Sedimentation of DNA of Dictyostelium discoideum lysed on alkaline sucrose gradients: Role of single-strand breaks in gamma ray lethality of sensitive and resistant strains

J. Mol. Biol. (1973)79,267-284

Sedimentation of DNA of Dictyostelium discoideum Lysed on Alkaline Sucrose Gradients : Role of Single-strand Breaks in Gamma Ray Lethality of Sensitive and Resistant Strains ALLY

T. Goumrt

AND

R. A. DEERING

Biophysics Department The Pennsylvania rgtate University University Park, Pa. 16802, U.S.A. (Received 15 January

1973, and in revised form 14 June 1973)

The nuclear and mitochondrial DNA of the amoebae of the cellular slime mold Dic@~telium dkxrideum have beenlabeled with [methyL3H]thymidine by allowing them to grow on Esohwichia coli MT- containing this label in its DNA. Neutral CsCl gradients were used to identify the labeled molecules. Alkaline sucrose sedimentation profiles of cells lysed directly on the gradients revealed two high molecular weight species, one of about 90 S (single-strand mol wt = 1.4~ lo*) identified by alkaline C&l rebanding as nuclear DNA, and another of 43 S (single-strand mol wt = 2.3 x lo’), identified aa mitochondrial DNA. These alkaline sucrose gradients were used to study the production of single-strand breaks and their rejoining in DNA of a gamma ray-resistant strain (NC-4; 10% survival dose for cell proliferation, D,, = 300 krad) and in two radiation-sensitive daughter mutants (~~-18, Die = 75 krad; e-13, D,, = 4 krad). With *OCo gamma rays, breaks were produced in nuclear and mitochondrial DNA at an efficiency of one break per 33 eV in all three strains. At doses up to about 100 krad, these single-strand breaks were closed equally well during post-irradiation incubation of NC-4, ys-18 and ~-13, even though their survivals were widely different, indicating no apparent correlation between parental strand rejoining and survival in the sensitive strains. At higher doses, post-irradiation treatment with 1 mg caffeine/ml sensitized NC-4 and retarded strand-rejoining, suggesting that lethality in this resistant strain may be related to strand breaks. It is concluded that single-strand rejoining is a necessary, but not sufficient, condition for radiation survival in this organism. The nature of the apparently unrepaired lesions leading to lethality in the sensitive strains is not known.

1. Introduction The cellular slime mold Dictyostelium considerable cellular

development

amoeba-like

discotieum

is a eukaryotic

micro-organism

of

value as a model system for the study of the control processes underlying

Its replicative

and differentiation.

during whioh the

phase,

cells feed on bacteria or soluble nutrient, and actively multiply by fission,

is clearly separated

from a developmental

there is aggregation

into multioellular

phase. During the developmental

masses, and differentiation

t Present address: Rookefeller University, New York, N.Y 10021., U.S.A. 267

phase

into two cell types,

268 spores end stalk dls

A. T. KHOTJRY AND

R. A. DEERINQ

(Newell, 1971; Banner, 1967). In addition, its relatively low

DNA content suggests a degree of complexity considerably less than that of the more highly developed eukaryotes, offering the possibility of an understanding of fundamental eukaryotic molecular phenomena. Generally, the cells are haploid, containing nuclear DNA with a total molecular weight of about 5 x lOlo distributed among seven chromosomes, and mitochondrial DNA with a total molecular weight of about 3 x lOlo. The complexity of each chromosome is thus only about two to three times that of the Escherichia wli genome (Sussman & Rayner, 1971; Fidel t Banner, 1972). The molecular mechanisms by which eukaryotic cells in various stages of their life cycle cope with deleterious agents such as radiation, mutagenic and carcinogenic chemicals are inadequately understood. In this laboratory we have begun a series of studies aimed at characterizing the molecular responses to such agents of D. diswideum in the actively dividing and developmental phases of growth, with particular attention to the involvement of DNA and its repair. The wild type strain (NC-4) of this slime mold is resistant to gamma rays, ultraviolet light and several alkylating agents as compared to several sensitive mutants, two of which, ys-18 and ys-13, have been characterized more extensively. The gamma ray doses required to give 10% survival for NC-4, p-18 and ys-13 are 300, 75 and 4 krad, respectively (Deering et al., 1970; Payez et al., 1972). We are investigating the molecular basis of these differences in sensitivity and have so far concentrated to a large extent on the responses to gamma rays, with some additional studies also in progress on the effects of ultraviolet light and alkylating agents (Guialis & Deering, unpublished results; Payez & Deering, 1972). One molecular lesion always observed after ionizing irradiation of cells is the DNA single-strand break (for a recent review, see Setlow & Setlow, 1972). Most cells have the ability to rejoin these breaks (Painter, 1970; Smith, 1971); some of those which cannot are radiation-sensitive (Kapp & Smith, 1970; McGrath & Williams, 1966), suggesting that the single-strand break may be at least one of the types of lethal lesions in these systems. The availability of mutants with widely differing radiation responses in the dose range appropriate for producing numerous single-strand breaks in DNA of lo7 molecular weight or larger, prompted us to investigate the production and closing of such breaks in the nuclear and mitochondrial DNAs of the resistant NC-4 and sensitive p strains. Lysis of cells directly on alkaline sucrose gradients (McGrath t Williams, 1966) has been used in several systems for dete rmining the sizes of single-strand DNA molecules after irradiation. Since our D. diswideum strains use bacteria as their sole nutrient, it was necessary to devise and evaluate a reliable technique for labeling the slime mold DNA without also retaining labeled DNA of bacterial origin in the cell lysates. Here we report: (1) the labeling of the nuclear and mitochondrial DNAs of D disc&&urn by growth on E. wli 15T- labeled in its DNA; (2) the identification of the sedimentation peaks obtained after lysis on alkaline gradients ; and (3) the application of these results to an evaluation of the production and closing of single-strand breaks in the nuclear and mitochondrial DNAs of the resistant and sensitive strains. The effect of caffeine (an often used inhibitor of “repair”, see Deering et al., 1970) on rejoining of strands in NC-4 as compared to its effect on survival is also determined.

