Insights into the role of Val45 and Gln182 of Escherichia coli MutY in DNA substrate binding and specificity
© Chang et al; licensee BioMed Central Ltd. 2009
Received: 13 August 2008
Accepted: 12 June 2009
Published: 12 June 2009
Escherichia coli MutY (EcMutY) reduces mutagenesis by removing adenines paired with guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG). V45 and Q182 of EcMutY are considered to be the key determinants of adenine specificity. Both residues are spatially close to each other in the active site and are conserved in MutY family proteins but not in Methanobacterium thermoautotrophicum Mig.MthI T/G mismatch DNA glycosylase (A50 and L187 at the corresponding respective positions).
Targeted mutagenesis study was performed to determine the substrate specificities of V45A, Q182L, and V45A/Q182L double mutant of EcMutY. All three mutants had significantly lower binding and glycosylase activities for A/G and A/8-oxoG mismatches than the wild-type enzyme. The double mutant exhibited an additive reduction in binding to both the A/G and A/GO in comparison to the single mutants. These mutants were also tested for binding and glycosylase activities with T/G- or T/8-oxoG-containing DNA. Both V45A and Q182L mutants had substantially increased affinities towards T/G, however, they did not exhibit any T/G or T/8-oxoG glycosylase activity. Surprisingly, the V45A/Q182L double mutant had similar binding affinities to T/G as the wild-type EcMutY. V45A, Q182L, and V45A/Q182L EcMutY mutants could not reduce the G:C to T:A mutation frequency of a mutY mutant. Expression of the V45A mutant protein caused a dominant negative phenotype with an increased G:C to A:T mutation frequency.
The substrate specificities are altered in V45A, Q182L, and V45A/Q182L EcMutY mutants. V45A and Q182L mutants had reduced binding and glycosylase activities for A/G and A/8-oxoG mismatches and increased affinities towards T/G mismatch. However, in contrast to a previous report that Mig.MthI thymine DNA glycosylase can be converted to a MutY-like adenine glycosylase by replacing two residues (A50V and L187Q), both V45A and Q182L EcMutY mutants did not exhibit any T/G or T/8-oxoG glycosylase activity. The dominant negative phenotype of V45A EcMutY mutant protein is probably caused by its increased binding affinity to T/G mismatch and thus inhibiting other repair pathways.
Reactive oxygen species are generated by endogenous processes such as mitochondrial oxidative phosphorylation as well as exogenously following exposure to ionizing radiation and chemicals . Oxidative DNA damage including strand breaks and oxidative base lesions are specifically repaired by base excision repair pathways . The first step in this pathway is carried out by a lesion-specific DNA glycosylase, which cleaves the N-glycosidic bond between the base and deoxyribose sugar . The most abundant and highly mutagenic oxidative DNA damage lesion is 8-oxo-7,8-dihydroguanine (8-oxo-G or GO) that can form base-pair with adenine or cytosine during DNA replication to produce a G:C to T:A transversion [4, 5]. In Escherichia coli, MutT, MutM (Fpg), MutY, MutS, and Nei (End VIII) are involved in defending against the mutagenic effects of 8-oxoG lesions [reviewed in  and . The MutT protein has pyrophosphohydrolase activity, which eliminates 8-oxo-dGTP from the nucleotide pool. MutM glycosylase (Fpg protein) removes both mutagenic GO adducts and ring-opened purine lesions paired with cytosines. MutS and MutY increase replication fidelity by removing the adenines misincorporated opposite GO or G [5, 7, 8], and thus reduce G:C to T:A transversions. Nei can excise GO, when GO is paired opposite a cytosine or adenine and can serve as a backup pathway to repair 8-oxoG in the absence of MutM and MutY [6, 9].
