Mutation Research, DNA Repair, 273 (1992,~ 281-288, '3 1992 Elsevier Science Publishers B.V. All rights reser-ved 0921-8777/92/$05.00

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MUTDNA 06479

Evidence for excision repair in promitochondrial DNA of anaerobic cells of Saccharomyces cerecisiae K. P a s u p a t h y a n d D.S. P r a d h a n Biochemisto" Dicision, Bhabha Atomic Research Centre, Bomb~: 400 085 (h~d;a) (Received 2t May 1991) (Revision received !6 August t9'9~) (Accepted t9 August |99i)

tk%'u'ords: l~;rimidine dimers: Endonuclease sensitive sites: Excision repair: Pho~oreaO.ivatiom Promkchondria: Respira~opy adaptation: Saccharomyces cere# isiae

Summary The respirator' adaptation (i.e., essentially mitochondrial biogenesis) in the excision repair-defective rad3-B,pe mutants of Saccharomyces cerecisiae undergoing transition from t~e anaerobic to the aerobic state is found to be far more sensitive to 254-nm ultraviolet radiation (UV) than that of the RAD wild-~ype strain. We confirm that mitochondria of aerobic cells of a PAD strain lack the excision repair capaci~ of UV-induced pyrir:fidine dimers at all doses tested (1-15 J / m : L tn centrasL in promitochondria of anaerobic cells of the wild-type strain excision repair appears to rake place. This process is very efficient at tow doses (at 0.5-5 J / m 2 100% of the UV endonuclease-sensitive sites disappear), whereas at high doses its efficiency is reduced by about 50%. "Une promitochondrial excision repair of pyrimidine dimers appears to be under nuclear control since it is blocked in the tad2 mutant. Fir~aIly photoreactiva|ion is found to be operating in nuclei, mitochondria and promitochondria.

De novo biogenesis of mitochondria i:~ Saccharomyces cerecisiae cells undergoing trm~sition from anaerobic to aerobic growth (respira~ory adaptation) is far more sensitive to 254-nm ultraviolet irradiation (UV) and chemical mutagens than cell viabili~' (Pasupathy and Pradhan, 1978, 1981). This impairment can be solely attributed to the susceptibility of DNA of promitochondrial structures, whose integrity seems to be essential for de novo biogenesis of mitochondria. The

gremcr sensitivi~, of mitochondriM than of nuclear DNA ¢o DNA-damaging agents can stem either from certain unique structural features of mitochondriai DNA or from a less efficient DNA-repair abitity of promitochondriai s~ructures. The studies described in the present communication were aimed at examining the factors ~;.~derlying the relatively greater susceptibili~- of DNA of promitochondrial structures of anaerobically grown S. ceret'isiae cells to UV exposure.

Materials and methods Correspondence: Dr. D.S. Pradham BiochemisL,3 DMsion. Bhabha Atomic Research Centre, Bombay 4(N 085 (tndia~.

Y e a s t 5traip~s

The following strains of S. cerevisiae were used.

282 They were all haploid, and differed in their sensitivities to UV. (I) Wild type: ATCC 3177. (2) Rad mutants~ which are UV-sensitive strains, were kindly gifted by Prof. R. Haynes, Department of Biology, York University, Ontario, and details are given below. (a) HT8-9A a ade 2-1 lys 2 tad 1-1; (b) HTg-14 B a d e 2-1 lys 2 his 3 ura t rad 2-20; (c) HTI2-6B tad 3-2.

to attain a dose rate o f 0 . 1 8 J/m2/s. Cell suspensions were contk,enusly stirred during irradiation. The suspensions of cells (both aerobically grown . . . . . . . . . . . . . . . . ,y grown) were beld under atmosobodc aii during irradiation. We did not feel it necessa~3~ to hold the suspensions of anaerobically grown cells under a nitrogen atmosphere during irradiation since anaerobic cells suspended in phosphate buffer did not exhibit any degree of respiratory adaptation in a non-growth medium.

Media For growth under anaerobic conditions, yeast cells were grown in a medium containing 1% yeast extract, 2% bactopeptone and 2% glucose: For growth under aerob!c conditions, the composition of the medium was the same as above except that the glucose concentration was 0.8%. The media are designated as YEPD-anaerobic medium and YEPD-aerobic medium, respectively. Growth under anaerobic conditions was carried out in batches of 400 ml medium in 500-ml Erlenmeyer flasks. Ox3,gen-free nitrogen was passed through the medium during growth without disturbing the layer of yeast cells at the bottom. Aerobic growth was carried out in batches of 200 ml in 500-ml Erlenmeyer flasks by aeration through a sparger.

Pre-labelling of nuclear and promitochondrial DNAs of S. cerecisiae In order to label DNAs of nuclei and mitochondria, 3H-uridine (specific activity 2.405 × 10 ~ Bq/mmole) was added to log-phase cells (1.85 × 105 Bq/ml medium) which were then grown for approximately 6-7 generations to a final titre of 5-7 X 10 7 cells/ml (cells at this stage were in the late log phase) in batches of 200 ml in 500-ml Erlenmeyer flasks, by aeration through a sparger.