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2. Materials and Methods (a) Cell strains D. dkcoCdeum NC-4 was obtained from M. Sussmsn. Gamma-sensitive mutants ~a-18 and ys-13 were isolated by Deering et a.?. (1970). For this study, E. coli B/r or 15T- was used as the sole source of nutrient for slime mold amoebae. (b) Solutions, viability acraayand stack maintenance The composition of the agar (BPM) and the buffer (PBS), the viability sssay, and the general proceduresfor the growth and maintenance of D. dakcoideum have been given by Deering et a.2.(1970). (c) chowth of labeled and unlabeled bacteria E. coli 15T- was grown for 3 h into stationary phase in minimal A medium (Marsden et al., 1972), supplemented with 5 pg thymidine/ml. To label the bacterial DNA, the thymidine was supplemented with [meth$-3H]thymidine (Amersham-Searle; spec. act. 20 Ci/mmol) to give a final spec. act. of 0.2 Ci/mmol, unless otherwise stated. Unlabeled E. coli wsa washed in buffer and stored at -62°C in the presence of 16% (v/v) glycerol at a concentrationof 5 x loll cells/ml. Labeled E. coli was washed with buffer and stored at lOlo cells/ml at 6°C. The techniques for growing and storing E. cola B/r have been described by Deering et al. (1970). E. coli B/r protein was labeled by growth in minimal A medium supplemented with 10 $?i/mmol of L-[3H]leucine (Amersham-Searle) and 7 rg unlabeled L-leucine/ml. (d) Labeling of D. discoideum DNA The DNA of D. dticoideum was labeled by overnight growth on E. coli 15T- labeled with [wat%hyl-3H]thymidine. The D. dkcoideum cells were then “chased” to eliminate residual label of bacterial origin. The chase consisted of washing the amoebae relatively free of bacteria by differential centrifugation (3 centrifugations of 2 min each at 150 g, resuspension in buffer, one final centrifugation to pellet amoebae) and resuspend&g them in unlabeled E. coli 15T-. After growth for 1.5 h, the amoebae were again washed free of bacteria and resuspendedin fresh unlabeled E. coli 16T-. After a second period of growth of 1.5 h, the labeled bacterial DNA had been successfullychased out, as described in Results. The total chase period was 2 x 2 h for e-13 rather than the 2 x 1.5 h used for NC-4 and ys-18 strains, due to the longer doubling time of this strain (5 h compared to 3 h for NC-4 and ~~-18). This method of labeling the DNA of D. diawi&eumresultedin the incorporationof approximately 17,000 cts/min/106 cells. It was important in all experiments that the bacteria used as nutrient were aerated for at least 5 h before use, in order to eliminate undesirablelags in the growth of the amoebae. (e) Labeling of D. discoideumprotein D. discotieuwaprotein was labeled by growth on E. co& B/r labeled with [3H]leucine. Growth and chase conditions were the same as those describedfor DNA labeling. ( f ) Irradiation

Irradiation was with a Gammacell 200 8oCo source (Atomic Energy of Canada, LM) at a dose rate of 8.5 krad/min, with aeration, at the temperature of ice-water. Unless otherwise noted, irradiation of D, discoideum was in the presenceof the bacteria used as nutrient. (g) Isolation of nuclei for CaCl gradient lysatee Nuclei were isolated using a modificationof the procedureof Sussman & Rayner (197 1). Amoebae were harvested in late log phase and washed free of bacteria as described previously. They were then washed once in cold distilled water and resuspendedin water at 2 x 10s cells/ml. 5 ml of “nuclear lysing solution” (0*04OhTriton X100, 0.004% spermine, and 0.25 M-sucrose)were added to 1 ml of a suspensionof amoebae. These amoebae were subjected to repeated pipetting until 90% were lysed. The unlysed cells were pelleted by

270

A. T. RHOURY

AND R. A. DEERING

centrifugation at 400 g for 6 min. The supernatant wss then centrifuged at 1000 g for 10 min to pellet the nuclei. The pellet was suspended in cold (S’C) wash solution (O-26 ~-sucrose, 0.001% spermine, 0.05 M-KCl), centrifuged again at 1600 g for 10 min, and the pellet resuspended in oold wash solution. (h) Preparation of csU&r and laucIeur 1ysote.sfor C&l grad* After the final pelleting in the normal washing procedure, amoebae to be lysed were resuspended in ice-cold 7% suerose (w/w), pelleted, resuspended in ioe-cold 7% sucrose, and sedimented once more. The cells were lysed at a concentration of 4 x 10s/ml in a lysing solution consisting of 77% (v/v) 0.1 M-EDTA (pH 8) and 23% (v/v) 20% sodiumN-lauryl sarcosine (w/w). Solid C&l wee added at a concentration of 0.27 g/ml of lysing solution and dissolved by gentle stirring. The solution wss then heated to 60% for 10 min. This procedure is essentially that of Firtel & Banner (1972). Nuolear lysates were prepared in the same way, except that the added washes in sucrose were omitted. (i) Alkaliraemevose gmdient 8~?ChWl&h A gradient of 4.8 ml of 5% to 18% (w/ v ) sucrose in the Beckman SW60.1 rotor wss used. Sedimentation of phage T4 DNA in this gradient showed that it was isokinetio. The two gradient solutions, modified from Elkind (1971) had the following composition: (1) 6% sucrose (w/v), 0,003 M-EDTA, O-9 M-NaCI, 0.1 aa-NaOH, pH 12.26, and (2) 18% sucrose (w/v), 0.003 M-EDTA, 0% M-NaCl, 0.16 M-N&OH, pH 11%. The gradients were overlaid with 0.25 ml of the following solution: 0.01 M-EDTA, 0+X M-N&C&0.45 M-NaOH, pH 13.3. Amoebae (6 to 7 x 10s; approx. 0.1 pg DNA), washed free of bacteria and suspended in 26 4 buffer, were layered on the gradient. Centrifugation began 30 min after layering of the cells on the gradients. Varying the number of cells from 2 x lo3 to 16 x 10s cells/gradient did not alter the sedimentation of the DNA. Temperatures greater than 26’C during the lysis period led to non-reproducible patterns. Delayiug the start of centrifugation more thsn 3 h after the amoebae were layered on the gradient resulted in smaller pieces of nuclear DNA. Unless otherwise indicated, centrifugation was at 30,000 mvs/ti for 90 min at 20°C. Essentially all counts were recovered from these gradients.