The N-terminal domain of MutY shares similar structure, including the helix-hairpin-helix (HhH) and Gly/Pro Asp loop motifs (Figure 1), with AlkA, EndoIII, and OGG1. The G/T mismatch glycosylase Mig.Mth I of Methanobacterium thermoautotrophicim is a member of the HhH superfamily  and is involved in reducing spontaneous deaminated bases from 5-methyl-cytosine residues. The substrate specificity of Mig.Mth I is in the order: U/G = T/G > G/G > T/C = U/C > A/G. In the binding pocket for the target base, Mig.Mth I shares six out of seven residues of EcMutY's adenine binding (R19, E37, L40, N140, Q182, M185, and D186). The only non-conserved residue in this binding pocket is Q182 of EcMutY, which corresponds to L187 of Mig.Mth I (Figure 1). Interestingly, Fondufe-Mittendorf et al.  could convert Mig.Mth I into a MutY-like glycosylase with altered substrate preference of Mig.Mth I from T/G to A/G by replacing two residues (A50V and L187Q) in the substrate binding pocket. Their data point to the potential importance of V45 and Q182 in EcMutY substrate recognition.
In this study, we have investigated the role of V45 and Q182 of EcMutY by targeted mutagenesis. V45A, Q182L, and V45A/Q182L mutant proteins have reduced binding affinity and glycosylase activity to DNA containing A/G or A/GO mismatches. These mutants are impaired to complement E. coli mutY mutants in vivo. The affinities to T/G dramatically increased in V45A and Q182L mutants, but not in the V45A/Q182L double mutant. However, V45A, Q182L, and V45A/Q182L mutant proteins do not exhibit glycosylase activity towards T/G or T/GO. Therefore, V45 and Q182 of EcMutY are important for the adenine-specific activity of EcMutY.
Results and discussion
Selection of EcMutY mutation
A/G and A/GO binding and glycosylase activity
Apparent dissociation constants (K d ) of MutY mutants.
1.3 ± 0.11a
6.1 ± 1.7 (0.21)b
8.9 ± 5.0 (0.15)
15 ± 2 (0.09)
0.0048 ± 0.0029
0.45 ± 0.06 (0.01)
0.051 ± 0.009 (0.09)
0.59 ± 0.12 (0.01)
14 ± 1
0.011 ± 0.004 (1273)
0.043 ± 0.014 (326)
8.4 ± 0.7 (1.7)
0.036 ± 0.013
0.024 ± 0.009 (1.5)
0.088 ± 0.061 (0.41)
0.013 ± 0.008 (2.77)
Rate constants (K2) of MutY mutants.
0.24 ± 0.03a
0.19 ± 0.03 (0.80)b
0.025 ± 0.016 (0.11)
0.032 ± 0.026 (0.14)
2.4 ± 0.3
1.3 ± 0.1 (0.54)
0.16 ± 0.02 (0.07)
0.37 ± 0.05 (0.15)
T/G and T/GO binding and glycosylase activity
We next measured the T/G and T/GO binding affinities of these mutants. Wild-type EcMutY showed a comparatively high K d (14 ± 1 nM) with a T/G mismatch (Table 1), however, this value is much lower than that of EcMutY with C:G homoduplex (315 ± 49 nM) . The K d values of V45A and Q182L mutants to T/G drastically decreased to 0.011 ± 0.004 nM and 0.043 ± 0.014 nM, representing a 1273 and 326-fold increase in binding affinity, respectively (Table 1). Surprisingly, V45A/Q182L double mutant showed a K d value of 8.4 ± 0.7 nM, which is close to that of wild-type EcMutY. The geometry or architecture of the substrate binding pocket in the V45A/Q182L double mutant may not favor thymine binding. For T/GO binding, V45A, Q182L, and V45A/Q182L mutants showed similar K d values as wild-type EcMutY (Table 1), indicating that the T/GO binding affinity of EcMutY does not involve V45 and Q182. This result is consistent with the findings of Li et al.  that tight T/GO binding is controlled by the C-terminal domain of EcMutY.
Because Mig.Mth I T/G glycosylase can be converted to an A/G glycosylase by replacing two residues (A50V and L187Q) , we examined the glycosylase activities of Vl45A, Q182L, and V45A/Q182L on T/G and T/GO. None of the EcMutY mutants showed any T/G and T/GO glycosylase activity at enzyme concentration up to 1 μM under different buffers and temperatures (data now shown). Therefore, recognition and glycosylase of EcMutY mutants to T/G and T/GO are controlled by different mechanisms.
