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Uracil-DNA glycosylase activity from Dictyostelium discoideum
Abstract
We have isolated and partially characterized a uracil-DNA glycosylase activity from the cellular slime mold, Dictyostelium discoideum. This glycosylase has a broad pH optimum (6.5-8.5) and is fully active in 10 mM EDTA or in 5 mM Mg2+. Its molecular weight by gel filtration is about 55 000. This enzyme activity may work in concert with previously described apurinic/apyrimidinic (AP) endonuclease activities in the excision repair of uracil from the DNA of this lower eukaryote.
... The mode of action of this enzyme has been shown to be the hydrolytic fission of the N-glycosidic bond between uracil base and the deoxyribose sugar (Lindahl et al., 1977) with the subsequent production of an abasic site in the DNA and free uracil base (Figure 1.2). UDGase activity has been demonstrated in a wide variety of organisms, and the enzyme has been studied extensively (Cone et al., 1977;Lindahl et al., 1977;Talpaert-Borlé et al., 1979;Leblanc et al., 1982;Crosby et al., 1981;Kaboev et al., 1981;Blaisdell and Warner, 1983;Colson and Verly, 1983;Kaboev et al., 1985;Guyer et al., 1986;Bensen and Warner, 1987;Caradonna et al., 1987;Morgan and Chlebek, 1989;Olsen et al., 1989;Williams and Pollack, 1990;Masters et al., 1991;Bones, 1993;Upton et al., 1993;Winters and Williams, 1993). The enzyme, from all sources so far studied, has been shown to be monomeric and active in the absence of divalent metal ions (and in the presence of chelators such as EDTA and EGTA) or other co-factors. ...
The work presented in this thesis describes experiments carried out in order to determine the three-dimensional structure of a DNA repair enzyme, uracil-DNA glycosylase. An open reading frame, UL2, in the herpes simplex virus type 1 genome, is known to encode a uracil-DNA glycosylase. By sequence homology, there are three candidate start codons which might express a functional uracil-DNA glycosylase. Expression from two of these was attempted in Escherichia coli, using plasmids designed for high level production of recombinant proteins. The second candidate start codon produces high levels of a soluble, functional uracil-DNA glycosylase in Escherichia coli both in a native form, and as part of a fusion protein. Both the fusion and the native form of the enzyme have been purified to apparent homogeneity, as has a recombinantly expressed insoluble Escherichia coli uracil-DNA glycosylase. Preliminary attempts were made at deriving structural and functional information from the soluble, native recombinant herpes simplex enzyme with the use of circular dichroism. This form of uracil-DNA glycosylase has subsequently been crystallised in two ways, firstly as the free enzyme, and secondly in a complex with a single stranded DNA oligonucleotide. Extensive optimisation of the crystallisation parameters have been carried out in conjunction with modifications to the original purification protocol, and large, single crystals of both free, and DNA bound forms, suitable for X-ray diffraction studies are now readily reproduced. A systematic search for isomorphous heavy atom derivatives has been carried out for both types of crystal, and preliminary phases have been obtained for the DNA-bound form of the enzyme. This has enabled the calculation of an electron density map in which protein secondary structure features can be located. Improvement of this map will reveal the molecular structure of the enzyme/ DNA complex.
... The pH optima obtained for vsUNG and vcUNG are relatively broad and in agreement with what has been reported for other char- acterized UNGs [38][39][40][41][42]. A shift in [NaCl] optimum was observed for both vsUNG and vcUNG, where the optimum conditions changed from low concentrations at high pH to higher concentrations at low pH. ...
The genes encoding uracil-DNA N-glycosylase (UNG) from the marine, psychrophilic bacterium Vibrio salmonicida and the mesophilic counterpart Vibrio cholerae have been cloned and expressed in Escherichia coli. The purified proteins have been characterized in order to reveal possible cold adapted features of the V. salmonicida UNG (vsUNG) compared to the V. cholerae UNG (vcUNG). Characterization experiments demonstrated that both enzymes possessed the highest activities at pH 7.0–7.5 and at salt concentrations in the range of 25–50 mM NaCl. Temperature optima for activity were determined to approximately 30 °C for vsUNG and 50 °C for vcUNG. Temperature stability of the enzymes was compared at 4 °C and 37 °C, and vsUNG was found to be more temperature labile than vcUNG. Kinetic studies performed at three different temperatures, 15 °C, 22 °C and 37 °C, demonstrated higher catalytic efficiency for vsUNG compared to vcUNG due to lower KM-values. The increased substrate affinity of vsUNG is probably caused by an increased number of positively charged residues in the DNA-binding site of the enzyme compared to vcUNG. Thus, activity and stability measurements reveal typical cold adapted features of vsUNG.