For neutral CsCl analysis, the following were added to a lysate of 4 x lo6 cells: 0.1 ml of unlabeled E. co&iand D. d4scoideurn DNA as carrier (10 pg each) ; 2 pg of 14C!-labeled E. wli DNA in 10 ~1 of SSC (SSC is 0.15 M-NaCI, 0.016 IK-sodium citrate), water and CsCl to give a final volume of 65 ml and a starting CsCl density of 1.670 g/cm3. Each gradient contained 6 ml. The gradients were ruu at 30,000 revs/min for 64 h at 20°C in a Be&men 6OTi Axed-angle rotor. A peristaltic pump was used to collect fractions through a capillary lowered to the bottom of the tube. (k) Alkahe C&l gradienb Whole cell lysates and material collected from alkaline sucrose gradients were centrifuged in alkaline C&l. Polyallomer tubes were used to avoid problems of DNA adsorption (Szybalski & Szybalski, 1971). In the analysis of peaks from sucrose gradients, the following components were mixed to make 6.5 ml of alkaline CsCl: 0.1 ml of alkaline sucrose containing the peak material to be analyzed; 0.66 ml of 0.4 M-KaHPO, adjusted to pH 13.3 with 60% (w/w) KOH; 10 d containing 0.2 pg E. di marker DNA; 0.1 ml containing 10 pg each of unlabeled D. thkddmm and E. wli DNA ; water and C&l to make a starting density of I.7 13 g/am3 (C&l contribution). 6 ml of this solution were used per gradient. Alkaline CsCl gradients of nuclear and mitochondrial DNA from previous neutral CsCl fractions were prepared in a similar manner. The pH value of the gradients at the end of the run wss 12.5. C&l gradients from the whole cell lysates were similar, except that the 0.64~ml initial sample contained the added components used in lysis (see se&ion (h), above). The starting density of C&l in this gradient was 1.727 g/om3. Centrifugation conditions and collection of fractions were the same as with neutral c&l.

ALKALINE

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OF SLIME MOLD DNA

(1) Isohtion of “C-labti

271

E. coli DNA

A modification of the procedure developed by Murmur (1961) was ueed. The resulting DNA had a spec. act. of about 3600 cts/min/pg, with optimal settings for the LX230 COUIltW.

(m) Preparation of [3H]thym&ndzbekd pluqe T4: size marker for alkaline su.cro~egradient-3 T4 phage containing DNA labeled with [meti&3H]thymidine was prepared by the method of Marsden et al. (1972). The phage suspensionfor centrifugationin alkalinesucrose gradients (25 ~1)was in buffer and containedabout 0.1 pg phage DNA (l-9 x lOlo phage/ml) and about 2000 cts/min. (n) Scintillation coun&ing Samples were counted using a scintillation fluid composed of 667 ml toluene, 333 ml T&on-X100,5*5 g PPO and 0.5 g dimethyl POPOP (Patterson & Greene, 1966). Fractions from alkaline sucrose gradients collected directly in l-dram shell vials and of volume 0.16 ml, were counted by adding 0.25 ml water and 3.0 ml scintillationfluid. This gave a clear emulsion for counting. The counting efficiency increased by only 7% from the bottom to the top of the gradients. Fractions from CsCl gradients (O-15ml), also collected directly in l-dram shell vials, were counted by adding 0.6 ml water and 3.5 ml scintillation fluid. These shell vials were then placed inside larger standard scintillationvials for counting in a Beckman LS230 counter.

3. Results (a) Labeling of D. discoideum DNA It was necessary initially to determine whether the procedure used was successful DNA of bacterial origin. This was accomplished by centrifugation of lysed D. &&deum in neutral CsCl after labeling, a~ shown in Figure 1. The gradients contained a density marker of [14C]thymine-labeled E. coli DNA. There was no significant number of 3H counts in the region of E. wli DNA, indicating that the chase procedure WM

in labeling D. discoideum DNA and whether the chase succeeded in eliminating

I-

..* . .. . ..’ _ .,

5

IO

15

20

25

3:

Froctmno

35

40

45

’ 0 50 TOP

FIQ. 1. Neutral CsCl gradientof a wholecelllysateof D. diecvideum NC-4 labeled with [m&yL3H]thymidine from E. coli lbT_ and then ohssed with unlabeled 16T- for two 14-h periods; “H label from amoebae (-a--a---); “C-labeled 1. coli marker DNA (--O--O--); lye&a of isolatednuolei(. * * A * * * A * * a).Densitiesare indicated es g/cm3. 18

A. T. KHOURY

272

AND

R. A. DEERING

result has also been confirmed in the work of Deering & Jensen (1973), who compared gradients for unchased and chased samples. Sussman & Rayner (1971) and Firtel & Banner (1972) have characterized the DNA of axenic strains of D. discoideum. The strains were obtained initially from strain NC-4 cultures by selection for the ability to grow in a nutrient medium without bacteria (axemc growth). The axenic strains are also capable of growth on bacteria. These workers showed that the DNA of these cells yielded a bimodal banding pattern on neutral CsCl gradients. By isolating the DNA of purified nuclei and mitochondria, they demonstrated that the higher density peak (l-682 g/cm3, considering E. cola’to be l-703, g/cm3) was mitochondrial in origin, and that the lower density peak (1.676 g/cm3) was nuclear in origin. Quantitatively, about 65% of the DNA was nuclear and 36% was mitochondrial. When strain NC-4 and its axetic Ax-3 derivative (obtained from William F. Loomis, Jr, University of California, San Diego, who originally isolated this strain) were grown on E. wli KT- labeled with [3H]thymidine, these two strains gave identical patterns of labeling on our fixed-angle CsCl gradients (Ax-3 experiments by William Ford, this laboratory). Figure 1 shows one such pattern for the NC-4 strain. We have estimated by micropyonometer measurements that the densities of our two labeled peaks are 1.687 and l-681 g/cm3, relative to E. wli at 1*7O3r,glom3. We believe this to be in good agreement with the published densities of the mitochondrial (1.682) and nuclear (l-676) DNAs, considering the difficulties inherent in accurately determining absolute densities in fixed-angle rotors. To further support this conclusion, a lysate of a purified nuclear preparation was equilibrated in a C&l gradient, with the resultii pattern shown by the dotted line in Figure 1. Although a shoulder of higher density material remains (presumably mitoohondrial contamination), this preparation is considerably enriched in material with a density of l-681 g/cm3. This supports our conclusion stated above, based on the literature and the densities, that our labeled peaks correspond to those previously reported as mitochondrial and nuclear DNA. Additional evidence that these peaks are both DNA and not a possible labeled polysacoharide (Sussman & Rayner, 1971) is provided by our observation that both their densities increase by O-06 g/cm3 in alkaline CsCl, as would be expected of DNA (Vmograd et al., 1963). A small amount of, as yet unidentified, labeled material, possibly a polysaccharide, banded near the top of our gradients. SUOC~SS~~~. This