The previous success to mutate Mig.Mth I into MutY-like enzyme  is in contrast to our failure to covert EcMutY to thymine glycosylase. It is possible that it is easier to catalyze adenine removal than thymine excision. It is interesting to point out that wild-type Mig.Mth I is a weak adenine glycosylase and Mig.Mth I with A50V/L187Q mutation becomes a stronger adenine glycosylase. However, wild-type EcMutY has no thymine glycosylase activity and V45A/Q182L mutant may be harder to gain this activity. In addition, the active site of Mig.Mth I is more relaxed for both purine and pyrimidine excision while MutY is more strict to excise purines. The difference in the base removal by these two enzymes may be contributed by other residues surrounding the active site.
By aligning MutY and Mig.Mth I sequences (Figure 1) and inspecting the structures of Mig.Mth I , EcMutY catalytic domain  with docked thymine (Figure 2), we predicted that several residues may contribute to their difference in activities. The thymine docked into the EcMutY active site preserves the proposed edge-on hydrogen bonding interaction with the conserved glutamate (E37, E42 in Mig.MthI) and can support a potential hydrogen bond with a tyrosine in a potential S120Y mutant (Figure 2A). It is clear from Figs. 2C and 2D that the MutY active site poorly accommodates the methyl group of thymine relative to the Mig.MthI active site. In the present study, one important mutation (Q182L) has been created. Based on our observations and the models shown in Figure 2, the subsequent mutations most likely to promote the conversion of MutY into a thymine glycosylase would be Q25R and L22F. These two mutations would complete the methyl-binding pocket and stabilize the interaction with thymine. In addition, an S120Y mutation, which would provide an additional hydrogen bond (i.e. to the O2 of thymine) might further enhance any thymine glycosylase activity resulting from the Q182L, Q25R, and L22F mutations. However, such predictions highlight the difficulty inherent in designing proteins with novel activities. In particular, it has been shown that Y126S mutation of Mig.Mth I inactivates T/G mismatch-specific glycosylase activity , however, the presence of Tyr in BsMutY indicates that Tyr may not be the only key residue for T/G catalytic activity. Similarly, BsMutY contains R31 to accommodate the methyl group of thymine relative to the Mig.MthI active site, but does not excise thymine.
Recently, a bifuncional MutY-like glycosylase, TthMutY, has been shown to have strong A/GO and G/GO activities, comparatively weak T/GO and A/G activities, and no T/G glycosylase activity . Unlike Mig.MthI, TthMutY and EcMutY contain extra C-terminal domains which facilitate their recognition of the mispaired GO base. V42 and Q170 of TthMutY are identical with the corresponding residues in EcMutY (V45 and Q182) but different from A50 and L187 of Mig.MthI (Figure 1). Although TthMutY has weak T/GO glycosylase activity, its L19 and Q39 residues, which correspond to F27 and R47 of Mig.MthI, respectively, are also conserved in EcMutY and BsMutY. This suggests that F27 and R47 of Mig.MthI may not be absolutely required for T/G or T/GO glycosylase activity. Interestingly, Y112S TthMutY mutant lost the thymine glycosylase activity supporting that Y126 of Mig.Mth I may be important for T/G catalytic activity. It remains to be tested whether S120Y, Q25R, and L22F mutants of EcMutY or in combination with V45A and Q182L can gain T/G glycosylase activity.
V45A, Q182L, and V45A/Q182L EcMutY mutants cannot complement the mutY mutation
Mutation frequencies of λDE3-containing E. coli mutM mutY mutant strains.
