Yeast mitochondrial DNA characterization after ultraviolet irradiation

267

Mutation Research, 73 (1980) 267--277

© Elsevier/North-HollandBiomedicalPress

YEAST MITOCHONDRIAL DNA CHARACTERIZATION AFTER ULTRAVIOLET IRRADIATION

SHARON

C. H I X O N a*, H A R O L D

L. F R A N K S

a and E T H E L M O U S T A C C H I

b

a Department of Biochemistry, Comprehensive Cancer Center, University of Alabama in Birmingham, University Station Birmingham, A L 35294 (U.S.A.) and h Fondation Curie-Institutdu Radium, Biologie, Bdtiment 110, Orsay 91 (France) (Received 28 March 1980) (Revision received 20 June 1980) (Accepted 25 June 1980)

Summary Yeast mitochondrial (mtDNA) 3H-labelled was isolated from exponential phase cells after ultraviolet light irradiation. Both the size and amount of mtDNA were found to be reduced during a 40-h liquid-holding (LH) period in non-growth medium following irradiation as compared to the mtDNA recovered from nonirradiated cells under similar conditions. After the LH period, previously irradiated cells were resuspended in growth medium containing [14C]adenine. Double labelled mtDNA (3H and 14C) was isolated from cell samples removed during new growth. A recovery in the amount and size of mtDNA was observed in irradiated cells during new growth. These biochemical studies agree with the observed loss and recovery of mtDNA genetic markers in UV-irradiated exponential phase yeast after a period of LH and new growth resp. Ultraviolet irradiation produces thymine dimers in nuclear and mtDNA in yeast (Waters and Moustacchi, .1975). Cells with mtDNA damage become respiratory
* T o w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .

268 Hixon et al., 1975). Genetic experiments have indicated a loss of m t D N A genetic markers following UV irradiation with a partial recovery of markers after liquid holding (LH) and new cell growth (Moustacchi et al., 1978; Heude and Moustacchi, 1979). In addition yeast mutants have been isolated that are sensitive to UV irradiation primarily for petite m u t a n t induction (Moustacchi, 1971; Moustacchi et al., 1976). The existence of such mutants implies that a recovery pathway may exist for UV-irradiation damage in mtDNA. During the liquid-holding period following the irradiation of exponential phase cells, a dose- and time-dependent degradation of m t D N A has been observed (Waters and Moustacchi, 1974b; Hixon and Moustacchi, 1978). This paper reports a further characterization of irradiated m t D N A isolated during the liquid-holding period and at time intervals during new growth in complete medium. An abstract of this report has been previously published (Hixon et al., 1979). Materials and methods

Radioactive labelling and cell irradiation The haploid strain N 123 (a h i s 1 ) o f Saccharomyces cerevisiae was used in these experiments. Cells were grown to exponential phase overnight in Difco yeast nitrogen base with 2% glucose and 40 pg/ml histidine at 30°C on a shaker. Following a 75-min incubation with 1 pg/ml cycloheximide (Grossman et al., 1969) [6-all]uracil (8 pCi/ml, spec. act. 30 Ci/mmole) was added. After a 2-h labelling period radioactivity was chased b y washing the cells with water containing 10 pg/ml nonradioactive uracil. Cells were then resuspended to 5 . 106/ml and irradiated at 254 nm with an incident dose of 5 J/m2/sec at 25 ° C. Cell suspensions were liquid held in water with 10/~g/ml uracil for 40 h. Aliquots (60 ml) were withdrawn and centrifuged at selected time intervals. The cell pellets were mixed with 5 • 109 nonradioactive carrier cells of N 123 and frozen in 10 ml of 50 mM succinate buffer containing 10 mM MgSO4, 3 mg/ml sulfonated polystyrene (Polysciences Inc.), 10% glycerol (v/v) and 1 mg/ml KCN (pH 5.0) for storage until samples were collected for DNA isolation. Growth conditions After LH for 40 h as described above, cells were collected b y centrifugation and resuspended to 5 • 107/ml in liquid growth medium (1% yeast extract, 2% peptone, 2% glucose) containing [8-14C]adenine, 2 pCi/ml, spec. act. 48 mCi/ mmole. Cell growth was monitored b y counting the cell number every 2 h using a h e m o c y t o m e t e r . At given time intervals during growth, cells (6 ml) were withdrawn, mixed with 5 • 109 carrier cells and frozen in succinate buffer for later DNA isolation. DNA preparation Cells were slowly thawed and washed once in 20 mM Tris, 10 mM EDTA, 0.35 M sucrose, 1 mg/ml KCN pH 8 before mixing with glass beads for cell disruption according to the "hand-shake" m e t h o d described by Lang et al. (1977). The broken cells were collected b y centrifugation ( 1 5 0 0 0 × g ) and

