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Originally published In Press as doi:10.1074/jbc.M405882200 on July 7, 2004

J. Biol. Chem., Vol. 279, Issue 37, 38177-38183, September 10, 2004
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Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase*{boxs}

Thomas M. Hitchcock{ddagger}, Liang Dong{ddagger}, Ellen E. Connor§, Lisiane B. Meira||, Leona D. Samson||, Michael D. Wyatt§, and Weiguo Cao{ddagger}**

From the {ddagger}Department of Genetics, Biochemistry & Life Science Studies, South Carolina Experiment Station, Clemson University, Clemson, South Carolina 29634, the §Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina 29208, and the ||Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142

Received for publication, May 26, 2004 , and in revised form, June 28, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N1-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase {beta}, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency ~80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N6-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA bases adenine (Ade), guanine (Gua), cytosine (Cyt), and 5-methylcytosine (5-MeC),1 are subject to deamination caused by endogenous and environmental agents (1–4). The rate of base deamination is accelerated by aberrant sequence contexts or high temperature (5, 6). Hypoxanthine (Hyp), xanthine (Xan), uracil (Ura), and thymine (Thy) are the corresponding deamination products derived from Ade, Gua, Cyt, and 5-MeC, respectively. Previous studies suggested that xanthine in DNA is prone to spontaneous depurination because of the instability of the C'–N glycosidic bond (1, 7); however, a more recent study indicates that Xan is a stable lesion in DNA under physiological conditions (8, 9). Treatment of deoxyguanosine or DNA by nitrous acid, nitric oxide, or 1-nitrosoindole-3-acetonitrile also yields an intracyclic guanine deamination product, oxanine (Oxa), in which the N1-nitrogen is substituted by an oxygen atom (10, 11). A chemical pathway leading to the formation of xanthine and oxanine from guanine by nitrous acid treatment has been proposed based on the isolation of a diazoate intermediate (12). Oxanine in DNA is as stable as guanine by comparison of spontaneous hydrolysis of the N-glycosidic bond (7).

All deaminated base lesions are promutagenic as demonstrated by in vitro DNA polymerase studies. Uracil, as the deamination product of cytosine, pairs with adenine, causing Cyt to Thy transitions (13, 14). The mutagenic potential of xanthine is more controversial. Earlier studies (15, 16) indicate that Thy can pair with Xan to produce G/C to A/T transitions. However, a more recent study (17) suggests that dXTP is not incorporated opposite Thy, although the incorporation efficiency of dXTP opposite Cyt is approximately 15,000-fold less than that of dGTP to Cyt. When Xan is placed in a DNA template, the Klenow fragment of Escherichia coli polymerase I inserts a dCTP to pair with Xan, whereas the reverse transcriptase from human immunodeficiency virus inserts a Cyt or Thy with similar efficiencies (9). dOTP can be incorporated to pair with both a Cyt or Thy in DNA by the Klenow fragment, thus generating G/C to A/T transitions (17).

Xanthine DNA glycosylase activities of E. coli AlkA and endo VIII proteins have been reported (9, 18). E. coli endo V has been shown to have deoxyxanthosine endonuclease activity (19, 20). Human alkyladenine glycosylase (AAG, also called MPG for methylpurine DNA glycosylase) has also been shown to have xanthine DNA glycosylase activity2 (9).