Mutation Frequency (RifR/108 cells)
Mutation frequency (Lac+/108 cells)
1. CC104 (WT)
4.1 ± 1.3
1.2 ± 1.0
2. CC104 mutMmutY
1464 ± 314
486 ± 65
3. CC104M-Y- + pET11aa
2055 ± 704
156 ± 102
4. CC104M-Y- + pET-MutYb
8.8 ± 4.4
0.9 ± 0.8
5. CC104M-Y- + pET-V45Ac
630 ± 177
6.3 ± 2.1
6. CC104M-Y- + pET-Q182Ld
181 ± 74
10.2 ± 8.7
7. CC104M-Y- + pET-V45A/Q182Le
728 ± 358
20 ± 13
CC104 strain was designed to screen G:C→T:A transversions at an essential residue in the active site of β-galactosidase encoded by lac Z gene, thus it allowed us to measure this type of mutation in EcMutY-V45A, EcMutY-Q182L and EcMutY-V45A/Q182L mutants. Similar to the results of rifampicin forward assay, the mutation frequency of the mutMmutY double mutant increased 400-fold over the wild-type cells (Table 3, right panel, lines 1 and 2). The EcMutY-V45A, EcMutY-Q182L and EcMutY-V45A/Q182L mutants showed partial defect in preventing G:C→T:A transversions as compared to the wild-type EcMutY. Strains expressing V45A, Q182L and V45A/Q182L mutants, respectively, showed 5, 8, and 17- fold higher mutation frequencies than CC104 wild-type (Table 3, right panel, lines 5–7). These increased G:C→T:A mutation frequencies are in agreement with the reduced glycosylase activities with A/G and A/GO mismatches in these mutants.
Wild-type cells expressing the V45A mutant protein have an increased G:C to A:T mutation frequency
Mutation distribution of rpoB in λDE3-containing E. coli CC102 (mutM+mutY+) harboring pET11a and pET11a-V45A.
No. of clones with rpoB mutation (%)
A:T → T:A
G:C → A:T
G:C → T:A
A:T → G:C
Lac+ reversion rate of λDE3-containing E. coli CC102 (mutM+mutY+) strains
1. CC102 + pET11a
0.45 ± 0.12
2. CC102 + pET-MutY
0.51 ± 0.16
3. CC102 + pET-V45A
1.60 ± 0.36
The substrate specificity of DNA glycosylases is very subtle to the change of amino acids located in the DNA binding pocket, and a single amino acid alteration may significantly alter its enzyme activity. Our results show that V45 and Q182 of EcMutY are important for the binding affinity and glycosylase activity to DNA containing A/G or A/GO mismatches. The V45A/Q182L double mutant exhibited an additive reduction in binding to both the A/G and A/GO as for the single mutants. Our unexpected results that EcMutY-V45A and EcMutY-Q182L show increased T/G binding affinity without gaining T/G glycosylase indicate the complicated nature of the DNA-enzyme interaction.
Escherichia coli DH5α (F-, φ80-dlacZΔM15, endA1, recA1, hsdR1, (rk-mk+), supE 44, thi-1, gry A96(Nalr), rel A1, Δ (lacZYA-argF) U169) was purchased from Invitrogen. PR70 (Su- smrlacZ X74 galU galK miA68:: Tn10 kan) was obtained from M. S. Fox. The miA68:: Tn10 kan allele in PR70 contains a transposon at the mutY gene at nucleotide 747 and produces a truncated MutY protein . CC102 [ara Δ (lac-proB)XIII thi F'-lacI378 lacZ461 proA+B+)  and CC104, which is identical to CC102 except for the mutation at residue 461 of β-galactosidase, were generous gifts from J. H. Miller. The derivative CC104 mutM mutY: mutM::mini-kan mutY::mini-Tn10 was also from J. H. Miller. λDE3 lysogenic strains were constructed according to the procedures described by Invitrogen. XL1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F proAB lacI, Z ΔM15 Tn10 (Tet)]) was purchased from Stratagene.
Construction of E. coli mutY mutants
The mutant mutY genes encoding proteins containing V45A, Q182L and V45A/Q182L were constructed by the QuickChange Site-Directed Mutagenesis Kit (Strategene). The mutagenesis PCR primers for the desired amino acid substitutions are listed below.
Primers, Chang564 and Chang565, are designed for the V45A mutant and Chang566 and Chang567 are designed for the Q182L mutant using plasmid pMYW-1  as the template. The plasmids containing the mutY mutant genes with V45A or Q182L mutation were named pMY-V45A and pMY-Q182L. The double mutant was derived from pMY-V45A using primers Chang566 and Chang 567. After the PCR reaction, the template plasmids were digested with DpnI restriction and transformed into XL1-Blue supercompetent cells (Strategene). The correct clones were confirmed by DNA sequencing.
