Mutation Research, 244 (1990) 115-121

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Elsevier MUTLET0347

Uracil-DNA glycosylase activity affects the mutagenicity of ethyl methanesulfonate" Evidence for an alternative pathway of alkylation mutagenesis Douglas F. Fix*, David R. Koehler and Barry W. Glickman Department of Biology, York University, 4700 Keele Street, North York, Ont. M3J 1P3 (Canada)

(Accepted21 December1989)

Keywords: UraciI-DNA glycosylase;Ethylmethanesulphonate;O~-Ethylguanine;Guanines,alkylated;Alkylationmutagenesis

Summary Mutagenesis induced by the alkylating agent ethyl methanesulfonate (EMS) is thought to occur primarily via mechanisms that involve direct mispairing at alkylated guanines, in particular, O6-ethyl guanine. Recent evidence indicates that alkylation of guanine at the 0-6 position might enhance the deamination of cytosine residues in the complementary strand. To determine whether such deamination of cytosine could play a role in the production of mutations by EMS, the efficacy of this agent was tested in uracil-DNA glycosylase deficient (Ung) strains of Escherichia coll. The Ung - strains showed a linear response with increasing doses of EMS. This response was independent of the u m u C gene product. In contrast, the Ung + strains yielded a dose-squared response that became linear at higher doses of EMS when the cells were defective for the u m u C gene product. These results support a model for mutagenesis involving the deamination of cytosines opposite O6-alkylated guanines followed by an error-prone repair event.

Simple alkylating agents represent a class of chemical compounds that are both mutagenic and carcinogenic through direct interactions with cellular DNA to produce base adducts (see Singer, 1975; Lawley, 1984). Numerous sites on DNA can

Correspondence: Prof. Barry W. Glickman, Department of Biology,York University,4700KeeleStreet, North York, Ont. M3J IP3 (Canada), Tel. (416) 736-5242; Fax (416) 736-5698. * Present address: Departmentof Microbiology,Southern Illinois University,Carbondale, IL 62901 (U.S.A.).

be alkylated but the mutagenic (and carcinogenic) capacity of these agents seems to correlate with their propensity to generate O6-alkyl guanine (O6-alkylG; Loveless, 1969; Pegg, 1984; Singer, 1986). This association has been deduced from in vitro and in vivo studies that illustrate the potential of O6oalkylG to base-pair with thymine during DNA replication. For example, in vitro replication experiments indicate that thymine and not cytosine is preferentially inserted opposite O6-methyl guanine (O6-MeG; Abbot and Saffhill, 1979; Loechler et al., 1984). Likewise, in vivo ex-

0165-7992/90/$ 03.50 © 1990ElsevierSciencePublishers B.V. (BiomedicalDivision)

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periments also demonstrate a correlation between the production of O6-alkylG and the induction of mutations and cancers (Frei et al., 1978; Heflich et al., 1982; Beranek et al., 1983). Further, studies of the specificity of mutations induced by alkylating chemicals demonstrated that G:C ~ A:T transitions, the predicted O6-alkyl directed base substitution, predominated (Prakash and Sherman, 1973; Coulondre and Miller, 1977; Burns et al., 1986, 1987; Lucchesi et al., 1986; Richardson et al., 1987a,b). In support of this mispairing model, NMR studies confirm that O6-MeG:thymine base pairs can form proper hydrogen-bonding interactions whereas O6-MeG:cytosine base pairs cannot (Williams and Shaw, 1987). Thus, there is sufficient experimental support for the model that O6-alkylG is a promutagenic lesion that exerts its mutagenic potential by direct mispairing during DNA replication. An additional pathway for the production of G:C ~ A:T transitions by alkylating chemicals has been suggested (Richards et al., 1984; Sowers et al., 1987), This hypothesis proposes that cytosines located in the DNA strand opposite O6-alkylG residues are more susceptible to deamination as a result of 'cross strand protonation'. The resulting uracil residues, if left unrepaired, would then direct the insertion of adenine, also resulting in a G:C A:T transition. Alternatively, the removal of uracil by uracil-DNA glycosylase (Lindahl, 1974) would produce an apyrimidinic (AP) site, which is susceptible to further base excision repair. It is possible that insertion of thymine opposite O6-EtG could occur during this base excision-repair process. Alternatively, AP sites could act as mutagenic substrates in the presence of the umuDC gene products (Schaaper et al., 1983; Walker, 1984). Thus, several alternative pathways might be involved in the production of mutations by alkylating chemicals. Ethyl methanesulfonate (EMS) is one example of a chemical agent that potentiates the alkylation of DNA bases (see Singer and Grunberger, 1983). EMS is an effective mutagen for E. coli and many eukaryotic cells (Schendel et al., 1978; Frei et al., 1978; Heflich et al., 1982; Beranek et al., 1983) and