E. coli AlkA and endo VIII contain some glycosylase activity to remove mutagenic oxanine from DNA, but it appears that a large excess of glycosylase proteins is needed to detect limited activities (18). To understand the mutagenicity and potential oxanine DNA glycosylase activities in mammalian systems, we investigated Oxa-containing incorporation by human DNA polymerase {beta}. Although dOTP was preferentially incorporated to pair with a Cyt-containing template, dCTP, dATP, dTTP, and to a much lesser extent, dGTP were incorporated to pair with an Oxa-containing template. To identify enzymes that may act on oxanine in mammalian cells, we examined oxanine DNA glycosylase activities of mammalian cell extracts and of known human DNA glycosylases. ODG activity was detected in cell extracts and purified human AAG. The absence of obvious ODG activity in Aag knock-out mouse spleen tissues suggests that AAG is a major oxanine glycosylase in mammalian cells. Human AAG can act on C/O, T/O, A/O, and G/O base pairs as well as single-stranded Oxa-containing DNA.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Separation of Nucleotides by RP-HPLC—Deoxyoxanosine 3'-triphosphate was generated by incubating 10 mM deoxyguanosine 3'-triphosphate (Sigma) with 100 mM NaNO2 in 3 M acetate buffer (pH 3.7) in a final volume of 100 µl at 37 °C for 2 h. The resulting deamination products were separated on a Nova-Pak C18 ODS column (4 µm, 3.9 x 150 mm inner diameter, Waters) by RP-HPLC using a linear gradient (0–20% acetonitrile, 20 min) in a 100 mM triethylammonium bicarbonate TEAB) buffer (Fluka) at pH 7.0, with a flow rate of 1 ml/min. Fractions (0.5 ml/tube) with absorbance at 260 nm were scanned from 220 to 360 nm using a 1700 UV-visible spectrophotometer (Shimadzu). Nucleotide identities were verified by comparing with unique UV signatures of known compounds (17).

Construction of Oxanosine-containing Oligonucleotide Substrates—A single-nucleotide gap substrate contained two deoxyoligonucleotides (1.5-fold in excess), EV38N.BD (23-mer) and EV35C.T (66-mer), with a 36-mer 5'-carboxyfluorescein (FAM)-labeled deoxyoligonucleotide (EV37N.BU) (see Fig. 1). A duplex 1-nucleotide gap was formed by incubating the three oligonucleotides at 85 °C for 3 min and allowing them to anneal at room temperature for at least 30 min. The single-nucleotide extension reaction was performed at 37 °C for 60 min in a 200-µl reaction mixture containing 20 µM dOTP, 5 µM duplex 1-nucleotide gap substrate, 1x EcoPol buffer (New England Biolabs), and 6 units of Klenow fragment (3'-exo-). The reaction mixture was concentrated 4-fold by Speedvac, supplemented with an equal volume of formamide, and heated to 55 °C for 5 min immediately prior to loading. The resulting 37-nucleotide extension product was separated from the substrate by electrophoresis on a 7 M urea-16% denaturing PAGE gel at 250 V for 18 h using a Bio-Rad Protean IIXi apparatus. The oligonucleotides were excised under UV shadowing and desalted by Sep-pak C18 column (Waters) (21). The Oxa-containing 37-mer was annealed to the 66-mer template (EV35C.T) and incubated at 37 °C for 60 min in a 150-µl reaction mixture containing 100 µM each dNTP, 5 µM substrate, 1x EcoPol buffer, and 6 units of Klenow fragment (3'-exo-). The resulting full-length 62-mer oligonucleotide was again purified by denaturing PAGE as described previously. This Oxa-containing strand (1 µM) was annealed with a 66-mer complementary strand (2 µM) to form C/O, T/O, A/O, and G/O base pairs.



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  		FIG. 1.
Construction of oxanosine-containing deoxyoligonucleotide substrate. A, RP-HPLC purification of dOTP. Deaminated dGTP samples (20 µl), generated as described under "Experimental Procedures," were injected into a C18 column and eluted with a 0–20% acetonitrile gradient in TEAB buffer (pH 7.0) over 20 min. The assignment of each peak was based on UV spectra (insets). B, schematic illustration of incorporation procedure. An oligonucleotide duplex was incubated with Klenow fragment and excess dOTP to yield a single Oxa-containing oligonucleotide (37-mer). The extension product was purified by denaturing PAGE, reannealed to the template strand, and extended by addition of regular dNTPs and Klenow fragment. C, sequence of Oxa-containing duplex oligonucleotide. D, representative experimental data are shown for Oxa substrate construction. Lane 1, 36-, 37-, and 38-mer length markers; lane 2, EV37.BU (36-mer); lane 3, single-nucleotide extension product (37-mer); lane 4, full-length extension product.