Measurement of mutation frequency
Overnight cultures (0.1 ml) of each strain were plated on LB agar plates containing 0.1 mg/ml rifampicin. The cell titer of each culture was determined by plating 0.1 ml of and 10-6 dilution onto LB agar plates. For each measurement, four independent cultures were plated, and the experiments were repeated at least three times. The mutation frequency was determined by calculating the ratio of Rifr cells to total cells. For LacZ+ reversion mutation assay, overnight cultures (0.2 ml) of each strain were plated on M9 agar plates containing 0.2% lactose and colonies were scored after three days. The ratio of LacZ+ cells to total CC104 cells was calculated to be the G:C→T:A transversion frequency. The ratio of LacZ+ cells to total CC102 cells was calculated to be the G:C→A:T transition frequency.
EcMutY protein expression and purification
E. coli strains PR70/DE3 harboring expression plasmids pMY-V45A, pMY-Q182L, and pMY-V45A/Q182L were grown in LB broth containing 50 μg/ml ampicillin at 37°C. At OD600 of 0.6, isopropyl β-D-thiogalactoside (IPTG) was added to the culture to a final concentration of 0.2 mM, and the culture was incubated at 20°C for 16 hrs. The EcMutY mutant proteins were purified by ammonium sulfate precipitation, phosphocellulose, hydroxylapatite, heparin, Hitrap-S column chromatographies as previously described for the wild-type MutY . The purified proteins were divided into small aliquots and stored at -80°C. Protein concentration was determined by Bradford Method.
The nucleotide sequences of 40-mer DNA substrates containing mismatches used in this study were:
(where X = A or T and Y = G or GO). The X-strand was labeled by [γ-32p]ATP on the 5' end and then annealed with the Y-strand. The annealed double-stranded oligonucleotides were converted to 44-mers by filling the sticky ends on both sides with Klenow fragment .
EcMutY binding and glycosylase assays
The EcMutY binding and glycosylase activities were assayed as previously described by Lu et al . The MutY binding reaction mixture contained 20 mM Tris-HCl, pH7.6, 80 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM EDTA, 2.9% glycerol, 20 ng of poly dI/dC, and 1.8 fmol of labeled DNA (0.09 nM) in 20 μl reactions. After 30 min incubation at 30°C, 3 μl of 50% glycerol was added to the mixtures, and the samples were loaded to a 4% polyacrylamide gel in TBE buffer [50 mM Tris-borate (pH 8.3) and 1 mM EDTA]. After electrophoresis, the gel was dried and exposed to a PhosphorImager screen. Enzyme-bound and free DNA bands were quantified on PhosphorImager and analyzed by ImageQuant (GE Health). To determine active site concentrations, binding experiments were performed with 8 nM of A/GO-containing DNA for wild-type, V45A, and Q182L EcMutY while 30 nM of the same DNA was tested for the V45A/Q182L mutant. The MutY concentrations used ranged from 2 to 400 nM as determined by the Bradford assay.
where b = Kd + [DNA]T + [P] active .
The glycosylase assay was carried out in a 10 μl reaction containing 1.8 fmol of DNA substrate (0.18 nM), 20 mM Tris-HCl (pH 7.6), 1 mM DTT, 1 mM EDTA, 2.9% glycerol and 50 μg/ml of bovine serum albumin. After incubation at indicated temperature for 30 min, reaction mixtures were supplemented with 1 μl of 1 M NaOH and heated at 90°C for 30 min. Five μl of formamide dye (90% formamide, 10 mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue) was added to the sample and 5 μl from this mixture was loaded onto a 14% polyacrylamide sequencing gel containing 7 M urea. For time course studies, enzyme reaction was initiated at 37°C or at 4°C for A/G or A/GO-containing DNA, respectively, and aliquot taken at different time points were immediately frozen in -70°C in the presence of 0.1 N NaOH, followed by heating at 90°C for 30 min before adding 5 μl formamide dye and loading to 14% 7 M urea sequencing gels. Bands corresponding to cleavage products and intact DNA were quantified from PhosphorImager images. Graphs and rate constants of glycosylase activities are generated by SigmaPlot for Windows Version 10.0 (Systat Software, Inc.).
