Partial Purification and Characterization of a Uracil-DNA Glycosylase from Wheat Germ*

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 resist-ant 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 ex- amined 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germ uracil-DNA glycosylase to completely remove available uracil from synthetic DNA substrates in which thymine had been replaced by uracil in varying percentages was also examined and found not to depend on percentage of uracil in the substrates.

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germ uracil-DNA glycosylase to completely remove available uracil from synthetic DNA substrates in which thymine had been replaced by uracil in varying percentages was also examined and found not to depend on percentage of uracil in the substrates.
Uracil is not a usual constituent of DNA in either eucaryotes or procaryotes. However, uracil can be introduced into nascent DNA if intracellular dUTP concentrations are abnormally high, allowing incorporation of this nucleotide into the growing chain by DNA polymerase (1). Uracil can also arise in mature DNA if cytosine bases are deaminated, either spontaneously (2) or by chemical mutagens such as bisulfite (3). Uracil incorporation results in Ade-Ura base pairs of approximately the same size and shape as Ade-Thy pairs, but cytosine deamination replaces Gua-Cyt with Gua-Ura pairings leading to transition mutations if uncorrected prior to the next round of replication.
UraciI-DNA glycosylases remove uracil where it occurs in DNA by hydrolytically cleaving the N-glycosylic bond between the base and sugar, leaving an apyrimidinic site which is subsequently recognized and repaired by other enzymes (4, 5). Uracil-DNA glycosylases have been purified from several bacterial and eucaryotic organisms including Escherichia coli (61, Bacillus suhtilis ('i), and human blast cells (8) and found to be generally similar in such physical and enzymatic parameters as molecular weight, substrate specificity, and lack of * This research was supported by Research Grant PCM 8109637 from the National Science Foundation. This is Paper 13094 from the Minnesota Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "ahertisement" in accordance with 18 U.S.C. Section 1734 sole1.y to indicate this fact. $ Funded by National Institute of Health Grant T32-GM07323. To whom further correspondence should he addressed. metal cofactor requirements. So far, no comparable repair activity has been purified and characterized in higher plant tissues.
This report details the characterization of one such uracil-DNA glvcosylase, purified over 1000-fold from wheat germ.
The plant enzyme has been found to resemble already characterized uracil-DNA glycosylases in most respects, yet differs from the E. coli enzyme in its response to the uracil-DNA glycosylase inhibitor induced in phage T5-infected bacteria (9), indicating that some variation exists between these two examples of eucaryotic and procaryotic DNA repair enzymes.
In addition, the ability of the wheat germ glycosylase to remove uracil from synthetic DNA substrates was examined as a function of percentage of uracil present in the DNA.

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Wheat Germ Uracil Glycosylase
phates. Reactions were typically incubated at 37 "C for about 1% h (longer reaction periods resulted in loss of radioactivity from polynucleotide) followed by addition of 200 pl of 0.1 M EDTA to halt polymerase activity. These preparations were dialyzed against 0.01 M Tris-HC1, pH 7.5,0.2 M EDTA overnight to remove unincorporated nucleotides and purified on a Bio-Gel A-1.5m column to remove small oligonucleotides. For the poly(dA-dT) substrates, where the dUTP-dTTP ratio was 100:0, there was an average of one base between uracils in the newly synthesized region; where the dUTP-dTTP ratio was 50:50, there was an average of three bases between uracils; and where the ratio was 5:95, there was an average of 39 bases between uracils.
Uracil-DNA Glycosylase Assay Procedure-The assay for glycosylase activity is the same as that described in Duncan et al. and Warner and Rockstroh (12,13) using columns (0.3 X 15 cm) containing Bio-Rad AG I-X8 formate packed to a height of 3 cm. Unless otherwise specified, the standard incubation conditions were 30 min at 37 "C in an assay mixture containing 30 pl of 1 M 4-(2-hydroxyethyl)-I-piperazineethanesulfonic acid, pH 7.5, 10 pl of 0.04 M EDTA, pH 7.8, 10 p1 of 0.1 M dithiothreitol, 10 pl of 1 mg/ml of bovine serum albumin, and varying amounts of T5 DNA-containing uracil in a final assay volume of 200-210 pl. Where indicated, the T5 DNA substrate was replaced with uracil-containing calf thymus DNA or poly(dA-dT). All reactions were initiated with the addition of enzyme and stopped with 500 pl of 0.1 M ammonium formate, pH 4.2. After addition of formate, the reaction mixture was cooled on ice and then applied to the columns. Counting was performed in Aquasol 2 (New England Nuclear) scintillation counting mixture in a Beckman LS 7000 liquid scintillation spectrometer. One unit of uracil-DNA glycosylase is defined as the amount of enzyme needed to release l nmol of uracil from phage T5 DNA substrate/min under standard conditions. Typically, 0.001-0.002 unit of enzyme was used/assay during the characterization.
