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Escherichia coli MutY protein has aguanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C->C:G transversions
Introduction
Materials And Methods
Bacterial strains
Enzymes and chemicals
Selection of mutator strains
Spontaneous mutation assays
Genetic mapping
Sub-cloning and sequencing of the mut39::miniTn10-tet gene
Preparation of GST fusion protein
Gel mobility shift assay
8-oxoG-containing DNA cleavage assay
Results
Isolation of mutator strains
Genetic mapping of the mutator locus in GC39 strain
Sub-cloning and sequencing of the mut39::miniTn10-tet gene
Spontaneous mutations in mutY and mut39 strains of E.coli
Construction of GST-MutY fusion plasmid and purification of MutY protein
Gel mobility shift assay
Cleavage activity of MutY protein on 8-oxo-G:G mispair-containing duplex DNA
Discussion
Acknowledgements
References
Escherichia coli MutY protein has aguanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C->C:G transversions
ABSTRACT
INTRODUCTION
Errors during DNA replication and spontaneous DNA damage are potential sources of spontaneous mutations (1-3). However, the critical cause of each spontaneous base substitution is not completely understood. In particular, the pathways leading to G:C->C:G transversions and their biological importance remain uncertain (3). Spontaneous G:C->C:G transversions are rare events. However, the frequency of the transversions significantly increases upon exposure to ionizing radiation and oxidizing chemicals (4-7). Active oxygen species can be produced as an inescapable by-product by the incomplete reduction of oxygen during aerobic metabolism, and their production is further enhanced by exposure to various oxidative stress conditions (8-10). Active oxygen species are potential causes for spontaneous DNA damage inducing G:C->C:G transversions (11-14). In fact, G:C->C:G transversions are predominant in spontaneous mutations in an Escherichia coli sodAsodB mutant (our unpublished results), which is fully defective in superoxide dismutase activities (15). Base mismatches during DNA replication are also potential sources of spontaneous mutations (3,16-19). The G:G mispair, for instance, could assume a relatively stable conformation in duplex DNA (20,21). If unrepaired, these mismatches and oxidative DNA damage could have mutagenic consequences. Therefore, low rates of spontaneous G:C->C:G transversions (3) would be achieved by the prevention and repair of oxidative base damage and by the correction of base mismatches.
Mutants with higher than normal rates of spontaneous mutations, termed mutators, have facilitated the understanding of mutational pathways. Mismatch repair directed by dam methylation and requiring the products of the mutH, mutL and mutS genes predominantly corrects transition mutations in E.coli (3,16-18,22,23). Several mutators, mutT, mutM, mutY, mutA and mutC, show elevated frequencies of specific transversions (24-28). However, these mutator strains do not have significantly enhanced levels of G:C->C:G transversions (3). This implies that additional repair systems correct mispairing that leads to the G:C->C:G transversions. Hence, we attempted to isolate and characterize additional mutators in order to clarify the pathways of G:C->C:G transversions.
We used a sensitive screening method to detect mutators that revert to defined mutations in lacZ by a limited number of base substitutions (3,24,25). Strains CC101-CC106 all carry a specific lacZ mutation affecting residue 461 in lacZ on an F[prime]lacproAB episome (29). In the CC103 strain, only a G:C->C:G change can restore the wild-type codon at position 461 in the lacZ gene (29). We mutagenized the CC103 strain by random miniTn10-tet insertion mutagenesis (30). The strain GC39 was selected by higher rates of papillae formation and reversion to Lac+. The sequence of the miniTn10-flanking region indicated that the mutator locus was identical to that of the mutY gene. Purified MutY protein bound to 7,8-dihydro-8-oxo-guanine (8-oxoG):G as well as to 8-oxoG:A mispairs. In addition, we found that the MutY protein had a DNA glycosylase activity to remove unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C->C:G transversions by removing the unmodified guanine from the 8-oxoG:G mispair in E.coli.
MATERIALS AND METHODS
Bacterial strains
Escherichia coli strains CC103 and CC104 are derivatives of P90C [ara[Delta](lacproB)XIII] with an F[prime]lacproAB episome (29). Each strain carries a different lacZ- mutation affecting residue 461 in [beta]-galactosidase (3,29). A series of isogenic mapping strains containing Tn10kan, Tn5 or Tn9 at specific sites in the genome (31) were used to map mutator loci on the E.coli chromosome. The transduction experiments with P1vir phage were carried out according to Miller (32) with a slight modification.
