Mismatch DNA Glycosylase Research Articles

Insights into the role of Val45 and Gln182 of Escherichia coli MutY in DNA substrate binding and specificity

BackgroundEscherichia 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).ResultsTargeted 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.ConclusionThe 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.

Ab Initio Comprehensive Conformational Analysis of 2‘-Deoxyuridine, the Biologically Significant DNA Minor Nucleoside, and Reconstruction of Its Low-Temperature Matrix Infrared Spectrum

A comprehensive conformational analysis of isolated 2'-deoxyuridine (dU), a minor DNA nucleoside, has been performed by means of ab initio calculations at the MP2/6-311++G (d,p)//DFT B3LYP/6-31G (d,p) level of theory. At 298.15 and 420 K, all 94 allowed conformers of dU are within 8.96 and 7.91 kcal/mol Gibbs energy ranges, respectively. Syn orientation for the base and South (S) conformers for the sugar dominate at 298.15 K: syn/anti=62.3%:37.7% and S/N=77.2%:22.8%. At 420 K in the majority of conformers, the base is anti oriented and the population of North (N) sugars increases: syn/anti=39.3%:60.7% and S/N=63.0%:37.0%. Values of all conformational parameters and correlations between them, as well as their correlations with valence bonds, and also correlations between valence bonds and angles were estimated. In general, 14 types of intramolecular H-bonds were detected (1-3 H-bonds per conformer, the total number 175), namely, C1'H...O2 (16 H-bonds), C2'H1...O5' (9), C2'H2...O2 (21), C3'H...O2 (21), C5'H1...O2 (14), C5'H2...O2 (11), C6H...O4' (37), C6H...O5' (22), C3'H...HC6 (4), O5'H...HC6 (2), O3'H...O5' (5), O5'H...O4' (1), O5'H...O3' (4), and O5'H...O2 (8). Geometric, vibrational, structural-topological, and energetic features of the OH...O intramolecular H-bonds in dU conformers were determined. The close similarity between energetic and geometric characteristics of dU and thymidine DNA-like conformers in anti and relevant syn conformations and their transition states of the anti-->syn interconversion implies that mismatch DNA glycosylase discriminates between the two nucleosides, mainly because of the difference in the shapes of their bases. Convolution of calculated IR spectra of all the dU conformers within the limits 3400-3700 cm(-1) appears to be consistent with its low-temperature matrix IR spectrum (Ivanov et al. Spectrochim. Acta, Part A 2003, 59, 1959), wavenumber discrepancy not exceeding 1%. It was concluded that, for a reliable reproduction of the experimental spectrum, the whole set of conformers should be taken into consideration. The suggested method makes reconstruction of the isolated nucleoside IR spectrum at a physiological interval of temperature reasonably possible.

A distinct TthMutY bifunctional glycosylase that hydrolyzes not only adenine but also thymine opposite 8-oxoguanine in the hyperthermophilic bacterium, Thermus thermophilus

Oxidative damage represents a major threat to genomic stability because the major product of DNA oxidation, 8-oxoguanine (GO), frequently mispairs with adenine during replication. We were interested in finding out how hyperthermophilic bacteria under goes the process of excising mispaired adenine from A/GO to deal with genomic oxidative damage. Herein we report the properties of an Escherichia coli MutY ( EcMutY) homolog, TthMutY, derived from a hyperthermophile Thermus thermophilus. TthMutY preferentially excises on A/GO and G/GO mispairs and has additional activities on T/GO and A/G mismatches. TthMutY has significant sequence homology to the A/G and T/G mismatch recognition motifs, respectively, of MutY and Mig. MthI. A substitution from Tyr112 to Ser or Ala (Y112S and Y112A) in the putative thymine-binding site of TthMutY showed significant decrease in DNA glycosylase activity. A mutant form of TthMutY, R134K, could form a Schiff base with DNA and fully retained its DNA glycosylase activity against A/GO and A/G mispair. Interestingly, although TthMutY cannot form a trapped complex with substrate in the presence of NaBH 4, it expressed AP lyase activity, suggesting Tyr112 in TthMutY may be the key residue for AP lyase activity. These results suggest that TthMutY may be an example of a novel class of bifunctional A/GO mismatch DNA glycosylase that can also remove thymine from T/GO mispair.

