Mutation Research, 150 (1985) 235-240 235 Elsevier

MTR 02031

Mutagen sensitivity of Drosophila melanogaster

VIII. The influence of the mei-41 D5, mus(1)lO1 D1, mus(1)lO2D1, mus(1)103 ol, mus(2)205A1, and mus(3)31 oD1 loci on alkylation-induced

mutagenesis

P. Dennis Smith and Ruth L. Dusenbery Department of Biology, Southern Methodist University, Dallas TX 75275 (U.S.A.)

(Received 15 January 1985) (Revision received 21 January 1985)

Alkylating agents (AAs) represent a major class of DNA damaging chemicals which can produce an extensive array of DNA lesions leading to cytotoxicity and mutagenicity (Singer and Kusmierek, 1982). In both prokaryotic and lower eukaryotic organisms, cellular DNA-repair mecha- nisms have evolved to protect the genome from the cytotoxic and mutagenic consequences of alkyla- tion damage (Lindahl, 1982). In these organisms, a major mechanism for the direct repair of alkyla- tion damage involves a base-excision-repair path- way in which, by a concerted series of reactions, a damaged region in a single strand of DNA is removed and replaced by DNA synthetic copying of the undamaged complementary strand ('repair replication') to restore the undamaged duplex.

Our laboratory has focused its attention on alkylation repair and mutagenesis in Drosophila melanogaster as a model genetic system for higher eukaryotes. Over 100 mutations have been isolated

Dedication: This manuscript is dedicated to Professor Frits Sobels for his foresight in recoEniTing the need for a journal devoted to mutation research and his perseverance in seeing it to such a successful state.

Abbreviations: AAs, alkylating, agents; EMS, ethyl methane- sulfonate; ENU, N-ethyl-N-nitrosourea; MMS, methyl methanesulfonate; MNU, N-methy-N-nitrosourea; mus, mutagen-sensitive; PDS, postreplication damage synthesis; SLRL, sex-linked recessive lethal; UV, ultraviolet light.

on the basis of somatic hypersensitivity to methyl methanesulfonate (MMS) which have identified 30 loci as putative DNA-repair genes (Smith et al., 1980). Primary cell cultures have been prepared from homozygous mutants representing 7 of these loci and have been examined for the ability to perform repair replication, as monitored by un- scheduled DNA synthesis, following damage by monofunctional alkylating agents. Of these 7 loci, 6 have been shown to be defective in alkylation-in- duced repair replication (R.L. Dusenbery, in pre- paration).

In the present report, we examine the effects of these loci on mutation induction by 4 AAs (MMS, MNU, EMS, ENU). Mutagenesis is assayed using the SLRL test following a procedure in which repair-proficient males are fed specific AAs and mated briefly to either repair-deficient or repair- proficient females. Comparison of the frequencies of mutations, induced in mutagen-treated mature sperm, between females differing in repair geno- types permits determination of the influence of specific repair genes on the mutation process.

Materials and methods

Repair-proficient Basc males were collected within 24 h of eclosion and aged with females for a 2-day period. After ageing, the males were sep- arated from the females, aged an additional 24 h,

0027-5107/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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starved for 3 h and fed either 0.1 mM MMS (Aldrich Chemical Co.), 0.5 mM MNU (Sigma Chemical Co.), 0.5 mM EMS (Eastman Kodak) or 0.5 mM ENU (Sigma Chemical Co.) for 24 h in M/30 phosphate buffer plus 5% sucrose by the method of Aaron et al. (1977).

Treated males were mated individually in vials to 2 repair-deficient females or appropriate control repair-proficient females for 3 days. From each parental male, 20 F1 cultures were established and, following the criteria of Wurgler et al. (1977), the F2 cultures were screened for the occurrence of SLRLs. Cultures were grown on standard Drosophila medium and incubated at 24 _+ 1°C. Repair-deficient strains were checked periodically during the course of the experiments for continued expression of mutagen sensitivity according to established procedures (Smith, 1976). Descriptions of non-mutagen-sensitive strains can be found in Lindsley and Grell (1977). Mutagen-sensitive strain descriptions can be found in (Smith et al., 1980).

