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J Biol Chem, Vol. 274, Issue 43, 31034-31038, October 22, 1999

The Role of the Escherichia coli Mug Protein in the Removal of Uracil and 3,N⁴-Ethenocytosine from DNA^*

Eugene Lutsenko and Ashok S. Bhagwat

From the Department of Chemistry, Wayne State University, Detroit, Michigan 48202

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The human thymine-DNA glycosylase has a sequence homolog in Escherichia coli that is described to excise uracils from U·G mismatches (Gallinari, P., and Jiricny, J. (1996) Nature 383, 735-738) and is named mismatched uracil glycosylase (Mug). It has also been described to remove 3,N⁴-ethenocytosine (C) from C·G mismatches (Saparbaev, M., and Laval, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8508-8513). We used a mug mutant to clarify the role of this protein in DNA repair and mutation avoidance. We find that inactivation of mug has no effect on C to T or 5-methylcytosine to T mutations in E. coli and that this contrasts with the effect of ung defect on C to T mutations and of vsr defect on 5-methylcytosine to T mutations. Even under conditions where it is overproduced in cells, Mug has little effect on the frequency of C to T mutations. Because uracil-DNA glycosylase (Ung) and Vsr are known to repair U·G and T·G mismatches, respectively, we conclude that Mug does not repair U·G or T·G mismatches in vivo. A defect in mug also has little effect on forward mutations, suggesting that Mug does not play a role in avoiding mutations due to endogenous damage to DNA in growing E. coli. Cell-free extracts from mug⁺ ung cells show very little ability to remove uracil from DNA, but can excise C. The latter activity is missing in extracts from mug cells, suggesting that Mug may be the only enzyme in E. coli that can remove this mutagenic adduct. Thus, the principal role of Mug in E. coli may be to help repair damage to DNA caused by exogenous chemical agents such as chloroacetaldehyde.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytosine is the most unstable of the four bases in DNA and deaminates hydrolytically to create U·G mismatches. If unrepaired, uracil can pair with an adenine during replication causing a C to T mutation. For this reason, cells contain uracil-DNA glycosylase (Ung), an enzyme that removes the uracil and initiates its replacement with cytosine. The importance of Ung in mutation avoidance is evidenced by the observation that ung strains of Escherichia coli (1) and yeast (2) accumulate C to T mutations.

Cytosines methylated at position 5 similarly deaminate to create T·G mismatches, which are not subject to repair by Ung. In E. coli, a specialized mismatch correction process called very short patch repair corrects these mispairs to C·G (3). The key enzyme in this repair pathway is a sequence-specific, mismatch-specific endonuclease, Vsr, which hydrolyzes the phosphodiester linkage preceding the mismatched T (4). No eukaryotic sequence homologs of this enzyme have been reported; instead a DNA glycosylase is thought to serve the same function (5). This enzyme excises thymines from T·G mismatches (6) and prefers mismatches that are followed by a G·C pairs (7, 8). This enzyme, thymine-DNA glycosylase (TDG),¹ could prevent mutations when 5-methylcytosines within CG dinucleotides deaminate to thymine.

The cDNA for TDG was cloned and its sequence was determined (9). Remarkably, a sequence homolog of this protein was found in E. coli and Serratia marcescence (10). The investigators who made this observation suggested that the bacterial homolog was a uracil-DNA glycosylase specific for U·G mismatches and named it mismatch-specific uracil-DNA glycosylase (Mug). Their conclusions were based on properties of truncated forms of TDG, and biochemical assays done using E. coli cell-free extracts. They further suggested that it may act as a backup enzyme for Ung and may be important in avoiding mutations during stationary phase of cell growth (10).