Molecular Wezht-Centrifugation through linear 10-2576 glycerol gradients was done using glycerol solutions containing 0.01 M potassium phosphate, pH 7.5, and 1 mM dithiothreitol. The samples were centrifuged for 42-43 h in an SW 50.1 head at 46,000 rpm in a Beckman L5-50 ultracentrifuge maintained at 2-4 "C. 150-pl fractions were collected and assayed for the presence of cytochrome c ( A d d , ovalbumin (AM), bovine serum albumin (AYw), and uracil-DNA glycosylase.
Gel filtration was performed in a column (1 X 25 cm) equilibrated prior to use with 0.01 M potassium phosphate, pH 7.5, 1 mM dithiothreitol, and 15% glycerol. Bovine serum albumin, ovalbumin, chymotrypsinogen, and cytochrome c protein standards were used to calibrate the column. The flow rate was maintained near 2.5 ml/h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the uracil-DNA glycosylase molecular weight was done on 11.25%) acrylamide gels according to the procedure of Laemmli and Favre (14). Protein staining was accomplished using the silver-staining technique described by Merril et al. (15). Wheat germ uracil-DNA glycosylase was prepared for electrophoresis by purifying the enzyme through the DEAE-cellulose stage as detailed under "Results." This preparation was loaded onto a hydroxylapatite column (1.0 X 2.0 cm) and eluted with a linear 0.01-0.4 M potassium phosphate, pH 7.5, gradient. Fractions containing enzymatic activity were examined by gel electrophoresis to correlate peak enzymatic activity with band intensity on the gel.
Miscellaneous-Partially purified phage T5 uracil-DNA glycosylase inhibitor and E . coli uracil-DNA glycosylase were the generous gifts of Richard Leder (University of Minnesota). Inhibitor was always added to assay mixtures before the enzyme.
Base analysis of the phage T5 DNA substrate and reaction products from glycosylase assays was carried out using descending paper chromatography for about 18 h as described by Warner et al. (IO). Correspondence between pyrimidine base standards and radioactivity was determined by visualizing the standards on the chromatograph under UV light and then cutting the paper into pieces (1 X 1 cm) and counting radioactivity.
All protein concentrations were determined using the Bradford (16) dye-binding technique. Exonuclease activity was determined using the procedure of Eichler and Lehman (17) with nondenatured E. coli [ 'HIDNA to measure double-stranded exonuclease activity or heat-denatured quick-cooled E. coli ['HIDNA to measure singlestranded exonuclease activity. Apurinic and nonspecific endonuclease activity was measured using the nicked DNA circle assay described by Kane and Linn (18) using untreated and acid-depurinated PM2 ['HIDNA.

Purification
Preparation of Crude Extract-All procedures were carried out at 0-4 "C unless otherwise stated. 500 g of wheat germ were suspended in 2 liters of buffer containing 0.01 M potassium phosphate, pH 7.3, 15% glycerol, and 1 mM dithiothreitol (PGD buffer) and homogenized for 1 min at high speed in a Waring blender. Cell wall debris was removed by centrifugation for 15 min at 10,000 X g. This supernatant was then filtered through several layers of cheesecloth. 150 ml of 5% (w/v) streptomycin sulfate were slowly added to the approximately 2 liters of turbid supernatant with gentle stirring and allowed to sit for 20 min on ice followed by centrifugation for 15 min at 10,000 X g to remove nucleic acids.