Sequencing of the rpoB gene
E. coli chromosomal DNA was isolated using a genomic DNA purification kit (Gentra System, Minneapolis, MN). The main group of mutations (cluster II) of the rpoB gene was PCR amplified using primers Chang440 (5'-CGTCGTATCCGTTCCGTTGG-3') and Chang441 (5'-TTCACCCGGATACATCTC GTC-3') as described and designed previously . The PCR product was purified with the QIAquick PCR purification kit (QIAGEN, Valencia, CA) and sequenced directly with Chang442 primer (5'-CGTGTAGAGCGTGCGGTGAAA-3').
- 8-oxoG or GO:
- DTT :
- EcMutY :
Escherichia coli MutY
- HhH :
- IPTG :
- k 2 :
- K d :
This work was supported by Public Health Service Grants GM 35132 and CA78391 from the National Institute of Health. We thank Drs. Maurice S. Fox and Jeffery H. Miller for providing E. coli strains. Special thanks to Dr. Eric Toth for his insightful discussion and effort to generate figure 2 and Dr. Gerald Wilson for his assistance in analysis of dissociation constants. We thank Bryan Dodson for critical reading of this manuscript.
- Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T: DNA Repair and Mutagenesis. 2005, Washington, D.C.: ASM PressGoogle Scholar
- Krokan HE, Nilsen H, Skorpen F, Otterlei M, Slupphaug G: Base excision repair of DNA in mammalian cells. FEBS Lett. 2000, 476: 73-77.View ArticleGoogle Scholar
- Mol CD, Parikh SS, Putnam CD, Lo TP, Tainer JA: DNA repair mechanisms for the recognition and removal of damaged DNA bases. Annu Rev Biophys Biomol Struct. 1999, 28: 101-128.View ArticleGoogle Scholar
- Lu A-L, Li X, Gu Y, Wright PM, Chang D-Y: Repair of oxidative DNA damage: mechanism and functions. Cell Biochem Biophy. 2001, 35: 141-170.View ArticleGoogle Scholar
- Lu A-L, Bai H, Shi G, Chang D-Y: MutY and MutY homologs (MYH) in genome maintenance. Front Biosci. 2006, 11: 3062-3080.View ArticleGoogle Scholar
- Hazra TK, Hill JW, Izumi T, Mitra S: Multiple DNA glycosylases for repair of 8-oxoguanine and their potential in vivo functions. Prog Nucleic Acid Res Mol Biol. 2001, 68: 193-205.View ArticleGoogle Scholar
- Michaels ML, Miller JH: The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxo-guanine). J Bacteriol. 1992, 174: 6321-6325.PubMed CentralGoogle Scholar
- Tchou J, Grollman AP: Repair of DNA containing the oxidatively-damaged base 8-hydroxyguanine. Mutat Res. 1993, 299: 277-287.View ArticleGoogle Scholar
- Blaisdell JO, Hatahet Z, Wallace SS: A novel role for Escherichia coli endonuclease VIII in prevention of spontaneous G → T transversions. J Bacteriol. 1999, 181: 6396-6402.PubMed CentralGoogle Scholar
- Li X, Wright PM, Lu A-L: The C-terminal domain of MutY glycosylase determines the 7,8-dihydro-8-oxo-guanine specificity and is crucial for mutation avoidance. J Biol Chem. 2000, 275: 8448-8455.View ArticleGoogle Scholar
- Gogos A, Cillo J, Clarke ND, Lu A-L: Specific recognition of A/G and A/8-oxoG mismatches by Escherichia coli MutY:removal of the C-terminal domain preferentially affects A/8-oxoG recognition. Biochemistry. 1996, 35: 16665-16671.View ArticleGoogle Scholar
- Manuel RC, Czerwinski EW, Lloyd RS: Identification of the structural and functional domains of MutY, an Escherichia coli DNA mismatch repair enzyme. J Biol Chem. 1996, 271: 16218-16226.View ArticleGoogle Scholar
