©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Studies on the Catalytic Mechanism of Five DNA Glycosylases
PROBING FOR ENZYME-DNA IMINO INTERMEDIATES (*)

(Received for publication, March 20, 1995; and in revised form, June 7, 1995)

Bin Sun (1) Katherine A. Latham (1) M. L. Dodson (1) R. Stephen Lloyd (1)(§)

From the Sealy Center for Molecular Science and the Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1071

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

DNA glycosylases catalyze scission of the N-glycosylic bond linking a damaged base to the DNA sugar phosphate backbone. Some of these enzymes carry out a concomitant abasic (apyrimidinic/apurinic (AP)) lyase reaction at a rate approximately equal to that of the glycosylase step. As a generalization of the mechanism described for T4 endonuclease V, a repair glycosylase/AP lyase that is specific for ultraviolet light-induced cis-syn pyrimidine dimers, a hypothesis concerning the mechanism of these repair glycosylases has been proposed. This hypothesis describes the initial action of all DNA glycosylases as a nucleophilic attack at the sugar C-1` of the damaged base nucleoside, resulting in scission of the N-glycosylic bond. It is proposed that the enzymes that are only glycosylases differ in the chemical nature of the attacking nucleophile from the glycosylase/AP lyases. Those DNA glycosylases, which carry out the AP lyase reaction at a rate approximately equal to the glycosylase step, are proposed to use an amino group as the nucleophile, resulting in an imino enzyme-DNA intermediate. The simple glycosylases, lacking the concomitant AP lyase activity, are proposed to use some nucleophile from the medium, e.g. an activated water molecule. This paper reports experimental tests of this hypothesis using five representative enzymes, and these data are consistent with this hypothesis.


INTRODUCTION

DNA glycosylases initiate the cellular DNA base excision repair pathway. In this pathway, damaged or inappropriate bases are removed from the DNA as free bases by cleavage of the N-glycosylic bond (reviewed in (1) and (2) ). These enzymes have been classified into two groups based on the types of products they generate (Fig. 1). The simple glycosylases are limited to the N-glycosylic bond cleavage, the product of which is an abasic site (Fig. 1B). The glycosylase/AP (^1)lyases carry out the glycosylase reaction and also catalyze a subsequent AP lyase activity at a rate approximately equal to the glycosylase rate (Fig. 1, C-E). T4 endonuclease V, Micrococcus luteus UV endonuclease, Escherichia coli endonuclease III, and E. coli formamidopyrimidine (Fapy) glycosylase (FPG) are members of the first group(3, 4, 5, 6, 7, 8, 9, 10) . Uracil DNA glycosylase (UDG) and adenine DNA glycosylase (MutY) from E. coli, mammalian N-methyl purine glycosylase (MPG), and 3-methyladenine DNA glycosylase of E. coli are examples of simple glycosylases (11, 12, 13, 14) . To date the underlying mechanistic differences between these two groups of enzymes have not been described.


Figure 1: Schematic representation of the overall reaction pathway for DNA glycosylases and glycosylase/AP lyases.



T4 endonuclease V, a repair glycosylase/lyase specific for ultraviolet light-induced cis-syn cyclobutane pyrimidine dimers, was the first DNA glycosylase to have its structure determined by x-ray crystallography and its active site residues identified(15, 16, 17, 18, 19, 20, 21, 22) . T4 endonuclease V uses the N-terminal threonine alpha-amino group as a nucleophile to attack the deoxyribose C-1` in the glycosylase step. The amino acid Glu is crucial for this attack and for the subsequent lyase reaction(17, 18, 20, 21) . The consequence of this mechanism is the formation of an imino enzyme-DNA intermediate as the product of the glycosylase step (Fig. 1C). This intermediate may be hydrolyzed, resulting in formation of an abasic site (Fig. 1D), or undergo a beta-elimination reaction, resulting in a lyase scission of the sugar-phosphate DNA backbone (Fig. 1E). The ratio of the rates of these two reaction legs would be responsible for the observed pH dependence of the relative yields of abasic sites and single strand breaks. Additional data to support the proposed mechanism of endonuclease V have recently been obtained by isolation of a stable covalent enzyme-DNA complex after reduction of the intermediate with sodium borohydride (NaBH(4))(19) .