Enzymes and chemicals
Phenyl-[beta]-d-galactoside (P-Gal) was obtained from Sigma Chemicals. Tetracycline hydrochloride, kanamycin and chloramphenicol were purchased from Wako Pure Chemicals. Ampicillin and rifampicin were the products of Meiji Seika and Boehringer-Mannheim, respectively. 5-Bromo-4-chloro-3-indolyl-d-galactoside (X-Gal), restriction enzymes and the BcaBest sequencing kit were obtained from Takara Shuzo. Taq DNA polymerase and isopropyl-1-thiol-[beta]-d-galactopyranoside (IPTG) were purchased from Toyobo. Oligonucleotide containing a single 8-oxoG residue was obtained from Trevigen, Inc. Plasmid pGEX-4T-3, glutathione-Sepharose 4B and thrombin protease were obtained from Pharmacia Biotech. [[alpha]-32P]dCTP (>111 TBq/mmol) and [[gamma]-32P]ATP (>259 TBq/mmol) were the products of ICN Biomedicals Inc.
Selection of mutator strains
Random Tn10 insertion mutagenesis was performed in E.coli CC103 by infection with a derivative of [lambda] phage, [lambda]1098, which carries a Tn10-derived transposon (miniTn10-tet) that can transpose the Tn10 from the phage into the E.coli chromosome (3,30). The cells of E.coli CC103 were grown in [lambda]ym broth (30) and infected with [lambda]1098 at the multiplicity of infection of ~5. The infected cells were plated on [lambda]ym agar plates containing 15 µg/ml tetracycline and incubated at 39.5°C for ~20 h. To select G:C->C:G mutator strains, ~40 000 colonies were replica-plated to glucose minimal plates (3) containing 15 µg/ml tetracycline, 40 µg/ml X-Gal and 500 µg/ml P-Gal. Plates were incubated at 37°C for ~10 days. We selected several colonies that had a rate of papillae formation significantly higher than normal. P1 lysates were prepared on these mutants and the transposon was again transduced into E.coli CC103 to ensure that only one transposon was present in each mutant derivative.
Spontaneous mutation assays
A single colony was inoculated into glucose minimal medium and cultured at 37°C to the stationary phase. The reversion to Lac+ was assayed as follows: 0.1 ml of cell suspensions were plated on minimal agar plates containing 1% lactose (lactose minimal plates) and then incubated at 37°C for ~10 days. The normal plating density was ~2 × 108 cells per plate. The mutation to rifampicin resistance was assayed as follows: 0.1 ml of cell suspensions (~2 × 108 cells) were plated on LB plates (3) containing 100 µg/ml rifampicin and then incubated for ~24 h. The number of mutant colonies on the plates was counted to estimate the mutation frequency.
Genetic mapping
The gene responsible for the mutator phenotypes was mapped on the E.coli chromosome using the P1 mapping kit, a series of isogenic mapping strains containing Tn10kan, Tn5 or Tn9 at specific sites in the genome (31). P1 transduction was performed according to Miller (32) with a slight modification. The concentrations of tetracycline, kanamycin and chloramphenicol, when added, were 15, 50 and 30 µg/ml, respectively. Kanamycin- or chloramphenicol-resistant colonies were tested for growth on tetracycline plates at 37°C. Individual KanrTets or CmrTets colonies were then tested for growth on Lac indicator plates (glucose minimal plates containing P-Gal and X-Gal) that monitor papillation.
Sub-cloning and sequencing of the mut39::miniTn10-tet gene
The chromosomal DNA of E.coli GC39 carrying themut39::miniTn10-tet was digested by BamHI and ligated into the BamHI site of pUC118. The resulting plasmids were introduced to E.coli JM109 and then plated on LB plates containing 50 µg/ml ampicillin and 15 µg/ml tetracycline. The nucleotide sequence of the miniTn10-mut39 junction was determined by the dideoxynucleotide chain termination method (33). Sequencing reactions were carried out with the BcaBestTM sequencing kit andthe primer 5[prime]-CCAAAATCATTAGGGGATTCATCAG-3[prime] (theminiTn10-tet end) (34). The sequencing products were resolved on denaturing 6% polyacrylamide gels. After electrophoresis, the gels were dried and then autoradiographed using Fuji RX films at -80°C.