- Manuel RC, Lloyd RS: Cloning, overexpression, and biochemical characterization of the catalytic domain of MutY. Biochemistry. 1997, 36: 11140-11152.View ArticleGoogle Scholar
- Noll DM, Gogos A, Granek JA, Clarke ND: The C-terminal domain of the adenine-DNA glycosylase MutY confers specificity for 8-oxoguanine.adenine mispairs and may have evolved from MutT, an 8-oxo-dGTPase. Biochemistry. 1999, 38: 6374-6379.View ArticleGoogle Scholar
- Chmiel NH, Golinelli MP, Francis AW, David SS: Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain. Nucl Acids Res. 2001, 29: 553-564.PubMed CentralView ArticleGoogle Scholar
- Guan Y, Manuel RC, Arvai AS, Parikh SS, Mol CD, Miller JH, Lloyd S, Tainer JA: MutY catalytic core, mutant and bound adenine structures define specificity for DNA repair enzyme superfamily. Nat Struct Biol. 1998, 5: 1058-1064.View ArticleGoogle Scholar
- Fromme JC, Banerjee A, Huang SJ, Verdine GL: Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase. Nature. 2004, 427: 652-656.View ArticleGoogle Scholar
- Manuel RC, Hitomi K, Arvai AS, House PG, Kurtz AJ, Dodson ML, McCullough AK, Tainer JA, Lloyd RS: Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase. J Biol Chem. 2004, 279: 46930-46939.View ArticleGoogle Scholar
- Horst JP, Fritz HJ: Counteracting the mutagenic effect of hydrolytic deamination of DNA 5-methycytosine residues at high temperature: DNA mismatch N-glycosylase Mig. Mth of the thermophilic archaron Methanobacterium thermoautotrophicum THF. EMBO J. 1996, 15: 5459-5469.PubMed CentralGoogle Scholar
- Fondufe-Mittendorf YN, Harer C, Kramer W, Fritz HJ: Two amino acid replacements change the substrate preference of DNA mismatch glycosylase Mig.MthI from T/G to A/G. Nucl Acids Res. 2002, 30: 614-621.PubMed CentralView ArticleGoogle Scholar
- Lu A-L: Isolation and analyses of MutY and MutY homologs (MYH). Methods Enzymology. 2006, 408: 64-78.View ArticleGoogle Scholar
- Lu A-L, Fawcett WP: Characterization of the recombinant MutY homolog, an adenine DNA glycosylase, from Schizosacchromyces pombe. J Biol Chem. 1998, 273: 25098-25105.View ArticleGoogle Scholar
- Mol CD, Arvai AS, Begley TJ, Cunningham RP, Tainer JA: Structure and activity of a thermostable thymine-DNA glycosylase: evidence for base twisting to remove mismatched normal DNA bases. J Mol Biol. 2002, 315: 373-384.View ArticleGoogle Scholar
- Back JH, Park JH, Chung JH, Kim DS, Han YS: A distinct TthMutY bifunctional glycosylase that hydrolyzes not only adenine but also thymine opposite 8-oxoguanine in the hyperthermophilic bacterium, Thermus thermophilus. DNA Repair (Amst). 2006, 5: 894-903.View ArticleGoogle Scholar
- Kim M, Huang T, Miller JH: Competition between MutY and mismatch repair at A·C mispair in vivo. J Bacteriol. 2003, 185: 4626-4629.PubMed CentralView ArticleGoogle Scholar
- Cupples CG, Miller JH: A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc Natl Acad Sci USA. 1989, 86: 5345-5349.PubMed CentralView ArticleGoogle Scholar
- Wright PM, Yu J, Cillo J, Lu A-L: The active site of the Escherichia coli MutY DNA adenine glycosylase. J Biol Chem. 1999, 274: 29011-29018.View ArticleGoogle Scholar
- Lu A-L: Repair of A/G and A/8-oxoG mismatches by MutY adenine DNA glycosylase. DNA Repair Protocols: Prokaryotic Systems. Edited by: Vaughan P. 2000, Totowa, NJ: Humana Press Inc, 3-16.View ArticleGoogle Scholar
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