Results

Previous studies examining the ability of mutagen-sensitive strains to repair damage in- duced by ultraviolet light demonstrated that these mutants could be divided into 3 groups based on their ability to perform excision repair or postrep- lication damage synthesis ('PDS') (Brown and Boyd, 1981). Mutants at the mei-41, mus 101 and

mus 310 loci are proficient for excision repair but show defects in PDS, mus 205 mutants are par- tially defective in excision repair and completely defective in PDS, while mutants at the mus 102

and mus 103 loci do not display defects in UV-in- duced repair functions. A number of the mutagen-sensitive strains which have been tested for their effects on alkylation-induced mutation frequencies (this report) have been analyzed as primary embryonic cell cultures for the capacity to perform repair replication following induction of alkylation damage. These data are summarized in Table 1 and a manuscript detailing these experi- ments is in preparation (R.L.D.).

Control strains, which do not exhibit mutagen sensitivity and which have served as the parental strains for the induction of mus mutations, are capable of performing unscheduled DNA synthe-

TABLE 1

REPAIR REPLICATION CAPACITY OF CONTROL AND

MUTAGEN-SENSITIVE PRIMARY EMBRYONIC CELL

CULTURES FOLLOWING DAMAGE BY MONOFUNC-

TIONAL ALKYLATING AGENTS

Genotype Alkylating agent

MMS MNU EMS ENU

,4. Con t ro l

y + + + NT w + + + +

cn q- + + +

cn b w NT NT NT NT

st NT NT NT NT

B. M u t a n t s

y m e i - 41 O S / y + Y B s + + + NT w m u s 101 o l . . . .

s t m u s 3 ] 0 D1 NT NT NT NT

cn b w m u s 205 AI - - - NT w m u s 103 °1 . . . .

w m u s 102 TM NT NT NT NT

+ , capable of repair replication.

- , incapable of repair replication. NT, not tested.

sis following treatment of cell cultures with MMS, MNU, EMS and ENU.

Although the cn bw and st parental strains have not been examined in the UDS assay, the similarity of mutagen sensitivity in these strains with other control strains would suggest a similar repair proficiency. The mei-41D5 mutant exhibits repair proficiency following alkylation damage consistent with the proficiency observed after UV damage. Interestingly, the mus 101 °1 mutant, which, on the basis of UV data, would be expected to be similarly excision-proficient, is unable to perform repair replication following alkylation damage. The m u s 2 0 5 A1 mutant, which exhibits partial UV excision deficiency, is similarly incapa- ble of alkylation-induced repair replication. Fi- nally, the m u s 1 0 3 D1 mutant, which was selected on the basis of MMS sensitivity and displays wild type sensitivities to UV, X-irradiation and nitro- gen mustard (Boyd et al., 1976), exhibits no defect in either UV-induced excision repair or PDS but, interestingly, fails to show repair replication fol- lowing alkylation damage. This is the first direct indication that the mus 103 locus is actually in- volved in DNA repair.

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TABLE 2

I N F L U E N C E OF M A T E R N A L REPAIR GENOTYPE ON THE F R E Q U E N C Y OF SEX-LINKED RECESSIVE LETHALS I N D U C E D BY 0.1 m M MMS

Genotype Lethals Total tests % SLRL % m u s / % m u s + * t ° .

w '" 18 1162 1.6+0.5 - w m u s 101 D1 67 1 035 6.1 + 0.7 3.8 (S) w m u s 102 D1 22 1163 1.9 + 0.7 1.2 (NS) w m u s 103 D1 42 1181 3 .6+0 2.2 (S) y 14 1173 1.2+0.2 - y m e i - 4 1 ° 5 / y + Y B s 10 1174 0.8+0.7 0.7 (NS) c n b w (*) 23 1160 2.0+1.2 - c n b w m u s 205 A1 90 1214 7.4 4- 0.2 3.7 (S) st (1 expt.) 19 589 3.2 - st m u s 310 91 47 828 6.2+ 1.3 1.9 (S)

S, significant at the 5% level by X 2 test. NS, not significant at the 5% level by X 2 test. (*), individual experiments inhomogenous.

Table 2 presents data on the influence of vari- ous maternal repair genotypes on mutation induc- tion by MMS, a strong nitrogen-site alkylating agent. For those strains which exhibit a defect in repair replication following alkylation treatment, mus 101DI, mus 103 D1 and mus 205 At, mutation frequencies are significantly elevated over ap- propriate repair-proficient control strains. For mus 310 D1, which has not yet been tested in the UDS assay for alkylation damage but which is clearly defective for PDS following UV damage, a signifi- cant increase in mutagenesis is also seen. Con- versely, for mei-41DS, which is capable of repair replication following alkylation damage, and mus

102 D1, for which no repair defect has yet been determined, no significant difference is observed between mutants and controls for mutation induc- tion.