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Fig. 1. (a) S e d i m e n t a t i o n of m t D N A i s o l a t e d f r o m i r r a d i a t e d 1 0 0 J / m 2 cells ~ m m e d i a t e l y a R e r i r r a d i a t i o n (~) a n d a f t e r 18 h (o) a n d 4 0 h (e) l i q u i d h o l d i n g i n w a t e r . (b) M t D N A f r o m n o n l r r a d i a t e d cells.

lysed by the addition of 3 ml of 0.25 M Tris, 0.1 EDTA, 1% sarcosyl (ICN) pH 8.0 with gentle mixing for 5 min at 60 ° C. Nucleic acids were separated from cell debris by extraction 2X with chloroform: isoamyl alcohol (24 : 1) and centrifugation 20 m i n at 12 000 ×g. The aqueous layer was withdrawn and treated with 0.I ml bovine pancreatic RNAase (Sigma, 1 mg/ml) and TI RNAase (Sigma, 500 units) I h at 37°C.,RNAase was pretreated 10min at 80°C. After overnight dialysis RNAase treatment was repeated followed by continued dialysisagainst 3 changes of 10 m M Tris, 1 m M EDTA, p H 7.0. Cesitun chloride solutions were prepared by the addition of 6.00 g CsCI to 5.85 ml of D N A solution. The D N A binding drug DAPI (4,'6-diamidino-2phenylindole) was added to a final concentration of 100 #g/ml to enhance the separation of nuclear and m t D N A (Williamson and FenneU, 1975). Tubes were centrifuged 48 h at 42 000 rpm in a fixed angle 65 rotor in an IEC-60 ultracentrifuge. Fractions (0.17 ml) were collected dropwise from the bottom of each tube. To identify the nuclear and m t D N A containing fractions 0.02 ml of each fraction was counted for radioactivity.

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Fig. 2. (a) Cell g r o w t h in c o m p l e t e m e d i u m in c o n t r o l (A) a n d i r r a d i a t e d ( e ) cells f o l l o w i n g i r r a d i a t i o n a n d 4 0 h o f l i q u i d h o l d i n g in w a t e r . (b) N o n i ~ a d i a t e d cells u p t a k e o f [ 1 4 C ] a d e n i n e ( ) during new g z o w t h w i t h s i m u l t a n e o u s m o n i t o r i n g o f p r e v i o u s l y l a b e l l e d [ 3 H ] D N A (. . . . . . ). CPM$ are t h e s u m m a t i o n o f c p m s u n d e r n u c l e a r (A) a n d m i t o c h o n d r i a l (z~)-DNA p e a k s f r o m p r e p a r a t / v e c e s i u m c h l o r i d e g r a d i e n t s . (c) I r r a d i a t e d cells u p t a k e o f [ 1 4 C ] a d e n i n e ( ) in t h e p r e s e n c e o f r e t a i n e d d e g r a d e d [ 3 H ] D N A (. . . . . . ). CPMs are o b t a i n e d as d e s c r i b e d a b o v e (b) f o r n u c l e a r ( e ) a n d m i t o e h o n d r l a l (o) D N A .

Sucrose gradien ts Cesium chloride fractions containing m t D N A were pooled and dialyzed overnight against 10 mM Tris 1 mM EDTA, pH 7.0. D N A solutions were then concentrated against ficoll (Sigma). D N A (0.3 ml) was carefully layered o n t o 10 ml neutral sucrose gradients (10--30% sucrose) in Tris--EDTA buffer, pH 7.0.

