181

Mutation Research, 50 (1978) 181--193

© Elsevier/North-Holland Biomedical Press

BIOCHEMICAL ANALYSIS OF DAMAGE INDUCED IN YEAST BY FORMALDEHYDE I. INDUCTION OF SINGLE-STRAND BREAKS IN DNA AND THEIR REPAIR

N. MAGAI~A-SCHWENCKE, B. EKERT and E. MOUSTACCHI Fondation Curie-Institut du Radium, Biologie, Bdtiments 110 et 112, 91405 - Orsay (France)

(Received 2 March 1977) (Revision received 15 September 1977) (Accepted 31 October 1977)

Summary Analysis of sedimentation profiles in alkaline sucrose gradients showed that, through a metabolic process, formaldehyde (FA) produced single-strand breaks in DNA of exponential phase cells of haploid wild-type Saccharomyces cerevisiae. The production of this type of lesion was dose-dependent. Strains defective in excision-repair of pyrimidine dimers induced by ultraviolet (UV) irradiation showed a reduced capacity to undergo single-strand breaks after treatment with FA. This indicates that the repair pathways of damage induced by UV and FA share a common step. Post-treatment incubation of wild-type cells in growth medium indicated a lag in cell division during which a slow recovery of DNA with a normal size was observed.

Introduction Formaldehyde (FA), a compound widely distributed in man's environment, is used mainly in the paper and synthetic resin industries; it is also used for sterilization purposes in agriculture and it is one of the products of fuel combustion. It is mutagenic in Escherichia coli [19], Neurospora [9], Saccharomyces cerevisiae [3] and Drosophila [18]; moreover, it induces recombination and gene conversion in Saccharomyces cerevisiae [4] and in Drosophila [1]. The sensitivity to FA of Escherichia coli mutants defective in excision-repair of ultraviolet-induced lesions [19] or in DNA polymerase I [21] is higher than that of the corresponding wild-type. The same is true for excision-deficient mutants of Saccharomyces cerevisiae [5] and for cell lines of Xeroderma

182

pigmentosum as compared with normal human fibroblasts in culture [6]. These observations indicate that, in cells, FA may act directly and/or through intermediary metabolism(s) on DNA, and further that a part of the induced damage is reparable by genetically controlled mechanisms. A number of experimental data are available on the molecular nature of lesions induced in vitro by FA on proteins, nucleic acids and their metabolic precursors [10]. On the contrary, the amount of information on the situation in vivo is limited and restricted to viruses [24] and prokaryotic cells [21--29]. Hence, we decided to study quantitatively the capacity of FA to induce damage in the cellular DNA of a simple eukaryotic organism Saccharomyces cerevisiae which has been the object of previous genetical studies with FA [3--5]. In this first communication, we provide evidence that, through a metabolic process, FA induces single-strand breaks in yeast DNA and that a part of this type of damage is reparable. Materials and methods

Strains. The haploid strain of Saccharomyces cerevisiae N123 (a his, RAD) and two UV-sensitive mutants rad~_3 and rad3.~swere used. These two mutants belong to the same pathway and are known to be defective in the excision of UV-induced pyrimidine dimers [28,23]. They both had a higher sensitivity to FA-induced killing [ 5]. Media. Cells were grown in YEPD (Yeast extract Difco 0.5%, Bacto peptone Difco 2%, glucose 2%, in distilled water) or in Y synthetic medium [20]. For plates, the YEPD medium was solidified with 2% Bacto-Agar Difco. Method. The protocol involved isotopic labeling of DNA, treatment with FA, conversion of cells to spheroplasts and lysis of spheroplasts on the top of alkaline sucrose gradients through which the DNA was subsequently sedimented. The inactivation of the cells' reproductive capacity was measured by plating aliquots on solidified YEPD. All treatments were performed on logphase cells. Strains whose DNA was to be analyzed were grown to stationary phase in Y synthetic medium at 30°C with continuous aeration. A small inoculum (3 × 10 s cells/ml) was then transferred into the same medium to which [6-3H] uracil (15 pCi/ml and specific activity of 20 Ci/mmole) or [2-14C]-adenine (10 pCi/ml and specific activity of 52 mCi/mmole) was added for labeling of the nucleic acids. Cells were allowed to perform 5--6 generations. They were then harvested at a density of a b o u t 5 X 106 cells/ml, washed twice with saline (NaC1 9%o ), resuspended in 0.1 M phosphate buffer (pH 7) at a final concentration of 1 × 107 cells/ml and sonicated for 30 sec for clump disruption. Cells were then treated in one of the following ways. (1) Immediately converted to spheroplasts (control). (2) Treated at 20°C with different concentrations of FA for the periods indicated in Results, then rapidly filtered through Millipore filters (HAWP 0.45 p) and thoroughly washed with saline. They were then resuspended and converted into spheroplasts. (3) Untreated and FA-treated cells were re-incubated in YEPD at 30 ° with aeration for different periods, and growth was followed by h e m o c y t o m e t e r countings. At different time intervals, cells were converted into spheroplasts. (4) Untreated and FA-treated cells