 
Mutagenicity of Oxanine as a Deoxynucleotide and in DNA—Single-nucleotide extension reactions were carried out at 37 °C for 30 min in a 10-µl volume containing 10 nM single-nucleotide gap substrate (EV35C.T annealed with EV38N.BD and EV37N.BU, see Fig. 1 for sequences), 50 µM dOTP or other dNTP, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 100 mM KCl, 0.4 mg/ml bovine serum albumin, 1 mM dithiothreitol (DTT), 10% glycerol, and 1 nM polymerase {beta}. Reactions were quenched by addition of an equal volume of GeneScan stop buffer. Electrophoresis and quantitative analyses were performed as described under "DNA Glycosylase Activity Assays" below. To study the potential incorporation of dNTP on an Oxa-containing template by human polymerase {beta}, extension reactions were carried out as described previously except that the substrate was a single-nucleotide gap with the Oxa-containing strand as a template.

Preparation of Mammalian Cell Extracts—Pig thymus tissues (390 g) were homogenized in a Waring blender with 200 ml of extraction buffer containing 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.2 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml pepstatin, and 0.5 µg/ml leupeptin at 4 °C. The extract was diluted by adding 500 ml of extraction buffer and then filtered through cheesecloth. Cell debris was removed by centrifugation at 15,000 x g for 10 min in a Beckman Coulter Avanti J-25 centrifuge. Proteins were precipitated by adding ammonium sulfate to 80% saturation. After dialysis using a Spectrum Spectra/Por 10-kDa membrane against the dialysis buffer containing 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.2 mM DTT, the supernatant was precipitated by stepwise ammonium sulfate saturation from 20 to 80%, with 10% increments.

Aag–/– and matched Aag+/+ mouse tissues were thawed on ice in 2.5 volumes of glycosylase assay buffer containing 20 mM Tris-HCl (pH 7.2), 100 mM KCl, 5 mM EDTA, and 5 mM 2-mercaptoethanol as described previously (22). Tissues were homogenized on ice in a 7-ml Dounce homogenizer (Wheaton). Cell debris was removed by centrifugation at 18,000 x g for 30 min in a refrigerated microcentrifuge. The supernatants were stored at –70 °C prior to glycosylase activity assays.

DNA Glycosylase Activity Assays—Purification of human AAG proteins was carried out as described previously (23, 24). DNA glycosylase cleavage assays were performed at 37 °C for 60 min in a 10-µl reaction mixture containing 10 nM oligonucleotide substrate, an indicated amount of glycosylase, 20 mM Tris-HCl (pH 7.2), 100 mM KCl, 5 mM EDTA, and 2 mM 2-mercaptoethanol as described previously (22). The resulting abasic sites were cleaved by incubation at 95 °C for 5 min after adding 0.5 µl of 1 N NaOH. Reactions were quenched by addition of an equal volume of GeneScan stop buffer. Samples (3.5 µl) were loaded onto a 7 M urea-10% denaturing polyacrylamide gel. Electrophoresis was conducted at 1500 V for 1.6 h using an ABI 377 sequencer (Applied Biosystems). Cleavage products and remaining substrates were quantified using GeneScan analysis software.

Gel Mobility Shift Assays—The binding reactions were performed at 37 °C for 30 min in a 10-µl volume containing 50 nM DNA substrate, 20 mM Tris-HCl (pH 7.2), 50 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.1 mg/ml bovine serum albumin, 10% glycerol, and the indicated amount of hAAG ({Delta}54-E125Q). Samples were supplemented with 5 µl of 50% glycerol and electrophoresed at 200 V on a 6% native polyacrylamide gel in 1x TB buffer (89 mM Tris base and 89 mM boric acid) supplemented with 5 mM EDTA. The bound and free DNA species were analyzed using a Typhoon 9400 Imager (Molecular Dynamics) with the settings, photomultiplier tube at 600 V, excitation at 495 nm, and emission at 535 nm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Preparation of dOTP and Oxa-containing DNA—Oxanine is a newly identified deamination product of guanine (11) (Fig. 1A). To generate dOTP, dGTP was treated with 100 mM NaNO2 in 3 M acetate buffer (pH 3.7). Similar to a previous report (17), this reaction produced approximately 40% dXTP and 10% dOTP, respectively (Fig. 1A). Peaks corresponding to dGTP, dXTP, and dOTP were identified by their unique UV spectra (Fig. 1A, insets). Deoxyoxanosine triphosphate was separated at base line level from other products by RP-HPLC, collected, lyophilized, and stored at –80 °C (17). The Oxa base was incorporated into DNA by a single-nucleotide gap fill-in reaction using Klenow fragment (Fig. 1, B and C). The Oxa-containing 37-mer was purified by denaturing PAGE, reannealed to the top template strand, and extended to full length. Extension products were verified by GeneScan analysis using 10% denaturing PAGE (Fig. 1D).