... It has been shown to act both in a processive 'sliding mechanism', where it locates sequential uracil residues prior to dissociation from the DNA, and a distributive 'random hit' mechanism (Higley and Lloyd, 1993; Purmal et al., 1994; Bennett et al., 1995). UDG has previously been isolated and characterized from rat liver (Domena and Mosbaugh, 1985), human cells and tissues (Wittwer and Krokan, 1985; Wittwer et al., 1989), calf-thymus (Talpaert- Borle et al., 1979), slime mold (Guyer et al., 1986), yeast (Crosby et al., 1981), wheat germ (Blaisdell and Warner, 1983), prokaryotes (Lindahl , 1974; Cone et al., 1977; Williams and Pollack , 1990; Sobek et al., 1996; Purnapatre and Varshney, 1998) and viruses (Winters and Williams, 1993; Focher et al., 1993). ...
Uracil-DNA glycosylase (UDG; UNG) has been purified 17000-fold from Atlantic cod liver (Gadus morhua). The enzyme has an apparent molecular mass of 25 kDa, as determined by gel filtration, and an isoelectric point above 9.0. Atlantic cUNG is inhibited by the specific UNG inhibitor (Ugi) from the Bacillus subtilis bacteriophage (PBS2), and has a 2-fold higher activity for single-stranded DNA than for double-stranded DNA. cUNG has an optimum activity between pH 7.0-9.0 and 25-50 mM NaCl, and a temperature optimum of 41 degrees C. Cod UNG was compared with the recombinant human UNG (rhUNG), and was found to have slightly higher relative activity at low temperatures compared with their respective optimum temperatures. Cod UNG is also more pH- and temperature labile than rhUNG. At pH 10.0, the recombinant human UNG had 66% residual activity compared with only 0.4% for the Atlantic cUNG. At 50 degrees C, cUNG had a half-life of 0.5 min compared with 8 min for the rhUNG. These activity and stability experiments reveal cold-adapted features in cUNG.
We have cloned an AP endonuclease gene (APEA) from Dictyostelium discoideum, along with 1.8 kb of the 5′ flanking region. There are no introns. The sequence predicts a protein of 361 amino acids, showing
high homology to the major human/Escherichia coli exonuclease III family of AP endonucleases. There is 47% identity and 64% similarity to the Ape endonuclease of human cells
using the C-terminal 257 amino acids of the Dictyostelium protein. The 104 amino acids on the N-terminus show only low homology with other AP endonucleases. Instead, this region shows
high homology with the acid-rich regions of proteins associated with chromatin, such as nucleolins and HMG proteins. The gene
is transcriptionally activated up to 7-fold after treatment of cells with sublethal levels of DNA damaging agents, including
ultraviolet light, MNNG and bleomycin. Induction does not occur following blocking of replication fork polymerases with aphidicolin.
It is not eliminated by treatment with kinase or phosphatase inhibitors. Four DNA damage-sensitive mutants all retained the
DNA damage-induced up-regulation.
DNA double strand breaks (DSBs) are a particularly cytotoxic variety of DNA lesion that can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ). HR utilises sequences homologous to the damage DNA template to facilitate repair. In contrast, NHEJ does not require homologous sequences for repair but instead functions by directly re-joining DNA ends. These pathways are critical to resolve DSBs generated intentionally during processes such as meiotic and site-specific recombination. However, they are also utilised to resolve potentially pathological DSBs generated by mutagens and errors during DNA replication. The importance of DSB repair is underscored by the findings that defects in these pathways results in chromosome instability that contributes to a variety of disease states including malignancy. The general principles of NHEJ are conserved in eukaryotes. As such, relatively simple model organisms have been instrumental in identifying components of these pathways and providing a mechanistic understanding of repair that has subsequently been applied to vertebrates. However, certain components of the NHEJ pathway are absent or show limited conservation in the most commonly used invertebrate models exploited to study DNA repair. Recently, however, it has become apparent that vertebrate DNA repair pathway components, including those involved in NHEJ, are unusually conserved in the amoeba Dictyostelium discoideum. Traditionally, this genetically tractable organism has been exploited to study the molecular basis of cell type specification, cell motility and chemotaxis. Here we discuss the use of this organism as an additional model to study DNA repair, with specific reference to NHEJ.