Exposure of cells to UV Anaerobically and aerobically grown cells were harvested, washed and resuspended in phosphate buffer, pH 7.0, at a density of 10 7 cells/mt. Cell suspensions were placed in Petri dishes of 9 cm diameter to give a layer of approximately 4 mm depth. The distance between the UV lamp (Philips TUV 15-W germicidal lamp monochromatic at 254 nm) and the layer was about 30 cm

Post-irradiation h, cubation Aliquots of anaerobically and aerobically grown irradiated and unirradiated ce!l suspensions were transferred to fresh YEPD-aerobic medium, aerated in the dark for 2 h and then exposed to visible light (Philips fluorescent lamp emitting light of 370-800 nm). ©ther aliquots of the cell suspensions were transferred to fresh YEPDaerobic medium and aerated in visible light for 2 h.

Resph'atoo, adaptation studies Cells of various S. cerecisiae strains grown anaerobically for 16 h in YEPD-anaerobic medium were harvested and suspended in phosphate buffer (pH 7.0) at a cell density, of 107 cells/ml. Aliquots of cell suspensions were exposed to different doses of UV. The irradiated and unirradiated ceil suspensions were transferred to a fresh growth medium containing 0.8% glucose (i.e., ";'EPD-aerobic medium) and aerated in the dark. Atiquots of cell suspensions were withdrawn at different time intervals to determine the o~,gen uptake capability using a GME oxygraph with a Clark-type electrode. The reaction was carried out as follows: the reaction mixture, in 1.5 ml. contained 67 mM potassium phosphate buffer (pH 7.4), 400 mM CaC12 and 400 mM AICI3. all in 1.2 ml; 0.2 ml of cell suspension and 0.1 ml of substrate (succinate 0.1 M). The o~cyge~t consumption was expressed as nmoles of 0 2 consumed/min/mg protein (Chance and Williams, 1955).

Isolation of subcelhdar organelles and theil DNAs Nuclei and mitochondria were isolated from aerobically grown cells (18 h) as described by

283

Gordon and Rabinowitz (1973). Promitochondria from anaerobically grown cells were isolated by the procedure of Criddle and Schatz (!969). D N A from subcellular organelles was prepared with a modification of the procedure of Rcynolds (1978).

Analysis of pyrimidine dimers (UV-endonucleasesensitit'e sites) b, [;NA The method based on the use of the UV endonuclease of Micrococcus haeus was according to the procedure of Reynolds (1978). The enzyme was partially purified from M. haeus (Carrier and Setlow; 1970) and was suspended in 0.002 M Tris-HC1 buffer, pH 7.5, containing 0.001 M MgCI,. The prelabelled nuclear and mitochondrial DNAs were isolated from cells after various treatments (irradiation, post-irradiation incubation, etc.). Aliquots of D N A solutions were treated with endonuclease. The number of endonuclease-sensitive sites, i.e., the number of single-strand breaks in DNA, was determined by sedimentation of treated D N A solutions through alkaline sucrose density gradients in a Beckman model L2-65 B ultracentrifuge with an SW 65 rotor at 40,000 rpm for 120 rain at 20°C. Each gradient was siphoned out, fractions were collected and exprebscd as per cent of total radioactivity loaded on to grodient (Nair and Pradhan t975). The resulting data were analysed with a computer program that calculates weight-average molecular weights from the distribution of radioactivity through a gradient essentially as described by Lehnert and Mm'oson (!97t). Gradients had previously been calibrated using T, and lambda phage DNA~ ns standards. The number of UV endonudease-sensitive si,es, manifested a:, strand breaks, was calculated according to Reynolds (1978). using the following formula:

Results

Studms on the progressive acquisition of respiratopy capacity of cells (RAD strain) during transition from anaerobic to aerobic growth and its inhibition by a low dose of UV (19 J / m - ' , which has no effect on survival) were reported earlier (Pasupathy and Pradhan, t978). Exposure of UV-irradiated ceils to visible light reversed this inhibition (during transition from the anaerobic to the aerobic state, spontaneous production of the cytoplasmic petite mu~qion was found to be negligible). A d o s e - r e s p o n s e curve for inhibition of respiratory adaptation in cells of the /CAD strain is shown in Fig. 1A and the survival curve for the same strain after UV exposure is shown in Fig. lB. The dose required for 90c~ UV-induced cell lethalib' in RAD is t70 J / m e and that required for 90c;~ UV-induced inhibition of respiratory. adaptation is i9 J / m -~ in the same strain. The study v~as extended to t a d mutants of 5;'. cererisiae diffc:dng in their sensitivity to UV. AIt the rad mutants used here have been shown to lack nuclear D N A excision repair synthesis following exposure to UV (Haynes and Kuntz, 1981). The do~e effects on cell snrx~ival and on respirat o ~ ' adaptation were determined, tt can be seen from Tabie 1 that the r e s p i r a t o a ' adaptation process in aH the UV-sensitive strains is nearly 20

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Fig. 1. Dose-resp~msc curve for inhibition of r~spiratoJ3" adaptation (At and :~up.'ival c u r v e (B) of witdqyp~ cells of S. ce~'etisiae after UV irradiathm.

284

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TABLE !

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SENSITIVFt'Y Of: R [ i S P I R A T O R Y A D A P T A T I O N A N D CE[,I, S U R V I V A L O F 5. cere~isiae STRAINS TO UV Smdn

Dose for 90'::; celt MIIing {J/l'a21

Dose for 9(K,'; reducdon of rcspiraIog'

Dose for 90c; reduction of res.pira{oO' adapla!hm

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P) 074 0.6 0,7

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Evidence for excision repair in promitochondrial DNA of anaerobic cells of Saccharomyces cerevisiae.

The respiratory adaptation (i.e., essentially mitochondrial biogenesis) in the excision repair-defective rad3-type mutants of Saccharomyces cerevisiae...
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