(b) Alkaline sucrose projlles of 3H-lubeEedD. discoideum amoebae Figure 2 shows a typical sedimentation profile of D. diswideum NC-4 amoebae lysed on alkaline sucrose gradients. For this particular preparative scale gradient, the thymidine specific activity was 5 Ci/mmol (instead of 0.2 Ci/mmol as for later experiments) and the samples counted are fractions from pooled, equivalent fractions of six gradients. The profile shows labeled material sedimenting in three regions, of approximately 9 S, 43 S and 90 S. Pooled preparations of each of these three species were retained, as indicated by the numbered regions on the gradient, and were stored at -52°C for later CsCl density analysis. The position to which phage T4 DNA (single-strand mol wt = 5.5 x 10’; Freifelder, 1970) sedimented is indicated in the Figure. With reference to this, molecular weights of the three sedimenting speaies of D. diecoideum could be calculated using the equation of Burgi & Hershey (1963) with Studier’s (1965) value of GC = 0~4,

ALKALINE

SEDIMENTATION 353 127

rl

218 105

120 83

550 61

OF SLIME 175 38

I

MOLD

DNA

273

2 0 Mol.wt (x10-? 16 s2(

i Boltom

Fraction

no.

IUP

FIU. 2. Alkaline sucrose proille of unirradiated D. diawideum NC-4. The numbered regions indicated those %&ions pooled for later identification. The 9 5, 43 S and 90 S regions referred to in the text oorrespond to regions 1,2 and 3, respeotively. The arrow indioates the position to which phage T4 DNA sediments : sedimentation is from right to left.

The number-average molec&r weight, M,, of the 90 S material, l-4 x 10s (average of 12 gradients), was calculated according to Palcio & Skarsgard (1972). The 43 S material appears to be fairly homogeneous ; the DNA sedimenting to the fraction with maximum radioactivity has a molecular weight of 2.3 x 107. Firtel & Banner (1972), from renaturation studies, have estimated the molecular weight of mitochondrial DNA from D. discoideum to be about 4 x 107. If the 43 S material represented half of a linear DNA helix, its double-stranded molecular weight would be 4-6 x 107. This moleculsx weight is similar to that of the mitochondrial DNA of other simple eukaryotes, such as yeast, Neuroqora and Tetrahyrrwna (Borst, 1972). Some mitochondrial DNAs 8re known to exist in the form of twisted ciroles, whioh gives them a higher s-value in alkaline sucrose (Dawid & Wolstenholme, 1967). Judging from the s-value in some preliminary neutral sucrose gradients of cell lysates of what we believe to be the material that bands at the 43 S position in alkaline gradients, it appears the 43 S material is not closed circular DNA. Fir&l& Banner (1972) did not 6nd circular DNA in eleotron micrographs of prep8rations of mitoohondrial DNA &fromD. dkwideum. !J!he material, designated 9 S for convenienoe, in actuality had a range of sedimentation values from 0 to about 16 S, as indicated by other, longer centrifugation runs. It is shown later that this “9 S ” material is probably a mixture of several components, one of which seems to be low molecular weight DNA (<5 x 10e). (c) Alhxzline CsCl density of sucrose gradient peak material The material recovered from the 9 S, 43 S end 90 S regions was oentrifirged in alkaline C&l with [14C]thymine-labeled E. wli marker DNA. Figure 3(a) shows the results. The 9 S material gave a broad bend of apparently small molecular weight DNA, which had a density intermediate between the 43 S end 90 S material. Additional material banded near the top of the gradient. This is apparently the material found banding near the top of the neutral CsCl gradients of whole cell lysetes (see Fig. 1). Also some material, apparently of too small a molecular weight to band, was distributed throughout the gradient. The 9 S material appears to be a mixture of low molecular weight DNA and one or more other types of molecules.

274

A. T. KHOURY

AND

R. A. DEERING

8

.k3

0

2

20

(b)

,1

0 0 $

E. co/i DNA

16

8

Bottom

Fraction

no

Top

Fro. 3. (a) Alkaline C&l gradients of the 9 S (* - * 0 * * * 0 * * a; 2000 cts/min total), 43 S (--O--O--; 3200 ots/min total) and 908 (-@-a---; 1800 cts/min total) mate&l isol&ed from the alkaline suaroeegradient shown in Fig. 2. (b) Alkaline CsCl gradients of nuolear (-a---•--; 1300 cts/min total) and mitochondrial DNA (--O--O--; 1000 cts/min total) isolated from the neutral C&l gradient shown in the inset. Also shown for comparison ia the pattern for isolated D. di.amidm nuolei (a * * A +- - A - . a; 1600 otfqmin total).

