DNA e:ctraction Crude phage lysates were treated with lysozyme (10 "g/m1.), DNase I (1 p,g/ml.), and RNase (1 p.g/ml.) (Worthington Biochemical Corp., Freehold, N.J.) for 1 hr at 37°C. PBS2·FUdR phage from 21. of crude, VB medium lysate was purified and concentrated by 3 cycles of differential centrifugation (Takahashi & Marmur, 1963) to yield 2'0 ml. of purified phage stock (2 to 5 X 1010 p.f.u./mI.) suspended in Tris buffer (0·15 M.NaCI -0·10 M.Tris) adjusted to pH 7·4 with HC1. To 1·0 mI. of the above purified phage stock was added 0·10 ml. of tenfold concentrated SSC (SSC = 0·15 M-Na.cI-0·02 M-sodium citrate, adjusted to pH 7'8) and 0·10 ml. 20% sodium dodecyl sulfate. The solution was gently stirred by hand at room temperature for 2 min, and then extracted twice at 4°C with 2 vol. of chloroform-butanol (4: 1). The DNA was precipitated from the aqueous)ayer with 2 vol. of abs. ethanol and redissolved in 0'40 mI. SSC. (e)

Irradiation and a88ay of phage

PBS2 and PBS2-FUdR phage (2·0 mI. suspension at 10 7 p.f.u./ml. in 0·15 M-NaCl-0·I0 MTris-O·005 M-MgCI2' pH 7·4) were irradiated simultaneously as thin layers in 5'7-cm diameter Petri dishes placed on a rotary shaker during irradiation at a distance of 38 cm from a 15-w germicidal lamp (General Electrio Co.). At specified time intervals, 0·05-m1. portions of irradiated phage were diluted into half-strength Spizizen's minimal medium (Spizizen, 1958) with glucose omitted, and supplemented with 0'10% yeast extract and 10 ,..g FeCla,6H 2 0/ml. (Takahashi, I., personal communication). Samples of appropriately diluted PBS2 and PBS2-FUdR phage wereassayed on B_ BUbtiliB strains 168M, SB19, and on the ultraviolet-sensitive strains 168 uvr- (Reiter & Strauss, 1965), and 168 her-9 (Okubo & Romig, 1965), acoording to procedures described previously (Takahashi, 1963). Exponential-phase cultures at a cell density of lOB/mI. were used as indicator bacteria in all cases_ PBS2 and PBS2-FUdR phage form more turbid plaques and plate at approximately 80% efficiency on the non-host-reactivating strains compared to that of the wild.type strains. PBS2 phage did not form plaques on strain mmr, another radiation-sensitive B. BUbtiliB mutant (Searashi & Strauss, 1965). Preadsorption and plating were performed under subdued lighting. (f) Density-grculient centrifugation Samples for analytical CsCl density-gradient centrifugation were placed in 2°, 12 rom, Kel-F lined cells and centrifuged at 44,770 rev·fmin at 25°C for 22 hr under the usual conditions (Erikson & Szybalski, 1964). Tracings of the ultraviolet photographs were prepared with a Joyce-Loebl mark IIIC double-beam microdensitometer equipped with a cylindrical condenser lens, and copied in India ink to eliminate background caused by graininess and imperfections of the photographic film. CsCI was optical grade, manufactured by Rare Metals Derivatives, Inc., Ambler, Penn. For preparative centrifugation, phage DNA in CsCl (buffered at pH 7'6 with SSC) at a final density of 1·72 gJcm3 (2·50 mI. sample overlayed with 2'50 mI. paraffin oil) was centrifuged at 30,000 rev./min for 70 hr at 15°C in the SW39 rotor of the Spinco model L ultracentrifuge. Fractions of O·}O mI. were collected sequentially from the bottom of the tube (Szybalski, 1960) and O.D. (260 mp,) measured in a 2-mm micro-cuvette of 151'1. capacity. For radioactivity assay, 20 ml. ANPO liquid scintillator (295'2 g naphthalene; 18·48 g 2,5,diphenyloxazole (PPO); 0·184 g exnaphthylphenyloxazole (ex-NPO); 1400 mI. xylene; 1400 mI. dioxane; 840 ml. abs. ethanol) was added directly to a mixture of 0·97 mI. water and a 0·02-ml. portion of each of the O·IO-mI. fractions collected from the preparative run. Control experiments indicated little quenching of 14C under these experimental conditions.