Estradiol receptor potentiates, in vitro, the activity of 5-methylcytosine DNA glycosylase

At a concentration of 5×10−9 M of hemi-methylated DNA (one order of magnitude below the Km), MCF-7 (a human breast carcinoma cell line) nuclear extracts potentiate the activity of 5-methylcytosine DNA glycosylase (5-MCDG, alias G/T mismatch DNA glycosylase). Depending on the ratio between MCF-7 nuclear extracts and 5-MCDG, there is an up to 10-fold increase in 5-MCDG activity. The potentiation of 5-MCDG by MCF-7 nuclear extracts requires an estradiol response element adjacent to the hemi-methylated site. Depletion of the estradiol receptor from MCF-7 nuclear extracts with specific antibodies abolishes the potentiation of 5-MCDG activity. The estradiol receptor present in MCF-7 nuclear extracts can be precipitated with antibodies directed against 5-MCDG. Reciprocally, antibodies directed against the estradiol receptor precipitate 5-MCDG. The results indicate the formation of a complex between the estradiol receptor and 5-MCDG.

5-Methylcytosine DNA glycosylase participates in the genome-wide loss of DNA methylation occurring during mouse myoblast differentiation.

Changes in gene expression during mouse myoblast differentiation were monitored by DNA microarray hybridisation. Four days after the onset of differentiation 2.37% of the genes increased in activity from a value of zero, whereas during the same time 1.68% of total genes had decreased expression. During the first 24 h of differentiation an average of 700 000 CpG sites per haploid genome were demethylated. Maximal loss of DNA methylation is attained after 2 days of differentiation, followed by a gradual remethylation. The highest demethylation is observed in highly repeated DNA sequences, followed by single copy sequences. When DNA replication is inhibited by aphidicolin or L-mimosine this genome-wide demethylation is still observed. During the first 3 h of differentiation there is an increase in the number of hemimethylated CpG sites, which disappear rapidly during the course of genome-wide hypomethylation. Transfection of cells with an antisense morpholino oligonucleotide to 5-methylcytosine DNA glycosylase (G/T mismatch DNA glycosylase) decreases both the activity of the enzyme and genome-wide demethylation. It is concluded that the genome-wide loss of DNA methylation in differentiating mouse myoblasts occurs in part by formation of hemimethylated CpG sites, which can serve as the substrate for 5-methylcytosine-DNA glycosylase.

Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene.

We have shown that the DNA demethylation complex isolated from chicken embryos has a G(.)T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kidney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG. A 15- to 20-fold overexpression of 5-MCDG results in the specific demethylation of a stably integrated ecdysone-retinoic acid responsive enhancer-promoter linked to a beta-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A (an analogue of ecdysone). The state of methylation of the transgene was investigated by Southern blot analysis and by the bisulfite genomic sequencing reaction. Demethylation occurs downstream of the hormone response elements. No genome-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promoter-enhancer of the reporter gene. An increase in 5-MCDG activity does not influence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy number during selection of the clones with antibiotics. Immunoprecipitation combined with Western blot analysis showed that an antibody directed against 5-MCDG precipitates a complex containing the retinoid X receptor alpha. The association between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may target demethylation of the transgene in this system.

5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence.