Ammonium Sulfate Precipitation-Following precipitation of the nucleic acids with streptomycin, the supernatant was brought to 50% saturation with solid ammonium sulfate while maintaining the p H at 7.3 and then allowed to sit on ice for 30 min with stirring. This suspension was centrifuged for 15 min at 15,000 X g. The supernatant from this first cut was then brought to 80% saturation with solid ammonium sulfate and stirred on ice for 90 min followed by centrifugation for 15 min at 15,000 X g. T h e 50-80% fraction was found to contain most uracil-DNA glycosylase activity and was redissolved in 750 ml of PGD buffer and dialyzed against this buffer overnight to remove ammonium sulfate. DEAE-cellulose Chromatography-The dialyzed redissolved pellet was divided into 250-ml portions for easier handling. Each portion was loaded onto a DEAE-cellulose column (8 X 30 cm) equilibrated with PGD buffer. The column was then washed with PGD. Uracil-DNA glycosylase activity eluted prior to the bulk of wheat germ protein without requiring a salt gradient. Peak fractions were collected, pooled, and dialyzed against PGD buffer. Phosphocellulose Chromatography-The combined fractions containing uracil-DNA glycosylase activity were next loaded onto a phosphocellulose column (2.7 X 30 cm) equilibrated with PGD buffer. The column was washed with PGD buffer and then eluted with a linear 0.01-0.4 M KC1 gradient. Fractions containing peak activity were pooled, and the protein was precipitated with a saturating concentration of solid ammonium sulfate followed by centrifugation at 15,000 X g for 15 min. T h e pellet was resuspended in 20 ml of PGD buffer and dialyzed against this buffer to remove ammonium sulfate.
Sephadex G-IO0 Chromatography-This solution was loaded onto a Sephadex G-100 column (27 X 30 cm) equilibrated with PGD buffer and eluted with the same buffer.
Fractions containing uracil DNA-glycosylase activity were pooled. Bio-Gel Hydroxylapatite Chromatography-The pooled uracil-DNA glycosylase fractions were chromatographed on a hydroxylapatite column (1.0 X 25.0 cm) equilibrated with P G D buffer. To elute enzyme activity, a linear 0.01-0.4 M potassium phosphate gradient was used. Peak glycosylase fractions were pooled, and the protein was precipitated with a saturating concentration of solid ammonium sulfate followed by centrifugation. T h e pellet was resuspended and dialyzed against PGD buffer.
Bio-Gel A-1.5m Chromatography-Tbe concentrated glycosylase sample was applied to a Bio-Gel A-1.5m column (1.8 X 19 cm) and eluted with PGD buffer. The pooled glycosylase fractions were then concentrated via Millipore filtration.
Second Phosphocellulose Chromatography-The concen-trated enzyme was applied to a phosphocellulose column (1 X 3 cm) previously equilibrated with PGD buffer and washed with 5 column volumes of this buffer prior to elution of enzyme activity with a linear 0.01-0.4 M potassium phosphate gradient. The pooled enzyme fractions showed little loss of activity when stored at -20 "C in 40% glycerol. The purification of the enzyme is summarized in Table I.
The possible presence of double-and single-stranded DNA exonucleases as well as nonspecific endonucleases was examined in the final enzyme preparation. These nucleases were found to be less than 0.2% of the uracil-DNA glycosylase activity for double-stranded exonucleases, less than 2.0% for single-stranded exonucleases, and less than 0.1% for nonspecific endonucleases. An apurinic endonuclease co-chromatographed with the wheat germ uracil-DNA glycosylase activity during initial stages of purification, but had resolved away from peak glycosylase activity by the second phosphocellulose column. Apurinic endonuclease activity was less than 0.1% of glycosylase activity after the final stage of purification.

Verification of Uracil Release
To determine which pyrimidine bases were released by the partially purified glycosylase activity, the enzyme was incubated with uracil-containing phage T5 DNA long enough to release virtually all of the available uracil. This substrate contains approximately 40% of its tritium in cytosine, 50% in thymine, and the remaining 10% in uracil, making it particularly sensitive to the release of either cytosine or thymine. Fig.   1 shows that all of the radioactivity migrates as a single peak exactly paralleling the mobility of the uracil standard. No radioactivity co-migrates with either cytosine or thymine standards on the chromatograph, demonstrating that the wheat germ glycosylase liberates exclusively uracil from among the three pyrimidines potentially available in the DNA substrate.

FIG.
1. Base analysis of reaction products. Uracil DNA-glycosylase reaction products were analyzed using descending paper chromatography with an 86% 1-butanol solvent system as previously described (10) by spotting radiolabeled reaction products along with 0.04 pmol each of cytosine, uracil, and thymine dissolved in HCI.
Physical Characterization of the Enzyme Molecular Weight-The molecular weight of the wheat germ uracil-DNA glycosylase was determined to be 27,000 by comparison with the elution position of four standards of known molecular weight on a Sephadex G-100 column (Fig.  2).
