Selective nicking of mammalian mitochondrial DNA in vivo: Photosensitization by incorporation of 5-bromodeoxyuridine

J. Mol. Biol. (1975) 99, 761-776

Selective Nicking of Mammalian Mitochondrial DNA in vivo: Photosensitization by Incorporation of 5-Bromodeoxyuridine ROBERT A. T,AWS~rAwAND ])&VII) A. CLAYTON

Laboratory of Experimental Ontology Department of Pathology Stanford University School of Medicine Stanford, Calif. 94305, U.S.A. (Received 7 January 1975, and in revisedform 29 May 1975) Mammalian thymidine kinase minus cells retain a mitochondrial specific thymidine kinase and consequently incorporate thymidine and thymidine analogues predominantly into mitoehondrial DNA. The extent of incorporation of exogenous thymidine or 5-bromodeoxyuridine in mitochondrial DNA is approximately fiftyfold greater than in nuclear DNA. This phenomenon makes it possible to selectively damage 5-bromouraeil-labeled mitochondrial DNA in v/re by exposing cells to long wavelength ultraviolet light. Under conditions which produce lesions in ~ 95% of the mitochondrial DNA population, the cell does not undergo further division. Lethality induced by light exposure in these cells is probably not attributable to nuclear DNA damage alone. Most of the mitoehondrial DNA molecules isolated from light-treated cells are nicked circles, many of which are subsequently degraded in rive. Neither repair of light-induced single-strand scissions in mitochondrial DNA nor replication of nicked molecules is observed in these experiments. 1. I n t r o d u c t i o n Mammalian mitochondria contain a covalently closed circular DNA genome which has been well characterized (for reviews see Borst, 1972; Clayton & Smith, 1975). We have previously demonstrated t h a t cells lacking the major soluble cytoplasmic thymidine t~inase (E.C. 2.7.1.21) retain a mitochondrial specific thymidine kinase (Berk & Clayton, 1973). Such cells have been utilized to study the details of mtDNA t replication in which radioactive thymidine was incorporated into m t D N A at a fiftyfold higher substitution rate t h a n into nuclear DNA (Berk & Clayton, 1974). The cell lines also incorporate BrdUrd with the same relative bias. The sensitivity of BrUra substituted DNA to long wavelength ultraviolet light has been well documented (for a review see Hutchinson, 1973). Single-strand breaks are the primary lesion induced b y light of wavelength ~ 3 0 0 urn. Exposure to light of this wavelength does not damage unsubstituted DNA. The specificity of photo-nic~ing in v/~o has been convincingly demonstrated by Boettiger & Ternin (1970) who were able to inactivate viral genes in chick fibroblasts without lethal damage to host cell nuclear DNA. Thus, in thymidine kinase minus cells, it should be possible to induce single-strand tAbbreviations used: mtDNA, mitoehondrial DNA; TK +, thymidine kinase plus; TK-, thymidine kinase minus; EthBr, ethidium bromide. 761

762

R. A. L A N S M A N A N D D. A. C L A Y T O N

b r e a k s in m t D N A a t a m u c h h i g h e r f r e q u e n c y t h a n i n n u c l e a r D N A , b y e x p o s i n g cells grown in BrdUrd to light of appropriate wavelengths. W e r e p o r t h e r e t h e effects o f such specific s t r a n d b r e a k a g e in m o u s e L-cell m t D N A . We have analyzed the structural properties of the damaged mtDNA and its ability to be repaired and undergo replication.

2. M a t e r i a l s a n d M e t h o d s (a) Celt lines and growth conditions A subclone of L M T K - , C2-1, (Berk & Clayton, 1974) was used as the T K - cell line. The T K + cell line used in control experiments was LA9 (Robberson & Clayton, 1972). B o t h lines were grown in suspension culture in Eagle's minimal essential m e d i u m (Flow Laboratories, Inc.) plus 10% calf serum, 100 I.U. penicillin]ml a n d 100 ~g streptomycin/ml. I n p r e p a r a t i o n for long wavelength ultraviolet light irradiation experiments, the cells were transferred to 100-ram plastic tissue culture plates (Falcon) a n d p r o p a g a t e d for 6 to 8 generations in the same m e d i u m except t h a t fetal calf serum replaced calf serum and s t a t e d amounts of B r d U r d (Sigma Chemical Co.) were added. Medium containing B r d U r d was k e p t a t 4°C in the d a r k a n d used within 10 d a y s of the addition of t h e analogue. (b) Ligh$ aploaratus and procedures The light sources were two Westinghouse FS20 fluorescent sun lamps with chrome fixtures with no additional reflector arrangement. A trough to hold a shielding solution of t h y m i d i n e (Boettiger & Temin, 1970) between the cells and lamps was constructed using Plexiglass sidewalls a n d supports a n d a Coming P y r e x b o t t o m (A3oo ---- 0.16, A2so -~ 2.0). The trough was filled to a height of 2 cm with a solution of 1.5 m g thymidine/ml, 10 mME D T A . A second sheet of P y r e x was leveled and s u p p o r t e d e x a c t l y 5.0 cm above the tops of the lamps, serving as a support for the plates of cells. A Gossen light meter was used to establish t h a t a uniform illnm~uation of 500 foot candles was obtained across the top plate. Medium was removed from plates of cells to be i r r a d i a t e d a n d replaced b y TD buffer (134 m~-NaC1, 5 m~-KC1, 0"7 m~-Na2HPO4, 2.5 m~-Trls (pH 7"5)). Plates were i r r a d i a t e d through t h e bottom. The t e m p e r a t u r e of the t h y m l d i n e solution in t h e trough a n d buffer in the plates rose <: I°C during 60 mln of continuous irradiation. (c) Purification of mit~chondria Medium was removed from plates a n d replaced b y cold TD buffer. Cells were detached mechanically a n d p a c k e d b y centrifugation. Cell pellets were suspended in 10 ml hypotonic buffer (10 mM-NaC1, 1.5 mM-CaC12, 10 mM-Tris (pH 7.5)) a n d incubated on ice for 8 rain. The swollen cells were t h e n disrupted using repeated gentle strokes with a Dounce homogenizer until > 9 5 % of t h e cells were broken (as judged b y phase contrast microscopy) leaving swollen, i n t a c t nuclei without visibly adhering cytoplasm. Special care was t a k e n during homogenization of i r r a d i a t e d cells since t h e y were often 2 to 3-fold larger t h a n normal a n d thus were more fragile. Concentrated (2.5 × ) mannitol-suerose buffer was a d d e d to t h e homogenate to a final concentration of 0-21 M-mannitol, 0.07 M-sucrose, 5 m ~ - E D T A , 5 mM-Tris (pH 7.5). Nuclei and unbroken cells were removed b y two sequential 5-rain centrifugations a t 2000 revs/min in a Sorvall GLC-1 centrifuge. The second s u p e r n a t a n t solution was layered on top of 10 ml of 1"5 M-sucrose (5 m ~ - E D T A , I0 mM-Tris (pH 7.5)) a n d centrifuged for 30 rain a t 22,000 revs/min in an SW27 rotor. Mitochondria were collected from the top of the 1.5 M-sucrose, diluted with mannitol-suorose, a n d pelleted b y centrifugation for 20 rain a t 15,000 revs[ rain in a Beckman JA-20 rotor. (d) Preparation of nuclear D N A Cell pellets containing 107 cells were suspended a n d homogenized in hypotonic buffer as described above. Homogenization was continued until no i n t a c t cells were visible under