A 1468 bp cDNA coding for the chicken homolog of the human MBD4 G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (416 amino acids) shows 46% identity with the human MBD4 and the conserved catalytic region at the C-terminal end (170 amino acids) has 90% identity. The non-conserved region of the avian protein has no consensus sequence for the methylated DNA binding domain. The recombinant proteins from human and chicken have G/T mismatch as well as 5-methylcytosine (5-MeC) DNA glycosylase activities. When tested by gel shift assays, human recombinant protein with or without the methylated DNA binding domain binds equally well to symmetrically, hemimethylated DNA and non-methylated DNA. However, the enzyme has only 5-MeC DNA glycosylase activity with the hemimethylated DNA. Footprinting of human MBD4 and of an N-terminal deletion mutant with partially depurinated and depyrimidinated substrate reveal a selective binding of the proteins to the modified substrate around the CpG. As for 5-MeC DNA glycosylase purified from chicken embryos, MBD4 does not use oligonucleotides containing mCpA, mCpT or mCpC as substrates. An mCpG within an A+T-rich oligonucleotide is a much better substrate than an A+T-poor sequence. The K:(m) of human MBD4 for hemimethylated DNA is approximately 10(-7) M with a V:(max) of approximately 10(-11) mol/h/microgram protein. Deletion mutations show that G/T mismatch and 5-MeC DNA glycosylase are located in the C-terminal conserved region. In sharp contrast to the 5-MeC DNA glycosylase isolated from the chicken embryo DNA demethylation complex, the two enzymatic activities of MBD4 are strongly inhibited by RNA. In situ hybridization with antisense RNA indicate that MBD4 is only located in dividing cells of differentiating embryonic tissues.

Differential Destabilization of the DNA Oligonucleotide Double Helix by a T·G Mismatch, 3,N4-Ethenocytosine, 3,N4-Ethanocytosine, or an 8-(Hydroxymethyl)-3,N4-ethenocytosine Adduct Incorporated into the Same Sequence Contexts

The T.G mismatch and the exocyclic adduct 3,N(4)-ethenocytosine (epsilonC) are repaired by the same enzyme, the human G/T(U) mismatch-DNA glycosylase (TDG). This enzyme removes the T, U, or epsilonC base from duplex DNA. The rate of cleavage was found to differ with the lesion and was also affected by neighbor sequences [Hang, B., Medina, M., Fraenkel-Conrat, H., and Singer, B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13561-13566]. Since sequence influences duplex stability, we determined the thermodynamic stability of T.G and epsilonC-containing 15-mer duplexes in which the bases flanking the lesion were systematically varied. The duplexes contained central 5'-TTXTT, 5'-AAXAA, 5'-CCXCC, or 5'-GGXGG sequences, where X is T, epsilonC, or two closely related structural derivatives of epsilonC: 3,N(4)-ethanocytosine (EC) and 8-(hydroxymethyl)-epsilonC (8-HM-epsilonC). Each of the four lesions, incorporated opposite G, decreased both the thermal (T(m)) and thermodynamic stability (DeltaG degrees (37)) of the 15-mer control duplexes. On the basis of the T(m) and DeltaG degrees (37) values, the order of destabilization of the TTXTT sequence in 15-mer duplexes was as follows: 8-HM-epsilonC > EC > epsilonC > T.G. The DeltaT(m) values range from -15.8 to -9.5 degrees C when C(t) = 8 microM. Duplexes with flanking AA or TT neighbors were more destabilized, by an average of 2 degrees C, than those with flanking GG or CC neighbors. The base opposite the modified base also influenced duplex stability. Within the TT context, of the four changed bases opposite the adducts, C had the greatest destabilizing effect, up to -18.4 degrees C. In contrast, a G opposite an adduct was generally the least destabilizing, and the smallest value was -3. 0 degrees C. Destabilizations were enthalpic in origin. Thus, this work shows that independently changing the modified base, the sequence, or the base opposite the lesion each affects the stability of the duplex, to significantly varying extents. The potential contribution of the thermodynamic stability to repair efficiency is discussed.

5-methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex.