3. Results and Discussion (a) Incorporation of 5-jtuorodeoxyuridine into phage PBS2 DNA

The incorporation of 5-fluorouracil in place of uracil into the RNA of coliphage MS2 and tobacco mosaic virus increases the buoyant density of RNA in OS2S0, (Shiroura et al., 1965; Lozeron & Szybalski, 1966). Thus, it appeared that density-

B. A. LOZERON AND W. SZYBALSKI

280

gradient centrifugation would provide a rapid and convenient method for detecting FUdR incorporation into PBS2 DNA. As shown in Fig. 1, PBS2 phage preparations A and B grown in the presence ofFUdR (traces c and e) and the DNA extracted from these purified phage preparations (traces d and f) band at significantly higher buoya.nt densities than that of normal PBS2 phage (1·435 gjcm3 , trace a) and PBS2 DNA 44,770 rev./min CsCI

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FIG. 1. Buoyant density profiles of phage PBS2 (traces a, c, e) and of the phage DNA (traces b,d,f). The phage was grown as described in Materials and Methods, either in FUdR·free VB synthetic mediwn (traces B, b) or in the presence of FUdR (preparation A: 500",g FUdR/ml. of Penassay broth, traces c, d: preparation B: 50 ",g FUdR/ml. of VB synthetic medium, traces e, f). The dashed line in trace e indicates the banding pattern upon addition of unlabeled PBS2 phage. The dashed lines in traces b, d, f indica.te the position of Oytophaga johnsorvii DNA a.dded as the density :rna.rker (1·6945 g/cm3 ). The buoyant densities ofPBS2 phage preparations were determined by pycnometric measurements.

(1·722 gjcm 3 , trace b). The conclusion that the increase in buoyant density ofFUdRgrown PBS2 DNA preparations indeed reflects the incorporation of FUdR in place of UdR in the DNA is further confirmed by the following [2- 140]FUdR incorporation data. Purified DNA from PBS2 preparation 0 (Fig. 2, analytical trace a), grown in the presence of [2. 14C]FUdR (specific activity, 3·1 X 106 ctsjminjmg) was analyzed in a preparative gradient (Fig. 2, preparative run) for 140. Radioactivity appea.rs almost exclusively in the "hea.vy" DNA fraction corresponding to a buoyant density of 1'732 to 1·748 gjcm 3 • A small fraction of radioactivity "tails" into the peak of normal phage DNA (fractions 13 and 14; Fig. 2) of buoyant density 1·722. Fractions 13 and 14, however, were shown by analytical centrifugation to contain contaminating amounts of heavy DNA which largely accounted for the tailing radioactivity in these fractions.

5-FLUOROURACIL-CONTAINING DNA

281

The heavy DNA was chromatographioally analyzed for [ 0]FU. DNA from PBS2 preparation D (Fig. 3, trace a, control) (2 p.g DNA supplemented with 50 p.g eaoh ofura.cil and FU as carriers) was hydrolyzed in 12 N-perchloric acid for 60 minutes at 100°0. The free bases were separated by two-dimensional paper chromatography (Gordon & Staehelin, 1959) and ultraviolet-absorbing material corresponding to uracil and FU eluted and analyzed for 140. Of the total radioactivity recovered 96% was eluted from the ultraviolet-ab80r~ing spot corresponding to FU. 14

(b) Buoyant de'fUlity of PB82 DNA ani/, its relatiO'fUlhip to tke percentage of FUdR

incorporation The buoyant density ofPBS2 DNA (1·722 g/cm 3 ) is 0·034 gJcm 3 higher tha.n would be the density of DNA with a similar base composition (28% G + 0; 1·688 g/cm 3 ;