Previous studies have also shown that E. coli endonuclease III cleaves the sugar-phosphate DNA backbone on the 3` side of an AP site via a beta-elimination reaction rather than by a phosphotransfer (endonuclease-type) activity(23, 24, 25) . Furthermore, the E. coli FPG protein has been reported to cut the DNA sugar-phosphate backbone by a beta, or -elimination reaction(26) .

As a generalization of these results, a hypothesis has been proposed (27) that the initial action of all DNA glycosylases proceeds by a nucleophilic attack at the sugar of the damaged base nucleoside, resulting in scission of the N-glycosylic bond. In this hypothesis, the DNA glycosylases, which carry out an accompanying AP lyase reaction, are proposed to use an amino group as the attacking nucleophile, resulting in an imino enzyme-DNA intermediate (Fig. 1C). Simple glycosylases, lacking this concomitant AP lyase activity, are proposed to use some nucleophile from the medium, e.g. an activated water molecule, to promote catalysis (Fig. 1B). In this second case, no covalent enzyme-DNA intermediate is predicted to occur.

An experimental consequence of this hypothesis is that it should be possible to trap the putative lyase covalent intermediate by reduction of the imino intermediate with NaBH(4). This would result in a covalent enzyme-DNA complex which should be resolvable by denaturing polyacrylamide gel electrophoresis. Enzyme inhibition and formation of stable covalent complexes are the primary end points of the protocol used in this study.


EXPERIMENTAL PROCEDURES

Materials

E. coli endonuclease III and FPG were gifts from Drs. J. Laval and T. O'Connor (Institut Gustave-Roussy, Villejuif Cedex, France). E. coli MutY was a gift of Dr. M. Michaels (Amgen Corp., Thousand Oaks, CA). E. coli UDG was purchased from Epicentre Technologies. Mouse N-methyl purine DNA glycosylase was a gift of Drs. R. Roy and S. Mitra (Sealy Center for Molecular Science, University of Texas Medical Branch). The synthetic oligonucleotides used in this study are described in Table 1. The Klenow fragment of DNA polymerase I (3` 5` exonuclease-deficient) was a gift from Drs. L. M. Bloom and M. F. Goodman (University of Southern California). Dr. L. Loeb (University of Washington) provided 8-oxo-dGTP. Dr. P. Doetsch and L. Augeri (Emory University) provided the oligonucleotide containing the 5,6-dihydrouracil.



Preparation of DNA Substrates

All oligonucleotide strands containing damaged or inappropriate bases were P-labeled on the 5`-end with T4 polynucleotide kinase (New England Biolabs) and annealed to their complementary strands to form the substrate duplex DNA. The oligonucleotide duplex containing a site-specific 8-oxo-deoxyguanine was made as follows: a 49-base oligonucleotide with the sequence of 5`AGGTATGAGGATGATTAGGAATTGGTGGCTCGTTGCAGGCATGGTAGCT-3` and a 20-base oligonucleotide with the sequence of 5`-AGCTACCATGCCTGCAACGA-3` were synthesized and purified. The 20-mer was labeled on the 5`-end with [-P]ATP and polynucleotide kinase, and after unincorporated label was removed, the 20-mer primer (0.2 nmol) was annealed to 0.2 nmol of the 49-mer template. The duplex was purified through a NENsorb cartridge (DuPont) per the supplier's instructions. An extension reaction was performed at 37 °C for 20 min in 10 µl of solution containing purified template/primer, 0.5 nmol of 8-oxo-dGTP, and 0.03 nmol of Klenow fragment. The extension buffer contained 50 mM Tris-HCl (pH 7.4), 50 mM KCl, 10 mM MgCl(2), and 5 mM 2-mercaptoethanol. The reaction was diluted to 30 µl with the extension buffer containing 30 nmol each of dATP, dCTP, and dTTP and 0.02 nmol of Klenow fragment. This reaction mixture was incubated for an additional 30 min. In order to decrease the incorporation of 8-oxo-dGTP at nonspecific sites, the template was designed so that no dGTP was required for primer extension. The first instance of a dTTP opposite a template dAMP was 8 bases after the position of 8-oxo-dGTP incorporation, so that denaturing PAGE would easily detect an 8-oxo-deoxyguanine mispaired with the template adenine. The extended 49-mer oligonucleotide strand and the 49-mer template were co-isolated by denaturing PAGE, co-purified through a NENsorb cartridge, and reannealed in the presence of an additional 0.1 nmol of template.