We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C and N-terminal parts have about 50% identity. As for the highly purified chicken embryo DNA demethylation complex the recombinant protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. The recombinant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylated CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for the recombinant protein expressed in E. coli and 3:1 for the purified enzyme from chicken embryos. The incubation of a recombinant CpG-rich RNA isolated from the purified DNA demethylation complex with the recombinant enzyme strongly inhibits G/T mismatch glycosylase while slightly stimulating the activity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG activities share the same areas of the N- and C-terminal parts of the protein. In reconstitution experiments RNA helicase in the presence of recombinant RNA and ATP potentiates the activity of 5-MCDG.

Mutagenic effects of gamma-rays and incorporated 8-[formula omitted]-purines on extracellular lambda phage: influence of mutY and mutM host mutations

The lethal and mutagenic effects on phage λcI857 of 60Co γ-rays and of decay of 3H incorporated into phage DNA both as 8-3H-deoxyadenosine and 8-3H-deoxyguanosine (using 8-3H-adenine as a labelled DNA precursor) were studied on four isogenic Escherichia coli strains: AB1157 M+Y+ (wild type, mutM+mutY+), AB1157 M−Y+ (mutM∷kan mutY+ mutant deficient in the formamidopyrimidine-DNA glycosylase MutM), AB1157 M+Y− (mutM+mutY mutant deficient in the A:G mismatch DNA glycosylase MutY), and AB1157 M−Y− (mutM∷kan mutY double mutant deficient in both DNA glycosylases). The main products of transmutation component of 3H decay in position 8 of purine residues are 8-oxo-7,8-dihydroadenine (8-oxoA) and 8-oxo-7,8-dihydroguanine (8-oxoG), the latter being responsible for the most part of the mutagenic effect. The lethal effects of both γ-rays and tritium decay virtually did not depend on the repair phenotypes of the host strains used. Therefore, the MutM and MutY glycosylases are not involved in the repair of lethal DNA damages induced by ionizing radiation or by the transmutation component of 3H decay in purine residues of phage DNA. The efficiencies of mutagenic action of 3H-purines Em (frequencies of c-mutations per one 3H decay in phage genome) were 2.4-, 3.8- and 55-fold higher in the M−Y+, M+Y− and M−Y− mutants, respectively, in comparison to the wild-type host. The mutagenic efficiencies Em for γ-rays were nearly identical in the M+Y+ and M−Y+ hosts, but were increased 1.8- and 8.3-fold, respectively, in the M+Y− and M−Y− mutants. These data suggest that: (1) the MutY and MutM DNA glycosylases are important for prevention of mutations caused not only by spontaneous oxidation of guanine residues, but also by ionizing radiation or by decay of 3H incorporated into purine bases of DNA; (2) the MutY and MutM enzymes functionally cooperate in elimination of mutagenic damages induced by these agents.

Profile of gene expression regulated by induced p53: connection to the TGF-β family

The transcription regulatory function of p53 was analyzed by using two inducible p53 systems in the human lung cancer cell line H1299. cDNA probes derived from RNA harvested 12 h after p53 induction were used to probe filters containing cDNA arrays. Over 20 genes were found to be significantly induced or suppressed by p53. The induced genes can be classified mainly as cell cycle inhibitors like p21waf, GADD45, apoptosis-related genes like Fas/APO1 and PIG3 or DNA repair genes like DDB2, DNA ligase and G/T mismatch DNA glycosylase. The suppressed genes include mainly cell cycle regulators like cyclin B1, cyclin H and kinases like c-abl, CLK1 and others. The most notable induced gene was MIC-1, encoding a TGF-β-related secretory protein, suggesting a potential paracrine component for p53 growth suppression.

A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch.

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N6-ethenoadenine (epsilonA) and 3,N4-ethenocytosine (epsilonC) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises epsilonA. The present work is directed toward identifying and purifying the human glycosylase activity releasing epsilonC. HeLa cells were subjected to multiple steps of column chromatography, including two epsilonC-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the epsilonC activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having epsilonC-DNA glycosylase activity. In addition, oligonucleotides containing epsilonC.G or G/T(U), could compete for epsilonC protein binding, further indicating that the epsilonC-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for epsilonC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.