PHOTOSENSITIZATION

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763

phase contrast microscopy. Nuclei were collected b y centrifugation a t 2000 revs/min for 5 rain in a GLC-1 centrifuge, t h e n resuspended a n d washed twice in t h e same buffer. Nuclei were then suspended in 10 ml S T E buffer (0.1 ~-NaCI, 0.01 M-EDTA, 0"05 M-Tris, p H 8"5), m a d e 1 ~/o in sodium dodecyl sulfate a n d thoroughly disrupted b y repeated passage t h r o u g h a 19-gauge hypodermic needle. 1.5 ml of this suspension was brought to a density of 1.685 to 1.695 g/cm 3 b y t h e a d d i t i o n of solid CsC1 a n d centrifuged for 40 h a t 30,000 revs/min, 20°C, in an SWS0.1 rotor.

(e) Purification of mitoehondriaZ DNA Mitochondrial pellets were suspended in 2.0 ml of S T E buffer a n d lysed b y the addition of 25% sodium dodecyl sulfate to a final concentration of 1%, followed b y a 3-min incub a t i o n a t 37°C. Solid CsCI was a d d e d to bring the lysate to a final density of 1.55 g/cm a a n d E t h B r was a d d e d to a final concentration of 400 gg/ml. Samples were then centrifuged for > 2 4 h a t 38,000 revs/min, 20°C, in a n SWS0.1 rotor. The tubes were p u n c t u r e d a n d 0.04-ml fractions collected through t h e bottom. Because of the heterogeneity of B r U r a substitution in m t D N A in these experiments, no discrete lower b a n d of closed circular D N A was visible b y fluorescence. The position of m t D N A in the gradient was assumed from t h e clearly visible fluorescent u p p e r b a n d containing contaminating nuclear DNA. Pronase digestion, when utilized, was performed b y adding previously self-digested Pronase (Calbiochem, B-grade) to a final concentration of 1.5 mg/ml to the mitochondrial sodium dodecyl sulfate lysate a n d incubating for 2 h a t 37°C. Collected lower b a n d m t D N A was p r e p a r e d for analytical CsCI gradient centrifugation b y extensive dialysis against STE buffer containing Dowex-50 cation exchange resin (Robberson & Clayton, 1972). After dialysis, t h e D N A was pelleted b y centrifugation for 8 h a t 30,000 revs/min, 5°C, in an SW50.1 rotor. (f) Agarose gel elec~rophoresis The m e t h o d is a modification of t h a t described b y Aaij & Beret (1972). A 2-layer gel system, 8 cm of 2 % agarose overlayered b y 0.4 cm of 0.6% agarose, was employed to maximize separation of open a n d closed circular m t D N A while ensuring t h a t open circular D N A fully entered t h e gels. The gel solutions a n d anode buffer contained 10 gg E t h B r / m l . D N A samples were pelleted through STE buffer which was carefully removed a n d replaced b y a buffer containing 10°//o sucrose, 0.5 mM-EDTA, 10 mM-Tris (pH 7-6). These samples (0.05 or 0.10 ml) were then layered on t o p of the gels u n d e r electrophoresis buffer. Since the samples were fractions of EthBr-CsC1 gradients a n d contained b o u n d E t h B r , resuspension a n d layering could be followed visually under ultra-violet light to minimize handling losses. Nevertheless, some losses occurred a n d were estimated b y adding samples o f 14C-labeled phage PM2 viral D N A to m t D N A samples before pelleting. The recovery of r a d i o a c t i v i t y from PM2 D N A could t h e n be monitored in the gels. Electrophoresis a t 5 mA/gel (0.5 cm internal diameter) was carried out a t room temp e r a t u r e for 5 to 6 h. (g) Det~nination of radioactivity Fractions (N0.04 ml) of the EthBr-CsC1 gradients were collected directly on W h a t m a n G F / A filters which were t h e n dried under h e a t lamps. The filters were counted in a Beckman LS230 liquid scintillation counter in a toluene-Omnifluor (New E n g l a n d Nuclear) cocktail. Agarose gels were divided into l - r a m slices which were placed in scintillation vials along with 0-5 ml of a 9:1 miYture of NCS solubilizer (Amersham-Searle) a n d water. The vials were t i g h t l y c a p p e d a n d shaken a t 37°C overnight. Omnifluor cocktail was then added. (h) I n vivo labeling of mitochondrial DNA Cells were radioactively labeled b y growth in m e d i u m containing either 1 gCi [5-methyl3H]thymidine/ml, 50 Ci/mmol; 2 pCi [5-3H]deoxycytidine/ml, 25 Ci/mmol; 0.01 /~Ci [5-methyl-l~C]thymidine/ml, 36 mCi/mmol; or 0-01 pCi [2-14C]deoxycytidine/ml, 50 mCi/ m m o l for t h e times indicated in the t e x t a n d Figure legends. T K - L-cells lack t h e cytoplasmic t h y m i d i n e kinase a n d do n o t concentrate t h y m i d i n e or incorporate significant 50