The DNA repair enzyme uracil-DNA glycosylase from Mycoplasma lactucae (831-C4) was purified 1,657-fold by using affinity chromatography and chromatofocusing techniques. The only substrate for the enzyme was DNA that contained uracil residues, and the Km of the enzyme was 1.05 +/- 0.12 microM for dUMP containing DNA. The product of the reaction was uracil, and it acted as a noncompetitive inhibitor of the uracil-DNA glycosylase with a Ki of 5.2 mM. The activity of the enzyme was insensitive to Mg2+, Mn2+, Zn2+, Ca2+, and Co2+ over the concentration range tested, and the activity was not inhibited by EDTA. The enzyme activity exhibited a biphasic response to monovalent cations and to polyamines. The enzyme had a pI of 6.4 and existed as a nonspherical monomeric protein with a molecular weight of 28,500 +/- 1,200. The uracil-DNA glycosylase from M. lactucae was inhibited by the uracil-DNA glycosylase inhibitor from bacteriophage PBS-2, but the amount of inhibitor required for 50% inhibition of the mycoplasmal enzyme was 2.2 and 8 times greater than that required to cause 50% inhibition of the uracil-DNA glycosylases from Escherichia coli and Bacillus subtilis, respectively. Previous studies have reported that some mollicutes lack uracil-DNA glycosylase activity, and the results of this study demonstrate that the uracil-DNA glycosylase from M. lactucae has a higher Km for uracil-containing DNA than those of the glycosylases of other procaryotic organisms. Thus, the low G + C content of the DNA from some mollicutes and the A.T-biased mutation pressure observed in these organisms may be related to their decreased capacity to remove uracil residues from DNA.
Publisher Summary This chapter reviews the DNA uracil repair and uracil–DNA glycosylases (UDG). The major source of DNA uracil in prokaryotic and eukaryotic cells is transient incorporation of dUMP during replication. This replicative uracil is quickly repaired by UDG, apyramidinic/apurinic (AP) endonucleases, and other enzymes of excision repair. The rate of dUMP incorporation depends mainly on the size of the intracellular dUTP pool, which vanes from 0.001 to 1% of the dTTP pool and seems to be closely regulated by physiological and genetic factors. The minor source of DNA uracil is spontaneous hydrolytic deamination of DNA cytosine, and the rate of cytosine deamination is several orders of magnitude lower than the rate of dUMP incorporation in proliferating cells. Normal capacity of UDG-driven repair is enough to repair both replicative and hydrolytic uracil, but saturation of UDG with replicative uracil leads to inefficient repair of hydrolytic uracil and to increase of C → T mutagenesis. The rate of mutagenesis is modulated by the size of the dUTP pool and by the capacity of the UDG-driven repair pathway.
Uracil-DNA glycosylase has been purified approximately 130,000-fold from extracts of human placenta. Although all of the uracil-DNA glycosylase activity coeluted through six chromatographic steps, at least four distinct peaks of activity were resolved in the final purification on a Mono S column. Each of the peaks containing uracil-DNA glycosylase activity contained two peptides of Mr = 29,000 and Mr = 26,500, respectively, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experimental evidence indicated that the Mr = 29,000 peptide was the uracil-DNA glycosylase enzyme. The amino-terminal sequence of each peptide was determined after blotting of the peptides from the gel onto Polybrene GF/C paper. The sequences were not related to each other, and neither was any significant homology to other proteins found. Uracil-DNA glycosylase had a molecular turnover number of approximately 600/min and apparent Km value of 2 microM. The enzyme is a basic protein and was stimulated about 10-fold by 60-70 mM NaCl whereas higher concentrations were inhibitory.
We have used a thymidine auxotroph of the simple eukaryote, Dictyostelium discoideum and alkaline sucrose gradients of isolated nuclei to study alterations in DNA synthesis following irradiation of replicating haploid cells with 254 nm UV light. Three responses were characterized using pulse-chase protocols: (1) Lags in DNA synthesis as measured by the amount of label incorporated were 4, 9, and 20 h after 10, 50, and 200 J/m2. (2) The DNA synthesized during a 15-min pulse immediately after irradiation was of lower single strand molecular weight: 7, 3.5, and 3 x 10(6) dalton after 0, 50, and 200 J/m2. (3) The time required for maturation of the nascent DNA to full-sized single strands of about 2 x 10(8) dalton was 45-50 min for unirradiated cells, 3 h after 10 J/m2, and 20 h after 200 J/m2. The DNA of the irradiated cells did not mature uniformly during these delays; instead, a period of no increase in size was followed by a rapid, nearly control rate of maturation. We conclude: (a) at least some UV lesions block elongation of replicons; (b) the elongation of the replicons and their subsequent joining to yield mature high molecular weight DNA occurs after most of the lesions are repaired; (c) the timing of the different aspects of recovery suggest that initiation of replication is also inhibited.
Recent approaches to the study of DNA repair in Dictyostelium discoideum are reviewed. Thymidine auxotrophs facilitate the uptake of labeled thymidine into DNA during its replication and repair. The tmpA600 mutation leads to a loss of thymidylate synthase activity, and tdrA600 results in increased transport of thymidine into the cell. In the HPS401 double mutant (tmpA600tdrA600), thymidine is taken up uniformly into the nuclear and mitochondrial DNAs at levels up to 50-fold that in the wild type. tmpA maps on linkage group III. tdrA is on IV or VI, which cosegregate in strains containing this mutation. Alkaline sucrose gradients of nuclei from HPS401 pulsed for 15 min with [3H]thymidine in axenic medium show that the initially labeled single-strand DNA is about 7 x 10(6) daltons, which may be the size of the replicon. This nascent DNA matures in about 45 minutes to 2 x 10(8) daltons. Ultraviolet light (254 nm) decreases the size of the nascent DNA and delays its maturation. In addition to studies of DNA repair utilizing repair-proficient and -deficient mutants of thymidine auxotrophs, we are currently using two approaches for cloning genes involved in repair: 1) genes are sought that can functionally complement repair defects in Saccharomyces cerevisiae following transformation with a D. discoideum DNA library in YEp 24(URA); 4-NQO is used for the selection of RAD transformants; and 2) we have characterized and purified to near-homogeneity two repair enzymes from D. discoideum--uracil-DNA glycosylase and AP-endonuclease. An N-terminal sequence has been determined for the glycosylase, and a synthetic oligonucleotide probe derived from this sequence will be used to screen for this gene. A similar approach is in progress for the AP-endonuclease.