Concentration Dependence Experiments

Each enzyme except UDG was incubated with its appropriate oligonucleotide substrate at 37 °C for 30 min in 20 µl of a solution containing reaction buffer A (25 mM NaH(2)PO(4) (pH 6.8), 1 mM EDTA, 100 mM KCl or NaCl, and 100 µg/ml bovine serum albumin). UDG reactions were performed in 70 mM HEPES (pH 8.0), 1 mM EDTA, and 1 mM 2-mercaptoethanol. The reactions of endonuclease III and FPG were terminated by adding 20 µl of loading buffer (95% (v/v) formamide, 20 mM EDTA, 0.02% (w/v) bromphenol blue, 0.02% (w/v) xylene cyanol). In the case of the simple glycosylases UDG, MutY, and MPG, the reactions were further treated by the addition of 1 µl of undiluted piperidine (Eastman Kodak Co.). After heating at 90 °C for 30 min, the samples were dried and then resuspended in 20 µl of loading buffer. The piperidine treatment was carried out to convert all AP sites resulting from the glycosylase step to single strand breaks. The reaction products were separated by urea-PAGE. The products of DNA cleavage were detected by autoradiography and quantitated using BioImage software (Millipore).

Enzyme Inhibition and Demonstration of Enzyme-DNA Covalent Complexes by NaBH(4)

Reactions were carried out as described above except that the reactions were performed in the presence of 100 mM NaCl or 100 mM NaBH(4) in addition to the original salt of reaction buffer A. A NaBH(4) stock solution (2 M) was freshly prepared immediately prior to use by dissolving the powder in water. The reaction products were separated by nondenaturing PAGE and SDS-PAGE.


RESULTS

Experimental Rationale

As outlined in the Introduction, a protocol for determining whether a DNA glycosylase uses an amino group as the nucleophile in the glycosylase step consists of treatment of the reaction mixtures with NaBH(4)(27, 28) . The reaction products are then analyzed under denaturing and nondenaturing conditions by PAGE to assay for covalent and noncovalent interactions, respectively.

DNA Glycosylase/AP Lyases

A significant number of saturated pyrimidines (e.g. thymine glycol, 5,6-dihydrouracil) and ring-opened pyrimidines are released by the glycosylase action of E. coli endonuclease III (reviewed in (6) and (29) ). As shown in Fig. 2, lanes 3-6, endonuclease III incises a 37-mer oligonucleotide duplex substrate containing a unique dihydrouracil in a dose-dependent manner. The addition of NaBH(4) to these reactions inhibited the nicking activity and resulted in partial trapping of the P-labeled DNA substrates in the wells (Fig. 2, lanes 7-10). In order to assay directly for the formation of a stable protein-DNA complex, reactions were performed with increasing concentrations of endonuclease III in the presence of NaBH(4), and the production of stable complexes was analyzed by SDS-PAGE (Fig. 3). At enzyme to DNA mole ratios ranging from a high of 46 to 1 (lane 3) to a low of 1.6 to 1 (lane 5), gel shifted bands were readily detected. These data are consistent with a mechanism in which the nucleophilic attack is carried out by a primary amino group within the enzyme.


Figure 2: Concentration dependence and inhibition by NaBH(4) of E. coli endonuclease III incising a 37-base pair oligonucleotide duplex that contains a site-specific dihydrouracil. The oligonucleotide strand containing dihydrouracil was 5`-end-labeled with P. The substrate (0.06 pmol) was reacted with endonuclease III, and the products were separated by 15% denaturing PAGE. Lane 1, oligonucleotide markers. Lane 2, 37-mer substrate alone. Lanes 3-6, reaction of the substrate with decreasing amounts of the enzyme (0.28, 0.06, 0.01, and 0.002 pmol from lane 3 to lane 6, respectively). Lanes 7-10 are similar to lanes 3-6, except that the reactions were performed in the presence of 100 mM NaBH(4).