Preparation of GST fusion protein
A plasmid expressing the glutathione-S-transferase (GST)-MutY fusion protein was constructed as follows: the plasmid pLC20-5 bearing E.coli mutY gene (35) was amplified with two PCR primers; one containing an EcoRI site followed by the sequence around the putative start codon (5[prime]-AACAACAGTGAATTCGGTGACCAT-3[prime]) and the other containing a SalI site followed by the sequence around the stop codon (5[prime]-ATATAGTCGACGTTGCAGGAAAGTA-3[prime]). A standard PCR was performed with 100 µl standard reaction mixture containing 20 mM Tris-HCl (pH 8.4), 1.5 mM MgC12, 200 µM each dNTP, 0.5 mM of primer and 5 U of Taq DNA polymerase. The amplified 1147 bp fragment was digested with EcoRI and SalI, and then the EcoRI/SalI fragment containing the whole coding region of the mutY gene was inserted into EcoRI/SalI-digested pGEX-4T-3. The resulting plasmid was named pGEX-MutY. The pGEX-MutY could reduce the frequency of G:C->T:A transversions in the mutMmutY mutant.
Escherichia coli BL21 was transformed with the plasmid pGEX-MutY. The cells were grown at 22-24°C in 1 l of LB medium containing 100 µg/ml ampicillin until the absorbance at 600 nm reached ~0.8. Expression of the GST-MutM fusion protein was induced by the addition of 0.1 mM IPTG, and growth was continued at 22-24°C for an additional 8 h. Cells were then harvested by centrifugation and resuspended in phosphate-buffered saline (PBS) containing 1% Triton X-100. A cell extract was prepared by sonication of the cell suspensions, followed by centrifugation at 15 000 g for 30 min at 4°C. The supernatant was applied to a glutathione-Sepharose 4B column. The column was washed with 10 column vol of PBS, and the bound protein was eluted with 15 mM glutathione in 50 mM Tris-HCl (pH 8.0). The eluted samples were concentrated by ultrafiltration, and the buffer was replaced by PBS. The GST-MutY fusion protein was cleaved by thrombin protease by incubation for 12-14 h at 4°C. Proteins were assayed by Coomassie blue staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Gel mobility shift assay
The oligonucleotide containing a single 8-oxoG residue was the product of Trevigen, Inc. Each of the 24mer oligonucleotides was labeled at the 5[prime]-end with [[gamma]-32P]ATP by T4 polynucleotide kinase and then annealed with its complementary oligonucleotides as previously described (36). The binding reactions were carried out in 25 mM Tris-HCl (pH 8.0), 0.5 mM dithiothreitol, 0.5 mM EDTA, 10% glycerol, 25 mM NaCl, 25 mM KCl, 10 mM ZnCl2, 0.125 mM each dNTP, 4 mM spermidine, 50 µg/ml calf thymus DNA and various amounts of MutY protein in a final volume of 5 µl. After an incubation for 30 min at 4°C, the reaction mixtures were electrophoresed on 12% non-denaturing polyacrylamide gels in TBE buffer (25 mM Tris, pH 8.0, 24 mM boric acid and 0.5 mM EDTA) at 100 V at room temperature. After electrophoresis, the gels were dried and then autoradiographed using Fuji RX films at -80°C.
8-oxoG-containing DNA cleavage assay
Duplex oligonucleotides containing a single 8-oxoG residue at position 10 were constructed as mentioned above (Fig.
RESULTS
Isolation of mutator strains
To isolate mutator strains with a higher than normal rate of G:C->C:G transversions, the E.coli strain CC103 was mutagenized by random miniTn10-tet insertion mutagenesis. In this strain, only a G:C->C:G change can restore the wild-type codon at position 461 in the lacZ gene on F[prime]lacproAB episome (29). Mutators were selected by screening the rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. Colonies grow until they exhaust the glucose. Lac+ revertants, which can utilize the P-Gal as a carbon source, grow out as papillae and are stained blue by X-Gal (3). We screened ~40 000 colonies and found five mutator strains that generate papillae at higher rates than normal (Fig.