Uracil residues are introduced into prokaryotic and eukaryotic deoxyribonucleic acid (DNA) as a normal physiological process during DNA synthesis, and by spontaneous chemical modification of cytosine residues in DNA; thus, the acquisition of uracil in cellular DNA is unavoidable. However, the rate of uracil accumulation may vary significantly, depending on the ratio of deoxyuridine triphosphate (dUTP) to deoxythymidine triphosphate (dTTP) in intracellular pools and on whether the cells are exposed to cytosinedeaminating agents. The biological consequences of uracil residues in DNA may have cytotoxic, mutagenic, or lethal effects. An uncontrolled accumulation of the uracil residues in DNA leads to various perturbations of molecular events, ranging from altered protein-nucleic acid interactions to uracil-DNA degradation. The importance of eliminating uracil from DNA is underscored, by the observation that the uracil-DNA repair pathway of almost every organism examined, is remarkably similar. It appears that not only is one nucleotide DNA repair evident in E. coli as well as in human cells, but also that uracil-DNA glycosylase is one of the most highly conserved polypeptides yet identified.
We have cloned an AP endonuclease gene (APEA) from Dictyostelium discoideum, along with 1.8 kb of the 5' flanking region. There are no introns. The sequence predicts a protein of 361 amino acids, showing high homology to the major human/Escherichia coli exonuclease III family of AP endonucleases. There is 47% identity and 64% similarity to the Ape endonuclease of human cells using the C-terminal 257 amino acids of the Dictyostelium protein. The 104 amino acids on the N-terminus show only low homology with other AP endonucleases. Instead, this region shows high homology with the acid-rich regions of proteins associated with chromatin, such as nucleolins and HMG proteins. The gene is transcriptionally activated up to 7-fold after treatment of cells with sublethal levels of DNA damaging agents, including ultraviolet light, MNNG and bleomycin. Induction does not occur following blocking of replication fork polymerases with aphidicolin. It is not eliminated by treatment with kinase or phosphatase inhibitors. Four DNA damage-sensitive mutants all retained the DNA damage-induced up-regulation.
Uracil DNA glycosylase hydrolyzes the N-glycosidic bond between sugar phosphate backbone and uracil residue appearing as the result of spontaneous deamination of cytosine or during wrong incorporation of dU residues during DNA synthesis. Uracil DNA glycosylases are very conservative enzymes. They have been recognized in all pro- and eukaryotic organisms and also in pox and herpes viruses. This review highlights the pathways of accumulation of uracil and its derivatives in DNA, the main physicochemical and biochemical properties of uracil DNA glycosylase, and regulation of its functioning. Special attention is paid to detailed mechanisms of recognition and removing of damaged (or wrong) base by uracil DNA glycosylase. These mechanisms have been validated by the methods of X-ray analysis and kinetic and thermodynamic approaches.
A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases. It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini. The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5. The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs. Neither RNA nor RNA-DNA hybrid are degraded by the enzyme. The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence. Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase. We suggest that this enzyme be designated as endonuclease V of E. coli.
Strains of Escherichia coli with a mutation in the sof (dnaS) locus show a higher than normal frequency of recombination (are hyper rec) and incorporate label into short (4-5S) DNA fragments following brief [3H]thymidine pulses [Konrad and Lehman, Proc. Natl. Acad. Sci. USA 72, 2150 (1975)]. These mutant strains have now been found to be defective in deoxyuridinetriphosphate diphosphohydrolase (dUTPase; deoxyuridinetriphosphatase, EC 3.6.1.23), the enzyme that catalyzes the hydrolysis of dUTP to dUMP and PPi. Reversion of one sof- mutation to sof+ restores dUTPase activity and abolishes the accumulation of labeled 4-5S DNA fragments. Mutants initially isolated as defective in dUTPase (dut-) are also hyper rec and show transient accumulation of short DNA fragments. Both the sof and dut mutations are located at 81 min on the E. coli map, closely linked to the pyrE locus. The sof and dut loci thus appear to be identical. A decrease in dUTPase as a consequence of a sof or dut mutation may result in the increased incorporation of uracil into DNA. Rapid removal of the uracil by an excision-repair process could then lead to the transient accumulation of short DNA fragments. It is possible that at least a portion of the Okazaki fragments seen in wild-type cells may originate in this way.