Figure 3: Probing for an E. coli endonuclease III-DNA substrate covalent complex by NaBH(4) reduction. The dihydrouracil containing substrate (0.06 pmol) was reacted with the enzyme in the presence of 100 mM NaCl or NaBH(4), and the products were subjected to 15% SDS-PAGE. Lane 1, substrate alone. Lane 2, reaction with 5.6 pmol of enzyme in the presence of NaCl. Lanes 3-6 resulted from the reactions of the substrate with decreasing enzyme amounts (2.8, 0.56, 0.11, and 0.02 pmol from lane 3 to lane 6, respectively) in the presence of NaBH(4).



E. coli formamidopyrimidine (Fapy) glycosylase (FPG) has been shown to recognize and remove ring-opened purines (Fapy bases) and 8-oxo-deoxyguanine in duplex DNA. FPG has been proposed to work via a concerted glycosylase beta,-elimination process (reviewed in (30) ). Previously, it had been proposed that an amino group on the enzyme was involved in a nucleophilic attack on the C-1` of 8-oxo-dG, with formation of a Schiff base intermediate between the enzyme and its substrate(30) . The FPG substrate used in this study, a 49-base oligonucleotide duplex containing a unique 8-oxo-deoxyguanine at position 21, was synthesized as described under ``Experimental Procedures.'' FPG was found to cleave this substrate at the damaged site in a dose-dependent manner as revealed by analysis of the products through denaturing PAGE (Fig. 4, lanes 3-6). The nicking activity of FPG was completely inhibited by the presence of NaBH(4) in the reaction mixture (Fig. 4, lanes 8-11). The P-labeled DNA substrates were trapped in the wells in a dose-dependent manner, suggesting the formation of an enzyme-DNA covalent complex between FPG and its substrate. Further analysis demonstrated that a shifted band was formed in the FPG reactions in the presence of NaBH(4), even when the reaction products were subjected to SDS-PAGE (Fig. 5), indicating the existence of a covalent FPGbulletDNA complex. Quantitative analysis using BioImage software revealed that, at two different FPG concentrations, the percentages of the FPG cleaved products in the absence of NaBH(4) (33.7 and 12.0% cleaved, as shown in Fig. 4, lanes 4 and 5) were similar to the percentages of the observed covalently trapped complex using the same two FPG concentrations in the presence of NaBH(4) (30.0 and 9.0% trapped, as shown in Fig. 5, lanes 7 and 8). This suggests that the breakage of the DNA backbone occurs via formation of a covalent enzyme-DNA complex. In contrast to what was observed for endonuclease III, the minimum ratio of enzyme to DNA that gave easily detectable gel shifted DNAs was 20 to 1. However, longer exposure of these gels revealed gel shifted DNAs at mole ratios of 4 to 1. It has not been determined whether this might be due to a high percentage of inactive enzyme in this preparation.


Figure 4: Concentration dependence and inhibition by NaBH(4) of E. coli FPG incising a 49-base pair oligonucleotide duplex that contains a site-specific 8-oxo-deoxyguanine. The oligonucleotide strand (approximately 0.05 pmol) containing 8-oxo-deoxyguanine at position 21 was 5`-end-labeled with P. The substrate was reacted with FPG in the presence of 100 mM NaCl or 100 mM NaBH(4), and the products were separated by 15% denaturing PAGE. Lane 1, markers. Lane 2, substrate alone. Lanes 3-6, reactions contained decreasing amounts of FPG (9.9, 5.0, 1.0, and 0.5 pmol from lane 3 to lane 6, respectively) in the presence of 100 mM NaCl. Lanes 7-11 are similar to lanes 2-6 except that the reactions were carried out in the presence of 100 mM NaBH(4).