The 90 S material banded in a single peak and the 43 S material in B dual peak of greater density. In order to identify conclusively the two peaks of larger s-values isolated &fromalkaline suorose, D. dh~ideum nuclear and mitochondrial DNA were IUII for comparison in alkaline C&l with a 14C-labeled E. cc& DNA marker. These nuclear and mitochondri~l DNAS were isolated using a neutral CsCl gradient of DNA from a lysate of D. discodeum cells in an experiment similar to that of Figure 1. This neutral gradient is shown as an inset in Figure 3(b). The indicated fractions were rebanded in alk&ne CsCl as shown. Also shown here is a peak obtained in another gradient when DNA from B nuolear lysate was banded in alkaline CsCl. Note that the 43 S material (a) bands at the same position as mitochondrial DNA (b) and the 90 S material (a) at the same position as nude&r DNA (b). Also, the 43 S and 90 S material band in broad and narrow peaks, as do the mitochondrial and nuclear peaks, respectively. The peaks of nuclear and mitoohondrial DNA from the neutral CsCl gradient are narrower than those resulting from the 43 S and 90 S material. This is believed to be because the sucrose gradient material was subjected to considerable handling and extended storage in the freezer after oolleotion, while subjected to radioactive decay.

ALKALINE These

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factors would reduce the molecular weight of the DNA, resulting in wider bands

in the subsequent alkaline CsCl gradients. These results, along with the size estimates, indicate that the 43 S sedimenting material is mitochondrial DNA and the 90 S sedimenting material is nuclear DNA. It is believed that the broad dual peak of the mitochondrial DNA in alkaline CsCl is an indication that the complementary strands of this DNA have different thymine +guanine contents (Primrose, 1971). If true, this is in agreement with findings on other mitochondrial DNAs (Borst, 1972). &irradiated cells of the three strains used here (NC-4, ys-18 and ys-13) all gave the same sedimentation pattern in alkaline sucrose. Deering & Jensen (1973) found that ionizing radiation caused a preferential decrease in the synthesis of nuclear DNA in D. discoidem as compared to mitoohondrial DNA. Alkaline sucrose gradients of cells pulsed with label after irradiation showed almost no 90 S material, which is consistent with the evidence that this peak is nuclear DNA. (d) Sedinzentation of protein lube1 To determine if the DNA from the amoebae was sedimenting freely, or in assooiation with protein, strain NC-4 was labeled with [3H]leucine, and these cells were subjected to the usual alkaline suomse sedimentation. Most of the label was at the top of the gradients; only a very small amount sedimented near the bottom of the tube. There was no peak of protein in the region of nuclear or mitoohondrial DNA, indicating that the sedimenting DNA molecules were relatively free of associated protein. (e) Gamma ray effect on sedimentation profiles: yield of strand breaks The effect of radiation on typical alkaline sucrose gradient profiles is shown in Figure 4. Included in this Figure as a control is a profile of unirradiated ~~3-13.The 8.4~krad dose (a) caused no detectable shift in the position of the mitochondrial DNA peak. The nuclear DNAs of NC-4 and ys-13 were reduced in size, by about the same amount. Also in Figure 4(a) is a profile of NC-4 irradiated with 84 krad. This dose caused the DNA peaks to shift too far towards the top of the gradient for successful molecular weight analysis ; thus, some gradients were spun for twice the normal time, as shown in (b). After 84 krad, the mitochondrial and nuclear DNAs co-sedimented as a single peak of lower molecular weight. This would be predicted from theoretical considerations (Lehmann & Ormerod, 1970). If two populations of DNA of different sizes are subjected to ionizing radiation, after a dose sufficient to put, on the average, five strand breaks in the smaller of the molecules, the two populations will have the same average molecular weight and a random size distribution. Centrifuging these gradients for twice the normal time separated the peak of nuclear plus mitochondrial DNA from the 9 S material. These results show that the DNA of the three strains undergoes approximately the same amount of breakage after irradiation. The amoebae used for the gradients shown in Figure 4 were washed free of bacteria before irradiation, rather than just before lysis, as was the case in other experiments. This allowed the amoebae to be lysed on the gradients within one minute after irradiation. Pal& & Skarsgard (1972) have used an analysis developed by Charlesby (1954) to determine if the size distribution in a sucrose gradient is random. If so, as in the case when DNA accumulates enough randomly placed chain breaks induced by radiation, this analysis gives the number-average molecular weight (M,) of the distribution.

A.

270

T. KHOURY

AND Mol.

353

218

wt( x KY) 550

120

I

I

R. A. DEERING

I

17.5

I

2.0

I

I

I

I

22

27

6-

O6-

(b)

3-

o-

7

2

17

I2 Froctm

no

FIG. 4. Effect of e°Co gamma rays on alkaline sucrose pattans. (a) Unirmdiated p-13 (-•-a-), ~6-13 given 8.4 kmd (* * * IJ . -. 0 * . *; 6% survival), NC-4 given 8.4 krad (-A-A-; 100% survival), and NC-4 irrmdiatedwith 84 kmd (-O-O-; 80 y0 survival). (b) Twice the normal sedimentation time: amoebae given 84 krad, NC-4 (--a-@---; 80% survival), p-18 (--O--O--; 9% survival), and ye-13 (* ** 0 ***0 -*0;0.06%survival).

0

40

80 Radiation

FIG. 6. Plot of l/M, (0); F-13 (A).

120 dose (krod

160

1

zlereuadose to determine efficiency of strand breakage: NC-4 (a);

~a-18

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277

This approach was partioul8rly useful for our gradients. In Pigure 4(a), the 8*4-k& dose has caused part of the nuclear DNA peak to merge with mitochondriel DNA; yet it is possible using this analysis to determine the M, value of the nuclear DNA from the portion of the peak that is visible. In a similar way, the M, value of the peak of co-sediment@ nuclear plus mitochondrial DNA in Figure 4(b) can be determined, even though it is partially merged with the 9 S materiel. A plot of l/M,, versus dose should give a straight line, the slope of which is equal to the number of breaks per dalton per rad, and the intercept of which is l/M, for no irradiation (L&t et al., 1967). Figure 5 shows such 8 plot for the DNA of D. diawideum. The points for doses greater than 10 krads are for co-sedimenting nuclear end mitochondrial DNA; the points at 8.4 krad are for nuclear DNA only. There was no indication of any difference in eEciency of strand breakage for these two species of DNA. The efficiency of strand breakage calculated from the slope, for each of the three strains, is one break per 33 eV. (f) Rejoining of &an& after ~rrad&ion Figure 6 shows that by l-5 hours after 8.4 krad (a) and 6 hours after 34 krad (b), strain NC-4 has rejoined the strands broken by irradiation, in both the nuolear and mitochondrial DNAs. (The cells were irradiated in the presence of bacteria ; the Mol. wt 1 x lo_6