Mutagenicity of Oxanine—We determined the possibility of incorporation of dOTP to DNA using a single-nucleotide gap substrate. In control reactions, human DNA polymerase {beta} extended Gua, Cyt, Ade, and Thy fully to form corresponding Watson-Crick base pairs (Fig. 2A). dOTP was only incorporated opposite a template Cyt with approximately 60% of the efficiency of dGTP. dOTP was not detectably incorporated opposite the other template bases (Fig. 2A). Similar results were obtained on a primer-template substrate when the downstream 23-mer was omitted (data not shown). These results suggest that human polymerase {beta} preferentially incorporates dOTP to pair with the cognate Cyt template.



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  		FIG. 2.
Mutagenicity of oxanine as a deoxynucleotide and in DNA. A, results of the addition of dOTP to DNA by human polymerase {beta}. Addition of dOTP and individual dNTP by human DNA polymerase {beta} was assayed as detailed under "Experimental Procedures." The N position in the top template strand was changed to Cyt, Gua, Thy, and Ade. Each template was tested by a Watson-Crick base pair dNTP and dOTP. F, carboxy-fluorescein fluorophore. B, extension of dNTP on an Oxa-containing template by human DNA polymerase {beta} is shown. H, HEX fluorophore (Integrated DNA Technologies).

 
To determine the mutagenicity of an Oxa base in DNA, a single-nucleotide gap substrate was formed by annealing the Oxa-containing template with two complementary top strands (Fig. 2B). Human polymerase {beta} incorporated all four nucleotides to pair with the Oxa template with varying efficiency (Fig. 2B). The incorporation of Cyt opposite Oxa was close to completion, whereas dATP and dTTP were incorporated ~80% as efficiently as dCTP (Fig. 2B). The incorporation of the Gua base was poor, generating ~16% misincorporation product. The Oxa base was not incorporated to pair with Oxa template by human polymerase {beta}.

Oxanine DNA Glycosylase Activities in Mammalian Cells— Given that small base modifications in general are recognized by DNA glycosylases (25), we decided to determine whether there was a DNA glycosylase with activity toward oxanine. Total proteins from pig thymus tissues were extracted and fractionated by stepwise ammonium sulfate precipitation. ODG activities were assayed using C/O and T/O substrates (Fig. 3). DNA cleavage corresponding to an approximately 36-mer fragment was observed primarily in 30–60% ammonium sulfate fractions for both C/O and T/O substrates, with the highest activities appearing at the 50% fraction. No DNA cleavage at a corresponding location was observed in the non-Oxa-containing DNA (Fig. 3, T/A lanes). The ODG activities were observed in similar fractions in calf liver tissues (data not shown). These results indicate that mammalian cells possess DNA glycosylases that contain ODG activities.



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  		FIG. 3.
Oxanine DNA glycosylase activities in pig thymus. The oxanine DNA glycosylase assays were conducted in a reaction mixture (10 µl) containing 10 nM fluorescently labeled oligonucleotide substrate (O/C, O/T, or T/A), 2 µl of ammonium sulfate-fractionated cell extract, 10 mM HEPES (pH 7.4), 10 mM DTT, 20% glycerol, and 2 mM EDTA. After incubating at 37 °C for 60 min, the reaction mixtures were supplemented with 0.5 µl of 1 N NaOH and incubated at 95 °C for 5 min to cleave the resulting apurinic/apyrimidic sites from glycosylase action. T/A is a perfectly matched double-stranded DNA without lesions or mismatches, in which both strands are 5' fluorescently labeled. The top and bottom strands are 66-mer and 60-mer, respectively (see Ref. 45 for details). M, 36-mer length marker. Sat., saturation.

