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1.
The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein was characterized and shown to form a stable complex with Escherichia coli uracil-DNA glycosylase (Ung). As determined by mass spectrometry, the Ugi protein had a molecular weight of 9,474. We confirmed this value by sedimentation equilibrium centrifugation and determined that Ugi exists as a monomeric protein in solution. Amino acid analysis performed on both Ugi and Ung proteins was in excellent agreement with the amino acid composition predicted from the respective nucleotide sequence of each gene. The Ung.Ugi complex was resolved from its constitutive components by nondenaturing polyacrylamide gel electrophoresis and shown to possess a 1:1 stoichiometry. Analytical ultracentrifugation studies revealed that the Ung.Ugi complex had a molecular weight of 35,400, consistent with the complex containing one molecule each of Ung and Ugi. The acidic isoelectric points of the protein species were 6.6 (Ung) and 4.2 (Ugi), whereas the Ung.Ugi complex had an isoelectric point of 4.9. Dissociation of the Ung.Ugi complex by SDS-polyacrylamide gel electrophoresis revealed no apparent alteration in the molecular weight of either polypeptide subsequent to binding. Furthermore, when the Ung.Ugi complex was treated with urea and resolved by urea-polyacrylamide gel electrophoresis, both uracil-DNA glycosylase and inhibitor activities were recovered from the dissociated complex. Thus, the complex seems to be reversible. In addition, we demonstrated that the Ugi interaction with Ung prevents enzyme binding to DNA and dissociates uracil-DNA glycosylase from a preformed DNA complex. 相似文献
2.
3.
Peter Karran Tomas Lindahl Ingrid Øfsteng Grethe B. Evensen Erling Seeberg 《Journal of molecular biology》1980,140(1):101-127
Two Escherichia coli K12 mutants defective in 3-methyladenine-DNA glycosylase have been isolated following mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine. The mutants, which are of independent origin and have been designated tag-1 and tag-2, contain greatly reduced amounts of 3-methyladenine-DNA glycosylase activity in cell-free extracts. The defect in the tag-1 strain is observed at 43 °C but not at 30 °C, and a partially purified enzyme from this strain is unusually heat-labile, indicating that the defect in the tag-1 strain is due to a mutation in the structural gene for 3-methyladenine-DNA glycosylase.We have shown that 3-methyladenine-DNA glycosylase is responsible for the rapid removal of 3-methyladenine from the DNA of E. coli cells treated with monofunctional alkylating agents. The active release of this base is greatly impaired in the mutant strains. Both tag mutant strains are abnormally sensitive to killing by monofunctional alkylating agents and are defective in the host cell reactivation of methyl methanesulphonate-treated bacteriophage A. The tag mutation does not confer an increased sensitivity to ultraviolet or X-irradiation, and host cell reactivation of irradiated λ is normal in these strains. Further, there was no increase in the rate of spontaneous mutation in a tag strain.Three-factor transductional crosses with nalA and nrdA have shown that the tag-2 mutation is located at 47.2 minutes on the map of the E. coli K12 chromosome. In the mapping experiments, the tag-1 mutation behaved differently and appeared to be located at 43 to 46 minutes, in a closely situated but non-adjacent gene. Possible implications of the non-identity of the tag-1 and tag-2 mutations are discussed. 相似文献
4.
Reddy P Jaruga P O'Connor T Rodriguez H Dizdaroglu M 《Protein expression and purification》2004,34(1):126-133
Formamidopyrimidine DNA glycosylase (Fpg) is a DNA glycosylase with an associated AP lyase activity. As a DNA repair enzyme, Fpg excises several modified bases from DNA associated with exposure to oxidizing agents such as free radicals. Experiments in many laboratories have been limited by the availability of the enzyme, and its production required at least a week of work to complete its purification. We have devised a new method that decreases the time and expense of purification of Fpg that should render this protein accessible to any laboratory. Fpg was subcloned into a gamma P(L) promoter-containing vector (pRE) and overproduced in the appropriate Escherichia coli host cells to about 25% of the total cellular protein. Fpg was purified to homogeneity in a simple two-step procedure with a 50% saving in time when compared to the previously known procedure. Comparative studies showed that the excision of 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine, and to a lesser extent, 8-hydroxyadenine was virtually identical for the Fpg purified using this method and for the Fpg purified by the original method. Therefore, this method should prove useful for a large number of laboratories and further research on oxidative DNA damage. 相似文献
5.