Figure 5: Probing for an FPG-substrate covalent complex by NaBH(4) reduction. The substrate (approximately 0.05 pmol) was reacted with FPG in the presence of 100 mM NaCl or NaBH(4), and the products were subjected to 15% SDS-PAGE. Lane 1, substrate alone. Lanes 2-6, reactions of the substrate with decreasing FPG amounts (5.0, 1.0, 0.2, 0.04, and 0.008 pmol from lane 2 to lane 6, respectively) in the presence of NaCl. Lanes 7-11 are similar to lanes 2-6 except that the reactions were carried out in the presence of NaBH(4).



Simple DNA Glycosylases (without Concomitant AP Lyase Activity)

E. coli MutY protein is an A/G or A/C mismatch adenine DNA glycosylase. Its amino acid sequence is homologous to that of E. coli endonuclease III, and, like endonuclease III, the protein is a [4Fe-4S] cluster protein(31) . Data presented in Fig. 6A indicate that E. coli MutY released adenines from A/G mismatched sites in a dose-dependent manner. Even though both glycosylase and AP lyase activities have previously been noted (31) , (^2)this preparation of E. coli MutY appears to act primarily as a simple DNA glycosylase. Like the DNA glycosylase/AP lyases, the catalytic activity of MutY (Fig. 6B) was inhibited by NaBH(4). This apparent inhibition could be due to NaBH(4) reducing the AP site, such that it is no longer a substrate for piperidine cleavage. When these reaction products were analyzed under nondenaturing gel conditions, both control and the NaBH(4)-treated MutY reactions showed a small percentage of gel shifted DNAs (data not shown). However, analyses of the same samples using denaturing SDS-PAGE did not show gel shifted bands (Fig. 7). In these experiments, the highest mole ratio of enzyme to DNA was 56 to 1. Since gel-shifted DNAs were detectable at lower enzyme/DNA ratios for the previous two glycosylase/AP lyases, these data suggest that no covalent intermediate was formed between MutY and its substrate.


Figure 6: Concentration dependence and inhibition by NaBH(4) of E. coli MutY incising a 50-base pair oligonucleotide duplex that contains a site-specific A/G mismatch. The oligonucleotide strand containing the adenine of an A/G mismatch at position 21 was 5`-end-labeled with P. The substrate (0.025 pmol) was reacted with MutY in the presence of 100 mM NaCl or NaBH(4), followed by ± piperidine treatment. The products were separated by 15% denaturing PAGE. A, lane 1 is the 50-mer substrate alone. Lanes 2-6 resulted from reactions containing decreasing amounts of MutY (0.56, 0.11, 0.02, 0.005, and 0.001 pmol from lane 2 to lane 6, respectively). Lanes 7-12 are similar to lanes 1-6 except that the reactions were followed by piperidine treatment. B, lane 1, markers. Lane 2 resulted from the reaction of a 50-mer A/T control with MutY followed by piperidine treatment. Lane 3 is substrate alone. Lane 4 resulted from the reaction of the substrate with MutY (0.56 pmol). Lanes 5 and 6 resulted from the reaction of the substrate with MutY (0.56 pmol in lane 5, 0.11 pmol in lane 6) in the presence of NaCl, followed by piperidine treatment. Lanes 7 and 8 are similar to lanes 5 and 6, except that the reactions were carried out in the presence of NaBH(4).




Figure 7: Probing for an enzyme-DNA substrate covalent complex by NaBH(4) treatment of E. coli MutY reactions. MutY was incubated with its substrate (0.05 pmol) in the presence of 100 mM NaCl or NaBH(4), and the products were subjected to 15% SDS-PAGE. Lane 1, substrate alone. Lane 2, reaction of the enzyme (2.8 pmol) with its substrate in the presence of NaCl. Lanes 3-5, reactions with decreasing enzyme amounts (2.82, 0.56, and 0.11 pmol from lane 3 to lane 5, respectively) in the presence of NaBH(4).