Uracil-DNA glycosidase, an enzyme that catalyzes the release of free uracil from uracil-containing DNA, has been purified 11,000-fold from E. coli cell extracts. The enzyme preparation was essentially homogenous, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme is a small monomeric protein of a molecular weight of 24,500 ± 1,000. The enzyme efficiently releases uracil from single-stranded DNA and also from double-stranded DNA with uracil residues hydrogen-bonded to either adenine or guanine residues. On the other hand, DNA molecules containing 5-bromouracil residues, pyrimidine dimers, or deaminated purine residues are not substrates. Uracil-DNA glycosidase does not cleave free dUMP at a detectable rate and shows little activity with uracil-containing oligonucleotides. It has no cofactor dependence and apparently acts by hydrolytic cleavage of the base-sugar bonds in dUMP residues, as there is no incorporation of phosphate or pyrimidines in the DNA when uracil is released. When the enzyme was incubated with covalently closed circular DNA containing a small number of uracil residues introduced by deamination of cytosine residues with bisulfite, alkali-labile sites, but no chain breaks were introduced in the DNA molecules. Such DNA molecules could subsequently be cleaved by an endonuclease that specifically attacks DNA at apurinic and apyrimidinic sites, E. coli endonuclease IV. These data indicate that uracil-DNA glycosidase functions in the repair of DNA containing accidentally introduced uracil residues.
An activity which released free uracil from dUMP-containing DNA was purified approximately 1,700-fold from extracts of Thermothrix thiopara, the first such activity to be isolated from extremely thermophilic bacteria. The enzyme appeared homogeneous, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a native molecular weight of 26,000 and existed as a monomer protein in water solution. The enzyme had an optimal activity at 70 degrees C, between pH 7.5 and 9.0, and in the presence of 0.2% Triton X-100. It had no cofactor requirement and was not inhibited by EDTA, but it was sensitive to N-ethylmaleimide. The purified enzyme did not contain any nuclease that acted on native or depurinated DNA. The Arrhenius activation energy was 76 kJ/mol between 30 and 50 degrees C and 11 kJ/mol between 50 and 70 degrees C. The rate of heat inactivation of the enzyme followed first-order kinetics with a half-life of 2 min at 70 degrees C. Ammonium sulfate and bovine serum albumin protected the enzyme from heat inactivation. One T. thiopara cell contains enough activity to release about 2 X 10(8) uracil residues from DNA during one generation time at 70 degrees C.
Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling.
Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated
sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted
growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity,
but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did
a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic
properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed
for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum
which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.
A uracil-DNA glycosylase has been purified over 1,000-fold from wheat germ, the first such repair activity isolated from a higher plant. The enzyme has a molecular weight of approximately 27,000 and is resistant to metal ion chelators, but inhibited by high concentrations of either mono or divalent cations. This glycosylase is unable to release uracil from the mononucleotides dUMP and dUTP or from wheat germ RNA. Twelve pyrimidine analogues which closely mimic uracil structurally and the nucleoside uridine were examined for their ability to inhibit glycosylase activity. However, only 5-azauracil and 6-aminouracil inhibited enzymatic release of uracil to the same degree as uracil itself. An inhibitor induced by bacteriophage T5 which inhibits Escherichia coli uracil-DNA glycosylase has been shown not to affect the glycosylase isolated from wheat germ, indicating that these two enzymes differ. The ability of the wheat germ uracil-DNA glycosylase to completely remove available uracil from synthetic DNA substrates in which thymine had been replaced by uracil in varying percentages was also examined and found not to depend on percentage of uracil in the substrates.
The bovine uracil-DNA glycosylase previously isolated from thymocyte nuclei was further purified by 1 order of magnitude with the aid of affinity chromatography. The final preparation was totally devoid of DNase and apurinic or apyrimidinic (AP) endonuclease activities, and it corresponded to purifications of 457-fold over the nuclear extract and of about 2000-fold over the crude tissue homogenate. Most of the general enzyme properties already described were confirmed. Furthermore, this mammalian uracil-DNA glycosylase was shown to bind specifically with polymerized and not with monomeric nucleotide compounds, while having a preference for double-stranded forms. It cleaved N-glycosyl linkages only at the deoxyuridyl units located in internal positions of polynucleotide chains. The enzyme also used RNA-DNA hybrids as functional substrates and was practically ineffective on deoxyuridyl residues at the 3'-ends of nucleic acids. The activity of the glycosylase was greatly impaired in assays with DNA substrates that contained amounts of AP sites exceeding 5 microM. The inhibitory concentrations of AP residues were about 100 times lower than those found equally effective for the other reaction product, i.e. free uracil, and were almost comparable to the Km values for deoxyuridyl nucleotides in the DNA substrates. This all appears as a modulation of the glycosylase catalysis by the relative amounts of its substrate and product structures in DNA. The data lead us to surmise that the removal of uracil from cellular DNA is functionally coupled to the expected elimination of the formed AP sites by specific endonucleases. Base-exchange and base-insertion experiments with the purified enzyme yielded negative results under various conditions. The glycosylase behaved essentially as a hydrolase which has no associated base-insertase properties and irreversibly excises uracil from DNA by a mechanism for channeling the process to the next steps of the repair pathway.