764

R . A . LANSMAN AND D. A. CLAYTON

amounts of exogenous thymidine into nuclear DNA (Berk & Clayton, 1973). As a consequence, the concentration of label in the meditun does not fall by more than 10% throughout long-labeling periods. (i) I n vivo l~5~i~,g of nz~cl~r DNA LA9 nuclear DNA was labeled, under the isotope conditions described in section (h), above, by growth of cells for > 3 generations in [z4C]thymidine or for 3 h in [3H]thymidine and 2× 10 -v ~-BrdUrd when the cells had been prelabeled with BrdUrd. The molarity of thymidine under the latter condition is 0.1 or 0.2 the molarity of BrdUrd in the medium. C2-1 nuclear DNA was labeled, under the isotope conditions described in section (h), above, by growth of cells for > 3 generations in [z~C]- or [3H]deoxyeytidine. (j) De~rrnination of buoyant d ~ i ~ s of DNA Analytical Cscl buoyant density equilibrium gradients were performed at 44,000 revs/ min, 20°C, in a Beckman model E ultracentrifuge equipped with a photoelectric scanner. Buoyant densities of mtDNA were calculated as previously described (Clayton & Vinograd, 1967) using a crab d(A-T) marker of e ---- 1-670. The extent of BrUra substitution was calculated on the assumption that full substitution would result in a ,t8 of 128 mg]ml since mouse mtDNA conta~na 32% thymine (0 = 1-692) (Clayton & Teplitz, 1972) and the buoyant separation between poly[d(A-T)] and poly[d(A-BrUra)] is 200 mg/ml (Wake & Baldwin, 1962). Preparative CsCl buoyant density equilibrium gradients were performed at 30,000 revs/ min, 20°C. The buoyant density differences between the centers of mass were estimated by assuming that 1 ml of the gradient represented a £p --~ 0.053. This assumption is derived from the fact that the equilibrium density gradient in an SWS0 rotor at ~ ----43,000 revs/m~n is approximately 0.10 g/cm4 (Radloff et al., 1967)t. Correcting for ~2r (r is 10.73 cm) for an SWS0.1 rotor at 30,000 revs/min yields a zip --~ 0.053 for 1 ml of column height if the I)NA is not buoyant at the extremes of the gradient. We have verified this assumption by analyzing the peak separation between mouse and human mtDNA. These DNAs have a ~8 of 8 mg/ml (Clayton & Teplitz, 1972; Clayton & Vinograd, 1967) and exhibit an 18-drop (~145/~1) separation under these eentrifugation conditions. Since mouse nuclear DNA has the same buoyant density and thus the same average thymine composition as mouse mtDl~A (Clayton & Teplitz, 1972), the determination of % substitution of BrUra for thymine is as described above. Thus, a one fraction ( ~ 8 /A) difference in these preparative gradients reprdsents an approximate substitution rate of 0.33~o.

3. R e s u l t s The cell line C2-1, a subclone of L M T K - , was chosen because it incorporates higher levels of [3H]thymidine into m t D N A than the parent line (Berk & Clayton, 1974). As expected, C2-1 also incorporates more BrdUrd into m t D N A t h a n the parent. Analytical ultracentrifugation studies of L M T K - grown in 40/~g BrdUrd/ml have previously ~hown a heterogeneous distribution of m t D N A with a mean b u o y a n t density of 1.705 indicating a mean substitution rate, BrUra for thymine, of ~10~/o (Clayton & Teplitz, 1972). Very little D N A with substitution higher than 25% was present. Figure 1 is an optical density trace of an analytical CsC1 b u o y a n t density gradient containing m t D N A from C2-1 cells grown for more than six generations in 40 and 200/~g BrdUrd/ml. Heterogeneous substitution is obtained at both concentrations. The mean density at 40/~g/ml (l~ig. l(a)) indicates 38% substitution, almost fourfold higher than t h a t obtained in L M T K - (Clayton & Tephtz, 1972). At 200 /~g/ml (Fig. l(b)), a mean substitution of 56~/o is obtained and the trace shows significant amounts of D N A in the density range expected for 20 to 80~/o substitution. t The density gradient cited in Radioff et at. (1967) is incorrectly printed as 0.010 g/cm4 instead of 0.10 g/cm% The correct gradient is displayed in Figure 1 of Radloff st al. (1967).

PHOTOSENSITIZATION

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765

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FIG. 1. Photoelectric O.D. scans of b u o y a n t n e u t r a l CsC1 gradients of closed circular m t D N A from C2-1 cells labeled w i t h (a) 40 ~g/ml a n d (b) 200 pg/ml B r d U r d for > 6 generations. The field is directed to t h e r i g h t a n d t h e peaks i n the left side of t h e p a n e l are crab d(A-T) m a r k e r DNA, 0 = 1.670. The abscissa denotes t h e b u o y a n t position expected for mouse m t D N A w i t h v a r y i n g degrees of B r U r a substitution. T h e samples were centrifuged a t 44,000 revs/mln~ 25°C for 36 h. The m e a n s u b s t i t u t i o n of B r U r a for t h y m i n e in m t D N A is 38% a n d 56~o for (a) a n d (b), respectively.