Because there was no evidence in previous work by any research group that such a backup enzyme existed, we tested this possibility. For this purpose, we studied the repair of U·G and T·G mismatches in mug⁺ and mug strains. Our results clearly indicate that Mug plays no role in the repair of U·G or T·G mismatches and may repair 3,N⁴-ethenocytosine·G mismatches as suggested recently by Sapabaev and Laval (11).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains and Plasmids-- JM253.140 (F^- araD139 (argF-lac)U169 rpsL150 relA1 deoC1 rbsR22 flhD5301 fruA25 mug::mini-Tn10) was kindly provided by J. Reiss (Princeton University). GM31 (dcm-6 thr-1 hisG4 leuB6 rpsL ara-14 supE44 lacY1 tonA31 tsx-78 galK2 galE2 xyl-5 thi-1 mtl-1) and RP4182 {(flaD-flaP)DE4 trp gal rpsL} are from our collection. BH156 is GM31 with ung-1 tyrA::Tn10 and was obtained from M. Lieb (University of Southern California School of Medicine, Los Angeles, CA). BH161 (BH156 with  DE3 lysogen) was constructed by A. Beletskii (Wayne State University) using a kit from Novagen (Madison, WI). Construction of BH157 and BH158 is described below. P1vir phage is from our collection. Plasmid pET11-d was purchased from Stratagene (La Jolla, CA).

Construction of BH157 and BH158-- BH157 and BH158 were constructed by the P1 transduction of mug::mini-Tn10 from JM253.140 into GM31 and BH156, respectively. LB medium supplemented with 0.2% glucose and 5 mM CaCl₂ was inoculated with an overnight culture of JMR253.140 and was shaken vigorously at 37 °C for 30 min. P1vir phage was added to the culture at a multiplicity of infection of 0.1, and the infected cells were further incubated 37 °C for 3 h with continued shaking. The cells were lysed with chloroform, the culture was centrifuged to clear the cell debris, and the supernatant was removed. The phage in the supernatant was titered and was used to infect GM31 or BH156. For the infection, 10 ml of overnight cultures were centrifuged, and the cell pellets were resuspended in 1 ml of a buffer containing 5 mM CaCl₂ and 10 mM MgSO₄. One hundred µl of the suspensions were infected with 10, 50, or 100 µl of the P1 phage, and the cultures were incubated at 37 °C for 30 min without shaking. One hundred µl of 1 M sodium citrate and 1 ml of LB were added to each tube, and the cells were incubated for 1 h at 37 °C with shaking. Following concentration by centrifugation, the cells were spread on LB plates containing 12 µg/ml tetracycline. The plates were incubated overnight at 37 °C, and three colonies from each transduction were studied further. The DNA from these colonies was amplified by PCR using the following primers: primer 1, 5'-GATCACCTATCTGCTGGAACAGTACGATCGTG-3'; and primer 2, 5'-CTGTATGTCTGCGATGAATCCGGAATG-3'. The colonies that gave rise to a larger PCR product than mug⁺ cells were chosen for further analysis.

Confirming the Disruption of mug-- The successful transfer of mug::mini-Tn10 allele was confirmed by Southern hybridization. The region flanking the mug gene in BH156 chromosomal DNA was amplified by PCR using the following primers 1 and 2 mentioned above. The 2.6-kilobase pair PCR product was excised from a low melting point agarose gel and labeled with nonradioactive digoxigenin using the digoxigenin High Prime DNA labeling and detection starter kit II (Roche Molecular Biochemicals). Chromosomal DNAs from BH156, BH157, and BH158 were digested with PvuII, and the fragments were separated in 0.7% agarose gel. The DNA was blotted onto a nylon membrane and hybridized with probe labeled with digoxigenin. After treatment with alkaline phosphotase-conjugated antibodies raised against digoxigenin, the membrane was incubated with solution containing the chemiluminescence substrate for the enzyme CSPD and exposed to x-ray film for 1 h. The autoradiograph showed that the mug⁺ strain BH156 contains a 3.0-kilobase pair PvuII fragment containing mug, whereas the corresponding fragment in BH157 and BH158 is 6 kilobase pairs. This confirms the disruption of mug in BH157 and BH158.