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Fig. 3. The s e d i m e n t a t i o n of m t D N A i s o l a t e d f r o m cells w i t h d r a w n d u r i n g n e w g r o w t h i n c o m p l e t e m e d i u m . T r i t i u m l a b e l (- . . . . . ) is f r o m D N A l a b e l l e d p r i o r t o l i q u i d h o l d i n g w h i l e [ 1 4 ( ~ ] D N A ( ) is n e w D N A s y n t h e s i z e d d u r i n g n e w g r o w t h in [ 1 4 C ] a d e n i n e c o m p l e t e m e d i u m . (a) N o n i r m d i a t e d cells L H 4 0 h a n d 8 h i n g r o w t h m e d i u m . (b) I r r a d i a t e d cells ( 1 0 0 J / m 2) p r i o r t o LH, 8 h i n g r o w t h m e d i u m . (c) Same as (b); 10 h i n g r o w t h m e d i u m .

Gradients were spun at 19 000 rpm (Fig. 1) or at 23 000 rpm (Figs. 3 and 4) for 18 h at 4°C. Fractions were collected dropwise (0.25 ml) from the bottom of the tubes. Gradients were calibrated with 23 $20 [3H]fd viral D N A (Miles) and [SH] 5 $20 R N A (Miles). All radioactive samples were added to 5 ml o f Ready Solv Beckman scintillation cocktail and counted on a Searle scintillation counter. Both 3H and 14C

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Fig. 4. D e s c r i p t i o n as in Fig. 3 w i t h S1 n u c l e a s e t r e a t m e n t p r i o r t o s e d i m e n t a t i o n . Markers o f 14C a n d 3 H in e a c h p a n e l i n d i c a t e t h e p e a k p o s i t i o n s o f t h e D N A i s o l a t e d f r o m t h e s a m e t i m e p o i n t b u t u n d i g e s t e d w i t h S1. (a) C o n t r o l u n i ~ a d i a t e d cells: L H f o l l o w e d b y 8 h in g r o w t h m e d i u m . ( b ) Irradiated cells ( 1 0 0 j / m 2 ) ; L H f o l l o w e d b y 12 h in g r o w t h m e d i u m .

were corrected to accurate values by processing the data using a computer program for dual labelling written by Mike Adamson. $1 nuclease treatment D N A was treated with the S1 nuclease from Aspergillus oryzae (Miles) prior to sucrose-gradient centrifugation. The reaction mixture contained 170 pg of single stranded calf-thymus D N A (Sigma), 0.084 ml of 2.5 mM zinc acetate, 0.2 ml o f acetate buffer pH 4.5, 100/zl radioactive D N A and 36 units of S1 enzyme

273 in a final volume of 0.4 ml. The reaction mixture was incubated for I h at 37°C. These conditions were found to degrade greater than 90% of single-stranded fd viral DNA with no detectable degradation of double-stranded yeast nuclear DNA. Losses of radioactivity were determined b y the retention of radioactive DNA collected on millipore filters after trichloroacetic acid precipitation. Results

Fig. 1 indicates the apparent S values on neutral sucrose gradients containing m t D N A isolated from irradiated and nonirradiated cells during the liquidholding (LH) period. The size of m t D N A from nonirradiated cells did n o t significantly change during the LH period. An immediate size reduction was seen in m t D N A isolated from cells directly after irradiation (100 J / m 2, 20--30% cell survival). This reduction in size was also accompanied by a gradual diminution in the total amount of m t D N A recovered from irradiated cells as previously d o c u m e n t e d (Hixon and Moustacchi, 1978). The approximate S values of the m t D N A were determined by comparing the sedimentation of fd viral DNA (23S) and 5 S R N A to the sedimentation of mtDNAs from irradiated and non-irradiated cells. The average S values of control and irradiated m t D N A after 40 h LH from 6 separate experiments were 27.2S + 2.9 S.D. and 19.9S + 4.2 S.D. resp. These values may be converted to approximate molecular weights b y employing the empirical formula S ° = 0.063 M °'a7 for native DNA (Doty et al., 1958; Eigner and Dory, 1965). These calculations result in a molecular weight of control m t D N A equal to 1 3 . 2 . 1 0 .6 and irradiated m t D N A 5.69 • 106 daltons which indicates an overall decrease in size of 56.9% 40 h after irradiation. Although yeast m t D N A has an approximate molecular weight of 50" 106 daltons as confirmed by electron microscopy and the summation of restriction-enzyme fragments, present isolation procedures yield broken molecules following extensive purification (Locker et al., 1974; Sanders et al., 1975, 1977). The inability to isolate intact m t D N A molecules from nonirradiated cells causes difficulty in determining a more accurate estimation of the average size reduction of m t D N A after irradiation. Cells which retained previously labelled 3H during the LH period were allowed to reinitiate growth in complete medium containing [14C]adenine. This experiment enables one to follow the fate of " o l d " DNA while " n e w " DNA is synthesized. Fig. 2a depicts the increase in cell number during new synthesis in growth medium. After a 2--3 h lag period nonirradiated cells began to grow completing a new generation b y 6 h. Irradiated cells lagged behind for an additional 2--3 h period before new growth appeared with a cell doubling by 9 h. The progression of radioactive incorporation of 14C with ongoing 3H turnover is shown in Figs. 2b and c. The cpm in Fig. 2 have been calculated from the summation of radioactivity under the nuclear and m t D N A peaks from CsC1 gradients (data n o t shown). No significant reincorporation of 3H appeared to occur in the control nuclear DNA during new cell growth. Therefore the experimental cpms have been standardized against the same constant 3H cpm for nuclear DNA for b o t h control and irradiated cells in the figures shown. In both irradiated and nonirradiated cells while an uptake in '4C denotes new ongoing