studies of mutational specificity have illustrated a preference for the induction of G:C --, A:T transitions (Coulondre and Miller, 1977; Burns et al., 1986), which may reflect the production of O6-ethyl guanine (O6-EtG). Consideration of the 'cross strand deamination' model, however, suggests that other mechanisms may also be involved in the mutagenicity of EMS. In this paper, we sought to explore these alternative models by examining mutagenesis induced by EMS in a strain of E. coli deficient for uracil-DNA glycosylase. In this strain, according to the model, uracil residues resulting from 'cross strand deamination' (Sowers et al., 1987) would go unrepaired, and mechanisms dependent on this process might be revealed. The results of these experiments support the possible participation of uracil in the mutagenicity of EMS. Materials and methods Bacterial strains The E. coli strains used in these experiments were derivatives of KMBL3835 (Table 1). Strain NR3951 was a uvrB derivative (Todd and Glickman, 1982). Strain NR8006 was an ung-1 derivative of NR3951 that has been previously described (Fix and Glickman, 1987). Strains NR80003 and NR6116 were umuC36 derivatives of

TABLE 1 I N A C T I V A T I O N C O N S T A N T S A N D log-log SLOPES FOR MUTATION Strain

Relevant markers

NR3951 NRS006 NR8003 NR6116

uvrB uvrB, ung-I uvrB, umuC-36 uvrB, ung-1, umuC-36

k ± S.E. -3.52+0.16 -2.74±0.13 -3.10=t:0.19 -2.49±0.14

m ± S.E. 1.89+0.08 0.89:t:0.07 1.31±0.23 0.98±0.18

Inactivation constants (k) were determined by linear regression using the exponential portion of the survival curves and the relationship S = e - kc, where C is the molar concentration of EMS. Log-log slopes for mutation (m) were also calculated by linear regression using the formula MF= aC~. These analyses were based o n two independent experiments for each strain. S.E., standard error.

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NR3951 and NR8006, respectively. The uvrB (excision-repair defective) strains were used in order to eliminate excision of O6-ethyl guanine residues by this pathway.

10 g 8

• NR3951uvrB O NRSO06uvrBung-1 • NRSO03uvrBu m u C 3 e ~ [~ NR6116 uwBumuC36ung-1

UmuC*

7

Ung

6

General methods

Dose-response kinetics for survival and mutagenesis were determined by treating log-phase cells (grown in LB broth, washed and resuspended in VB salts) with concentrations of 0, 1, 2, 3 and 4°70 EMS (Eastman) for 30 min at 37°C. Following exposure, cells were again washed, resuspended in VB salts and plated onto minimal media containing phenyl-/3-D-galactose for selection of L a c I mutants. Appropriate dilutions o f the cell suspensions were also plated onto LB agar for determination of viability. Results Each of the 4 strains used in this study exhibited typical dose-dependent decreases in viability with an initial shoulder region followed by an exponentially linear decline. The final slopes in this latter region, as determined by linear regression, are given in Table 1. The results indicated the U n g strains to be marginally more resistant to the lethal effects of EMS than were the Ung ÷ strains. The kinetics for mutation induction were very intriguing. First, the mutation frequency (MF) data were plotted as a function of increasing EMS concentration (Fig. 1). All the strains yielded a dosedependent increase in MF, but the kinetics appeared to be different. The U n g - strains gave the poorest mutational response, followed by the U v r B - strain (NR3951). The U v r B - U m u C strain (NR8003) showed the greatest increase in MF. This is consistent with the results of Schendel et al. (1978), suggesting an error-free 'SOS'dependent repair component of EMS mutagenesis. Another possibility for the enhanced MF observed in the U m u C - Ung + strain relies on the idea that the u m u C gene allows the continuation of replication past sites of poor base pairing (Bridges and Woodgate, 1985). When the base pairing properties of O6-alkylG are considered (Williams and

5 4 3 2 1

I 1

D

~

B

i 2

t 3

t 4

Percent

u.g-

EMS

Fig. 1. Mutation frequencies plotted as a function of percent EMS concentration. NR3951 (uvrB; filled squares); NR8006 (uvrB, ung; open squares); NR8003 (uvrB, umuC; filled triangles); NR6116 (uvrB, ung, umuC; open triangles). Each data point represents the average of two independent experiments.