Conversely, for mutagenesis by ENU (Table 3), a strong alkylating agent for oxygen species with relatively low nitrogen alkylation efficiency, de- fects in repair-replication ability are not reflected in enhanced mutagenesis: For all tested repair-de- ficient strains, no significant difference in muta- tion frequency is observed between these strains and their appropriate controls. We will return to this observation in the Discussion.

For MNU (Table 4), another strong oxygen-al-

TABLE 3

I N F L U E N C E OF M A T E R N A L REPAIR GENOTYPE ON THE F R E Q U E N C Y OF SEX-LINKED RECESSIVE LETHALS I N D U C E D BY 0.5 m M E NU

Genotype Lethals Total tests % SLRL % m u s / % m u s +

w 150 1169 12.8 +0.8 - w m u s 101 D1 142 1 069 13.3 + 2.3 1.04 (NS) w m u s 102 DI 172 1118 15.4+ 1.6 1.20 (NS) w m u s 103 D1 145 1173 12.4+ 1.8 0.97 (NS) y 76 1135 6.7 + 1.6 - y m e i - 41 D S / y + y B s 70 1126 6.2 + 0.4 0.93 (NS) cn b w (*) 88 1170 7.5 + 3.2 - cn b w m u s 205 ^1 69 1173 5.9 + 1.2 0.79 (NS)

S, significant at the 5% level by X 2 test. NS, not significant at the 5% level by X 2 test .

(*), individual experiments inhomogenous.

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TABLE 4

I N F L U E N C E OF M A T E R N A L REPAIR GENOTYPE ON THE F R E Q U E N C Y OF SEX-LINKED RECESSIVE LETHALS I N D U C E D BY 0.5 m M M N U

Genotype Lethals Total tests % SLRL % r n u s / % m u s +

w 57 1187 4.8 + 0.8 - w m u s 101 D1 49 1 101 4.5 5:0.7 0.9 (NS) w m u s 102 D1 62 1 131 5.5+1.8 1.2 (NS) w m u s 103 D1 (*) 53 1189 4.5 5:1.8 0.9 (NS) y 31 1183 2.65:0.9 - y m e i - 4 1 D S / y ÷ y B s 30 1163 2.6 5:0.7 1.0 (NS) c n b w 20 1154 1.7 + 0.8 - c n b w m u s 205 A1 41 1175 3.5 5:0.1 2.1 (S) st 17 1164 1.5 5:0.5 - st m u s 310 D1 62 1088 5.75:0.2 3.8 (S)

S, significant at the 5% level by X 2 test. NS, not significant at the 5% level by X 2 test. (*), individual experiments inhomogeneous.

kylating agent which also has significant nitrogen- alkylation ability, the genotypic response is inter- mediate between MMS and ENU. As for MMS, the mus 205 A1 mutant and the mus 310 D1 mutant show enhanced mutation induction, while for mus 101D1 and mus 103 Dr, no significant difference is observed. Finally, for EMS (Table 5), the pattern of enhanced mutability is again intermediate be- tween MMS and ENU. These experiments, though generally consistent, are somewhat less mechanisti- cally predictable, perhaps reflecting the lowered probability of in vivo ethylation in comparison to methylation rather than a fundamental difference in mechanism. Nevertheless, these data demon-

strate very clearly that alkylation repair-defective mutants have significantly enhanced mutability when DNA damage is induced by alkylating agents which have a high specificity for nitrogen sites.

Discussion

Our interpretation of present results rest upon two assumptions. First, we assume that alkylation damage induced in mature sperm of Drosophila is identical to that described in vitro and other in vivo systems (Singer and Grunberger, 1983). Sec- ond, we assume that a base-excision pathway, which includes a repair replication step, similar to

TABLE 5

I N F L U E N C E OF M A T E R N A L REPAIR GENOTYPE ON THE F R E Q U E N C Y OF SEX-LINKED RECESSIVE LETHALS I N D U C E D BY 0.5 m M EMS

Genotype Lethals Total tests % SLRL % m u s / ~ 6 m u s +

w 38 1173 3.2 5:0.2 - w m u s 101 D1 43 1039 4.1 +0 .4 1.3 (NS) w m u s 102 D1 39 1121 3.5 5:0.8 1.1 (NS) w m u s 103 D1 56 1186 4.7 5:0.4 1.5 (NS) y 50 1139 4.4 5:0.2 - y rnei- 4 1 0 5 / y + y B s 21 851 2.5 5:0.5 0.6 (S) cn b w 49 1158 4.25:1.1 - cn b w m u s 205 A1 59 1160 5.1 5:0.3 1.2 (NS) st 5,t 1171 4.6 5:0.6 - s t m u s 3 1 0 D1 74 885 8.45:0.1 1.8 (S)

