Effect of methyl methanesulfonate on survival of radiation-sensitive strains of Dictyostelium discoideum

415

Mutation Research, 73 (1980) 415--418 © Elsevier/North-Holland Biomedical Press

Short Communication EFFECT OF METHYL METHANESULFONATE ON SURVIVAL OF RADIATION-SENSITIVE STRAINS OF Dictyostelium discoideum

GREGORY PODGORSKI and R.A. DEERING

Biophysics Program, The Pennsylvania State University, University Park, PA 16802 (U.S.A.) (Received 18 December 1979) (Revision received 17 June 1980) (Accepted 25 June 1980)

Mutations in the cellular slime mold, Dictyostelium discoideum, conferring increased sensitivity to v-rays and/or ultraviolet light have been assigned to 9 genetic loci (Welker and Deering, 1978; Coukell and Cameron, 1979). To further characterize the nature of the DNA-repair defects leading to sensitivity in these mutants, we have determined cell survival following methyl methanesulfonate treatment. A report comparing the sensitivities of strains NC4(rad ÷) and Vs13(radB13)to a series of alkylating agents has appeared previously (Payer et al., 1972). Log-phase cells (2--8 X 106/ml) grown in liquid culture with Escherichia coil B/r were washed and resuspended in phosphate-buffered salts (PBS) according to the procedures of Deering et al. (1970). Methyl methanesulfonate (MMS) (Eastman Kodak Chemical) and 4-nitroquinoline 1-oxide (4NQO) (K and K Chemical) solutions were freshly prepared and diluted in PBS to the desired concentrations, and added to cell suspensions (1 × 107/ml) shaking at 23°C in sealed round-bottom, screw-cap tubes. Treatment was terminated 45 rain after addition of the chemical by 20-fold dilution in PBS followed immediately by 3 centrifugations (2 rain, 340 g) in PBS. The total cell count was determined with a Coulter counter before dilution and plating on nutrient plating medium in association with E. coli B/r (Deering et al., 1970). Clones appearing in the bacterial lawn were scored after a maximum of 7 days incubation at 23°C, and survival determined as the ratio of viable cells obtained from plate counts to the total cell count. The sensitivity of each strain was determined in a minimum of 2 independent experiments. Fig. 1 presents survival data following MMS treatment of NC4(rad÷) and 8 radiation-sensitive strains carrying various tad mutations. All strains used in this work were haploid. The origins and genotypes of these strains are shown in Table 1. Mutant strains originally isolated on the basis of UV-light and/or v-ray sensitivity also were sensitive to MMS when compared to the wild-type (NC4). The mutants 7s13(radB13), and A73(radI24) were extremely sensitive to MMS,

416

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I I I 50 40 50 MMS(raM;45rain)

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Fig. 1, Survival of radiation-resistant and -sensitive strains f o l l o w i n g a 45-min t r e a t m e n t w i t h MMS. T h e strains axe: e, NC4(rad+); o, 7s20(radA20); o 9"sl3(radBl3); u, TW8(radC35); A ~,sl4(radDl4); o, ~/s21(radF21); 0, TsS(radGS); e , 7 s l S ( r a d H I S ) ; ~, A~3(radI24).

while ~/s20(radA20), "[s14(radD14), ~/s21(radF21), and ~fs5(radG5) showed sensitivities intermediate between the most sensitive strain (7s13) and NC4(rad÷). TW8(radC35) and NC4(rad÷) responded identically to MMS at greater than 20% survival, but below that level, TW8(radC35) became more sensitive. Strain M28(radE100) has been shown to have the same sensitivity to MMS as the wild-type (rad÷), while being UV-light sensitive and slightly ~f-ray sensitive (Coukell and Cameron, 1979).

TABLE 1 ORIGIN AND R A D I A T I O N SENSITIVITY MARKERS C A R R I E D BY R E L E V A N T STRAINS SWain

Paxental radiationresistant

zad maxker

( rad +) strain NC4 TS12 TW8 A~f3 7s5 ~/sl 3

~?s14 ~'s18 7s20 7s21

NC4 TS12 AX3 NC4 N C4 NC4 NC4 NC4 NC4

Fr om Welker and Deering (1978).

+ + C35 I24 G5 B13 D14 H18 A20 F21

417 TABLE 2 C O M P A R I S O N O F S E N S I T I V I T I E S T O MMS, T ' R A Y A N D U V - L I G H T T R E A T M E N T Strain

N C 4 (rad ÷) TW8 (radC35) ~s18 (radHl8) 7s5 (radG5) ~s20 (radA20) 7s14 (radDl4) ~/s21 ( r a d F 2 1 ) ATs ( r a d I 2 4 ) 7s13 (radB13)

D10

DI0 mutant/Dl0 rad +

MMS (raM)

7-rays (k.rad) a

( j / m 2) a

MMS

64 61 51 24 20 20 20 5 2

300 280 70 50 9 40 40 6 4

155 43 49 25 7 18 18 5 2

-0.95 0.80 0.37 0.31 0.31 0.31 0.08 0.03

T-rays

-0.93 0.23 0.17 0.03 0.13 0.13 0.02 0.01

UV

-0.28 0.32 0.16 0.04 0.12 0.12 0.03 0.01

a V a l u e s o f D 10 f o l l o w i n g T - r a y a n d U V - l l g h t treatment were taken f r o m W e l k e r a n d D e e r i n g ( 1 9 7 8 ) .