Ultracentrifugation on glycerol gradients was also used to obtain a molecular weight by relating the sedimentation behavior of three standards with known so^., values to that of wheat germ glycosylase (data not shown). If the partial specific volume of the glycosylase is assumed to be close to 0.725, as is true for many proteins, the molecular weight was calculated to be about 28,000 (19). By correlating glycosylase activity present in fractions collected from a hydroxylapatite column with band intensity when these same fractions are examined on a sodium dodecyl sulfate-polyacrylamide gel, it is possible to match a particular band with the wheat germ glycosylase activity. As Fig. 3 shows, the band correlating best with glycosylase activity (indicated by the arrow) has a molecular weight of approxi- mately 25,000, which agrees with the sizes obtained by gel filtration and ultracentrifugation for this enzyme. Assuming the designated band does represent the glycosylase, Fig. 3 also shows that the activity is a relatively minor component among the plant's complement of proteins.
Temperature a n d pH Optima-The wheat germ glycosylase is moderately stable a t elevated temperatures, retaining about 50% of full activity after 1% h of incubation at 45 "C. One minute at 60 "C abolishes all activity.
The pH profile (Fig. 4) shows measurable activity between 6.5 and 8.5, indicating the enzyme has a broad tolerance of varying pH conditions. This profile also shows that at a given pH, the enzyme exhibits greatest activity in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered medium.
Effects ofMetal Ions and Metal Ion Chelators-The wheat germ uracil-DNA glycosylase is strongly inhibited by high concentrations of monovalent cations (Fig. 5A). The inhibition patterns seen when the glycosylase is exposed to K', Na+, and Li' are alike, indicating that inhibition is due to increased ionic strength in the reaction medium rather than the specific effects of a particular ionic species upon enzyme activity. The patterns for the two divalent species, Mg" and Ca'+ ( Fig. 5B 1, are also similar, again showing that ionic strength is the important factor inhibiting glycosylase activity. Fig. 5C presents the effects of the ionic chelator EDTA on glycosylase activity. The chealtor does not appear to inhibit activity at concentrations less than 20 mM. However, at 50 mM, EDTA shows significant inhibition, but this effect is amplified by addition of MgC12, indicating that the ionic loo0 r  strength of EDTA rather than its chelating ability is the important factor inhibiting enzyme activity. Furthermore, no requirement for metal ions was observed during purification and storage. The nonionic chelator 8-hydroxyquinoline was also tested on the wheat germ uracil-DNA glycosylase at concentrations up to its solubility limit in an aqueous buffer (about 3 mM) and found to have no inhibitory effect upon enzymatic activity.
N-ethylmaleimide, a sulfhydryl group-blocking agent, was tested on the glycosylase and only reduced activity to 75% of control levels at 50 mM, showing that sulfhydryl groups probably do not play an important part at the active site of the glycosylase.

Uracil Analogues a n d Glycosylase Inhibitors
Uracil Analogues- Fig. 6 shows the inhibitory effects of 14 nucleoside and pyrimidine analogues closely resembling uracil structurally. Wheat germ glycosylase was inhibited by uracil, 6-aminouracil, or 5-azauracil, while pyrimidines as similar to uracil as 6-azauracil, 5-fluorouracil, and 5-aminouracil do not appear to markedly decrease glycosylase activity below control levels. Thymine and cytosine, the two pyrimidines normally found in DNA, also do not inhibit activity. All compounds were present at 1 mM.
Effects of T5-induced Uracil-DNA Glycosylase Inhibitor-When bacteriophage T5 infects E . coli, it induces an inhibitor of host uracil-DNA glycosylase activity (9), presumably to somehow expedite the infection process. In an attempt to determine whether this inhibitor had similar effects on wheat germ glycosylase, the action of the uracil glycosylase inhibitor induced by phage T5 was compared on both wheat germ and E. coli glycosylases. Fig. 7 shows clearly that amounts of inhibitor which drastically diminish the activity of E. coli glycosylase have no apparent effect on the plant enzyme. Because the substrate is the same in both cases, this is evidence for a structural difference between the E. coli and wheat germ enzymes allowing inhibitor-enzyme interaction with the bacterial glycosylase, but preventing it with the plant enzyme.