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FIG. 2. Incorporation of [2_14C]FUdR into the DNA of phage PBS2, as determined by changes in the buoyant density in the CsCI gradient. The phage was grown in the presence of 50 p.g [2_HC]FUdR (3-1 X 108 ctsfminfmg) per ml. of VB synthetic medimn and purified by 3 cycles oflow- and high-speed centrifugation. The distribution of the released DNA (preparation 0) was measured by the absorbance at 260 mIL (-0- and trace a) and by radioactivity assay (-e-). The buoyant density profiles of individual fractions 9 to 12 are represented by traces b to e. Preparation 0 was contaminated with B. BUbtili8 host DNA (1·703 g/cm3 ) and RNA (broken line on uppermost trace), since the crude lysate was not treated withDN~and RNase. The e~t of ~2-lfO] FUdR in~rroration into phage DNA (fraction 11) is listed m Table 1. The dashed lines m traces a to e mdicate the position of C. jo1mBomi DNA. added as the density marker.

282

H. A. LOZERON AND W. SZYBALSKI

Erikson & Szybalski, 1964) but containing thymine instead of uracil. These figures indicate that the addition of methyl groups in the 5 position of uracil decreases the buoyant density of DNA; addition of one methyl group to each base should result in a decrease in buoyant density by approximately 0·1 gJcm 3 • The additionoffluorinein the 5 position of uracil, on the other hand, increa..~es the density of DNA (Figs 1 to 3, Table 1). The percentage of FUdR incorporated is directly related to the buoyant density increase of the DNA by a factor of approximately 1'7% of UdR replacement per 1 mgJcm 3 (0·6 mgJcm 3 per 1% UdR replacement), within the range of substitution from 0 to 25% (Table 1).

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FIG. 3. Distribution of FUdR·labeled PBS2 phage (PBS2.FUdR) and its DNA in the CsCl gradient. Preparation D of PBS2·FUdR phage was prepared as described for preparation 0 (Fig. 2) and centrifuged in the CsCI gradient. Fractions of 0·05 mI. were collected and their absorbance determined (-e-). The buoyant density profile of DNA released from preparation D by heating for 4 min to 62°0 in the presence of 0·3% Sarkosyl NL30 (Geigy Industrial Chemicals, Ardsley, N.Y. is represented by trace a; traces b.c, and d represent DNA released from fractions 23, 21 and 18-19, respectively. The extent of [2· 14 C]FUdR (3'6 X 10. cts/min/mg) incorporation into phage DNA (fraction 21; 1·729 g/cm3 ) is listed in Table 1. The dashed lines in traces a tod indicate the position of C. joh'YUJonii DNA added as the density marker.

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284

H. A. LOZERON AND W. SZYBALSKI

(c) Viability oj PBS2-FUdR phage and heterogeneity oj 5-jluorodeoxyuridine substitution

Viable titers of crude lysates of PBS2-FUdR phage grown in the presence of Penassa.y broth and 500 /Lg FUdR/ml., added at the time of infection, were 30% of controls (1010 p.f.u./ml.) in which no FUdR was present. Only a moderate FUdR incorporation of 10% was atta.ined under these conditions (Fig. 1, tracing d). FUdR substitution is considerably higher when VB synthetic medium is used (Table 1.) Under these conditions, however, the analog is exceedingly inhibitory to the formation of viable progeny phage, and only if the addition of 50/Lg FUdR/ml. is delayed until 20 minutes after infection do measurable amounts of viable progeny appear. The viable titer of the crude PBS2-FUdR lysate corresponding to PBS2 FUdR·DNA preparation C (Fig. 2), for example, was only 0'8% of the control lysate in which no FUdR was present, indicating that phage would have undergone only one or a maximum of two cycles of replication. Analytical density-gradient recentrifugation (Fig. 2, traces b, c, d and e) of sequentially collected fractions of PBS2 FUdR·DNA preparation C from a preparative gradient (Fig. 2, preparative run; control (trace a) represents an analytical run of this preparation) shows the presence of two broad classes of PBS2 FUdR·DNA molecules banding at 1·742 to 1·745 and 1·732 to 1·735 gJcm 3 • These data would be compatible with a model ofsemi-conservative replication (Meselson & Stahl, 1958) in which these fractions represent unifilarly and bifilarly FUdR-labeled DNA molecules. PBS2-FUdR·DNA preparations B (Fig. I, trace f) and D (Fig. 3, control (trace a», which had been purified from crude lysates 44,770 rev./minll 22 hr CsC! Jl

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FIG. 4. Buoyant density profiles of normal (traces a, b) and FUdR-la.beled (c, d) PBS2 DNA, in the native (NN; a, c) and denatured (dN; b, d) states. DNA was prepared as described in Fig. 3. An FUdR-DNA fraction of density 1·729 g/cm3 (traces c, d) was prepared by preparative CsCl gradient centrifugation. The DNA's were denatured by 1 min exposure to 0·1 N-NaOH followed by neutralization with 1 N.KH 2PO,. The dashed lines indicate the position of native 0, johnsonii DNA.