Figure 1. The GC39 mutator strain displayed more papillae than did the CC103 and CC103mutM strains. (A) Escherichia coli CC103, (B) CC103mutM and (C) CC103mut39 were inoculated to glucose minimal plates containing 40 µg/ml X-Gal and 500 µg/ml P-Gal. Plates were incubated at 37°C for 6 days. Of the five G:C->C:G mutator strains isolated, strain GC39 showed the highest rate of papillae formation, all colonies of which had >20 papillae on glucose minimal plates containing P-Gal and X-Gal (Table 1). Two tests were run to further assay the properties of the mutator strains. The five mutator strains were placed into three groups based on three tests for papillae formation, generation of Lac+ and rifampicin resistance (Table 1). Strain GC39 was put into Group I. The spontaneous reversion frequency to Lac+ was increased ~50-100-fold in strain GC39, compared with that of the wild-type CC103. The spontaneous mutation to rifampicin resistance was also increased ~10-20-fold in the GC39 strain over the control level. The group II mutators also stimulated the Lac+ reversion and Rifr mutation in the two strains at relatively high rates (Table 1). Therefore, we selected the GC39 strain for further studies. Table 1. 
Group
Strain
Papillation
Mutants per 108 cells
Lac+
Rifr
I
GC39
++
148
188
II
GC9
++
125
1550
GC24
++
101
1460
III
GC40
+
15
1280
GC41
+
28
2560
-
CC103
-
3
11
Genetic mapping of the mutator locus in GC39 strain
We used a kit of Singer's P1 mapping for E.coli consisting of >90 donor strains to determine the location of the mutator locus on the chromosome of E.coli (31). Sections of the E.coli chromosome were transferred to E.coli GC39 with P1 phage. Antibiotic-resistant transductants (Kanr or Cmr) were replica-plated on LB plates containing tetracycline to determine the rate of cotransduction. The cotransduction frequencies of Kanr Tets were 90, 23 and 19% for nupG3157::Tn10kan at 66.9 min, metC3158::Tn10kan at 67.9 min and zgd3156::Tn10kan at 66.0 min, respectively. The location of transposon insertion sites was recalibrated by using cotransduction frequencies for nearby genes, as done previously (37,38). KanrTets colonies were further replica-plated on glucose minimal plates containing P-Gal and X-Gal. They showed a significant reduction of papillae formation. No transductants containing DNA from other donor strains reduced the rate of papillation. These results indicated that the gene responsible for the mutator phenotypes, named mut39, was localized around 67 min on the E.coli chromosome.
Sub-cloning and sequencing of the mut39::miniTn10-tet gene
The nucleotide sequence of the junction between the mut39 gene and the miniTn10 insertion was determined using the sequence 5[prime]-CCAAAATCATTAGGGGATTCATCAG-3[prime] (nucleotides 1-25 from the miniTn10-tet end) (34) as a primer. The sequence determined was identical to the complementary strand of mutY gene (26,39). The sequences were compared with the databases of the National Center of Biotechnology Information by using the BLAST server. No other sequences could be found with significant sequence matches.
In addition, the expression of mutY+ gene markedly reduced the spontaneous frequency in the mutY and mut39 mutants of E.coli (Table 2). These results indicated that mut39 was the same gene as the mutY gene.
Table 2.