C2-1 is normally grown in suspension medium without BrdUrd. The cells have a doubling time of 19 hours in suspension or monolayer cultures and a plating efficiency of 80 to 90~. C2-1 has been carried for periods of time up to three months iu 200 ~g BrdUrd/ml with no increase in doubling time nor any decrease in colony-forming ability. Clearly, high levels of BrdUrd substitution in mtDNA and the low level substitution that occurs in nuclear DNA (see below) do not significantly affect the expression of any of the genetic information required for the propagation of these cells. Preparative CsC1 buoyant density gradient centrifugation has been used to detect low levels of BrdUrd substitution in nuclear ])NA. Cells grown in BrdUrd were labeled with [3H]thymidine or [3H]deoxyeytidine a n d mixed with control cells grown without BrdUrd and labeled with [14C]thymidine or [14C]deoxycytidine. The distribution of the two labels was determined in one-drop ( ~ 8 ~1) fractions from gradients containing DNA from nuclei isolated from the mixes. The data from three such mixes are shown in Figure 2: TK + L-ceUs (LA9) grown in 10 -7 M-BrdUrd (Fig. 2(a)) and 2×10 -7 M-BrdUrd (Fig. 2(b)); and C2-1 cells grown in 200 ~g/ml (7×10 -~ ~) BrdUrd (Fig. 2(c)). The substitution in each case has been calculated from the positions of the centers of mass of the distributions of the two labels. There is no difference in the buoyant position of nuclear DNA labeled with [3HI- compared to [x4C]thymidine in the absence of BrdUrd (data not shown). The mean density shift

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F[o. 2. Neutral CsCI equilibrium density gradients of L-cell nuclear D N A from cells grown in the presence a n d absence of BrdUrd. (a) Nuclear D N A from a mixture of LA9 cells grown for > 6 generations in 10- 7 ~ - B r d U r d and labeled with [SH]~hymidine and cells grown in BrdUrd-free medium and labeled with [14C]thymidine; (b) same as (a), except BrUra-labeled cells were grown in 2 × 10- 7 •-BrdUrd; (o) nuclear D N A from a mixture of C2-1 cells grown in 7 × 10 -4 M-BrdUrd and labeled with [3H]deoxyeytidine and cells grown in BrdUrd-free medium a n d labeled with [x4C]deoxyeytidine. These gradients were centrifuged in an SWS0.1 rotor a t 30,000 revs/min, 20°C, for 60 h. One-drop fractions ( N 8 ~1) were dripped onto GF/A filters and assayed for radioactivity.

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PHOTOSENSITIZATION

OF MITOCHONDRIAL

DNA

767

obtained in C2-1, 2.5 fractions or ~-~).0010 g/cm8, indicates ~0.83% substitution by BrUra. This value is slightly higher than the ~0.66% substitution calculated for the TK + cells at 10 -7 M.BrdUrd and lower than the ~-~1.5% substitution in TK + cells at 2×10 -7 M-BrdUrd. The LA9 cells with slml]~r nuclear substitution to that obtained in C2-1 serve as controls in irradiation experiments to help distinguish between the effects of damage to nuclear DNA versus mtDNA. The fluorescent sun lamp system described in Materials and Methods is a modification of that employed by Boettiger & Temln (1970) and is designed to obtain large doses of near u.v. while minimizing the transmission of all wavelengths below 300 nm. The ability of C2-1 and LA9 cells grown at different BrdUrd concentrations to form colonies after varying periods of irradiation is shown in Figure 3(a). Exposure of cells grown in BrdUrd-free medium to light for periods up to 30 minutes has no effect on viability. A single exposure of 60 minutes reduces viability by 50%, but two 30minute exposures separated by several hours of incubation in growth medium have no effect. The observation that BrdUrd-free cells fail to swell in hypotonic buffer after a 60 minute irradiation suggests that long exposures lead to membrane damage. I

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FIG. 3. Survival of T K - a n d T K + cells after exposure to light. (a) Plates containing 6 to 7 X 10 e cells were irradiated as described in Materials a n d Methods. I m m e d i a t e l y after irradiation, cells were trypsinized, pelleted, suspended in fresh medium a n d diluted in series. Samples containing from 7 × 101 to 7 x 104 cells were placed in 60-ram plates a n d incubated for 7 days. Colonies containing > 2 0 cells were counted as survivors. Cell lines tested included C2-1 grown w i t h o u t B r d U r d ( O ) ; C2-1 grown > 6 generations in 200 pg B r d U r d / m l ( Q ) ; LA9 grown > 6 generations in 1 × 1 0 -7 ~r-BrdUrd CA); a n d LA9 in 2 × 1 0 - ¢ M-BrdUrd CA). Unirradiated cells from all 4 populations gave > 90% survivors. The period of irradiation is indicated on t h e abscissa. (b) Plates containing 1.5 to 2.0 X 10 e C2-1 cells grown > 6 generations in B r d U r d were irradiated for 30 m i n (/~ ) or incubated in the dark ( • ) . Medium was t h e n replaced a n d incubation continued for 54 h. Some of t h e plates, irradiated at zero hour, were given a second 30-mln light t r e a t m e n t a t 12 h (O). A t the indicated times (abscissa) the cells were trypsinized, removed from t h e plates b y agitation and incubated in t h e cold in t h e presence of 0.05% t r y p a n blue for N 5 rain. Vital cells were counted as those which excluded t h e dye. No more t h a n 5 % of irradiated or control cells were stained.