5.FLUOROURACIL·CONTAINING DNA

285

of considerably higher infectivity corresponding to 2 to 2'5% of the controls and, hence, represent phage that had replicated through at least several cycles, show only one broad peak of FUdR·labeled DNA which is slightly skewed toward the heavier buoyant density. This material seems to be free of detectable amounts of unifiIarly FUdR.labeled DNA as judged from the analytical banding pattern of PBS2 FUdR DNA purified from preparation D and denatured in alkali according to the procedures in the legend to Fig. 4. One observes only one broad peak (Fig. 4, trace d (dN)), whioh bands at a buoyant density 0·014 g/om3 higher than that of native FUdR·DNA (1.729 g/om3 ) (trace 0 (NN)) and is free of any peak or shoulder of denatured FUdR. free DNA expeoted to band at the density of 1·736 g/cm3 • The spike of ultraviolet· absorbing material banding at a buoyant density slightly greater than 1'743 g/om 3 (trace d) appears to represent a precipitate of denatured FUdR·DNA. This DNA preparation, therefore, oonsists of a broad speotrum of heterogeneously substituted, bifilarly, FUdR·labeled DNA molecules. A comparison of the ratios between p.f.u.'s and the areas under the curves of the control (1·435 g/om3 ) and PBS2·FUdR preparation B (1'439 g/om 3 ) of Fig. I (traces a and e) indicates that this phage preparation contains at least 60% viable phage pa.rticles. As shown in Fig. 1 (preparation B, traces e and f), the buoyant density increment of the intact PBS2.FUdR phage is approximately one half that of the FUdR·DNA isolated from the same phage. Furthermore, the heavy, intermediate and light PBS2. FUdR phage fractions collected from the preparative CsC! gradient run of Fig. 3 (fraotions 18 and 19, 21, 23) yield FUdR·DNA's which band at respeotive lesser increments of buoya.nt density (traces d, c and b). It is evident that (I) the PBS2 phage prepara.tion is heterogeneously substituted and Can be partially resolved in a

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286

H. A. LOZERON "ND W. SZYBALSKI

preparative CsCI density gradient and (2) the greater buoyant density of PBS2.FUdR pha.ge relative to normal PBS2 phage is the direct result of FUdR incorporation into the DNA. (d) Analysis of the 'light' phage fraction

A small fraction of phage in normal PBS2 preparations (Fig. 1, trace a) bands at a significantly lower buoyant density (1·415 g/cm 3 ) in CsCI than that of normal PBS2 phage (1·435 g/cm 3). This fraction, designated here as 'light' phage, makes up arelative. ly higher proportion (25 to 35 %) of PBS2·FUdR phage preparations grown in the VB medium (Fig. 1, preparation B (trace e» and is shown to contain, almost exclusively, normal PBS2 DNA (Fig. 5, trace a). On the other hand, the purified phage fraction PBS2.FUdR of buoyant density 1·439 g/cm 3 (Fig. 1, trace e) gives rise to only PBS2·FUdR DNA of buoyant density 1·732 g/cm 3 (Fig. 5, trace b); no normal PBS2 DNA is detected in this fraction. These data show that (1) the contaminating amounts of normal PBS2 DNA in PBS2.FUdR·DNA preparations (Fig. 1, trace fj Fig. 2, trace a) arise from the 'light' phage fraction and (2) no detectable amount of normal PBS2 phage is synthesized in the presence of FUdR under our experimental conditions. It would appear, therefore, that this 'light' .phage fraction containing only normal DNA does not represent phage newly synthesized in the presence of FUdR but is a phage fraction that arises from the original inoculum. The viability of this particular fraction has not been tested, since initial experiments indicated that the viability of PBS2 in CsCI was rapidly lost. The low buoyant density of this fraction suggests, however, that these particles have a relatively high protein to DNA ratio compared to that of normal PBS2 phage and, consequently, most probably represent functiona.lly inactive phage with incomplete phage genomes. (e) Ultraviolet 8enaitivity and koBt-cell reactivation oj PBS2 phaiJe