Construction of an Overproducer of Mug-- The open reading frame of mug⁺ gene was amplified from chromosomal DNA of RP4182 using the following primers: primer 3, 5'-CCCGCTCTATCGCGGATCAGGCGCGCA-3'; and primer 4, 5'-CCCCCCCATGGTTGAGGATATTTTGGCTCCAGGG-3'. The amplification was done with the Pfu DNA polymerase, and the ~500-base pair PCR product was isolated from a low melting agarose gel. The DNA was digested with NcoI and MboI (compatible with BamHI), and ligated to pET11-d expression vector digested with NcoI and BamHI. Two clones with the expected inserts were picked for further analysis. The plasmids were transformed into BL21(DE3), and one transformant from each plasmid was grown in LB medium containing 1 mM IPTG. The cells were broken by sonication, and the cell debris was removed by centrifugation. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue. Cell-free extract from one of the two clones showed the presence of a new protein of the expected size. This plasmid clone, pF168, was used in further studies. To confirm that pF168 bears a wild-type copy of the mug gene, both strands of the insert were sequenced using the following primers: primer 5, 5'-CTAGTT ATTGCTCAGCGGTGGCAGC-3'; and primer 6, 5'-TATAGGGGAATTGTGAGCGGATAAC-3'. The sequence of the cloned mug⁺ was compared with the sequence in the GenBank^TM data base (GenBank^TM accession number U28379), and the two sequences were identical.

Genetic Reversion Assays-- The genetic reversion assays were performed as described previously (12), except in the case where the cells carried the plasmid pF168. In this case, E. coli strain (BH161), carrying the overproducer pF168, was electroporated with pAKS2. The latter plasmid contains the kan allele cloned in pACYC184 (13). Following plating, three independent colonies were picked and grown in 10 ml of LB containing 50 µg/ml carbenicillin and 20 µg/ml chloramphenicol at 37 °C until the A₅₅₀ reached 0.3. One hundred µl of each culture was used to inoculate 10 ml of prewarmed LB containing appropriate antibiotics and 30 µM IPTG. The cells were again grown till A₅₅₀ reached 0.3. The cells were centrifuged at 3000 × g for 10 min, and the cell pellet was resuspended in 1 ml of LB. Appropriate dilutions of these cultures were spread on LB plates to determine the number of viable cells, and the remaining culture was spread on kanamycin plates to determine the number of revertants.

The principal source of variation in mutation frequency data is the existence of mutational "jackpot" (14). We eliminated such data points from our data sets using the following procedure: the data point suspected of being from a jackpot was set aside and the mean and S.D. of the remaining data points were calculated. If the suspected data point was greater than 3 times the S.D. away from the mean, it was declared to be a jackpot and eliminated from the data set. If the data point was within 3 S.D. of the mean, it was included in the set, and the mean and S.D. of the complete data set were used in further analysis.

Forward Mutation Assays-- Appropriate E. coli strains were grown from single colonies in 5 ml of LB for 24 h at 37 °C with shaking. To determine the frequency of 5-fluorocytosine-resistant cells, the cultures were centrifuged to pellet the cells, and the pellets were washed twice in 5 ml of M63 minimal medium. The cells were ultimately resuspended in 1 ml of M63 medium and were spread on LB plates to determine the total number of viable cells. They were also spread on M63 minimal plates supplemented with 0.1% of casamino acids, 10 µg/ml 5-fluorocytosine, and 20 µg/ml each leucine, threonine, and histidine to determine the number of 5-fluorocytosine-resistant cells. The plates were incubated for 24 h at 37 °C. The mutant frequency is the number of 5-fluorocytosine resistant cells divided by the total number of viable cells.

To determine frequency of rifampicin-resistant cells, the overnight cultures were centrifuged to pellet the cells, and the pellets were resuspended in 1 ml of LB. The cells were spread on LB plates or LB plates containing 100 µg/ml rifampicin. The mutant frequency is the number of rifampicin-resistant cells divided by the total number of viable cells.

Biochemical Assays for Mug-- Five ml of overnight cultures of the appropriate E. coli strain were used to inoculate 250 ml of LB and grown at 37 °C to an A₅₅₀ of 1.0. The cells were harvested by centrifugation, and the pellet was washed with 50 ml of a buffer containing 25 mM HEPES, pH 7.6, and 1 mM ETDA. The cells were again harvested by centrifugation, and the pellet was resuspended in 20 ml of the lysis buffer (25 mM HEPES, pH 7.6, 0.5 mM ETDA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). Cells were kept on ice for 30 min and sonicated five times with 30-s pulses. Between pulses, they were chilled on ice for 30 s. The cell debris was removed by centrifugation for 30 min at 12,000 rpm 4 °C, and the supernatant was divided in aliquots. The aliquots were stored at 70 °C.