A uracil-DNA-glycosylase from Micrococcus luteus has been purified more than 3,000-fold. The enzyme preparation appears homogeneous, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is devoid of nonspecific endonucleases, specific endonucleases for apurinic and apyrimidinic sites, 3-methyladenine or 7-methylguanine-DNA-glycosylases. It behaves as a monomer protein of 19,400 daltons. It has an isoelectric point of 7.0 +/- 0.1. It has an optimal activity between pH 5.0 and 7.0. It has no cofactor requirement and is not inhibited by EDTA. Uracil-DNA-glycosylase is highly specific for DNA containing dUMP residues, releasing uracil as product of the reaction. It is 2-fold more active on single-stranded DNA than on double-stranded DNA. If it releases uracil dimers from ultraviolet-irradiated PBS1 DNA, it is at the threshold of the detection. The apparent Km is 7 X 10(-8) M, and uracil acts as a noncompetitive inhibitor with a Ki of 3.2 X 10(-4) M. Cis-syn cyclogbutadiuracil also is a potent inhibitor, while some analogs, produced by x-irradiation of uracil and thymine, are weak inhibitors. Spermine, between 10 and 400 microM, increases the enzymatic activity by 50% and is not inhibitory at other concentrations. Spermidine activates the enzyme at concentrations of 40 to 120 microM, but becomes inhibitory at 200 and 400 microM. A new finding is that drugs which intercalate in DNA, such as ethidium bromide and ellipticine, cause a 2- to 2.5-fold activation of this enzyme activity. The concentrations giving maximal activation depend on the drug. The enzyme does not behave as a processive enzyme during uracil excision.
Uracil-DNA glycosylase was partially purified from HeLa cells. Various substrates containing [3H]dUMP residues were prepared by nick-translatiqn of calf thymus DNA. The standard substrate was double-stranded DNA with
[3H]dUMP located internally in the chain. Compared to the release of uracil from this substrate, a 3-fold increase in the rate
was seen with single-stranded DNA, and a 20-fold reduction in the rate was observed when the [3H]dUMP-residue was located at the 3′end. The rate of [3H]uracil release decreased progressively when one, two or three of the dNMP residues were replaced by the corresponding rNMP;
in the extreme case when the substrate contained [3H]dUMP in addition to rCMP, rGMP and rAMP, the rate of [3H]uracil release was less than 3% of that of the control. The enzyme was inhibited to the same extent by uracil and the uracil
analogs 6-aminouracil and 5-azauracil, but very weakly, or not at all, by 5 other analogs. Our results suggest strongly that
uracil-DNA glycosylase has a high degree of selectivity for uracil in dUMP residues located internally in DNA chains and that
the recognition of the correct substrate also depends on the residues flanking dUMP being deoxyribonucleotides.
This chapter discusses that since DNA is the carrier of genetic information and spontaneous mutations occur only at low frequency, cellular DNA has often been regarded as an essentially stable entity. Recent developments have necessitated a revision of this view. With the discovery of insertion elements, it became clear that certain segments of DNA can move between many different chromosomal sites. Further, the susceptibility of DNA to heat-induced degradation at moderate temperatures and neutral pH leads to hydrolytic decay at a much faster rate than that expected from spontaneous mutation frequencies. The latter, somewhat paradoxical, observation can be rationalized by postulating the existence of efficient repair mechanisms to maintain the integrity of DNA. The chapter also discusses that several enzymes that act specifically on hydrolytically-damaged nucleotide residues in DNA have recently been discovered, purified, and characterized, and they are the main subject of the present review. Some of these enzymes, the DNA glycosylases, belong to a previously unrecognized class of enzymes that cleave base–sugar bonds in DNA. In addition to their role in surveying and removing DNA damage that would otherwise lead to unacceptable spontaneous mutation frequencies, the same enzymes may also play an important role in the repair of cellular lesions introduced by ionizing radiation or by exposure to chemical mutagens such as alkylating agents, nitrous acid, or bisulfite.