274 synthesis of mtDNA, an active turnover in the " o l d " 3H-radioactive label was seen. In nonirradiated cells after 8 h of growth the proportion of 14C radioactivity distributed to m t D N A is 22% which accurately reflects the percentage of m t D N A of the total DNA in this strain. However, in the irradiated cells, after 12 h of growth, the percentage of the total 14C label partitioned into m t D N A is 50% suggesting that m t D N A has a 2-fold higher specific activity during new DNA synthesis in previously irradiated cells. This observation suggests that in irradiated cells the m t D N A lost during LH is replaced during new cell growth to provide the proper a m o u n t of m t D N A per cell characteristic of this strain (Hall et al., 1976). Mitochondrial DNA containing radioactive label in "old"-3H and "new"-~4C DNA was isolated from cells at different time intervals during new growth. Both " o l d " and " n e w " m t D N A from control cells sedimented with identical S values in neutral sucrose gradients (Fig. 3a). However, m t D N A from irradiated cells retained the previously labelled [3H]DNA with an average molecular weight smaller that that seen in newly synthesized [~4C]DNA (Figs. 3b,c). This shift of [~4C]DNA toward the normal size distribution was in spite of any noticeable growth advantage of normal cells vs. petite m u t a n t cells during the first 12 h of new growth as monitored by plating the cell population every 2 h during growth. The 5 S ~4C material indicated in Figs. 3 and 4 could be removed by alkali digestion and trichloroacetic acid precipitation of individual sucrose fractions followed by collection onto glass fiber filters prior to scintillation counting. The large excess of [~4C]RNA produced during new growth was therefore not completely removed by RNAase digestion prior to cesium chloride gradient centrifugation. The presence of nicks or gaps in these 2 populations of 3H and ~4C containing DNA was assayed by treatment $1 nuclease (Karran et al., 1977). This nuclease has been shown to cut DNA in single-stranded areas to produce doublestranded fragments of DNA. The activity of the enzyme and reaction conditions were monitored in each experiment by the digestion of single-stranded fd viral DNA. The sedimentation of control (8 h in growth medium) and irradiated m t D N A (12 h in growth medium) after S1 treatment from one typical experiment are shown in Fig. 4. Control mtDNAs both " o l d " and " n e w " were shifted b y one fraction in the sucrose gradient after treatment with the S1 nuclease. DNA recovered from previously irradiated cells was more sensitive to S1 treatment. Prelabelled [3H]DNA was r e d u c e d by 3 fractions while newly synthesized [14C]DNA was reduced by 2 fractions. From calculated S values the percentage of size reduction effected by S1 nuclease treatment was 13% in nonirradiated [3H]- and [14C]DNA shown in Fig. 4a. Irradiated [3H] DNA was reduced in size 47.8%, [~4C]DNA 30% (Fig. 4b). These values may be used to calculate the number of double-stranded DNA cuts per average DNA molecule. If x = the number of cuts/molecule, assuming for simplicity that these cuts are equidistant, the following equation may be used to calculate a value for x. x x+l

-

fraction of size reduction

A calculation of x yields 0.15 cuts/molecule in non-irradiated [3H]-and [14C]DNA. For irradiated [ 3 H ] D N A x = 0.92; [14C]DNA x = 0.43.