The DNA oligonucleotides containing uracil (oligo vsr-U) or 3,N⁴-ethenocytosine (C1) were labeled with ³²P at the 5' end and hybridized to the unlabeled oligomer, oligo dcm-94b, at a molar ratio 1:10 to form the duplexes with a single U:G or etheno-C:G mismatch. The duplexes were subjected to treatments with different dilutions of cell-free extracts for 30 min at 37 °C in a treatment buffer (20 mM Tris-HCl, 10 mM EDTA) and then were treated with 0.1 M NaOH or FA-PY glycosylase to cleave at the AP sites for another 30 min at 37 °C. The FA-PY glycosylase protein was purified in collaboration with B. Taffe (Wayne State University). The products were separated in a 20% sequencing gel and identified by autoradiography and by scanning with a phosphorimager.

The sequences of the oligos used in these experiments were as follows: vsr-U, 5'-GACTGGCTGCTACUAGGCGAAGTGCC-3'; C1, 5'-GACTGGCTGCTAC(C)AGGCGAAGTGCC-3'; and dcm-94b, 5'-GGCACTTCGCCTGGTAGCAGCCAGTC.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Uracil Excision by Mug-- Duplex DNA containing a U·G mismatch was treated with cell-free extract prepared from ung mug⁺ cells. The DNA was further treated with NaOH to convert the abasic sites created by the extract to strand breaks, and the products were separated on a denaturing gel. The reactions were carried out in pairs, one with the Ung inhibitor UGI (15) and the other without the inhibitor. The use of UGI in one reaction assures that any uracil excision activity seen in that reaction must be due to an enzyme other than Ung.

The cell extract contained a weak uracil glycosylase activity (Fig. 1A, lane 4) that was resistant to UGI (lane 5). This activity was reproducible and is probably the same activity reported by Gallinari and Jiricny (10). Compared with this weak activity, the uracil excision activity in extracts prepared from ung⁺ mug⁺ cells was much easier to detect. Whereas 8 µg of cell-free extract from ung cells had barely detectable activity, 38 ng of extract from ung⁺ cells cleaved all the uracil in DNA (Fig. 1B, lane 7). The latter activity was completely inhibited by UGI confirming that it was due to Ung. Based on these and other results we conclude that uracil excision activity in E. coli due to Ung is at least 400 times greater than uracil excision due to any other enzyme including Mug.

View larger version (54K): [in this window] [in a new window]   Fig. 1.   Excision of uracil with cell-free extracts. The ability of extracts from ung+ and ung cells to excise uracils from U·G mismatches was tested. The substrate was a duplex containing the oligos vsr-U and dcm-94b, and the former oligomer was labeled at the 5' end. The reaction in which purified Ung was used without UGI served as a positive control, and the position of the resulting truncated oligomer is marked with an arrowhead. Following the uracil excision reaction, DNA was treated with NaOH to cut at the AP site and the products were separated on a denaturing gel. The total amounts of protein in the extracts and the presence or absence of UGI in the reaction is noted above each lane.

Role of Mug in the Repair of U·G or T·G Mismatches-- We used a genetic reversion system involving a defective kanamycin resistance gene (13) to assess the role of Mug in avoiding spontaneous C to T mutations. The reversion to kanamycin resistance results exclusively from C to T mutations at a site for cytosine methylation and either a 5-methylcytosine to T change or C to U to T change can be studied with this system in appropriate genetic backgrounds. T·G and U·G mismatches are, respectively, the intermediates in these mutagenic pathways, and hence any excision of T or U by Mug should reduce C to T mutations.

We compared the antimutagenic effects of Mug to those of Ung and Vsr using this assay. The presence of Mug did not affect mutations by either pathway (Table I). Whereas the presence of Ung reduced the reversion frequency by a factor of ~10, Mug did not significantly affect the frequency. We have previously shown that in a mug⁺ strain, Vsr reduces the frequency of 5-methylcytosine to T mutations by a factor of about 4 (16). In contrast, Mug reduced these mutations only slightly, and this reduction was not statistically significant (Table I).

In the experiments discussed above, cells were dividing at the time of their selection for kanamycin resistance. To assess the role of Mug in stationary phase of cell growth, cells were shaken at 37 °C for ~24 h and then plated to select for kanamycin-resistant revertants. Growing the cells to stationary phase increased the reversion frequency by a factor of ~2 compared with growing cells, but there was no significant effect of Mug on the mutant frequencies (Table I). In contrast, the Ung defect again increased the mutant frequency by a factor of approximately 10. Based on these results, we conclude that Mug does not play a significant role in the repair of U·G or T·G mismatches in E. coli.