Properties of 3-methyladenine-DNA glycosylase from Escherichia coli. 总被引:21,自引:0,他引:21
An Escherichia coli enzyme that releases 3-methyladenine and 3-ethyladenine in free form from alkylated DNA has been purified 2800-fold in 7% yield. The enzyme does not liberate several other alkylation products from DNA, including 7-methylguanine,O6-methylguanine, 7-methyladenine, N6-methyladenine, 7-ethylguanine, O6-ethylguanine, and the arylalkylated purine derivatives obtained by treatment of DNA with 7-bromomethyl-12-methylbenz[a]anthracene. The reaction of the enzyme with alkylated DNA leads to the introduction of apurinic sites but no chain breaks (less than one incision per ten apurinic sites), and there is no detectable nuclease activity with native DNA, depurinated DNA, ultraviolet-irradiated DNA, or X-irradiated DNA as potential substrates. The enzyme is termed 3-methyladenine-DNA glycosylase. It is a small protein, Mr = 19 000, that does not require divalent metal ions, phosphate, or other cofactors in order to cleave base-sugar bonds in alkylated DNA. 相似文献
6.
Liu P Theruvathu JA Darwanto A Lao VV Pascal T Goddard W Sowers LC 《The Journal of biological chemistry》2008,283(14):8829-8836
The repair of the multitude of single-base lesions formed daily in cells of all living organisms is accomplished primarily by the base excision repair pathway that initiates repair through a series of lesion-selective glycosylases. In this article, single-turnover kinetics have been measured on a series of oligonucleotide substrates containing both uracil and purine analogs for the Escherichia coli mispaired uracil glycosylase (MUG). The relative rates of glycosylase cleavage have been correlated with the free energy of helix formation and with the size and electronic inductive properties of a series of uracil 5-substituents. Data are presented that MUG can exploit the reduced thermodynamic stability of mispairs to distinguish U:A from U:G pairs. Discrimination against the removal of thymine results primarily from the electron-donating property of the thymine 5-methyl substituent, whereas the size of the methyl group relative to a hydrogen atom is a secondary factor. A series of parameters have been obtained that allow prediction of relative MUG cleavage rates that correlate well with observed relative rates that vary over 5 orders of magnitude for the series of base analogs examined. We propose that these parameters may be common among DNA glycosylases; however, specific glycosylases may focus more or less on each of the parameters identified. The presence of a series of glycosylases that focus on different lesion properties, all coexisting within the same cell, would provide a robust and partially redundant repair system necessary for the maintenance of the genome. 相似文献
7.
《Biophysical journal》2022,121(7):1276-1288
Polymerase chain reaction (PCR) is a powerful tool to diagnose infectious diseases. Uracil DNA glycosylase (UDG) is broadly used to remove carryover contamination in PCR. However, UDG can contribute to false negative results when not inactivated completely, leading to DNA degradation during the amplification step. In this study, we designed novel thermolabile UDG derivatives by supercomputing molecular dynamic simulations and residual network analysis. Based on enzyme activity analysis, thermolability, thermal stability, and biochemical experiments of Escherichia coli-derived UDG and 22 derivatives, we uncovered that the UDG D43A mutant eliminated the false negative problem, demonstrated high efficiency, and offered great benefit for use in PCR diagnosis. We further obtained structural and thermodynamic insights into the role of the D43A mutation, including perturbed protein structure near D43; weakened pairwise interactions of D43 with K42, N46, and R80; and decreased melting temperature and native fraction of the UDG D43A mutant compared with wild-type UDG. 相似文献
8.
D'Auria S Alfieri F Staiano M Pelella F Rossi M Scirè A Tanfani F Bertoli E Grycznyski Z Lakowicz JR 《Biotechnology progress》2004,20(1):330-337
The effect of temperature and glucose binding on the structure of the galactose/glucose-binding protein from Escherichia coli was investigated by circular dichroism, Fourier transform infrared spectroscopy, and steady-state and time-resolved fluorescence. The data showed that the glucose binding induces a moderate change of the secondary structure content of the protein and increases the protein thermal stability. The infrared spectroscopy data showed that some protein stretches, involved in alpha-helices and beta strand conformations, are particularly sensitive to temperature. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein is well represented by a three-exponential model and that in the presence of glucose the protein adopts a structure less accessible to the solvent. The new insights on the structural properties of the galactose/glucose-binding protein can contribute to a better understanding of the protein functions and represent fundamental information for the development of biotechnological applications of the protein. 相似文献
9.