Substrate Specificity
Ability to Release Uracil from dUMP and dUTP-To explore the ability of the wheat germ enzyme to release uracil from mononucleotides as well as polynucleotides, uracil release from [6-"HIdUMP and [5-:'H]dUTP by the glycosylase was measured. Under conditions such that over 50% of available uracil from the T5 DNA substrate was liberated, no release of free uracil from either mononucleotide was detect-FIG. 6. Effects of uracil analogues. The effects of all analogues were measured under the same standard assay conditions with 1 mM concentrations of a given analogue present in the reaction medium. The structure of the analogue is shown directly over the bur corresponding to the assay containing that analogue.   (Table 11). Thus, this enzyme has a requirement for a polynucleotide or oligonucleotide substrate. Ability to Release Uracil from RNA-E. coli RNA labeled in its uracil bases was exposed to wheat germ enzyme to test the ability of this glycosylase to release uracil from RNA. No uracil release was detectable under conditions which allowed the glycosylase to release more than 50% of available uracil from a DNA substrate and at RNA concentrations which partially inhibited uracil release from the DNA substrate (Table 11). Therefore, under assay conditions where ["HIRNA can inhibit and so possibly bind to wheat germ uracil glycosylase, the enzyme is incapable of releasing uracil from the same RNA species.
Inhibition by Wheat Germ RNA Species-The results described above indicate that while uracil is not released from RNA by glycosylase activity, RNA will inhibit this activity. To further explore the point, inhibitory effects of unlabeled calf thymus DNA and wheat germ RNA species were compared. As Fig. 8 shows, similar concentrations of RNA and DNA inhibit glycosylase activity on labeled calf thymus DNA to approximately the same extent, showing that either polynucleotide can effectively block access to the active site and that the DNA and RNA may bind in the same manner to the enzyme.
Preference between Single-stranded and Double-stranded DNA-Because the mechanism of uracil-DNA glycosylases may involve selective denaturation of its DNA substrate in the region of the uracil to be excised, the activity of wheat germ enzyme was tested in parallel reactions on both heatdenatured DNA (100 "C for 10 min) and nondenatured double-stranded DNA. Under similar conditions, the glycosylase has a reaction rate approximately 50% faster on denatured DNA. Nevertheless, nondenatured DNA was used in all other assays to characterize the enzyme because this is presumably the in vivo substrate.  Fig. 9 shows the results of an examination of the kinetics of uracil inhibition of wheat germ glycosylase activity. The pattern obtained is typical of a noncompetitive mode of inhibition by uracil with a family of straight lines converging to a common intercept on the l/[S] axis. Therefore, uracil may be able to combine with either the free glycosylase or the glycosylase-DNA complex. The apparent K,,, of the uninhibited reaction with T5 DNA substrate is 8.3 X lo-' M and the K , is 4.0 X

M.
Completeness of Reaction-To determine whether the distance between uracils in the DNA substrate affected the extent of reaction, the double-stranded co-polymer poly(dA-dT) into which uracil had been incorporated to varying degrees (see "Experimental Procedures") was exposed to the wheat germ uracil-DNA glycosylase. Fig. 10 shows the ability of the enzyme to remove all available uracil from three poly(dA-dT) substrates in which 5, 50, and 100% of the thymine residues in newly synthesized regions have been replaced by uracil. In all three substrates, a large majority of the available uracil was removed by enzyme action at the reaction's completion. The small differences in extent of reaction between the three substrates are probably due to the presence of small oligonucleotides refractory to uracil-DNA glycosylase attack. When substrates were not purified by gel filtration prior to enzyme assay, substantial differences in apparent extent of reaction were observed, almost certainly due to the presence of such oligonucleotides. as a function of duration of assay.

DISCUSSION
A wheat germ uracil-DNA glycosylase has been isolated and purified over 1000-fold. This is the first enzyme of its class to be isolated from a higher plant source, Several facets of its activity have been examined which included assessing the inhibitory effect of a large variety of uracil analogues, comparing the effect of T5-induced E. coli uracil-DNA glycosylase inhibitor on the wheat germ and bacterial glycosylases, determining a number of physical characteristics of this higher plant enzyme, and examining the effect of uracil distribution along a DNA substrate on the ability of the wheat germ glycosylase to remove that uracil from the substrate.