The MMS treatment required to reduce survival to the 10% level (Dl0), and the ratio of mutant Dl0 to NC4(rad ÷) D10 are presented in Table 2. The "),-ray and UV-light results, included in this table for comparison, are taken from the work of Welker and Deering (1978). Comparable sensitivity of a repair, deficient strain relative to the wild-type after exposure to a group of damaging agents implies the repair pathway affected in the mutant operates to similar extents on DNA modified by agents within the group. With the exception of TW8(radC35) and ~/s20(radA20), the order of relative MMS sensitivity among these strains was paralleled by T-ray and UV-light sensitivity (Table 2). This suggests appreciable overlap in repair pathways functioning on damage induced by these agents. Excluding Ts20(radA20) and TW8(radC35), the Dlo mutant/ Dl0 tad ÷ ratios following MMS treatment were 2--4 times greater than those obtained from T-ray and UV-light irradiated cells. This may indicate that the functions affected in these mutants, while capable of operating on damage induced by all agents listed in Table 2, are required to a greater degree in the repair of radiation
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NC4 to v-rays (Welker and Deering, 1978) and MMS below 20% survival (Fig, 1), this difference may reflect genetic variation between these strains unrelated to the radC35 mutation. This can be concluded from v-ray (Welker and Deering, 1978) and MMS (data not shown) survival data showing that TS12, the parental strain of TW8, is also more sensitive than NC4 at high radiation and chemical exposures. Many procaryotic (Felkner and Kadlubar, 1968; Kondo et al., 1970) and eucaryotic (Takebe et al., 1972; Prakash, 1976) mutants blocked at an early step in excision repair of UV-light-induced damage are sensitive to 4NQO as well as UV-light. However, when NC4 (rad ÷) and TW8 (radC35) were treated with 4NQO, both strains showed about the same resistance to this chemical, requiring a 45-min exposure to 45 pg/ml 4NQO to reduce survival to the 10% level. The radC ÷ gene product, therefore, appears highly specific for the repair of UV-light-modified DNA. Results presented here point out the complexity of repair processes in D. discoideum. As in Saccharomyces cerevisiae (Prakash, 1976), parallel responses between MMS and ionizing radiation, or 4NQO and UV-light, are not always observed. We thank Drs. M.B. Coukell and A.M. Cameron for communicating results prior to publication. This investigation was supported by Public Health Services Research Grant GM-16620 from the National Institute of General Medical Sciences. References C o u k e l l , M.B., a n d A . M . C a m e r o n ( 1 9 7 9 ) RadE, a n e w r a d i a t i o n - s e n s i t i v e l o c u s in Dictyosteliurn discoideum, J. G e n . M i c r o b i o l . , 1 1 4 , 2 4 7 - - 2 5 6 . D e e r i n g , R . A . , M.S. S m i t h , B . K . T h o m p s o n a n d A . C . A d o l f ( 1 9 7 0 ) G a m m a - r a y - r e s i s t a n t a n d -sensitive s t r a i n s o f slime m o l d (Dictyostelium discoideum), R a d i a l . R e s . , 4 3 , 7 1 1 - - 7 2 8 . Felkner0 I.C., a n d F. K a d l u b a r ( 1 9 6 8 ) P a r a i l a l b e t w e e n u l t r a v i o l e t l i g h t a n d 4 - n i t r o q u i n o l i n e - l - o x i d e s e n s i t i v i t y i n Bacillus subtilis, J. B a c t e r i o l . , 9 6 , 1 4 4 8 - - 1 4 4 9 . K o n d o , S., H. I c h i k a w a , K. I w o a n d T. K a t o ( 1 9 7 0 ) B a s e - c h a n g e m u t a g e n e s i s a n d p r o p h a g e i n d u c t i o n in s t r a i n s o f Escherichia coli w i t h d i f f e r e n t D N A r e p a i x c a p a c i t i e s , G e n e t i c s , 6 6 , 1 8 7 - - 2 1 7 . Payez, J.F., R.A. Deering and J.O. Freim Jr. (1972) Lethal effects of aikylating agents or ultraviolet light o n t h e v - r a y r e s i s t a n t p a r e n t s t r a i n a n d a v - r a y sensitive s t r a i n o f t h e c e l l u l a r slime m o l d , Dictyostelium discoideum, M u t a t i o n R e s . , 1 5 , 8 2 - - 8 5 . P r a k a s h , L. ( 1 9 7 6 ) T h e r e l a t i o n b e t w e e n r e p a i r o f D N A a n d r a d i a t i o n a n d c h e m i c a l m u t a g e n e s i s in Saccharomyces cerevisiae, M u t a t i o n R e s . , 4 1 , 2 4 1 - - 2 4 8 . T a k e b e , H., J. F u ~ y a m a , Y. Miki a n d S. K o n d o ( 1 9 7 2 ) H i g h s e n s i t i v i t y o f x e r o d e r m a p i g m e n t o s u m cells to the carcinogen 4-nitroqulnoline 1-oxide, Mutation Res., 15, 98--100. W e l k e r , D . L . , a n d R . A . D e e r i n g ( 1 9 7 8 ) G e n e t i c s o f r a d i a t i o n s e n s i t i v i t y in t h e slime m o l d Dictyostelium discotdeum, J. G e n . M i c x o b i o l . , 1 0 9 , 1 1 - - 2 3 . W e l k e r , D . L . , a n d R . A . D e e r i n g ( 1 9 7 9 ) In vivo n i c k i n g a n d r e j o i n i n g o f n u c l e a r D N A i n u l t r a v i o l e t o i r r a d i a t e d r a d i a t i o n - r e s i s t a n t a n d sensitive s t r a i n s o f Dictyostelium discoldeum, Mol. G e n . G e n e t . , 1 6 7 , 259--263.