1

6-

(b) 6-

Bottom

Fraction

no

TOP

FIG. 6. Rejoining of stmnds in NC-4. (a) Alkaline morose gradients of cells unirradi&ed (-•--.-), irmdiated 8.4 krad and inoubated 0.6 h (--O--O--) or 1.6 h (. . - 0 -. - 0 . - a). (b) Unirradiatad (-•--•--), irradiated 84 krad and inoubated 0.6 h (--O--O--) or 6 h (. . . q . . . q . . .).

278

A. T. KHOURY

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shortest incubation time shown, 0.5 of an hour, reflects the time it took to wash the amoebae free of bacteria after irradiation.) Other experiments showed that strain NC-4 also closed its strand breaks up to doses of 340 krad (5% survival). At this dose, closing of breaks took 12 to 15 hours. After 460 krad (0.1% survival), NC-4 did not close all of its strand breaks by 20 hours after irradiation, at which time 70% of the cells were lysed. Mol.wt ( x &‘, 353 I

218 I

120 I

55 0 I

175 I

2.0 I

(a)

i

6-

O

oQ.o.~o.o.oo.o-o.o.o.o.o.o.o~ I I I 2 7 I2 I7 22 Bottom Fraction no.

I 27 Top

FIG. 7. Rejoiuing of strands in ~8-13. (a) U&radiated (-O-O-), irradiated 8.4 krad and inoubatedO$h(--0--()--)orl*lh(**. q .** . . .). (b) Unirradiated (-a--•-), irradiated 84 kr8d and iucubated O-7 h (--O--O--) or 10.6 h (* . . 0 * - * 0 * * *).

Figure 7 shows that strain ys-13, the extremely radiation-sensitive mutant, also closed its single-strand breaks after 8.4 krad (a) and 64 krad (b), which gave survival levels of 6% and 0*06%, respectively. Strain ys-18 closed its breaks by 7.5 hours after a dose of 84 krad (9% survival). The usual way to analyze the rate of strand rejoining would be to plot Mn verse time after irradiation. This approach cannot be used here because there is more than one sedimenting species in the gradient patterns. Instead, a different type of analysis was devised. It was assumed that any DNA which appeared in the gradient proflles at fractions 17 or lower was of nuclear origin. Consequently, as a measure of the degree of strand rejoining in nuclear DNA, we have determined the percentage of total counts that sedimented between fractions 3 and 17 as a function of incubation time after irradiation. Fraotion 17 would be equivalent to a molecular weight of 6 x 10’. In the controls, 36% of the label sediment&i in this range. Figure 8 shows curves

ALKALINE

SEDIMENTATION

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MOLD

DNA

279

. zo-

,

I

I

I

I

I

I

I

0

40 -

0

I

I

40 -

.-.

I

(cl

.

l_

-_-.-.-_.

30 /

.:’, 0 d/ ..‘, :‘I3 Y

20

.0..’ __-, __ - -

-- _--o

‘0 &/ 0

:/

0

0

4 Hours after

irradiotlon

Fra. 8. Surnmary plots of peroentage of total counts sedimenting between fractions 3 and 17 after various radktion doses. (e) 8.4 kred; (b) 84 kmd; (c) 187 kmd. NC-4 (-•-a---), p-18 (--O__O__), 9-13 (. . . 0.. . 0.. *). Line drawn across the plot at 36% is the average value for unirradiated sample.

ofstrands in the three strains. Figure 8(a) gives the results for 8.4 krad. Strain p-13 appears to close the breaks at least as fast, and possibly faster, than strain NC-4 ; as far as can be detected by this technique both strains seem to complete strand rejoining. All the samples, by l-5 hours after irradiation, had a nuclear M, value equal to that of the control. Figure 8(b) gives a similar plot for 84 krad. All three strains reconstructed large pieces of DNA in approximately the same time, and apparently completed strand rejoining by about seven hours. Though not evident in Figure 8(b), analysis of M, values shows that strain p-13 took slightly longer to complete the rejoining than did strains NC-4 and p-18. Figure 8(c) indicates that NC-4 closes strand breaks in nuclear DNA faster after 167 krad than does ys-18 or ys-13, and is the only strain to complete strand rejoining in the interval studied. There are approximately 500 copies of mitochondrial DNA in D. diawideum. To determine if mitochondrial DNA closed its strand breaks, the M, value between fractions 19 and 24 was calculated. If the number of breaks remaining was under 0.17 per molecule (which is equivalent to at least five of six single-strand molecules having no breaks) it was considered that strand rejoining in mitochondrial DNA was complete. This calculation wss attempted only if most of the nuclear DNA had been obtained from this analysis to comparerejoining

280

A.

T. KHOURY

AND

R.

A. DEERINC

rejoined sticiently to sediment out of the region of the gradient containing mitochondrial DNA (greater than 25% of the label having a molecular weight greater than 6 x 107). It was found that the three strains completed mitochondrial strand rejoining by five hours after 84 krad and by about eight hours after 167 krad.

(g) Effect

ofcaffeine

on rejoining

If strain NC-4 was incubated after irradiation in the presence of one mg caffeine/ml, the viability decreased with inoreasing incubation time. For example, after a dose of 16’7 krad, survival decreased from 60% immediately after irradiation to 0.05% after 13 hours incubation in caffeine. Strain ~~13 was not sensitized by caffeine (see Deering et al., 1970).

353 WI-

218

Mol.

wt (x d)

120

550

175

20

f!

! i

! \

!