183 were re-incubated in water at 30°C with aeration for 24 h (liquid-holding conditions) and were then transformed into spheroplasts. A n~wly developed technique that allows the formation of spheroplasts from cells with an augmented resistance of their cell wall to enzymic digestion was used after liquid holding [25]. In some experiments, spheroplasts were prepared and then treated at 20°C with FA in the stabilizing medium at pH 7. The treated spheroplasts were then washed by centrifugation at 4°C with the same medium. Conversion to spheroplasts and alkaline sucrose gradient sedimentation. The method of Petes and Fangrnan [20] was adopted for the formation of spheroplasts and was modified for the alkaline sucrose gradients. The spheroplasting protocol required a b o u t 45 min. A suspension of spheroplasts {0.1 ml, 5 X 107 spheroplasts/ml) was layered directly upon a linear 15--30% sucrose gradient (4.6 ml). The gradient solution contained 1% sodium dodecyl sarcosinate (Sarkosyl), 0.015 M sodium EDTA, 0.01 M Tris, 0.9 M NaC1 and 0.1 M NaOH. The pH was adjusted to 12. Sarkosy~ (0.1 ml of 5%) was added on top of the gradients and spheroplasts were allowed to lyse for 15 rain at room temperature. Centrifugations were performed at 5 ° in an SW-50 Beckman rotor for 23 h at 1 4 0 0 0 rpm. 14C-Labeled T4 bacteriophage DNA was used as a sedimentation marker. To release the DNA, the phages were incubated for 10 rain at 60°C with 5% Sarkosyl (Geigy, Switzerland). Fractions of 0.2 ml were collected from the top of the gradient. R N A was hydrolyzed by incubation of the fractions at 37°C for 18 h with 1 ml of 0.6 M NaOH supplemented with uracil (1 mg/ml). The samples were chilled, neutralized with HC1 and precipitated with 0.5 ml of 50% trichloroacetic acid. Precipitates were collected on glass-fiber filters (Whatman GF/C), washed 3 times with 5% TCA and twice with 95% ethanol. The filters were dried and counted in a toluene-based scintillation fluid (4 g PPO, 0.1 g POPOP per liter). The number average molecular weight of single-stranded DNA was calculated As Mn = i/~ (Ci/Mi), where i is the fraction number and Ci is the percentage of recovered counts per minute having a relative molecular weight Mi. The relative molecular weight of DNA in a fraction was calculated according to Freifelder [12] b y comparing the distance sedimented by the yeast DNA (d2) with the distance sedimented b y T4 DNA {dl) and using the equation dl/d2 = (M1/ M~) °'3s. T4 DNA is assumed to have a molecular weight of 1.2 X 10 s daltons [16], and 6 X 107 daltons when single stranded. The position of this marker DNA is indicated in all the following sedimentation profiles. Results

( A ) Production o f single-strand breaks after treatment with FA in a wild-type strain (1) Treatment o f spheroplasts The characteristic sedimentation profile in alkaline sucrose gradient for the DNA of untreated spheroplasts from haploid wild-type cells is given in Fig. la. The average molecular weight of the material represented in the main peak was

184

.°L

la

lb 20

T4

!~--

l

15

T4

1

Io

,< 0

o
D..

10 0

g 5

1

5

10 FRACTION

I 15

I 20 NUMBER

I TOP

i 5

I I0 FRACTION

I

15

I 20 NUMBER

I tOP

187

2d

2c

1

T4

1"4

10

1

5

10 FRACTION

15

20

TOP

5

10 15 20 F R A C T I O N NUMBER

NUMBER

TOP

2e

!

15

L

I 5

J 10

I 15

FRACTION

J 20

L TOP

NUMBER

Fig. 2. S e d i m e n t a t i o n p r o f i l e s o f y e a s t D N A in alkaline s u c r o s e g r a d i e n t s f r o m cells t r e a t e d f o r 1 5 rain w i t h d i f f e r e n t c o n c e n t r a t i o n s o f F A . (a) U n t r e a t e d . (b) 8.2 m M F A . (c) 17.5 m M F A . (d) 33 m M F A . (e) 66 m M F A .

188

15

3

I

I 5

I 10

I 15

I 20

top

FRACTION NUMBER F i g . 3. S e d i m e n t a t i o n p r o f i l e of yeast D N A in alkaline sucrose gradient from r a d l . 3 strain treated with F A . e, u n t r e a t e d ; s , t r e a t e d f o r 1 5 m i n w i t h 6 6 m M F A .

dient. When the cells were held in water after the FA treatment the initial size of the D N A was not recovered even after 24 h of liquid holding. However, a part of the D N A was found in an intermediary position between the top and the main peaks (Fig. 5a). The cell survival was unchanged by such treatment. When FA-treated cells were incubated for 4, 8 or 12 h in growth medium an increasing amount of material was progressively represented in the position of the main undegraded peak (Figs. 5b, c and d). Besides this population of D N A 10

~,~

10~

,/ ~0

! 4

I 8

I 12

I 16

I 20

I 24

I 28

HOURS OF INCUBATION

F i g . 4. G r o w t h o f w i l d - t y p e c e l l s i n Y E P D a f t e r t r e a t m e n t w i t h F A f o r 1 5 m i n . o , u n t r e a t e d ; ~7, t r e a t e d with 33 m M F A ; a, treated with 66 m M F A . The cell number was determined by microscopic counting with a calibrated Burcker chamber.

361Sa 240



T4 >i> m i-~J .

Biochemical analysis of damage induced in yeast by formaldehyde. I. Induction of single-strand breaks in DNA and their repair.

181 Mutation Research, 50 (1978) 181--193 © Elsevier/North-Holland Biomedical Press BIOCHEMICAL ANALYSIS OF DAMAGE INDUCED IN YEAST BY FORMALDEHYDE...
693KB Sizes 0 Downloads 0 Views