E. coli uracil-DNA glycosylase (UDG) is responsible for the removal of uracil from DNA(11) . Uracil can arise in double-stranded DNA by incorporation of dUTP in place of dTTP or by deamination of cytosine. UDG is strictly a simple glycosylase, lacking detectable AP lyase activity. E. coli UDG was shown to catalyze the removal of uracil in a dose-dependent fashion (Fig. 8A). UDG was also inhibited by NaBH(4), and, as was the case for MutY, there was no evidence for trapping of a covalent dead-end intermediate (Fig. 8B). These denaturing gel electrophoresis data were confirmed when no shifted band was observed in nondenaturing PAGE (Fig. 9). Although the highest mole ratio of enzyme to DNA shown in Fig. 9was 5 to 1, additional experiments have confirmed that no covalent intermediates are detectable at enzyme to DNA ratios as high as 25 to 1 (data not shown). Therefore, there is no evidence for the formation of a covalent enzyme-substrate intermediate in the case of UDG.


Figure 8: Concentration dependence and inhibition by NaBH(4) of E. coli UDG incising a 49-base pair oligonucleotide duplex that contains a site-specific uracil. The oligonucleotide strand containing the uracil at position 21 was 5`-end-labeled with P. The substrate (0.026 pmol) was reacted with UDG in the presence of 100 mM NaCl or NaBH(4), followed by ± piperidine treatment, and the products were separated by 15% denaturing PAGE. A, lane 1 is the 49-mer substrate alone. Lanes 2-6 resulted from reactions containing decreasing amounts of UDG (0.12, 0.025, 0.005, 0.001, and 0.0002 pmol from lane 2 to lane 6, respectively). Lanes 7-12 are similar to lanes 1-6 except that the reactions were followed by piperidine treatment. B, lane 1, markers. Lane 2 resulted from the reaction of a 49-mer control (without uracil) with UDG followed by piperidine treatment. Lane 3 is substrate alone. Lane 4 resulted from the reaction of the substrate with UDG (0.12 pmol). Lanes 5 and 6 resulted from the reaction of the substrate with UDG (0.12 pmol in lane 5, 0.025 pmol in lane 6) in the presence of NaCl, followed by piperidine treatment. Lanes 7 and 8 are similar to lanes 5 and 6, except that the reactions were carried out in the presence of NaBH(4).




Figure 9: Probing for an enzyme-DNA substrate covalent complex by NaBH(4) treatment of E. coli UDG reactions. UDG was incubated with its substrate (0.03 pmol) in the presence of 100 mM NaCl or NaBH(4), and the products were subjected to 10% nondenaturing PAGE. Lane 1, substrate alone. Lane 2, reaction of the enzyme with its substrate in the presence of NaCl. Lanes 3-5, reactions with decreasing enzyme amounts (0.12, 0.03, and 0.005 pmol from lane 3 to lane 5, respectively) in the presence of NaBH(4).



Mammalian MPGs have been reported to have a broad substrate specificity, as does the E. coli 3-methyl adenine DNA glycosylase II, the product of the alkA gene. Substrates include all three N-alkylpurines but not O-2-alkylpyrimidines in DNA(32) . Recent studies have established that both AlkA and mammalian MPGs are capable of removing etheno adducts of A, G, and C from DNA(33) . In the current study, a slow cleavage of the sugar-phosphate DNA backbone of an etheno-A-containing substrate was observed for mouse MPG, although this rate was at least 2 orders of magnitude slower than the rate of the glycosylase step (Fig. 10). Whether this result reflects an intrinsic enzyme activity or a minor contamination is not clear. Consistent with a recent report(32) , 1-N^6-ethenoadenine-containing DNA was shown to be a specific substrate for mouse MPG (Fig. 10). MPG was also observed to be completely inhibited by NaBH(4) (Fig. 11), and like UDG and MutY, there was no evidence for the existence of a covalent MPG enzyme-substrate intermediate, even at enzyme to DNA ratios as high as 180 to 1 (Fig. 11, lanes 3-5). Again, the absence of the formation of gel shifted DNAs is indicative of a nucleophilic attack by some other group than a primary amine.