Hazra TK Izumi T Venkataraman R Kow YW Dizdaroglu M Mitra S 《The Journal of biological chemistry》2000,275(36):27762-27767
8-Oxoguanine (G*), induced by reactive oxygen species, is mutagenic because it mispairs with A. The major G*-DNA glycosylase (OGG), namely, OGG1 in eukaryotes, or MutM in Escherichia coli, excises G* when paired in DNA with C, G, and T, but not A, presumably because removal of G* from a G*.A pair would be mutagenic. However, repair of G* will prevent mutation when it is incorporated in the nascent strand opposite A. This could be carried out by a second OGG, OGG2, identified in yeast and human cells. We have characterized a new OGG activity in E. coli and then identified it to be endonuclease VIII (Nei), discovered as a damaged pyrimidine-specific DNA glycosylase. Nei shares sequence homology and reaction mechanism with MutM and is similar to human OGG2 in being able to excise G* when paired with A (or G). Kinetic analysis of wild type Nei showed that it has significant activity for excising G* relative to dihydrouracil. The presence of OGG2 type enzyme in both E. coli and eukaryotes, which is at least as efficient in excising G* from a G*.A (or G) pair as from a G*.C pair, supports the possibility of G* repair in the nascent DNA strand. 相似文献
10.
Manuel RC Hitomi K Arvai AS House PG Kurtz AJ Dodson ML McCullough AK Tainer JA Lloyd RS 《The Journal of biological chemistry》2004,279(45):46930-46939
The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary beta-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-A structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys142 and Lys20. Analyses of the crystallographic structure also suggest a role for Glu161 in the apurinic/apyrimidinic lyase chemistry. The beta-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes beta and beta-delta elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre- and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants. 相似文献
11.
Escherichia coli MutY is an adenine DNA glycosylase active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches. Although MutY can form a covalent intermediate with its DNA substrates, its possession of 3' apurinic lyase activity is controversial. To study the reaction mechanism of MutY, the conserved Asp-138 was mutated to Asn and the reactivity of this mutant MutY protein determined. The glycosylase activity was completely abolished in the D138N MutY mutant. The D138N mutant and wild-type MutY protein also possessed different DNA binding activities with various mismatches. Several lysine residues were identified in the proximity of the active site by analyzing the imino-covalent MutY-DNA intermediate. Mutation of Lys-157 and Lys-158 both individually and combined, had no effect on MutY activities but the K142A mutant protein was unable to form Schiff base intermediates with DNA substrates. However, the MutY K142A mutant could still bind DNA substrates and had adenine glycosylase activity. Surprisingly, the K142A mutant MutY, but not the wild-type enzyme, could promote a beta/delta-elimination on apurinic DNA. Our results suggest that Asp-138 acts as a general base to deprotonate either the epsilon-amine group of Lys-142 or to activate a water molecule and the resulting apurinic DNA then reacts with Lys-142 to form the Schiff base intermediate with DNA. With the K142A mutant, Asp-138 activates a water molecule to attack the C1' of the adenosine; the resulting apurinic DNA is cleaved through beta/delta-elimination without Schiff base formation. 相似文献
12.
Hypoxanthine-DNA glycosylase from Escherichia coli. Partial purification and properties 总被引:1,自引:0,他引:1
Hypoxanthine-DNA glycosylase from Escherichia coli was partially purified by ammonium sulfate fractionation and by chromatography on Sephacryl S-200, DEAE-cellulose, and phosphocellulose P-11 columns. Analysis of the enzymatic reaction products was carried out on a minicolumn of DEAE-cellulose and/or by paper chromatography, by following the release of the free base [3H]hypoxanthine from [3H]dIMP-containing phi X174 DNA. In native conditions, the enzyme has a molecular mass of 60 +/- 4 kDa, as determined by gel filtration on Sephadex G-150 and Sephacryl S-200 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a major polypeptide band of an apparent molecular mass of 56 kDa, and glycerol gradient centrifugation indicated a sedimentation coefficient of 4.0 S. Hypoxanthine-DNA glycosylase from E. coli has an obligatory requirement for Mg2+ and is totally inhibited in the presence of EDTA. Co2+ can only partially replace Mg2+. The enzyme is inhibited by hypoxanthine which at 4 mM causes 85% inhibition. The optimal pH range of the enzymatic activity is 5.5-7.8, and the apparent Km value is 2.5 x 10(-7) M. 相似文献
13.