Mutagenesis has remained an intriguing aspect of genetics since the beginning of this century, and its analysis has proceeded hand in hand with the elucidation of gene replication and expression. Interest in this area has further heightened with the growing awareness that numerous environmental agents may cause mutations in humans. These mutations may lead to metabolic as well as neoplastic diseases. Advances during the past 15 years have revealed two major classes of mutagenic mechanisms: directly induced base mispairing, and misrepair. Alkylating agents for instance, generate many different reaction products in DNA, but only two of these (O6-alkylguanine and O4-alkylthymine) are likely candidates for directly induced mispairing. He has also turned out to be an important mutagen, one that presents a particular serious challenge to large genomes; it converts cytosine to uracil and guanine to an analogue of cytosine. DNA lesions that interrupt DNA chain elongation, including many of other products of alkylation, often trigger an error-prone postreplication repair process. Current evidence suggests that this process involves in incorrect insertion of bases into gaps in progeny-strand DNA opposite such a lesion. Mutagenic mechanisms are subject to powerful genetic controls that include the activities of DNA polymerases in the selection of deoxynucleoside triphosphates and the removal of incorrectly inserted nucleotides.
We have detected in crude extracts of Bacillus subtilis an N-glycosidase activity which catalyzes the release of free uracil from DNA of the subtilis phage PBS2 labeled with [3H]uridine. This DNA contains deoxyuridine instead of thymidine. The enzyme is active in the presence of 1.0 mM EDTA and under these conditions Escherichia coli or T7 DNA labeled with [3H]thymidine is not degraded to labeled acid-soluble products. The activity resembles an N-glycosidase from E. coli which releases free uracil from DNA containing deaminated cytosine residues. Both enzymes in crude extracts are active in the presence of EDTA, do not require dialyzable co-factors, and have the same pH optimum. They differ in that the enzyme from E. coli is more sensitive to heat, sulfhydryl reagents, and salt. The enzyme from B. subtilis is inactive on DNA containing 5-bromouracil or hydroxymethyluracil. Extracts of PBS2-infected B. subtilis lose the N-glycosidase activity within 4 min after infection and contain a factor that inhibits the N-glycosidase activity within 4 min after infection and contain a factor that inhibits the N-glycosidase activity in extracts of uninfected cells in vitro.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Two apurinic/apyrimidinic- (AP-) specific endonuclease activities have been isolated from the cells of Dictyostelium discoideum by fractionation on DEAE-cellulose, CM-cellulose and Sephadex G-75. These activities, designated A and B, have apparent molecular weights of 49000 and 40000, respectively. Although their precise reaction optima differ somewhat, both A and B quantitatively nick AP DNA best at pH 8.0-8.5 in low salt (less than 100 mM NaCl); both require Mg2+. These activities are apparently specific only for AP sites in DNA. The low activities observed on heavily ultraviolet-irradiated DNA, gamma-irradiated DNA and osmium tetroxide-treated DNA are consistent with the small numbers of secondary AP sites expected in these DNAs. Both A and B produce single-strand nicks in AP DNA that result in termini that serve as good primers for Escherichia coli polymerase I. Hence, A and B appear to be Class II AP endonucleases which yield 3'-OH termini at nicks on the 5' side of baseless sugars. It is unclear whether A and B are independently coded proteins, different post-translational modifications of the same gene product, or whether one is an artifact arising from the isolation. Many of the properties of these D. discoideum AP endonuclease activities are similar to those of the predominant AP endonucleases observed in bacterial, plant and animal cells. They will be of use in the characterization of excision repair in this organism.
Rat liver uracil-DNA glycosylase has been purified from nuclear extracts over 3000-fold to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a monomeric protein with a polypeptide molecular weight of approximately 35 000. It has a native molecular weight of 33 000 as determined by gel filtration chromatography and a sedimentation coefficient of 2.6 S in glycerol gradients. The nuclear enzyme has an alkaline pH optimum and a pI value of 9.3. Nuclear uracil-DNA glycosylase catalyzes the release of free uracil from both single-stranded and double-stranded DNA with the former being the preferred substrate. The enzyme is unable to recognize dUTP, dUMP, or poly(dA-dT) containing a 3'-terminal uracil residue as a substrate. However, internalization of terminal uracil residues by limited chain elongation produced a substrate for the glycosylase. Another species of uracil-DNA glycosylase has been partially purified from mitochondria. This activity differs from the nuclear enzyme in that it has (i) distinctive chromatographic properties, (ii) a lower native molecular weight of 20 000 as determined by molecular sieving, (iii) a distinct NaCl inhibition profile, and (iv) a longer half-life during thermal denaturation.