:

6-

Fraction no

FIU. 9. Effeot of oeffeine on &rend rejoining. Alkaline suorormgradients of NC-4 irredi8ted with 167 krad (60% surviv8l) snd inoubatad in the presence or 8bbsenceof 1 mg caffeine/ml. Unirmdi8ted (-a---a-), 167 kr8d plue 0.6 h (--A-A--), 167 kmd plus 9 h (. * * q - * * 0 - - .), 167 kmd plus 9 h in 1 mg o&Gne/ml (--O--O--).

Figure 9 gives a comparison of gradient profiles of NC-4 cells incubated in the presence or absence of caffeine after 167 krad. The patterns show an inhibition by caffeine of rejoining of nuclear DNA strands. After nine hours inoubation with no caffeine, NC-4 had closed virtually all 70 of the strand breaks per sedimenting nuclear DNA molecule. In the presence of caffeine, an estimate of the position of nuclear DNA shows that 68 of 70 breaks per sedimenting molecule were closed. Rejoining was not complete, however. Figure 10 summarizes the effect of oaffeine on cells irradiated with 167 krad. Caffeine slowed rejoining and did not allow its completion by 17 hours after irradiation. If the caffeine was washed out at 13.2 hours, strand rejoining proceeded more quickly. This ooourred even though the survival of NC-4 had dropped to 0.05% by that time.

ALKALINE

SEDIMENTATION

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Caffeine

I

0

4

I

8 Time after

rrodiahon

MOLD

281

removed

I

I

12

16 (h

DNA

-I

)

FIQ. 10. Summary amve of a&eine &eat on strand rejoiuing after 167 krad to NC-4. No aaffeine (-•-a----); 1 mg caffeine/ml (--O--O--); incubated in caffeine for 13.2 h and then caffeine removed (* * * C, * * - @ * * -). The broken line drawn horizontally is the 36% average value for oontrols.

4. Discussion The efficienoy of single-strand breakage by c°Co gamma rays in this system was one break per 33 eV. This is within the range of published values (Setlow & Setlow, 1972; Town et al., 1972), although it is higher than the value of one break per 60 eV obtained by averaging the results of many publications (Town et al., 1972). This may imply that all strand breaks produced by ionizing radiation in this system were observed, and that they were not closed by a very fast strand-rejoining system before analysis (Town et al., 1972). On the other hand, if some of the observed breaks were enzymatically produced at the site of some other lesion, in addition to those directly produced by the radiation, then this would increase the apparent number of breaks per eV. Guialis & Deering (unpublished results) have results suggesting that enzymatically produced breaks can appear very soon after ultraviolet irradiation, even at ice-water temperatures, leaving open the possibility that there may be a contribution from this source after treatment with gamma rays as well. The dose of 8.4 krad causes no killing of NC-4 and almost complete killing of p-13 (5% survival). Figures 6 to 8 indicate that after this dose, NC-4 and ~~3-13close singlestrand breaks in the nuclear DNA within the same time after irradiation. A higher dose, 84 krad, was chosen since it gave very high survival of NC-4 (80%) while resulting in extensive killing of p-18 (9% survival). Figure 8 shows that NC-4 and p-18 have the ability to close single-strand breaks equally well after this dose. p-13, whose survival is only 0.06% after 84 krad, also seems to complete strand rejoining, although in a slightly longer time than NC-4 and ya-18. These results strongly indicate that inability to close single-strand breaks is not the cause of the gamma ray sensitivity of ys-18 and ys-13, at least for doses up to about 100 krad. After 167 krad, p-18 and ys-13 rejoin single-strand breaks in nuclear DNA more slowly than does NC-4. However, this dose is far in excess of that necessary to kill

282

A. T. KHOURY

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essentially all cells in the sensitive strains (0.02% survival for ys-18 and approx. 0905% for p-13) and, therefore, may not be relevant to the problem. Rejoining at these low survival levels may be disturbed by general metabolic deterioration rather than by deficiency of specific rejoining processes. A dose of 340 krad kills about 95% of NC-4 cells, yet this strain can still close virtually all of the single-strand breaks if given long enough (12 to 15 h). The strands are possibly not rejoined fast enough to ensure cell survival. This implies that for NC-4 (in contrast to ys-18 and ys-13), radiation induced lethality may be a direct consequence of too many single-strand breaks resulting from this very high dose, which are then not repaired rapidly enough. Any conclusions that strand rejoining is “complete” in our experiments must be qualified (as must all studies of cellular DNA size by the technique used in this paper) by the realization that the nuclear DNA observed in the gradients may be only about 0.04 of the intact chromosomal single-stranded DNA. Consequently, after much rejoining the intact DNA might still retain a few breaks undetectable by the present technique. More sensitive methods would be required to detect such residual breaks if they existed. Thus, our conclusions on the relation of strand rejoining to radiation sensitivity must be limited to the breaks that are detectable by the alkaline suorose gradient technique used here. The same qualification must be applied to virtually all published work on rejoining of strand breaks. A oomparison of strand rejoining in D. diswideum with other systems must be limited to the 8*4-krad dose, since most other work is in the dose range of 2 to 50 krad. After 8.4 krad, the single-strand breaks in D. diswideum are closed by about 1.5 hours. Among the mammalian cells, lymphoma cells close 80% of the radiationinduced single-strand breaks in 1.2 hours in the dose range 5 to 50 krad (Ormerod & Stevens, 1971). Murine leukemia cells close 80% of their breaks in about 20 minutes after 20 krad (Lett et al., 1967). Chinese hamster cells complete strand rejoining in about 20 minutes after I.5 to 5 krad (Elkind & Kamper, 1970). Bacterial systems, such as E. coli and Micrococcus radiodurana have been shown to complete rejoining after 20 and 50 krad, respectively, in about one hour (McGrath & Williams, 1966; Smith, 1971; Alexander et al., 1970). Thus the times required for rejoining in D. diswideum are comparable to other organisms. The partial inhibition of single-strand break closure by caffeine, and its concurrent sensitization with regard to cell survival, suggests that unclosed single-strand breaks may contribute to lethality in NC-4. Caffeine may slow rejoining, thus allowing some lethal “fixation” process to occur. The major, general conclusion that can be drawn from these results is that the closing of single-strand breaks in nuclear DNA is probably a necessary condition for survival, but not always a sufficient one. Other, as yet unidentified, lesions apparently contribute markedly to lethality if they cannot be repaired, as may be the case for strains p-18 and ~~3-13.Likewise, there is no apparent correlation between mitochondrial DNA breaks and lethality in this organism. It is of interest that ys-13 shows no oxygen-nitrogen effect for survival, whereas NC-4 and ys-18 do show this effect (Deering et al., 1970). This implies that the lethal lesion in this strain is produced equally well in the presence or absence of oxygen. A candidate for such a lesion might be the one recently reported by Paterson & Setlow (1972), whiah is not a strand break and is not sensitive to the presence of oxygen. Single-strand break production is sensitive to oxygen (see Paterson & Setlow, 1972).