768

R. A. LANSMAN

AND

D. A. CLAYTO N

In all subsequent experiments exposures of 30 minutes or less were employed. LA9 (TK +) cells grown in 10 -7 and 2X10 -~ M-BrdUrd have reduced viability after irradiation; one 30-mlnute exposure lowers survival rates to 40 and 20%, respectively. The effect of llght on the viability of C2-1 cells grown at 200/~g/ml is much more dramatic. The survival rate is 5 X 10- 8 after 30 minutes, 80-fold lower than that found in LA9 cells with similar nuclear BrUra substitution. Cells propagated without BrdUrd were not sensitized to light by incubation in 200 pg BrdUrd/ml for two hours before irradiation. Conversely, transferring BrdUrd-grown cells to BrdUrd-free medium for the two hours preceding irradiation did not enhance colony-forming ability. These results suggest that the difference in sensitivity between T K - and TK + cells is not caused by differing amounts of free BrdUrd in the cytoplasm. The most obvious difference between the two cell lines, grown as described, is the BrUra substitution in mtDNA. In the experiment shown in Figure 3(b), C2-1 cells grown in BrdUrd were briefly trypslnized, suspended, counted and checked for viability by exposure to trypan blue stain after one or two 30-minute light treatments followed by various periods of incubation in culture medium. The number of cells on plates which received irradiation remained essentially constant for 48 hours. Less than 5% of the irradiated cells divided. During this period the cells enlarged beyond normal size, but less than 5 % failed to exclude trypan blue. The amount of mtDNA in BrdUrd-grown cells has been quantitated by labeling with [SH]deoxycytidine, instead of thymid~e, so that labeling could be ca.rr_ied out during growth in high concentrations of BrdUrd. Figure 4 shows the radioactivity distribution in EthBr-CsC1 gradients of mtDNA prepared from C2-1 cells after more than five generations of growth in 200/~g/ml BrdUrd, 12 hours of incubation in BrdUrd plus [3H]deoxycytidine and irradiation for 0, 15 and 30 minutes. The DNA from unirradiated cells is resolved into two peaks, closed circular mtI)NA with heterogeneous BrUra substitution (lower band, fractions 3 to 20) and contaminating, unsubstituted nuclear X)NA. In the samples from irradiated cells, a distinct lower I

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FIG. 4., EthBr-CsC1 equilibrium density gradients of m t D N A isolated from C2-1 cells grown > 5 generations i n 200/~g B r d U r d / m l a n d labeled w i t h [3H]deoxyeytidine for 12 h prior to irradiation. Cells were irradiated for zero ( - - O - - O - - ) 1 15 ( - - S - - O - - ) ' a n d 30 ( - - A - - A - - ) rain prior to isolation of m t D N A .

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Fraction no. Fig. 5. (a) E t h B r - C s C I equdtibrium density gradients of m t D N A isolated from C2-1 eegs grown 5 generations in 200/~g B r d U r d / m l a n d labeled w i t h [3H]deoxyeytidine for 12 h. Ceils were t h e n irradiated for 30 m i n a n d m t D N A was isolated after zero ( - - 0 - - 0 - - ) ; 12 ( - - O - - O - - ) ; 24 (--l--l--) a n d 36 ( - - F ' l - - F 1 - - ) h of i n c u b a t i o n i n fresh m e d i u m c o n f i n i n g 200/~g B r d U r d / m l . (b) E t h B r - C s C l equilibrium density gradients of m t D N A isolated from C2-1 cells grown as described in (a). Cells were t h e n irradiated for zero ( - - 0 - - 0 - - ) ; 15 ( - - O - - O - - ) a n d 30 (--l--l--) m i n a n d i n c u b a t e d in BrdUrd-free m e d i u m oontsining [~H]thymidine for 12h.

770

R. A. LANSMAN AND D. A. CLAYTON

band is not present. The mtzDNA present is presumably non-closed circular BrUrasubstituted DNA at an intermediate position in the gradient. The conversion to non-covalently closed forms is not quantitative. The total radioactivity in BrUrasubstituted mtDNA is 8O~o in the 15-minute sample and 50~/o after a 30-minute light treatment. Of the several possible explanations for reduced recovery of mtDNA in irradiated ceils, the most obvious is that the yield of mitochondria isolated from irradiated cells is reduced. Careful analysis of the recovery of mitochondrial protein from irradiated cells demonstrates that no such reduction occurs (Lansman & Clayton, 1975). Two other phenomena may account for the loss of mtDNA. The first is that irradiation has resulted in covalent binding of mtDl~A to other molecules resulting in altered buoyant properties in EthBr-CsC1 gradients. The occurrence and extent of this reaction are demonstrated below. The second possibility is that damaged mtDNA molecules are degraded in rive after irradiation. Degradation of damaged mtDNA subsequent to irradiation is demonstrated in Figure 5(a). Cells grown in BrdUrd and labeled with [3H]deoxycytidine were irradiated for 30-minutes. Mitochondria were prepared from cells chilled and harvested immediately after irradiation and from cells which were incubated in fresh BrdUrd medium for 12, 24 and 36 hours after irradiation. The radioactivity in fractions 26 to 32 represents contaminating nuclear DNA while the majority of radioactivity in fractions 19 to 25 is non-closed circular mtDNA. Within 12 hours after irradiation, 70 to 7 5 ~ of this DNA is not isolatable, while the remainder is stable throughout the course of the experiment. Fractions 3 to 18 should contain closed circular mtDNA. Little, ff any, of the DNA which appears in the upper band after irradiation is transferred back to the lower band during the incubation period. This experiment indicates that there is no repair of damage to mtDNA caused by irradiation. It should be noted that there is a progressive decrease in the total amount of contaminating nuclear DNA radioactivity in the upper bands as a function of time after irradiation. This is most likely due to decreased mass contamination of mitochondrial preparations witch nuclear DNA from cultures in which cells are not dividing. We believe that most nuclear DNA contamination in preparations of mitochondria come from cells in mitosis at the time of homogenization, since very little nuclear DNA is routinely observed in mtDNA preparations isolated from stationary ceil cultures (unpublished data). In the experiment shown in Figure 5(b), C2-1 cells grown in BrdUrd and irradiated for 0, 15 and 30 minutes were placed for 12 hours in fresh BrdUrd-free medium contA.inlng [aH]thymidine. In the control cells, radioactivity was found in a broad lower band at the position expected for molecules containing one unsubstituted strand (see Fig. 4). The level of incorporation into mtDNA from irradiated cells is reduced to less than 5 ~ of control values after 15 minutes and less than 1~/o after 80 minutes. These data suggest that little or no DNA is synthesized in irradiated cells which can be identified as mtDNA either by BrUra substitution or a closed circular topology. This result is in contrast to that obtained in an experiment aimed at detecting repair synthesis and/or replication of nuclear DNA in irradiated C2-1 cells. The cells were labeled for two hours with [SH]deoxycytidine and at various times after irriadiation ~ t a l incorporation was measured as alkali-resistant, acid-precipitable material. Incorporation was less than 10~o of that found in controls during the first two hours after a 30 minute light treatment, but increased rapidly to a level threefold h i g h e r