Ultraviolet irradiation of DNA produces a variety of pyrimidine base damages. The activities of Escherichia coli endonuclease III and a human lymphoblast endonuclease that incises ultraviolet-irradiated DNA at modified cytosine moieties were compared. Both the bacterial and human enzymes release this cytosine photoproduct as a free base. These glycosylase activities are linear with times of reaction, quantities of enzyme, and irradiation dosages of the substrates. Both enzyme activities are similarly inhibited by the addition of monovalent and divalent cations. Analysis by DNA sequencing identified loci of endonucleolytic incision as cytosines. These are neither cyclobutane pyrimidine dimers, 6-(1,2-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4(1H,3H)-pyrimidinediones, nor apyrimidinic sites. This cytosine photoproduct is separable from unmodified cytosine by high-performance liquid chromatography. This separation should facilitate identification of this modified cytosine and elucidation of its biological significance. 相似文献
14.
A new assay specific for uracil-DNA glycosylase is described, Escherichia coli mutants partially and totally deficient in uracil-DNA glycosylase activity have been isolated by using this assay in mass-screening procedures. These have been designated ung mutants. The ung gene maps between tyrA and nadB on the E. coli chromosome. T4 phage containing uracil in their DNA grow on the most glycosylase-deficient hosts but are unable to grow on wild-type bacteria. This provides a simple spot test for the ung genotype. The ung mutants show slightly higher rates of spontaneous mutation to antibiotic resistance. Taken together, these results suggest a central role for uracil-DNA glycosylase in the initiation of an excision repair pathway for the exclusion of uracil from DNA. 相似文献
15.
Uracil DNA glycosylase inhibitor (Ugi), a protein of 9.4 kDa consists of a five-stranded antiparallel beta sheet flanked on either side by single alpha helices, forms an exclusive complex with uracil DNA glycosylases (UDGs) that is stable in 8M urea. We report on the mutational analysis of various structural elements in Ugi, two of which (hydrophobic pocket and the beta1 edge) establish key interactions with Escherichia coli UDG. The point mutations in helix alpha1 (amino acid residues 3-14) do not affect the stability of the UDG-Ugi complexes in urea. And, while the complex of the deltaN13 mutant with UDG is stable in only approximately 4M urea, its overall structure and thermostability are maintained. The identity of P37, stacked between P26 and W68, was not important for the maintenance of the hydrophobic pocket or for the stability of the complex. However, the M24K mutation at the rim of the hydrophobic pocket lowered the stability of the complex in 6M urea. On the other hand, non-conservative mutations E49G, D61G (cancels the only ionic interaction with UDG) and N76K, in three of the loops connecting the beta strands, conferred no such phenotype. The L23R and S21P mutations (beta1 edge) at the UDG-Ugi interface, and the N35D mutation far from the interface resulted in poor stability of the complex. However, the stability of the complexes was restored in the L23A, S21T and N35A mutations. These analyses and the studies on the exchange of Ugi mutants in preformed complexes with the substrate or the native Ugi have provided insights into the two-step mechanism of UDG-Ugi complex formation. Finally, we discuss the application of the Ugi isolates in overproduction of UDG mutants, toxic to cells. 相似文献
16.
Kuznetsov NA Koval VV Zharkov DO Vorobjev YN Nevinsky GA Douglas KT Fedorova OS 《Biochemistry》2007,46(2):424-435
Formamidopyrimidine-DNA glycosylase (Fpg) is responsible for removal of 8-oxoguanine (8-oxoG) and other oxidized purine lesions from DNA and can also excise some oxidatively modified pyrimidines [such as dihydrouracil (DHU)]. Fpg is also specific for a base opposite the lesion, efficiently excising 8-oxoG paired with C but not with A. We have applied stopped-flow kinetics using intrinsic tryptophan fluorescence of the enzyme and fluorescence of 2-aminopurine-labeled DNA to analyze the conformational dynamics of Escherichia coli Fpg during processing of good substrates (8-oxoG.C), poor substrates (8-oxoG.A), and substrates of unclear specificity (such as DHU and 8-oxoG opposite T or G). The analysis of fluorescence traces allows us to conclude that when the enzyme encounters its true substrate, 8-oxoG.C, the complex enters the productive catalytic reaction after approximately 50 ms, partitioning the substrate away from the competing dissociation process, while poor substrates linger in the initial encounter complex for longer. Several intermediate ES complexes were attributed to different structures that exist along the reaction pathway. A likely sequence of events is that the damaged base is first destabilized by the enzyme binding and then everted from DNA, followed by insertion of several amino acid residues into DNA and isomerization of the enzyme into a pre-excision complex. We conclude that rejection of the incorrect substrates occurs mostly at the early stage of formation of the pre-eversion recognition complex, supporting the role of indirect readout in damage recognition. 相似文献
17.
Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli 总被引:12,自引:0,他引:12
K Sakumi Y Nakabeppu Y Yamamoto S Kawabata S Iwanaga M Sekiguchi 《The Journal of biological chemistry》1986,261(33):15761-15766
We constructed a recombinant plasmid carrying a gene that suppresses tag mutation. To overproduce its gene product, a 0.8-kilobase DNA fragment which carries the gene was placed under the control of the lac promoter in pUC8. 3-Methyladenine-DNA glycosylase activity in cells carrying such plasmids (pCY5) was 450-fold higher than that of wild type strain, on exposure to isopropyl-beta-D-thiogalactopyranoside. From an extract of such cells, the enzyme was purified to apparent physical homogeneity, and the amino acid composition and the amino-terminal amino acid sequence of the enzyme were determined. The data were in accord with nucleotide sequence of the gene, determined by the dideoxy method. It was deduced that 3-methyladenine-DNA glycosylase I comprises 187 amino acids and its molecular weight is 21,100, consistent with the value estimated from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein. Only 3-methyladenine was excised from methylated DNA by the purified glycosylase. These results show that the tag is the structural gene for 3-methyladenine-DNA glycosylase I. 相似文献
18.
Cloning of Micrococcus luteus 3-methyladenine-DNA glycosylase genes in Escherichia coli 总被引:1,自引:0,他引:1
The 3-methyladenine-DNA glycosylase (m3ADG) excises 3-methyladenine (m3A) residues formed in DNA after treatment with alkylating agents. In Escherichia coli, the repair of this type of damage depends on the products of the genes tagA and/or alkA, which code for m3ADG I (20 kDa) and II (30 kDa), respectively. The tagA- and alkA--single mutants are sensitive to alkylating agents, the double mutant much more so. We have cloned two genes of Micrococcus luteus that can partly substitute the function of the E. coli tagA- and alkA- genes. An M. luteus genome bank was made by shotgun cloning of EcoRI + BamHI-digested DNA into pBR322. Two hybrid plasmids were identified that confer methylmethane sulfonate (MMS) resistance to the tagA- ada+ mutant and a capacity to reactivate MMS-treated bacteriophage lambda. Each hybrid plasmid directed the synthesis of 21-kDa m3ADG in E. coli tagA- ada-, which were not inhibited by 4 mM m3A. However, the restriction maps of the two cloned genes were different, and they showed no sequence homology as judged by the lack of cross hybridization. 相似文献
19.
Distribution of double-helix thermal stability of Escherichia coli and eukaryotic DNAs was analyzed. The results confirmed the previous propositions based on the study of the stability distribution in phage DNAs: (1) stability fluctuation appears near the boundaries of protein coding regions (PCRs) and non protein coding regions (NPCRs); (2) PCRs have less fluctuation than NPCRs. The present analysis also revealed that the local G + C content is lower in the beginning of PCRs of E. coli than the average G + C content of PCR and that deviations in the amino acid composition and the third letter usage PCRs are involved in the low G + C content; the biological meaning of this is discussed in relation to mRNA structure. 相似文献
20.
Uracil–DNA glycosylase (Ung) is a DNA repair enzyme that excises uracil bases from DNA, where they appear through deamination of cytosine or incorporation from a cellular dUTP pool. DNA repair enzymes often use one-dimensional diffusion along DNA to accelerate target search; however, this mechanism remains poorly investigated mechanistically. We used oligonucleotide substrates containing two uracil residues in defined positions to characterize one-dimensional search of DNA by Escherichia coli Ung. Mg2+ ions suppressed the search in double-stranded DNA to a higher extent than K+ likely due to tight binding of Mg2+ to DNA phosphates. Ung was able to efficiently overcome short single-stranded gaps within double-stranded DNA. Varying the distance between the lesions and fitting the data to a theoretical model of DNA random walk, we estimated the characteristic one-dimensional search distance of ∼100 nucleotides and translocation rate constant of ∼2 × 106 s−1. 相似文献