Shaw, 1987), thymine pairs as the 'correct' base whereas cytosine is an 'incorrect' base. Thus, if the u m u C gene product allows 'incorrect' bases to be inserted, a cell proficient for the u m u C function might more readily insert cytosine. This would occur less often in cells lacking the u m u C function. To better evaluate the differences in the kinetics of mutation induction in the various strains, the MF data were plotted on a log-log scale in order to distinguish linear from higher power relationships. It is thought that linear relationships reflect the predominance of a direct mispairing mechanism whereas dose-squared relationships imply the need for subsequent indirect functions; i.e., error-prone repair processes (Bridges, 1966). The log-log slopes were calculated by linear regression and these data are given in Table 1. Strain NR3951 (uvrB) gave a log-log slope of nearly 2, indicating a dose-squared response. The U n g - strain (NR8006), however, gave a log-log slope of nearly 1, indicating a linear response with dose. This suggests that uracil-DNA glycosylase activity can alter the mutational consequences of EMS. Further analysis revealed the U v r B - U m u C - strain (NR8003) to give a log-log slope value somewhat greater than 1 (when the slope was calculated over the entire dose range employed), and the U v r B - U m u C - U n g - strain (NR6116) yielded a linear response (Table 1). Thus,

118 in the absence o f glycosylase, the response was similar whether or not the strain was defective for the umuC gene product. However, in the presence o f glycosylase, a degree of dependence on umuC function was clearly observed. To further characterize the differences in kinetics of the EMS-induced mutations, the results were plotted in a different way. The experimentally determined mutation frequencies were divided by the spontaneous mutation frequencies to give a value we will refer to as the Relative Mutation Frequency (RMF). More simply, this represents the fold-increase above background that was observed at each of the EMS concentrations employed. The R M F determined for each dose was then divided by the concentration of EMS. This methodology is similar to that used by Pollard et al. (1977) to investigate the kinetics of UV-induced mutagenesis. In such an analysis, a truly linea~ response would be reduced to a straight line with zero slope whereas a non-linear response would be revealed by a more complex function. The results of this analysis are presented in Fig. 2. As expected from the log-log slopes, the U n g strains NRS006 and NR6116 gave straight lines with zero slope. Also as expected, the parent strain NR3951 gave a linearly increasing function. Most interesting were the results from the U m u C - strain NR8003. At lower doses, the data for this strain were quite similar to those obtained with the

20 •

NR3951 uvrB

G NR8006 uvr8 ung°l ~0

15

S

• ..°oo~o~..moc.

j UmuC

"5

),

Ung*

UmuC*

5 Ung"

0

1

2

3

4

Percent EMS

Fig. 2. Mutation frequencies divided by the spontaneous frequencies and by the EMS concentration. These values (RMF/EMS) were then plotted as a function of percent EMS concentration. Symbols as in Fig. 1.

U v r B - strain NR3951. However, at EMS concentrations of 2°7o or greater, the response approached that seen with the U n g - strains. It appears, therefore, that at concentrations less than 2070, the mutation induction in the U m u C - strain follows dose-squared kinetics, whereas, at concentrations greater than 20/0, the response becomes more linear. Consequently, the method of Pollard et al. (1977), allows the mutation response to be factored into umuC-independent and umuC-dependent components.

Discussion The analysis of dose-response relationships, particularly in repair-defective strains, can often provide insights into the mechanisms of mutagenesis. In this paper, we examined the effect of increasing concentrations of EMS on mutation induction in the lacI gene of E. coli. These studies included U n g - strains in an attempt to determine whether uracil residues, which have been suggested to result f r o m 'cross-strand deamination' opposite O6-EtG alkylation products (Richards et al., 1984; Sowers et al., 1987), might provide additional mutagenic pathways. The results indicated that in the absence of u r a c i l - D N A glycosylase, mutation induction followed linear dose-response kinetics. In contrast, in the presence of a functional ung gene product, the response was dose-squared. In addition, a dependence on umuC gene function was observed at the higher EMS concentrations. These data indicate the presence of uracil after EMS treatment. A model that attempts to accommodate these findings proposes that the m a j o r mechanism through which EMS-induced mutation occurs involves direct mispairing of the O6-ethylG lesion and only at higher concentrations of EMS does the umuC gene product play a role. The description applied by Schendel et al. (1978) that mutationinduction by alkylating agents reflects a 'race' between error-free and error-prone repair mechanisms and D N A replication seems appropriate. In this instance, each time an O6-EtG is formed, there is a potential for the deamination of the opposing cytosine. At this point, the 'race'