P H O T O S E N S I T I Z A T I O N OF M I T O C H O N D R I A L DNA

771

than controls from 6 to 12 hours. The rate of incorporation fell below t h a t of controls after 24 hours and essentially to zero after 36 hours, as might be expected since the cells did not divide. I t seems likely t h a t the high levels of incorporation observed between 6 and 12 hours are associated with the process of repair of light-induced single-strand breaks into nuclear DNA (Ben-Hur & E]kind, 1972). The extent of replication of nuclear DNA during this time period has not been determined. Two ~iuds of experiments indicate t h a t other ~iuds of lesions, in addition t o singlestrand breaks, are induced b y light in BrUra-substituted mtDNA. Mitochondria from light-treated and control cells were isolated, as described, to the point of ]ysis. The samples were then split and fractions digested extensively with Pronase. The digestion procedure did not increase the yield of mtDNA from unirradiated controls though it aid cause some nicking as evidenced b y a shift in radioactivity from lower to upper band. Si~fificant increases (20 to 30%) were observed in the recovery of radioactivity from cells which were irradiated for 30 minutes (Fig. 6). Both upper (fractions 23 to 34) and lower band (fractions 5 to 22) radioactivity were increased. I t seems likely from this result t h a t both closed circular and nicked circular m t D N A molecules were linked to protein b y a detergent-stable bond after exposure to light. Such a phenomenon has been observed for BrUra-substituted nuclear DNA in mammalian cells (V~reintraub, 1973). In a separate experiment, cells were labeled with [SH]lysine and mtDNA was isolated and analyzed in an EthBr-CsC1 density gradient. I n unlrradiated controls, no radioactivity was found in regions of the gradient containing DNA. However, after a 30-mluute irradiation, a peak of radioactivity was found in the position expected for upper band mtDNA. The nature of the labeled material and its possible relationship to m t D N A remain to be elucidated. Agarose gel eleetrophoresis in the presence of E t h B r has proved a better method than buoyant density gradient centrifugation for analyzing the intact and damaged

I

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Fie-. 6. EthBr-CsC1 equilibrium density gradients of mtDNA isolated from C2-1 cells grown >5 generations in 200 ~g BrdUrd/ml and labeled with [SH]deoxycy~idiue for 12 h prior ~o isolation. Cells were irradiated for 30 min and mtDNA isolated with (-- O - - O - - ) and without ( - - I - - m - - ) Pronase treatment, mtDNA isolated from an equal number of control cells (zero irradiation) are shown for comparison ( - - 0 - - 0 - - ) .

772

R. A. LANSMAN AND D. A. CLAYTON

mtDNA from light-treated cells. Closed and open circular molecules are well resolved and both are cleanly separated from contaminating nuclear DNA. As is shown b y the control sample in Figure 7, open circular mtDNA remains near the interface between 0.6 and 2.00/o agarose while closed circular molecules migrate about 10 mm into the 2 . 0 ~ agarose. Linear DNA (not shown) migrates at least 25 m m into the gel under these conditions. A 15-minute light exposure reduces the yield of closed molecules and increases the amount of radioactivity at the origin of the gel and also in a peak migrating slightly faster than the open circles in the control sample. The DNA at the origin m a y fail to enter the gel because it is cross-linked to protein. Rapid migration of some of the nicked species m a y indicate that light-damaged i

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FIG. 7. Agarose gel eleetrophoreses of mtDNA isolated from C2-1 cells grown > 5 generations in 200 ~g BrdUrd/ml and labeled with [~Hldeoxyeytidine for 12 h prior to irradiation. Ceils were irradiated for zero (-- O - - O --); 15 ( - - O - - Q - - ) and 2 x 30 ( - - I - - , i ) min prior to isolation of mtDNA. Migration is directed to the right. "Zero" and "IS-rain" cells were harvested immediately after irradiation. The 2 x 10-rain mtDNA sample was isolated 12 h after the first 30-min irradiation (see Table 1). moiecules are partially degraded to gapped circles or possible single-stranded circles. After two 30-minute light treatments there is no distinct peak of closed circular mtDNA. I n other gel experiments in which BrUra-substituted DNA from lighttreated cells has been separated from contaminating nuclear DNA in a preliminary centrifugation step, it was possible to look for linear m t D N A produced b y the action of light. Little, if any, was found (data not shown). This observation is in agreement with the prediction that double-strand breaks are not produced by light of the frequencies employed (Hutcbln~on, 1973). The gel system allows us to quantitate the relative amount of closed circular and total mtDNA after various light exposures. The data from the gels shown.in Figure 7 and gels not shown are s u m m ~ i z e d in Table 1. When cells receive two 30-minute

PHOTOSENSITIZATION OF MITOCHONDRIAL DNA

773

TmsT.v. 1

Agarose gel electro19horeti~analyses of amoun~ and form of mitochondrial DNA izolatable after light treatment of cells~ Irradiation time (min)

Closed circular mtDNA (cts/min)

0 (control) 15 30 2X30

2810 (100)~ 652 (23) 175 (6) 67 (2.3)

Open circular mtDNA (cts/min) 665 2190 1002 312

(100) (329) (151) (47)

Closed circular ~ open circular mtDNA (ors/rain) 3475 2842 1177 379

(100) (82) (34) (11)

t The data represent total radioactivity in the gel shown in Fig. 7 and similar gel profiles after correction for background of 12 cts/min per sample. :~ Numbers in parentheses refer to percentage of each m t D N A form(s) present relative to the control. I n the experiments with 0, 15 and 30 minutes irradiation times, m t D N A was isolated immediately after the irradiation period. I n the 2 × 30-min experiments, the cells were irradiated for 30 rain, incubaf~d in fresh BrdUrd-contalnlng medium for 8 h, irradiated again for 30 rain and harvested 4 h later (12 h after the initial irradiation).