Effects of Overexpression of Mug on U·G Repair-- It seemed possible that the inability of mug⁺ ung cells to repair U·G mismatches was due to inadequate expression of Mug in the cells. To see whether overexpression of Mug in the cells can reduce C to T mutations, the mug⁺ gene was cloned in a multicopy plasmid and expressed from a bacteriophage T7 promoter. A plasmid carrying this construct was introduced into mug⁺ ung cells along with the tester plasmid containing the kan gene. The expression of Mug was optimized by varying the concentration of IPTG used to induce the promoter, and the level of Mug in the cells was monitored by gel electrophoresis. The amount of free Mug in the cells increased with IPTG concentration, reaching a maximum between 20 and 100 µM of the inducer (Fig. 2). The level of soluble Mug did not increase at higher concentrations of the inducer, probably because Mug tends to aggregate at high concentrations.² The cell viability is also low at concentrations above 40 µM. Consequently, the assays were done at 30 µM IPTG.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Induction of Mug from a T7 promoter. SDS-polyacrylamide gel electrophoresis gel of extracts prepared from BH161 cells containing a plasmid are shown. The amount of IPTG used to induce the T7 promoter is shown above each lane. Lanes 1 and 2, pET11-d; lanes 3-8, pF168. The position of the induced protein is marked with an arrowhead.

Cell extracts containing high levels of Mug are able to excise uracils from DNA (Fig. 3, lane 4). In this case, nearly all the uracil was excised from U·G mismatches regardless of the presence of UGI in the reaction (Fig. 3, lane 5). Purified Mug is also able to excise uracil from U·G mismatches (not shown).

View larger version (55K): [in this window] [in a new window]   Fig. 3.   Uracil removal activity in a Mug overproducer. The excision of uracil by purified Ung and by cell-free extract from BH161 with pF168 are shown. The position of the resulting product is marked with an arrowhead.

Surprisingly, induction of mug⁺ had little effect on the frequency of C to T mutations. In one data set there was a slight decrease in mutant frequency as a result of Mug overproduction, but this effect was not reproducible (Table II). The mutant frequencies reported in this table are lower than that in Table I, because in this case the kan gene was on a low copy number plasmid. Induction of the T7 promoter with lower concentrations of IPTG or with 40 µM IPTG also did not affect the mutant frequency (not shown), suggesting that even at high concentrations, Mug cannot effectively substitute Ung to repair U·G mismatches in DNA.

Effect of Mug on Forward Mutations-- We wondered whether Mug could repair endogenous DNA damage other than U·G and T·G mismatches. If this were true, a mug mutation would have a mutator phenotype. We tested this possibility by comparing the frequencies of rifampicin-resistant and 5-fluorocytosine-resistant mutants in mug⁺ and mug strains.

The results were largely negative (Table III). The frequency of rifampicin-resistant mutants was slightly higher in a mug strain, but the the frequency of 5-fluorocytosine-resistant mutants was the same in the two genetic backgrounds. It is possible that Mug does prevent a small number of mutations and that this is not evident in the 5-fluorocytosine-resistant mutation assay because the background frequency of mutations is high in this assay (Table III). In any case, mug is at best a very weak mutator and hence is unlikely to be important for correcting endogenous damage to DNA in E. coli.

3,N⁴-Ethenocytosine Excision by Mug-- A wide range of chemicals react with bases in nucleic acids to form ethenobases. These chemicals include vinyl compounds, haloaldehydes, -haloketones, haloalkanes, halothioketenes, halo- ketenes, and products of lipid peroxidation (17, 18). After the work presented above was completed, Saparbaev and Laval purified a protein from E. coli that removes 3,N⁴-ethenocytosine (C) from DNA and showed that it was Mug (11). Purified Mug excised C from duplex DNA with C·G pairs, but not from single-stranded DNA. It also excised uracils, but its catalytic efficiency for the removal of C excision was 50 times higher than for the removal of uracil from U·G mismatches (11).