275 Discussion The mtDNA isolated from irradiated cells after LH is reduced in average size and amount present by approx. 50--60% after 100 J/m 2 (Hixon and Moustacchi, 1978). These experiments suggest that portions of all the mtDNA molecules were degraded after UV rather than 60% of the population undergoing complete degradation. The degradation of mtDNA in response to UV may indicate a normal aspect of mtDNA synthesis which is triggered by irradiation. A similar turnover of DNA previously labelled with tritium appeared to take place in nonirradiated cells during new growth as shown in Fig. 2b. The degradation of irradiated mtDNA after UV is unique for mtDNA since high doses of UV in yeast have resulted in losses of up to only 10% of the nuclear DNA (Hatzfeld, 1973). The observed decrease in size and overall reduction in the amount of mtDNA per cell agree with genetic experiments in exponential phase cells which indicate a loss of genetic markers from mtDNA after irradiation. This marker loss has been shown to occur in an ordered fashion around the mtDNA genome with a preferential loss of the oxi 3 region (Moustacchi et al., 1978; Heude and Moustacchi, 1979). Genetic experiments have also demonstrated that in exponential phase cells a recovery of original information takes place among the petite mutant population when genetic markers are assayed following LH and cell growth as compared to immediately after UV irradiation. This genetic data correlates with the biochemical observation of a slow restoration of the size of mtDNA molecules during the new growth of previously irradiated cells. In addition, new DNA synthesized during early rounds of replication following irradiation appears to be more intact than the previously irradiated DNA. This may indicate a preferential replication of selected molecules that retain more information than the majority of those molecules that axe reduced in size. The idea that mtDNA replication may involve a sizing step has previously been suggested (Borst and Grivell, 1978). The fate of the UV-induced dimers is yet to be determined in these molecules. Stationary phase cells do not excise dimers from mtDNA during LH (Waters and Moustacchi, 1974b). Exponential phase cells axe also thought to retain the same percentage of dimers per DNA nucleotides following LH (Moustacchi, personal communication). Thus the degradation of mtDNA during LH is probably not a part of the excision-repair pathway known to exist for nuclear DNA. Treatment with the S1 nuclease was applied in order to assay for any differences in nicks or gaps in control and irradiated mtDNA during new synthesis. Only a few sites were subject to cleavage by the enzyme. However, the most sensitive DNA for cleavage were the irradiated mtDNA molecules retained after degradation. Although the 81 nuclease is not known to cleave DNA at thymine dimers, a concentration of dimers in a high A--T rich region might cause distortion sufficient to melt out a single-stranded region subject to S1 nuclease action. Experiments now underway to quantitate UVinduced dimers in "new" and "old" mtDNA during new synthesis using the Micrococcus luteus UV endonuclease (Carrier and Setlow, 1970) should indicate the final fate of these dimers under conditions whereby a partial recovery mechanism for the petite mutation has been demonstrated from microbiological and genetic experiments.

276

Acknowledgements This research has been aided by grant IN-66 P from the American Cancer Society and a Medical Center Faculty Research Grant to S. Hixon. We wish to thank Adil Ocak, Sevim Ocak and Giesele Thomas for their excellent technical assistance. References B o r s t , P., a n d L . A . Grivell ( 1 9 7 8 ) T h e m i t o c h o n d r i a l g e n o m e o f y e a s t , Cell, 1 5 , 7 0 5 - - 7 2 3 . C a r r i e r , W.C., a n d R . B . S e t l o w ( 1 9 7 0 ) E n d o n u c l e a s e f r o m Micrococcus luteus w h i c h h a s a c t i v i t y t o w a r d u l t r a v i o l e t - i r r a d i a t e d d e o x y r i b o n u c l e i c a c i d : p u r i f i c a t i o n a n d p r o p e r t i e s , J. B a e t e r i o l . , 1 0 2 , 1 7 8 - - 1 8 6 . D o t y , P., B.B. McGill a n d S.A. R i c e ( 1 9 5 8 ) T h e p r o p e r t i e s o f s o n i c f r a g m e n t s o f d e o x y r i b o s e n u c l e i c a c i d , P r o c . N a t . A c a d . Sci. ( U . S . A . ) , 4 4 , 4 3 2 - - 4 3 8 . 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