S, significant at the 5% level by X 2 test. NS, not significant at the 5% level by X 2 test.

pathways observed in other organisms, functions in Drosophila melanogaster. Such pathways are initiated by DNA glycosylase activity which cleaves the glycosylic bond between an alkylated base and its deoxyribose moiety to produce an 'abasic site'. Endonuclease activity specific for abasic sites cleaves the sugar-phosphate backbone, and repair synthesis replaces a region of DNA at the damage site. At present, the role of postincision exonucleo- lytic activity is somewhat unclear, but the par- ticipation of a DNA ligase activity to complete the base-excision process is known.

In Table 6, we summarize data from Singer and Grunberger (1983) on in vitro alkylation of purines in double-stranded DNA. Of the 6 alkylation ad- ducts described, 3-alkA, 7-alkA, 3-alkG and 7-alkG are known to be repaired by base-excision path- ways in bacterial and mammalian systems and O6-alkG is known to be repaired by an al- kyltransferase reaction (Lindahl, 1982). From these data, we would conclude that each of the alkylat- ing agents used should be able to induce repair replication in repair-proficient cells and that mutants deficient in alkylation excision repair should be unable to do so. Our data on unsched- uled DNA synthesis, summarized in Table 1, indi- cate that this expectation is true for control strains and that the mus 101 D1, mus 103 D1 and mus 205 A1 mutants appear to be defective in alkylation excision repair.

Interpretation of our mutagenesis data for these strains is best done by comparison of the muta- genic consequences of the exposure of our control and mutant strains to MMS and ENU. For MMS, it may be noted that nearly 98% of the purine

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adducts identified in other systems, are on posi- tions on DNA bases which induce excision repair and are not directly involved in base pairing, while, for ENU, only 16% of the adducts occupy excision-inducing non-base pairing positions. Al- ternatively, the O6-alkylguanine lesion, which is not repaired by excision repair, occupies a base pairing position, and is believed to constitute the major adduct for misreplication mutagenesis, rep- resents only 0.3% of the MMS-induced purine adducts but nearly half of the ENU-induced purine adducts. In addition, studies of the relative stabil- ity of purine adducts in DNA (reviewed in Singer and Grunberger, 1983) suggest that, under mam- malian physiological conditions, 3- and 7-methyl- deoxyadenosine are relatively unstable, exhibiting half-lives of 26 h and 3 h, respectively.

For the musl01D1, musl03 D1, mus205 A1 and mus310 TM strains, MMS-induced mutagenesis is significantly enhanced 2-4-fold while ENU-in- duced mutagenesis is not enhanced at all. We hypothesize, for ENU, that the majority of muta- tions are induced during the early cleavage stages following fertilization by miscoding events at the 0 6 position during rapid replication and that no difference in mutation frequency would be ex- pected to be observed statistically between repli- cation repair-defective and control strains. Alter- natively, for MMS damage,we hypothesize that depurination events, induced by a glycosylase ac- tivity or occurring spontaneously at relatively un- stable sites, produce apurinic sites which cannot be repaired in our repair-replication-defective strains and which, consequently give rise to enhanced mutagenesis by some form of postreplication

TABLE 6

IN VITRO ALKYLATION OF DOUBLE-STRANDED NUCLEIC ACIDS

Percent of total alkylation

Adenine Guanine

1 3 7 3 0 6 7

MMS 3.8 10.4 1.8 0.6 0.26 85 MNU 1.3 9 1.7 0.8 6.3 67 EMS 1.7 4.9 1.1 0.9 2 65 ENU 0.2 4.0 0.3 0.6 7.8 11.5

Adapted from Table IV-14, B. Singer and D. Grunberger (1983) Molecular Biology of Mutagens and Carcinogens, Plenum, New York, p. 77.

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damage synthesis that restores the integrity of the DNA duplex at the expense of error-prone muta- genesis.

Acknowledgements

The authors are indebted to Dr. S.F. Cooper, Elka Nutt and Catherine Caspar for technical assistance. This project has been supported by grant ES-01101 from the National Institute of Environmental Health Sciences. Thanks to Susan Stone and Ann Nurre for assistance with typing the manuscript.

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