Of the 14 uracil analogues tested, only 6-aminouracil and 5azauracil elicited inhibition comparable to uracil itself at 1 mM concentrations. 5-Aminouracil, 6-azauracil, and several other structurally similar pyrimidines with modifications a t the 5 or 6 position of the ring had no effect. It is intriguing that a position alteration of the ring nitrogen in 5-azauracil from the 5 to 6 positions totally abolishes the inhibitory effect of this analogue, whereas if the amino group at the 6 position of 6-aminouracil is shifted to the 5 position, inhibition is lost. Clearly, whatever charge or steric constraints govern recognition of the analogues at the site of inhibition are exacting. This agrees with recent work describing a uracil-DNA glycosylase purified from HeLa cells (11) which was also significantly inhibited only by uracil and these two uracil analogues and very slightly by 5-aminouracil. Comparable, although less extensive, studies have shown that E. coli glycosylase is uninhibited by 5-aminouracil (6) and a human blast cell glycosylase is unaffected by 5-fluorouracil or 6-azauracil (8). This very high specificity is expected as it prevents removal of thymine and cytosine from DNA and uracil from RNA.
Although both wheat germ and E. coli uracil-DNA glycosylase are inhibited by uracil, the response of these enzymes to T5-induced uracil-DNA glycosylase inhibitor is strikingly different, showing that there are at least some structural differences between these two examples of eucaryotic and procaryotic repair activities preventing effective interaction of the inhibitor with one, but allowing it with the other.
The size of the wheat germ uracil-DNA glycosylase appears similar to that of all bacterial and human uracil-DNA glycosylases examined so far. The molecular weight, determined both by gel filtration and glycerol gradient centrifugation, is approximately 27,000, slightly larger than procaryotic enzymes (6,7), very similar to the Saccharomyces cereuisiae glycosylase (20), and smaller than the human blast cell enzyme (8).
The wheat germ glycosylase displays a broad pH optimum and is not overly sensitive to either ionic or nonionic metal ion chelators such as EDTA or 8-hydroxyquinoline. The inhibition caused by high concentrations of EDTA is probably due to the ionic strength of this chelator rather than its metal ion-binding properties because addition of MgC12 only further decreases enzymatic activity. The E. coli and HeLa enzymes also are not notably affected by low levels of EDTA as shown by Lindahl et al. (6) and Krokan and Wittwer (ll), respectively. Therefore, uracil-DNA glycosylases are probably not metalloenzymes.
The inhibitory effects of mono and divalent cations at relatively high concentration on wheat germ glycosylase activity are also accounted for by increased ionic strength of the reaction medium. It is known that as salt concentrations increase, the stability of the DNA duplex increases with a concomitant decrease in nucleic acid-protein complex stability for single-stranded DNA-binding proteins (21,22), perhaps explaining the uniform falloff in uracil-DNA glycosylase activity observed with increased monovalent, divalent, and ionic chelator concentration.
Some clues about the mode of substrate recognition of the wheat germ glycosylase are given by its inability to release uracil from the mononucleotide dUMP or dUTP as well as the generally similar interaction of RNA and DNA with the enzyme implied by the parallel inhibition patterns for the two polynucleotides. This could indicate that the substrate recognition of the glycosylase and binding site has a bipartite structure such that it is relatively nonspecific for polynucleic acids, being capable of binding single-stranded DNA and double-stranded DNA or RNA, but has a subsite which is extremely specific for uracil bases attached to 2"deoxyribose residues on that polynucleotide as evidenced by the analogue studies. The noncompetitive inhibition pattern seen on T5 DNA is consistent with the enzyme having two sites important for enzymatic activity.
The K , calculated for the wheat germ uracil-DNA glycosylase (8.3 X lo-' M) is similar to the values obtained for E. coli, B. subtilis, and human lymphoblast enzymes, which vary from 3.0 X 10" to 1.0 X IO-" M (4). The apparent K , of the wheat germ glycosylase (4.0 X M) derived from a double reciprocal noncompetitive uracil inhibition pattern is also within the range of values found for other enzymes of this class. Thus, the kinetic characteristics of the wheat germ enzyme are like other uracil-DNA glycosylases for which these characteristics have been examined.
By examining the effect of amount of uracil in the DNA substrate on the ability of the wheat germ glycosylase to excise the base, it was demonstrated that the extent of uracil removal is unaffected by uracil proximity in DNA substrates containing uracil residues as close as every other base or as far apart as one base in every 40. In both cases, the substrate underwent near total removal of available uracil, indicating that the enzyme requires at most one base between uracils excised on a poly(dA-dT) substrate.
Further characterization of the wheat germ enzyme's active site and mechanism of action will require more direct classical methods of enzyme study and perhaps sequencing of a glycosylase gene allowing the primary structure of the protein to be determined.