E. coli phages Tl, T2 and lambda survive ultraviolet irradiation to a considerably greater degree when plated on wild-type strains than when plated on the respective ultraviolet-sensitive mutant strains (Ellison, Feiner, & Hill, 1960; Howard-Flanders & Theriot, 1962;Harm, 1963). Ultraviolet-resistant, wild-type bacteria are considered to possess the capability to repair ultraviolet damage to their DNA (dark reactivation) via an enzymic repair mechanism, which often also effects repair of the ultravioletirradiated phage (host-cell reactivation) plated on these strains (Sauerbier, 19624; Harm, 1963; Metzger, 1964). Thus, the defective ability to repair ultraviolet-irradia.tion damage, manifested by the ultraviolet· sensitive strains, accounts for the differential survival ra.tes of ultraviolet· inactivated phage plated on the ultravioletresistant and sensitive strains. The enzymic excision of one of the major types of ultraviolet· induced irradiation damage, thymine dimer (Benkers & Berends, 1960; Setlow & Setlow, 1962), common to both the bacterial and viral genome, may in part account for one of the steps in the dark reversal phenomenon (Boyce & HowardFlanders, 1964; Setlow & Carrier, 1964). Other repair mechanisms must be envisioned, however, for in B. subtilis, not only the thymine· containing phage SP3 but also phage SPOI (SP82), which contains hydroxymethyluracil in place of thymine, appears to be susceptible to dark repair mechanisms (Okubo & Romig, 1965; Reiter & Strauss, 1965; Mahler, 1965). It was of interest, therefore, to determine if ultraviolet-irradiated PBS2 phage, of whioh the normal DNA contains uracil in place of the usual thymine

5·FLUOROURACIL·CONTAINING DNA

287

(Ta.kaha.shi & Marmur, 1963), a.lso might be subject to host-cell reactivation in B. subtiliB. The survival curves of ultraviolet-irradia.t,ed normal PBS2 and PBS2·FUdR phage, plated on the wild.type strains of B. subtilis SB19, 168M, and on the non-host· reactivating strains 168 uvr- (Reiter & Strauss, 1965) and 168 hcr-9 (Okubo & Romig, 1965) are plotted in Figs 6, 7(a) and 7(b). Before proceeding to a discussion on the ultra.violet-sensitiza.tion effects resulting from the FUdR substitution into the phage DNA, some over·all aspects of these ultraviolet· survival curves should be considered. The ultraviolet-survival curves of PBS2 and PBS2·FUdR phage obtained with the non.host-reactivating mutants are very nearly exponential, whereas when the-same ultraviolet.irradiated PBS2 stocks a.re plated on the wild-type parent strains, their inactivation rate is much slower, with the survival curve exhibiting two distinct slopes. On examination of the ultraviolet· survival curves of normal PBS2 phage in Figs 6 and 7, the experimentally determined, ultraviolet-resistant fraction, which is the extrapolated ordinate intercept of the final portion of this curve, is 20% of the total viable phage population. Our proffered interpretation of these ultravioletsurvival curves is similar to that proposed for the TI phage-E. coli host system (Sauerbier, 1962b; Harm, 1963). The abrupt decrease in slope of the PBS2·survival curves obtained with the wild-type parent strains may be interpreted as due to dark repair by a 20% fraction of the wild.type host cells. Other interpretations were considered but for reasons discussed below were eliminated. (1) The possibility that multiplicity reactivation was responsible for the decrease in slope at the low survival levels was precluded by the fact that m.o.i.'s of less than one were used throughout the entire ultraviolet dose.range in these experiments.