Uracil-DNA glycosylase activity in HeLa S3 cells was found in nuclei (70%), mitochondria (15%) and cytosol (15%) after fractionation in hypotonic buffers. After fractionation in isotonic buffers the activity in cytosol was increased, apparently as a consequence of leakage from the nuclei. Both in the nuclear and the mitochondrial fraction, a major 50 and a minor 18 kDa form were found after gel filtration in the presence of 0.5 M NaCl. However, after glycerol, gradient sedimentation or gel filtration in the presence of 2 M NaCl or 20% glycerol most of the 50 kDa form dissociated into a 22 kDa form, which was also the smallest catalytically active form found after partial trypsin digestion. The dissociation of the 50 kDa form was reversible. Biochemical properties of the nuclear and mitochondrial forms were very similar. Thus, they had similar apparent Km values, pH optima, heat sensitivities and activation energies, and were stimulated 2-5-fold by 40-60 mM monovalent salt.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Deoxyuridine can become resident in the DNA of prokaryotic and eukaryotic cells via two general mechanisms - deamination of cytosine to uracil, and nucleotide pool changes that lead to misincorporation of deoxyuridine in place of thymidine. In this paper we have examined the chemical basis of deamination reactions in DNA and discussed a possible mechanism for an increased rate of deamination by means of cross-strand protonation of cytosine by alkylated guanine. In addition, we have examined the genetic and drug-induced conditions that lead to dUMP misincorporation into DNA in place of thymidine and have presented experimental evidence indicating that the antifolate-induced lesion is a general drug-dose dependent lesion of human blood cells. Finally, the toxic and genetic impact of this lesion has been evaluated within the context of a review of the repair mechanisms elicited by dUMP in DNA.
Various DNA glycosylases exist, which initiate the first step in base-excision repair. A summary of the kinetic and physical characteristics of three classes of DNA glycosylases are presented here. The first class discussed, include glycosylases which recognize alkylated DNA. Various data from enzymes derived from both prokaryotic and eukaryotic sources is discussed. The second class deals with a glycosylase that recognizes and initiates the excision of pyrimidine dimers in DNA. To date, this enzyme has only been uncovered from two sources, Micrococcus luteus and the T4 bacteriophage of E. coli. The third class consists of the most studied of the glycosylases, the uracil-DNA glycosylase enzymes. Various characteristics are presented for the uracil-DNA glycosylases derived from various sources. Recent information from our laboratory is presented implicating that herpes simplex virus may mediate a uracil-DNA glycosylase activity in productivity infected cells.
A line of human lymphoid cells was tested for the presence of dUMP in DNA with or without treatment with the dihydrofolate reductase inhibitor, methotrexate. Cells treated with methotrexate and labeled with [(3)H]dUrd contained dUMP in DNA in readily detectable amounts ( approximately 0.8 pmol of dUMP per mumol of total DNA nucleotide), and this was increased approximately 3-fold if the cells were also treated with Ura at the same time. No dUMP (<1 fmol/mumol of DNA) could be detected by these methods in DNA from cells not treated with methotrexate, regardless of whether Ura was present or absent. The presence of dUMP in DNA from cells treated with methotrexate is a result of the great increase in intracellular concentration of dUTP and the fall in dTTP that accompany inhibition of thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) by the drug. These changes are apparently sufficient to overcome the normal mechanisms that exclude dUMP from DNA, and the enhancement by Ura reflects suppression of one of the mechanisms, Ura removal from DNA by the enzyme Ura-DNA glycosylase. The results suggest an active lesion of DNA in cells in which thymidylate synthetase is inhibited. Under these conditions there appears to be a cyclic incorporation and removal of dUMP resulting from reinsertion of dUMP during gap repair at sites of Ura removal. This consequence of the normal excision-repair process, which occurs when intracellular levels of dUTP approach those of dTTP, may have effects related to the cytotoxicity of drug inhibitors of thymidylate synthetase, clinical deficiencies of folate and vitamin B-12, and thymineless death, in general.
A sensitive endonuclease assay was used to study the fate of pyrimidine dimers introduced by ultraviolet irradiation into
the nuclear deoxyribonucleic acid of the cellular slime mold Dictyostelium discoideum. Analysis of the frequency of T4 endonuclease
V-induced single-strand breaks by alkaline sucrose gradient sedimentation showed that strain NC4 (rad+) removed greater than
98% of the dimers induced by irradiation at 40 J/m2 (254 nm) within 215 min after irradiation. HPS104 (radC44), a mutant sensitive
to ultraviolet irradiation, removed 91% under these conditions, although at a significantly slower rate than NC4: only 8%
were removed during the 10- to 15-min period immediately after irradiation, whereas NC4 excised 64% during this interval.
HPS104 thus appears to be deficient in the activity(ies) responsible for rapidly incising ultraviolet-irradiated nuclear deoxyribonucleic
acid at the sites of pyrimidine dimers.
THE findings that the thermal denaturation temperature (Tm) of DNA from a wide variety of bacterial, plant and animal sources as well as the buoyant density in a cæsium chloride gradient are linearly related to the guanine plus cytosine (G plus C) base composition1-2 have made it possible to search for the existence of DNA samples which would show an anomalous behaviour with respect to these two parameters. Native DNA preparations which show no correlation between their buoyant density and Tm would be expected to have components other than those usually encountered in DNA3; this situation might arise with a high degree of probability in the DNA isolated from bacteriophages or viruses, which because of their specialized behaviour would be expected to possess distinctive structural features.