| Strain | Number of Lac+ revertants per 108 cells |
| CC103 | 1 |
| CC103mutY | 120 |
| CC104mutY/pGEX-MutY | 18 |
| CC103mutM | 2 |
| CC103mut39 | 146 |
| CC103mut39/pGEX-MutY | 13 |
| CC103mutMmut39 | 149 |
| CC104 | 6 |
| CC104mut39 | 232 |
| CC104mutY | 204 |
| CC104mutY/pGEX-MutY | 18 |
Spontaneous mutations in mutY and mut39 strains of E.coli
Strain GC39 was initially isolated by increased rates of G:C->C:G transversions in E.coli CC103 (Table 1). We examined whether the mut39 mutation can affect the rate of Lac+ reversion in the E.coli strain CC104. In this strain, the G:C->T:A change can restore the wild-type codon at position 461 in the lacZ gene (29). The CC104 cells were infected with P1 phage multiplied in E.coli GC39 and plated on lactose minimal plates, followed by an incubation at 37°C for ~2 days. The mut39 mutation resulted in an enhanced frequency of spontaneous G:C->T:A transversions (Table 2). The properties of E.coli CC104mut39::miniTn10 were comparable with those of the CC104mutY::Tn10 strain (25). In addition, the introduction of the mutY::Tn10 mutation into E.coli CC103 also resulted in increased rates of papillae formation and reversion to Lac+ (Table 2).
The MutM and MutY DNA glycosylases are both parts of the 8-oxoG repair system in E.coli, which prevents the generation of G:C->T:A mutations caused by spontaneous 8-oxoG formation in DNA (13,14,28). The mutM and mutY mutants show an increased rate of spontaneous G:C->T:A mutations compared with the wild-type strain (25,28,40). When mutM and mutY were combined, the mutator activity is more striking (25,28,40). In contrast, the mutM mutation did not increase the spontaneous G:C->C:G transversions in CC103. Furthermore, the addition of the mutM mutation to the CC103mut39 led to no significant increase in mutation rates (Table 2).
Construction of GST-MutY fusion plasmid and purification of MutY protein
The entire open reading frame of the mutY gene was amplified by PCR and sub-cloned into pGEX-4T-3 to obtain a GST-MutY fusion construct. The sequence was checked to verify that no mutations had been generated by the PCR. The construct pGEX-MutY obtained was able to reduce the high rates of spontaneous G:C->T:A transversions in CC104mutMmutY. The fusion protein was over-expressed in E.coli BL21/pGEX-MutY treated with IPTG and then purified by means of a glutathione-Sepharose 4B column chromatography. The synthesis of MutY was confirmed by SDS-PAGE. The 39 KDa MutY protein was present on the gels after cleavage of the GST-MutY fusion protein with thrombin protease.
Gel mobility shift assay
The gel mobility shift assay was done to assess the binding activity of E.coli MutY protein to a series of double-stranded oligonucleotides containing various base mispairs. The 24mer oligonucleotides, including the one containing 8-oxoG, were synthesized and 32P-labeled at the 5[prime]-terminus with T4 polynucleotide kinase in the presence of [[gamma]-32P]ATP. The sequences of the oligonucleotides are shown in Figure
Figure 2. Sequences of oligonucleotides used to compare substrate specificity. O, 8-oxoG.
Cleavage activity of MutY protein on 8-oxo-G:G mispair-containing duplex DNA
Double-stranded oligonucleotides (24mer) containing a set of four DNA mispairs (G:G, 8-oxoG:G, 8-oxoG:A and G:A) at position 10 were constructed by annealing oligonucleotide 2 with complementary oligonucleotide 4 (labeled 2/4), 3/4, 3/5 and 2/5, respectively, and used as a substrate for MutY DNA glycosylase activity assay. Each duplex oligonucleotide was incubated at 37°C with purified MutY protein. Reactions were terminated by the addition of NaOH and subsequently heated at 95°C for 5 min. This served to cleave any AP sites generated by the DNA glycosylase. The progress of the glycosylase reaction can therefore be monitored by a change in the migration of the product. The results are shown in Figure
A
 B  |
Figure 3. Binding activity of purified MutY protein to double-stranded oligonucleotides with various mispairs. (A) 32P-labeled double-stranded oligonucleotides (10 fmol) containing G:C (lanes 1-3), 8-oxoG:A (lanes 4-6), G:G (lanes 7-9), 8-oxoG:G (lanes 10-12) and G:A (lanes 13-15) were incubated with MutY protein purified from the GST-MutY in the presence of sonicated calf thymus DNA. After incubation for 30 min at 4°C, the reaction mixtures were electrophoresed on 12% non-denaturing polyacrylamide gels in TBE buffer at 100 V. After electrophoresis, the gels were dried and then autoradiographed using Fuji RX films at -80°C. F, free oligonucleotide; B1, MutY protein-oligonucleotide complex; B2, additional MutY protein-oligonucleotide complex. Lanes 1, 4, 7, 10 and 13, without MutY; lanes 2, 5, 8, 11 and 14, with 40 ng MutY; lanes 3, 6, 9, 12 and 15, with 160 ng MutY. (B) Increasing amounts of the MutY protein were incubated with double-stranded oligonucleotides. The extent of the MutY binding to the 8-oxoG:A ([solid circle]) and 8-oxoG:G ([solid square]) was quantified by densitometry.