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283

Possibly ~~4.13,and maybe ys-18 to some extent, lacks the ability to repair (used here in a quite general, unrestricted sense) this oxygen-insensitive lesion. Another possibility is that ys-13 is not killed directly by the suggested non-strandbreak lesion, but by a secondary effect, such as a gap in daughter-strand DNA opposite the lesion, as suggested by Hopwood (1972), and paralleling equivalent observations with pyrimidine dimers (see Smith, 1971). Then it would follow that the repair mechanism that is possibly deficient in ~54-13 is one that somehow repairs daughter-strand DNA gaps, maybe a mechanism analogous to ret repair in E. cdi (see Smith, 1971). Caffeine slightly enhances the survival of ys-13 (Deering et al., 1970). It may do so because it retards DNA synthesis (Lehmann, 1972), thereby giving the cell more time to correct the proposed lesions before they result in daughter-strand gaps. This is also consistent with the observation of Cleveland & Deering (1972) that survival of ys-13 is aided by post-irradiation holding in the absence of nutrients before plating. This investigation was supported by Public Health Service grant GM16620 from the National Institute of General Medical Sciences. We thank W. Ford for help in the nuclear isolations, H. Mersden and Z. Khoury for help with phage T4 preparations, and D. Jensen and L. Silan for able technical assistance. REFERENCES Alexander, P., Dean, C. J., Lehmann, A. R., Ormerod, M. G., Feldschreiber, P. t Serianni, R. W. (1970). In Radiation Protection and Sensitization (Moroson, H. & Quintillianni, M., eds), Barnes and Noble, New York. Bonner, J. T. (1967). The Cellular Slime Molds, 2nd edn, Princeton University Press, Princeton. Borst, P. (1972). Annu. Rev. Biochem. 41, 333-376. Burgi, E. & Hershey, A. D. (1963). Biophye. J. 3, 309-321. Charlesby, A. (1954). Proc. Roy. Sot. London (Ser. A), 224, 120-128. Cleveland, R. F. & Deering, R. A. (1972). Int. J. Radiat. Biol. 22, 245256. Dawid, I. G. & Wolstenholme, D. R. (1967). J. Mol. B&d. 28, 233-245. Deering, R. A. & Jensen, D. (1973). Biophy8. J. In the press. Deering, R. A., Smith, M. S., Thompson, B. K. & Adolf, A. C. (1970). Rudiut. Res. 43, 711-728. Elkind, M. M. (1971). Biophye. J. 11, 602-520. Elkind, M. M. & Kamper, C. (1970). Biophya. J. 10,237-245. Firtel, R. A. & Bonner, J. (1972). J. Mol. Biol. 66, 339-361. Freifelder, D. (1970). J. Mol. Biol. 54, 567-577. Hopwood, L. E. (1972). Radiat. Rea. (A&r.) 51, 530. Kapp, D. S. & Smith, K. C. (1970). J. Bacterial. 103, 49-54. Lehmann, A. R. (1972). BtiphyEys.J. 12, 13161325. Lehmann, A. R. & Ormerod, M. G. (1970). B&him. Biophya. Acta, 217, 268-277. Lett, J. T., Caldwell, I., Dean, C. J. & Alexander, P. (1967). Nature (London), 214, 790792. McGrath, R. A. & Williams, R. W. (1966). Nature (London), 212, 534-535. Marmur, J. (1961). J. Mol. BioZ. 3, 208-218. Marsden, H., Ginoza, W. & Pollard, E. C. (1972). J. Vi’irol. 9, 100&1016. Newell, P. C. (1971). In Essays in Biochemistry (Campbell, P. N. & Dickens, F., eds), vol. 7, p. 87, Academic Press, New York. Ormerod, M. G. & Stevens, U. (1971). Biochim. Biophye. Acta, 232, 72-82. Painter, R. B. (1970). Current Topics in Radiation Research Quarterly, 7, 45-70. Palcic, B. & Skarsgard, L. D. (1972). Int. J. Radiat. BioZ. 21, 417-433. Paterson, M. C. & Setlow, R. B. (1972). Proc. Nat. Acud. Sci., U.S.A. 69, 2927-2931. Patterson, M. S. & Greene, R. C. (1965). And. Chem. 37, 854-857.

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Payez, J. F. & Deering, R. A. (1972). Mu&&. Rec. 16, 318-321. Payez, J. F., Deering, R. A. & Freim, J. O., Jr (1972). M&z& Rec. 15, 82-85. Primrose, S. B. (1971). Biochkn. Biophya. Acta, 247, 29-37. Setlow, R. B. & Setlow, J. K. (1972). Anw. Rev. Biophys. Bioeng. 1, 293-346. Smith, K. C. (1971). Photophyeiol. 6, 209-278. Studier, F. W. (1965). J. Mol. Biol. 11, 373-390. Summan, R. & Rayner, E. P. (1971). Arch. B&hem. Biophye. 144, 127-137. Szybalski, W. & Szybdski, E. H. (1971). In Procedures in Nucleic Acid Research (Cantoni, G. L. & Davies, D. R., eda), vol. 2, p. 311, Harper & Row, New York. Town, C. D., Smith, K. C. t Kaplan, H. S. (1972). R&at. Re.9. 52, 99-114. Vinograd, J., Morris, J., Davidson, N. & Dove, W. F., Jr (1963). Proc. Nat. Acud. Sk, U.S.A. 49, 12-17.