light exposures separated by eight hours of incubation in medium containing BrdUrd, the radioactivity in total mtDNA recovered is reduced to 10 to 1 2 ~ of controls. Only ~ 2 % of control levels of closed circular mtDNA remain in these cells. 4. Discussion The lack of complete BrUra substitution in mtDNA of T K - cells grown in high BrdUrd concentrations indicates that both BrdUTP, synthesized from exogenous BrdUrd utilizing mitochondrial thymidine kinase, and TTP, synthesized endogeneously, are utilized in mtDNA synthesis (Berk & Clayton, 1973). The heterogeneous substitution obtained in mtDNA from T K - cells indicates that differential utilization of these/~wo sources occurs in the cell population. At least part of the heterogeneity results from phenotypic differences among the cells. During more than 500 cell divisions and 16 months after the original cloning to obtain C2-1, the mean substitution decreased from 60 to ~ 4 0 ~ . It has been necessary to return to samples of C2-1 frozen soon after cloning to regain the levels of substitution indicated in Figure 1. Recloning of the C2-1 population yielded subclones which have mean substitutions ranging from 20 to 70~. We have no explanation of the kind of selective forces that might maintain these differences in a population of cells being propagated in medium free of BrdUrd. BrUra substitution has been measured in subclones after the ~ 3 0 generations which are required to obtain sufficient mtDNA for analysis. In all cases, the type of heterogeneous substitution shown in Figure 1 has been obtained. The rapid appearance of heterogeneity after cloning suggests that DNA molecules of different substitution may result from cell-cycle fluctuations in the level of endogenous TTP in the cytoplasm of phenotypically identical cells, mtDNA molecules synthesized when triphosphate pools in the cytoplasm are low may have a higher substitution than molecules synthesized during S phase when endogenous cytoplasmic TTP pools are higher. Recent experiments in this laboratory have

774

R.A.

LANSMAN

AND

D. A. C L A Y T O N

demonstrated that molecules containing one strand with 100% substitution are synthesized in cells in which TMP synthesis and nuclear DNA replication have been blocked by the addition of FdUrd to medium conta~nlng high concentrations of BrdUrd (Bogenhagen & Clayton, 1976). If T K - cells incorporated exogenous BrdUrd only into mtDNA, leaving nuclear DNA totally unsubstituted, it would be possible to damage extensively every mtDNA molecule in every cell in a population without damaging the nuclear DNA. This would provide an ideal system for observing the effects of depriving cells of the products of the mitochondrial genome without the use of chemical inhibitors. Unfortunately, when C2-1 cells are grown in concentrations of BrdUrd high enough to ensure significant substitution in all mtDNA molecules, a small amount of BrUra is also incorporated into nuclear DNA ( < 1% substitution). The source of BrdUTP used in nuclear DNA synthesis in these cells is not known. Likely possibilities are a residual non-mitochondrial klnase which phosphorylates BrdUrd at low rates or leakage of BrdUMP through mitochondrial membranes. Neither hypothesis leads to an obvious genetic selection or biochemical manipulation which would allow us to eliminate nuclear substitution. Given the substitution levels in mtDNA (56%) and nuclear DNA (N0.8%) obtained in C2-1 cells grown at 200 ~g BrdUrd/ml, it is possible to estimate that a light dose large enough to inflict at least one single-strand break in 90~/o of the mtDNA molecules will also cause about 5 × 10~ single-strand breaks in nuclear DNA per cell. This estimation assumes that each incorporated BrUra base in the cell's DNA has an equal probability of interaction with light and that a random distribution of single-strand breakage results. Though a large amount of nuclear damage is inflicted, the consequences for the cell appear to be minor relative to the effects of damage in 90% of the mtDNA molecules. 20 to 40% of a population of TK + cells with the same nuclear substitution survive the same amount of irradiation. This suggests that damage to nuclear DNA at these doses is largely repaired. Thus, the specific effects of damage to mtDNA can be observed in these experiments since the cell does not appear to repair this damage in mtDNA (Fig. 5(a)). Previous experiments using short wavelength u.v. light have shown that pyrimidine dlmers are not excised from mtDNA and that molecules containing dimers are neither replicated nor degraded (Clayton et aL, 1974). I t is most likely that the cell does not employ a repair system for mtDNA since u.v. doses high enough to induce one or more pyrimidine dimers in all of the mtDNA molecules in a cell are lethal due to the extent of damage to nuclear DNA. The experiments reported here demonstrate that single-strand breaks induced by light in BrUra-containing mtDNA are also not repaired. Restoration of a closed circular topology in nicked circular DNA molecules is a stringent test of the ability of a system to repair single-strand breaks. No transfer of upper band radioactivity to the lower band region in EthBr-Cscl gradients has been detected in this work. Instead, most upper band radioactivity in mtDNA preparations from irradiated cells is lost within 12 hours of irradiation. We believe that this loss cannot be attributed to a failure to isolate mitochondria from irradiated cells. Recovery of mitochondrial protein is not reduced in heavily irradiated cells for as long as 48 hours after irradiation. Electron micrographs of irradiated cells give no indication that the mitochondrial inner membranes are damaged in a way that would lead one to expect that mtDNA would not be coisolated with bulk mitochondr/al protein (Lansman & Clayton, unpublished data).