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begins. If the error-free repair enzyme O6-alkylguanine-DNA methyltransferase reaches the O6-EtG before deamination, mutation is avoided. If, however, deamination occurs first, a mutation could become fixed by replication across the uracil. Thus, Ung- strains are presented with two mutagenic options: (1) unrepaired O6-EtG residues could mispair directly during replication or (2) uracils could direct the insertion of adenine. Such models would likely be expected to show a linear relationship with increasing EMS concentration. Both mutational pathways would produce G:C A:T transitions. In the presence of a functional ung gene product, however, uracil residues could be removed, resulting in an AP site. Normally, an AP endonuclease would initiate resynthesis and DNA polymerase plus ligase would complete the repair process. Following this model, mutation would not only require an alkylation and deamination event but also an error-prone 'processing' event. This could explain the dose-squared response observed at lower doses in the UmuC ÷ background. At higher doses it is possible that a significant fraction of the AP sites might persist until replication. Such lesions would require processing dependent upon the u m u C gene product. However, this would only be expected at doses where base excision repair is saturated and the induction of the 'SOS' errorprone response has already occurred. Thus, in a U m u C - strain, the MF response would become more linear at high mutagen doses. This is what was observed. A study by Foster and Davis (1987) of mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in cells defective for exonuclease III (Xth - ), an AP endonuclease, revealed that 42% of the mutations at G:C base pairs in Xth- cells (but UmuC ÷) were transitions, rather than transversions as might have been predicted from the replicative bypass of AP sites. The authors suggest that this may indicate the insertion of thymine rather than adenine opposite AP sites that would probably be due to the depurination of N7-alkylG products. However, an alternative explanation could involve the deamination of cytosine opposite

O6-alkylG followed by the removal of uracil by uracil-DNA glycosylase. The resultant AP sites would persist in the Xth- host and upon replication direct the UmuC+-dependent insertion of adenine, which occurs 3-fold more frequently than the insertion of thymine (Kunkel, 1984). This would also yield a G:C ~ A:T transition. The finding that mutation frequencies were lower in the Ung strains was initially puzzling since the inability to remove uracil residues might be expected to enhance the mutation frequency in the Ung- strains. However, the 'race' described may help account for this observation. If one assumes that in the Ung- strains mutations result primarily during DNA replication (as suggested by the linear dose-response profile), then these mutations would arise subsequent to the repair of O6-alkylG residues by methyltransferase. It is therefore possible that many of the mutations are in fact the result of incorporation of adenine opposite uracil residues rather than thymine opposite O6-alkyl guanines. On the other hand, the Ung ÷ strains would likely initiate base excision repair before DNA replication. Consequently, mutations may in fact be fixed prior to replication. In addition, base excision repair may reduce the capacity of alkyltransferase to repair O6-alkylG residues, since these lesions would now be located in single-stranded DNA (Lindahl et al., 1982). Thus, the probability that particular O6-alkylG residues might give rise to a mutation would be enhanced in the Ung ÷ strains, simply because there are now several pathways for error. In summary, we have shown an influence of the ung gene product on the dose-dependence of EMSinduced mutagenesis. Furthermore, the presence of the u m u C gene product appears to become significant at higher concentrations of the mutagen. We suggest that our data is consistent with the idea that uracil residues accumulate opposite O6-EtG alkylation products by a process termed 'cross-strand deamination' (Sowers et al., 1987) and that these uracils provide the means for alternative processing schemes that enhance the overall mutagenic potential of O6-ethyl guanine residues.

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Acknowledgements The authors wish to thank Alia Ahmed for technical assistance and Drs. A. Gordon and D. Sedwick for the benefit of their discussions. This research was supported by the Natural Science and Engineering Research Council of Canada (NSERC) for financial support. D. Koehler was the recipient of an NSERC undergraduate research fellowship.

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Todd, P.A., and B.W. Glickman (1982) Mutational specificity of UV light in Escherichia coli: Indications for a role of DNA secondary structure, Proc. Natl. Acad. Sci. (U.S.A.), 79, 4123-4127. Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev., 48, 60-93. Williams, L.D., and B.R. Shaw (1987) Protonated base pairs explain the ambiguous pairing properties of O6-methylguanine, Proc. Natl. Acad. Sci. (U.S.A.), 84, 1779-1783. Communicated by F.H. Sobels