Figure 10: Concentration dependence of mouse MPG incising a 25-base pair oligonucleotide duplex that contains a site-specific ethenoadenine adduct. The oligonucleotide strand containing the ethenoadenine at position 6 was 5`-end-labeled with P. The substrate (0.05 pmol) was reacted with MPG, followed by ± piperidine treatment, and the products were separated by 20% denaturing PAGE. Lane 1, markers. Lane 2, 25-mer substrate alone. Lanes 3-7 resulted from reactions containing decreasing amounts of MPG (8.9, 1.8, 0.36, 0.07, and 0.01 pmol from lane 3 to lane 7, respectively). Lanes 8-13 are similar to lanes 2-7, except that the reactions were followed by piperidine treatment.




Figure 11: Probing for an enzyme-DNA substrate covalent complex by NaBH(4) treatment of mouse MPG reactions. MPG was incubated with its substrate (0.05 pmol) in the presence of 100 mM NaCl or NaBH(4), and the products were subjected to 10% nondenaturing PAGE. Lane 1, substrate alone. Lane 2, reaction of the enzyme with its substrate in the presence of NaCl. Lanes 3-5, reactions with decreasing enzyme amounts (8.9, 1.8, and 0.36 pmol from lane 3 to lane 5) in the presence of NaBH(4).



In summary, the two glycosylase/AP lyase enzymes tested (endonuclease III and FPG from E. coli) could be covalently trapped to their DNA substrates by treatment with NaBH(4), whereas the three simple glycosylases (E. coli UDG and MutY and mouse MPG) could not.


DISCUSSION

Fig. 1is a schematic representation of our current hypothesis describing catalysis for both DNA glycosylases and DNA glycosylase/AP lyases. A provides an overview of the basic features that are necessary to initiate repair at either damaged bases or abasic sites. The initial interaction between the enzyme and DNA is a random collision process (A, step 1), and the successful binding to nontarget DNA generally involves electrostatic interactions. In order to locate target sites (damaged bases (A, step 2) or abasic sites (A, step 3)), many of the DNA glycosylases and glycosylase/AP lyases translocate along DNA through a random, one-dimensional process, referred to as scanning or facilitated diffusion (reviewed in (2) ).

Upon binding to a damaged base, the simple DNA glycosylases catalyze the release of the damaged or inappropriate base by nucleophilic attack by an activated water molecule (A, step 4), leading to an abasic site and enzyme release. This model predicts that, due to the nature of the attacking nucleophile (B), no covalent intermediate should be trappable by NaBH(4) in the case of the simple glycosylases. Data presented in Fig. 6-11 for MutY, UDG, and MPG all support this prediction. However, direct evidence for this mechanism has been obtained for UDG from herpes simplex virus type-1, as revealed by x-ray crystallographic analyses of the enzyme in the presence and absence of DNA(34) . The crystal structure model shows a well ordered water molecule ideally positioned, and interacting with appropriate protein side chains, to serve as the nucleophile for the flipped-out (extrahelical) uracil base. The crystal structure and mutational analysis of human UDG also indicate that His may abstract a proton from a water molecule, creating an OH nucleophile that would then carry out the attack at C-1`(35) . These co-crystal structures provide strong support, in the case of UDG, for our mechanistic model of simple glycosylases.