The relative effects of MutY glycosylase on the 8-oxoG:G and 8-oxoG:A mispairs were determined. Increasing amounts of MutY protein were incubated with the 8-oxoG:G- and8-oxoG:A-containing oligonucleotides for 8 and 1 h, respectively. The results are shown in Figure
DISCUSSION
The mut39 locus responsible for the mutator phenotypes in GC39 was located around 67 min on the E.coli chromosome. The mutY gene is also mapped near 67 min (25). The sequence of the miniTn10-flanking region was identical to that of the mutY gene. The results indicated that mut39 is an allele of the mutY gene. This was supported by the finding that G:C->T:A transversions were also accumulated in E.coli CC104mut39, as in the CC104mutY strains. In addition, the introduction of mutY::Tn10 mutation into E.coli CC103 resulted in increased rates of reversion to Lac+.
Figure 4. Cleavage of double-stranded oligonucleotides containing a single 8-oxoG mispaired with C, G and A by the MutY protein. Substrate oligonucleotides (20 fmol) containing various mispairs were incubated with 420 ng of MutY protein in 10 µl of the reaction buffer containing 20 mM Tris-HCl (pH 7.6), 0.5 µg of BSA and 10 mM EDTA at 37°C for 14 h. After electrophoresis at 1800 V, the gels were dried and then autoradiographed using Fuji RX films at -80°C. Lanes 1, 3, 5, 7, 9 and 11, without MutY; lanes 2, 4, 6, 8, 10 and 12, with MutY. *, 32P-labeled strand at the 5[prime]-terminus. Figure 5. Time course of the MutY glycosylase reaction on 8-oxoG:G substrate. 32P-labeled oligonucleotide 3 was annealed with the complementary oligonucleotide 4. Substrate double-stranded oligonucleotides (20 fmol) containing 8-oxoG:G were incubated with 60 ng of MutY protein in 10 µl of the reaction buffer at 37°C for 0-10 h and analyzed as described in Figure 4. Purines undergo oxidation of the ring atoms leading to various chemical modifications. The highly mutagenic 8-oxoG is formed in large quantities (14,44). The 8-oxoG has strong mispairing properties, and both bacterial and eukaryotic DNA polymerases insert A opposite 8-oxoG at a high frequency (45-47). Consequently, the presence of 8-oxoG residues in the template induces G:C->T:A transversions (28,40,44,48). Studies with E.coli have shown that three different repair activities, MutM, MutY and MutT, cooperate to prevent mutations from being formed at 8-oxoG lesions. MutM DNA glycosylase removes purines with either ruptured or intact (but oxidized) imidazole rings, such as fapy and 8-oxoG, from DNA (13,43,49,50). MutY is also a DNA glycosylase which efficiently removes an adenine incorporated opposite an 8-oxoguanine (13,26,27,51). Such mispairs are formed if 8-oxoG remains in the template during DNA replication. The enzyme removes adenine from A:G mispairs as well (20). The MutT protein hydrolyses 8-oxo-dGTP to 8-oxo-dGMP, thus preventing 8-oxo-dGTP from being incorporated during replication (52). Strains defective in mutY and mutM functions accumulate spontaneous G:C->T:A transversion mutations (25,28,40). The present experiments showed that spontaneous G:C->C:G transversion mutations were also accumulated in strains defective in mutY function (Table 2). The results enable two main conclusions to be drawn, as represented in the model (Fig. Figure 6. Relative activity of MutY glycosylase on 8-oxoG:A and 8-oxoG:A substrates. Twenty fmol substrate oligonucleotides containing 8-oxoG:G (lanes 1-8) and 8-oxoG:A (lanes 9-16) mispairs were incubated with various amounts of MutY protein in 10 µl of the reaction buffer containing 20 mM Tris-HCl (pH 7.6), 0.5 µg of BSA and 10 mM EDTA for 1 and 8 h, respectively. After electrophoresis at 1800 V, the gels were dried and then autoradiographed using Fuji RX films at -80°C. Lanes 1, 8, 9 and 16, no enzyme; lanes 2 and 10, 1.2 ng; lanes 3 and 11, 2.4 ng; lanes 4 and 12, 6 ng; lanes 