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FIG. 7. (a). (b) Effeot of host reactivation of the ultraviolet survival of nonnal ( o .• )and FUdB· labeled (6.,.; preparation C, IIe6 Fig. 2) phage PBS2. The phages were plated either on wild·typeB. subtiliB strains (open symbols; (a) SB19 (b) 168;M) oronnon.host.reactivating strains (filled symbols; (a) 168 t.wr-. (b) 168 her·9) after pre.adaorptiOD to exponentially growing cultures. Each symbol represents an average of three experiments.

(2) The two·component curve could be the refleotion of two phage populations of differing intrinsic ultraviolet sensitivities, but no evidence is available to suggest that this is the ca.se. The ultra.violet.resistant portion does not appear to represent the 'light' pha.ge fraction for, as discussed in section (d), this phage fraction may very probably be regarded as non-infectious. Furthermore, the 'light' phage fraction, even if considered infectious, could not account for the ultraviolet-resistant fraction. for the proportion of 'light' phage in the population is much less than 20% (refer to Yxg. I, control, tra.ce al. Also. the extrapolated intercepts of the ultraviolet.survival curves of the normal and PBS2.FUdR phage preparations A and B of Fig. 6, for example, are quite consistent, even though the proportions of light phage present in these respective preparations are quite different (Fig. 1, traces a, c and e). Finally. evidence a.gainst a. 20%, intrinsic ultraviolet.resistant phage fraction is based on the observation that the ultraviolet-surviva.l curve of phage pla.ted on the ultraviolet. sensitive ba.c~rial hosts is missing the ultra.violet·resistant component. (f) Effect oJ FUdR incorporation into PBS2 DNA on ultraviolet 8eruitivity and Jw8t·oeU

reactivation The incorporation of FU into the RNA of tobacco mosa.ic virus sensitizes the intact virus a.nd its infectious nucleic acid to ultraviolet light (Be6a.revic, Djordjevic & ~uti6. 1963; Lozeron & Gordon, 1964). The present finding that FUdR could be incorporated into the DNAofPBS2phageprovided a unique opportunity to study the radiobiologioal effects resultixxg from FU incorporation into the DNA. The ultraviolet· survival curves of PBS2 and PBS2.FUdR preparations A and B. plated on the wild.type SB19 host. a.re plotted in Fig. 6. Based on b. values (Fig. 1).

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289

11 %(A) or 18% (B) of uracil is replaced by FUdR. The experimentally observed ultraviolet sensitization, represented by both the initial and fin.a.l portions of the survival curves, is correlated with the extent of FUdR substitution. The ultraviolet-survival curves of normal PBS2 and PBS2·FUdR phage prep&ration C (FUdR substitution = 24%), plated on both wild-type and the non-host-reactivating strain uvr- (Reiter & Strauss, 1965), &re plotted in Fig. 7(a). Comp&rable data &re obtained from the ultraviolet-survival curves of PBS2 and PBS2-FUdR phage plated on 168M and on the independently isolated, non-host-reactivating strain hcr-9 (Okubo & Romig, 1965) (Fig. 7(b». PBS2·FUdR phage appe&l'8 to be slightly but consistently more ultraviolet-sensitive than normal PBS2 plated on the uvr- a.nd hcr-9 stra.ins. The fact that the ultraviolet-resistant component of the PBS2-FUdR survival curves obtained with the wild-type bacterial strains is still present would suggest that PBS2-FUdR phage is still susceptible to considerable host-cell reactivation. In comparison, it is of interest to note that following ultraviolet irradiation, host-cell reactivation of bromodeoxyuridine.containing DNA is very effectively if not completely blocked (Stahl, Craseman, Okun, Fox & Laird, 1961; HOWard-Flanders, Boyce & Theriot, 1962). We are muoh indebted to Drs I. Takahashi, W. R. ROmig and B. Strauss for the phage and bacterial stocks usedin these studies, and to Dr C. Heidelberger for the generous gift of(2. 1'C]FUdR. We also wish to thank Dr I. Takahashi for critical reading of the manu. script and to acknowledge the expert assistance of Mr M. Fiandt and Mr D. Zuhse with the analytical esCI gradient centrifuga.tion. This work was supported by grants from the Na.tional Science Foundation (B-14976), the National Cancer Institute (CA-07175) and the Alexander and Margaret Stewart Trust Fund.

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