For the DNA glycosylase/AP lyases, the progress of the reaction scheme is more complex (A, steps 5-7) and can be illustrated by conclusions drawn from previous studies on endonuclease V. However, all of the data accumulated for FPG and endonuclease III described in this paper (Fig. 2-5) are in complete agreement with the endonuclease V studies. The catalytic activities of T4 endonuclease V have been extensively studied (reviewed in (27) and (28) ) and are schematically diagrammed in Fig. 1(A, steps 5-7, and C-E). The mechanism first proposed by Schrock and Lloyd (15) proceeds via a nucleophilic attack by the N-terminal alpha-amino group of the enzyme upon the C-1` carbon of the sugar on the 5`-most nucleoside in the cyclobutane pyrimidine dimer substrate. A protonated Schiff base intermediate is subsequently formed involving the N-terminal alpha-amino group and the sugar (A, step 5, and C). The fate of this intermediate is to be hydrolyzed either before (A, step 6, and D) or after the DNA undergoes a beta-elimination reaction (A, step 7, and E). This reaction results in either an AP site (A, step 6, and D) or scission of the sugar-phosphate backbone (A, step 7, and E), respectively. The propensity of the enzyme to catalyze a single strand break can be thought of as a competition between reaction E and reverse reaction D, which should depend on the pH of the medium. The rationalized mechanism of action for the DNA glycosylases tested in this study describes all DNA glycosylases as catalyzing their N-glycosylase reactions via nucleophilic attack on the sugar of their damaged base substrates (Fig. 1A). The bifurcation between the glycosylase/AP lyases and the simple glycosylases lies with the chemical nature of the attacking nucleophile. The glycosylase/AP lyases are hypothesized to utilize primary amines in that role and to form protonated Schiff base intermediates, such as that described for T4 endonuclease V. The simple glycosylases, on the other hand, are proposed to use nucleophiles from the medium for nucleophilic attack and not to form covalent enzyme-DNA intermediates.

The ability of endonuclease III and FPG to form covalent enzyme-substrate complexes in the presence of NaBH(4) and substrate DNA ( Fig. 3and Fig. 5) provides substantial support for the hypothesis that imino intermediates are involved in the catalytic mechanism of these enzymes. As predicted by our hypothesis, no enzyme-substrate complexes were detected in the simple glycosylases MutY (Fig. 7), UDG (Fig. 9), or MPG (Fig. 11), indicating that an imino intermediate is not likely to be involved in these enzymes' catalytic mechanisms. We suggest that the beta-elimination reaction does not occur in the reactions of the simple glycosylases because the electron withdrawal effects of the protonated Schiff base intermediate (Fig. 1B) are required for the 2`-hydrogen to be labilized.

Further evidence to support this hypothesis has come from other reports. The synthetic tripeptide, Lys-Trp-Lys, has been proposed to cleave DNA containing an AP site through a Schiff base intermediate formed by nucleophilic attack of one of the lysine -amino groups on the aldehydic carbon (C-1`) of the abasic site deoxyribose(36, 37) . This hypothesis has been supported by trapping the Lys-Trp-Lys-DNA intermediate by reduction with sodium cyanoborohydride. Also, the mechanism of cleavage at an AP site by endonuclease III has previously been proposed by Kow and Wallace (25) to proceed by a beta-elimination mechanism. Similarly, the reaction of FPG has been previously proposed to proceed through a beta,-elimination mechanism by way of the formation of a Schiff base intermediate(30) . In addition, proton abstraction during beta-elimination has been shown to occur by a syn stereochemical course for both endonuclease III and T4 endonuclease V. This is opposite to the stereochemistry observed for hydroxide ion-mediated backbone cleavage at an abasic site. In an additional mechanistic analogy between the reactions of endonucleases III and V, the crystal structure of endonuclease III (38) shows that Asp is located close in space to Lys, suggesting that Asp may play a similar role to that of the Glu of endonuclease V in stabilizing the charge of the imino intermediate(18) .

It should be emphasized that all data presented in this publication are consistent with our original hypothesis(27) . An understanding of the unified mechanism for DNA repair glycosylases will facilitate the identification of active site residues for other enzymes of this class, and will, hopefully, pave the way for an eventual understanding of the structure-function relationships within this important class of enzymes.


FOOTNOTES

*
This work was supported by National Institutes of Health NIH Grants ES04091 and ES06676 and American Cancer Society GrantFRA381. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 409-772-2179; Fax: 409-772-1790; rslloyd{at}scms.utmb.edu.

(^1)
The abbreviations used are: AP, apyrimidinic/apurinic; FPG, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy) DNA glycosylase; UDG, uracil-DNA glycosylase; MPG, N-methyl purine DNA glycosylase; MutY, adenine-DNA glycosylase for A/C or A/G mispair; PAGE, polyacrylamide gel electrophoresis.

(^2)
R. S. Lloyd and R. Manuel, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Gary Latham for helpful discussions in synthesizing the 49-mer oligonucleotide duplex containing the site-specific 8-oxo-deoxyguanine.


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