5 and 13, 30 ng; lanes 6 and 14, 45 ng; lanes 7 and 15, 60 ng. *, 32P-labeled strand at the 5[prime]-terminus. The mutM mutation did not result in enhanced rates of Lac+ reversion in strains CC103 and CC103mutY (Table 2). MutM protein is able to remove 8-oxoG from 8-oxoG:G mispair in vitro (43,47). The removal of 8-oxoG from 8-oxoG:G mispair by MutM protein might lead to the occurrence of G:C->C:G transversions, because dCMP is incorporated opposite guanine remaining in DNA during repair replication. Hence, it is reasonable that the activity of the MutM protein to remove 8-oxoG from the mispair is repressed before the MutY protein removes guanine from the 8-oxoG:G mispair in E.coli cells. The MutY protein might be implicated in this regulation for the MutM activity, as suggested by Bridges et al. (14). Braun et al. (53) also suggested that due to the action of nucleotide excision repair, oxidatively damaged G* of a G*:G mismatch is removed, and this will lead to G:C->C:G transversions. Nghiem et al. (25), using a detection method relying on a papillation technique, found that the mutY mutation resulted in an enhanced rate of spontaneous G:C->T:A transversions, whereas it did not enhance other types of base substitution. In their experiments, cells were incubated on glucose minimal plates for ~3 days to visualize blue papillae. In the present experiments, cells were incubated for ~10 days. Under such conditions, some turnover of cells and a slow increase in cell count are demonstrated (14). The delayed mutation accumulation of G:C->C:G transversions was also described by Mackay et al. (54). Lesions such as 8-oxoG may in fact be produced continuously and direct the misincorporation of dAMP and dGMP opposite the lesion in the template DNA. However, the repair of 8-oxoG:G mispair by MutY protein might be less effective than that of 8-oxoG:A mispair. Figure 7. 8-oxoG-related mutagenesis pathways leading to G:C->C:G transversions and the repair mechanism by the MutY protein. Oxidative damage can lead to 8-oxoG lesions in DNA. The asterisk indicates 8-oxoG. If the 8-oxoG lesion is not removed before DNA replication, translesional synthesis by DNA polymerase frequently leads to the misincorporation of G in addition to A opposite the 8-oxoG lesion. MutY protein removes the misincorporated G from the 8-oxoG:G mispairs. 8-oxoG:C mispairs formed during DNA repair replication can be removed by the MutM protein. In contrast, if the MutM protein removes 8-oxoG from the 8-oxoG:G mispair before the action of the MutY protein, the misincorporated G remains, leading to G:C->C:G transversions. The human homolog of the E.coli mutY gene (hMYH) has been cloned and sequenced (55). The human gene encodes a protein of 535 amino acids (55), which is in good agreement with the size of a polypeptide detected in HeLa cells that cross-reacted with the antibody against E.coli MutY protein (56). The human hMYH gene has 41% identity to the E.coli MutY protein (55), suggesting its important functions in the repair of oxidative damage to DNA and in the prevention of mutations from oxidative lesions. We are currently studying whether the human gene is able to reduce the frequency of spontaneous G:C->C:G transversions and whether the human MutY protein has a guanine-DNA glycosylase to remove unmodified guanine from 8-oxoG:G mispair.
ACKNOWLEDGEMENTS
We are grateful to Drs J. H. Miller, B. Weiss (Michigan State University, Ann Arbor, USA), A. Nishimura (The National Institute of Genetics, Mishima, Japan) and K. Yamamoto (Tohoku University, Sendai, Japan) for kindly supplying E.coli strains, phage and plasmids. We also thank Yukio Kasai for assistance during the present experiments. This study was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan.
REFERENCES
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