PHOTOSENSITIZATION OF MITOCHONDRIAL DNA

775

These considerations lead us to believe that loss of mtDNA reflects changes both by in vivo degradation of damaged molecules and cross-linkiug of BrUra-substituted mtDNA to protein. It is unclear why damaged molecules are susceptible to degradation, while rcplicative intermediates with extensive single-strand regions are a normal constituent of mammalian mtDNA populations (Berk & Clayton, 1974; Robberson et al., 1972). Single-strand breaks occur when the uracyfil radical, resulting from light-induced debromination, attacks a ribose carbon atom of the adjunct nucleotide (Hutchlnaon, 1973). Alternatively the radical can attack other molecules in close proximity. Weintraub (1973) has demonstrated cross-linking of DNA to histones after light treatment. Presumably, any protein bound to DNA can participate in the same reaction. The experiments described in the legend to Figure 6 suggest, rather than prove, that significant cross-linking of mtDNA to protein occurs during light exposure. Pronase digestion after the detergent lysis of isolated mitochondria might increase the yield of mtDNA in a gradient by reducing the number of molecules which are non-covalently trapped in the cesium-dodecyl suffate-protein "pad" which forms at the top of the gradient. Were this the case, we would expect Pronase to increase the yield in samples from control and fight-treated cells alike. Since the yield from controls is not increased, it seems most likely that the increase from light-treated samples consists of mtDNA molecules which, prior to Pronase treatment, are crossliul~ed to protein, reducing their buoyant density out of the range of the gradient. Perhaps it is more likely that the protein-DNA complexes aggregate in the 1% sodium dodecyl sulfate-5 M-CsC1 gradient suspension. We have also found that radioactivity from cells labeled with [3H]lysine is found in an EthBr-CsC1 density gradient in a band with approximately the same buoyant density as open circular or linear DNA. Since the radioactivity was found only in mitochondria prepared from light-treated cells, we assume that it represents lysine-containing material which was covalently linked to DNA by the light treatment. The fact that damaged mtDNA molecules are degraded and are not repaired, replicated nor replaced by augmented replication of undamaged mtDNA allows one to obtain a cell population which has only 2% of the normal amount of closed circular mtDNA. Since the cells remain intact through this treatment and continue to synthesize, if not replicate, nuclear DNA and continue to synthesize protein at near normal rates, it is possible to use the cells experimentally for at least 48 hours after irradiation (Lansman & Clayton, 1975). The most surprising behavior of the irradiated cells is that less than 5% divide. One interpretation of this observation is that a mtDNA-coded RNA or protein species has a positive control function with regard to cell division. However, it is known that cells grown in EthBr at high enough concentrations to completely inhibit replication and transcription of mtDNA complete at least one cell doubling in the presence of the drug (Smith et al., 1971 ; Attardi et al., 1970). HeLa cells have also been shown to go through at least two doublings in medium containing enough chloramphenicol to inhibit mitochondrial protein synthesis > 9 0 % (Sterrie & Attardi, 1972). These facts make it difficult to explain the lack of cell division in light-treated ceils in terms of an unsatisfied requirement for a mitochondrial gene product. C2-1 cells, grown long-term in 200 t~g BrdUrd/ml and exposed to fight twice for 30 minutes or a total of 60 minutes, may suffer damage that is not directly related to the integrity of mtDNA. Single-strand breaks in nuclear DNA in the C2-1 cell line

776

R . A . LANSMAN AND D. A. CLAYTON

m a y be more lethal t h a n they are in the T K + cells used as controls because the cell lines m a y differ in ability to repair these breaks. We have not attempted to study nuclear repair. Non-DNA damage also seems possible though it has been shown that light does not damage cells after short incubations in 200 pg BrdUrd/ml when the intraeellular concentration of free BrdUrd should be the same as it is long-term experimental cells. This research was supported by grants, NP-9 from the American Cancer Society, and CA.12312 from the National Cancer Institute. One of us (R. A. L.) is a postdoctoral fellow of the American Cancer Society (PG-868) and the other author (I). A. C.) is a Senior Dernham Fellow of the American Cancer Society, California Division (D-203). We thank A. J. Berk and C. A. Smith for critically reviewing this manuscript prior to publication. REFERENCES Aaij, C. & Borst, P. (1972). Biochim. Biophys. Acta, 269, 192-200. Attardi, G., Aloni, Y., Attardi, B., Ojala, D., Pica-Mattoccia, L., Robberson, D. L. & Storrie, B. (1970). Cold Spring Harbor Syrup. Quant. Biol. 35, 599-619. Ben-Hut, E. & Elkind, M. 1K. (1972). Biophys. J. 12, 636-647. Berk, A. J. & Clayton, D. A. (1973). J. Biol. Chem. 248, 2722-2729. Berk, A. J. & Clayton, D. A. (1974). J. Mot. Biol. 86, 801-824. Boettiger, D. & Temln~ H. 1~. (1970). Nature (London), 228, 622-624. Bogenhagen, D. & Clayton, D. A. (1976). J. Biol. Chem. In press. Borst, P. (1972). Annu. Bey. Bioehem. 41, 333-376. Clayton, D. A. & Vinograd, J. (1967). -Na2ure (London), 216, 652-657. Clayton, D. A. & Teplitz, R. L. (1972). J. Cell Sci. 10, 487-493. Clayton, D. A. & Smith, C. A. (1975). Int. Rev. Exp. Path. 14, 1-67. Clayton, D. A., Doda, J. N. & Friedberg, E. C. (1974). Proc. _Nat. Acad. Sci., U.S.A. 71, 2777-2781. Hutchinson, F. (1973). Quart. l~ev. Biophys. 6, 201-246. Lansman, R. A. & Clayton, D. A. (1975). J. Mot. Biol. 99, 777-793. Radloff, R., Bauer, W. & Vinograd, J. (1967). Proc. _Nat. Acad. ~ci., U.S.A. 57, 1514-1521. Robberson, I). L. & Clayton, D. A. (1972). Proc. _Nat. Acad. Sci., U.S.A. 69, 3810-3814. Robberson, D. L., Kasa/natsu, H. & Vinograd, J. (1972). Proc. _Nat. Acad. Sci., U.S.A. 69, 737-741. Smith, C. A., Jordan, J. M. & Vinograd, J. (1971). J. Mol. Biol. 59, 255-272. Sterrie, B. & Attardi, G. (1972). J. Mol. Biol. 71, 177-199. Wake, R. G. & Baldwin, R. L. (1962). J. Mol. Biol. 5, 201-216. Weintraub, H. (1973). Cold Spring Harbor Syrup. Quant. Biol. 38, 247-256.