We have confirmed the ability of Mug to excise C from DNA. Cell-free extracts from cells containing overproduction of Mug and purified Mug excised C from DNA (not shown). We also wanted to find out whether C removal activities other than Mug existed in E. coli. For this purpose, we used the mug strain described above.

When duplex DNA containing a C·G pair was treated with various cell-free extracts in a manner similar to that described for U·G mispairs, removal of C was readily detected in mug⁺ extracts (Fig. 4). This treatment converted the labeled substrate to shorter products of expected length or products that were ~1 nucleotide longer or shorter (Fig. 4, lane 2). Although the shorter product is likely to have resulted from the action of an AP endonuclease in the extract to the 5' side of the abasic site created by Mug, the source of the longer product is not known. Regardless, these results confirm the existence of C removal activity in E. coli (11). Furthermore, extract prepared from mug cells did not possess the ability to create abasic sites at the C (Fig. 4, lane 3). Thus mug appears to code for the principal C excision activity in E. coli.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Ethenocytosine removal activity in cell-free extracts. The ability of extracts from mug+ and mug cells to excise ethenocytosine from C·G pairs was tested. The substrate was a duplex containing the oligos C1 and dcm-94b, and the former oligomer was labeled at the 5' end. The reaction conditions were similar to those described in legend to Fig. 1, except that the FA-PY glycosylase protein was used to cleave the abasic site. The expected position of the final reaction product was identified by excising uracil from U·G pairs using Ung and electrophoresing the reaction products in an adjacent lane (not shown). The expected position of the product is marked with an arrowhead.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have shown here that although concentrated cell extracts containing Mug can be used to show a small amount of excision of uracils from DNA, this enzyme does not act as a U·G correction enzyme in growing or stationary E. coli. Furthermore, Mug does not appear to play a significant role in repairing any other DNA damage that occurs spontaneously in the cells. Mug is clearly more efficient at excising C from C·G pairs, and this may be the only activity of the kind in E. coli.

It is surprising that despite its ability to excise uracils from U·G mispairs, overproduction of Mug from a strong T7 promoter does not result in the reduction of C to T mutations (Table II). A possible reason for this apparent inactivity of Mug in vivo is that Mug may aggregate to form inclusion bodies. Consistent with this hypothesis we have found that purified Mug rapidly aggregates to form stable high molecular weight complexes. As a result, very little Mug may be available to repair the mismatches despite overproduction.

An alternate possibility is that C to U deaminations mostly occur in single-stranded regions of the genome and that Mug is unable to excise these uracils because of its strict requirement for U·G mismatches. In contrast, Ung acts preferentially on uracils in single-stranded DNA (19, 20) and should efficiently repair such uracils. If so, Mug is poorly suited to be a backup enzyme for Ung.

At this time, the biological role of Mug in E. coli remains a matter of speculation. If the role is in the removal of C from DNAand this is very likelythen the lack of a strong mutator phenotype for mug is not surprising. Defects in genes that code for enzymes that repair alkylated bases also do not have a mutator phenotype. This has been interpreted to mean that there is little alkylation damage to DNA bases in exponentially growing E. coli. By analogy, it is likely that there is very little endogenous C, or any other damaged base that may be removed by Mug, in E. coli.

It is important to note that some of the enzymes involved in the repair of DNA damage caused by exogeneous agents are induced by the damaging treatment. These include the the nucleotide excision proteins and the 3-methyl adenine glycosylase II, AlkA. It would be interesting to know whether mug⁺ is similarly inducible in response to damage to cellular DNA.

	ACKNOWLEDGEMENTS

We are grateful to J. Reiss (Princeton University) for providing a bacterial strain and to A. Beletskii (Wayne State University) for constructing a phage lambda lysogen.

	FOOTNOTES

^* The work presented here was supported by National Institutes of Health Grant GM53273.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: 463 Chemistry, Dept. of Chemistry, Wayne State University, Detroit, MI 48202. Tel.: 313-577-2547; Fax: 313-577-8822; E-mail: axb@chem.wayne.edu.

² E. Lutsenko and A. S. Bhagwat, unpublished results.

	ABBREVIATIONS

The abbreviations used are: TDG, thymine-DNA glycosylase; C, 3,N⁴-ethenocytosine; Ung, uracil-DNA glycosylase; Mug, mismatched uracil glycosylase; IPTG, isopropyl--D-thiogalactoside; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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