 
To identify which human DNA glycosylases excise oxanine, we tested purified glycosylases that are known to recognize damaged purines, including hOGG1, hNEIL1, hNEIL2, and hAAG. E. coli endo VIII, FaPy-DNA glycosolase (MutM), hOGG1, hNEIL1, and hNEIL2 but not mouse methylpurine DNA glycosylase are known to form cross-linking products with oxanine (26, 27). Under our assay conditions, human AAG was the only enzyme that showed oxanine DNA glycosylase activity (Fig. 4A). AAG versions with N-terminal 54 ({Delta}54) and 79 ({Delta}79) amino acid truncations are commonly used for biochemical studies (24, 28, 29); an AAG protein with a truncation of the first 100 amino acids still retains glycosylase activity (30). The 54 ({Delta}54) and 79 ({Delta}79) versions of hAAG showed comparable ODG activity. An inosine-containing substrate (T/I) was used as a positive control, which was cleaved near completion (Fig. 4A). To rule out the possibility that the observed cleavage activities may come from E. coli expression host contamination, assays were performed with an active site mutant {Delta}54-E125Q. This mutant is catalytically inactive because of the change of a proposed general base residue Glu to Gln (24, 28, 31, 32). No DNA cleavage was observed from {Delta}54-E125Q, indicating that the ODG activities are intrinsic to hAAG (Fig. 4A). N169S is a mutant that enhances DNA cleavage activities (24, 33). As expected, ODG activities were detected with this mutant.



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  		FIG. 4.
ODG activity of purified hAAG and in-cell extracts from Aag KO mice. The T/I substrate consisted of a 5'-carboxyfluorescein-labeled top strand (66-mer) and a 5'-tetrachloro 6-carboxy fluorescein (TET)-labeled inosine-containing bottom strand (60-mer) with the inosine located at position 37 (45). A, ODG activity of hAAG. {Delta}54, N-terminal 54-amino acid deletion of hAAG; {Delta}79, N-terminal 79-amino acid deletion of hAAG. B, ODG activities in cell extracts from Aag KO mice. The DNA glycosylase assays were carried out at 37 °C for 60 min in a 10-µl volume containing 9 µl of Aag–/– or matched Aag+/+ cell extract and 1 µl of 100 nM substrate. The G/U substrate is identical to the T/I substrate except that the T/I base pair is substituted by G/U. M, 36-mer length marker.

 
To verify that the AAG is the authentic oxanine glycosylase in mammalian cells, we assayed oxanine cleavage in mouse Aag knock-out (KO) (Aag–/–) tissues. G/U, T/I, and C/I substrates were used to examine assay methodology in both KO and age-matched wild type spleen tissues. As expected, uracil DNA glycosylase activities were readily detected in both KO and wild type tissues (Fig. 4B). Cleavage of inosine in the T/I base pair was primarily observed in the wild type spleen tissue, consistent with previous reports (22, 34) that AAG is a major hypoxanthine DNA glycosylase in mammalian cells. In keeping with the preferential cleavage of the T/I base pair by purified mammalian AAG (28, 33, 35–37), little C/I cleavage activity was observed in the wild type cell extract (Fig. 4B). The band above the specific cleavage band at 36-mer was probably generated by other unidentified cleavage activities in cell extracts. Cleavage of both T/O and C/O substrates was detected in the wild type tissue, but little cleavage was observed in the KO tissue. These results suggest that AAG is a major oxanine DNA glycosylase in mammalian systems.

ODG Activity from hAAG—Initial experiments showed that hAAG was active toward any Oxa-containing base pair (Fig. 4A). To compare the effect of opposite bases on cleavage, we studied the cleavage kinetics using substrate in excess (enzyme:substrate (E:S) ratio of 1:10), E:S in equal molar ratio (E:S = 1:1), and enzyme in excess (E:S = 10:1). When the substrate was in excess, cleavage of N/O (N = Cyt, Thy, Ade, Gua) base pairs was approximately 2%, similar to the cleavage of the T/I substrate (Fig. 5, A and C). When the E:S ratio was raised to 1:1, the cleavage of N/O or T/I reached ~10%. There was no significant difference in cleavage rate among the four Oxa-containing base pairs (data not shown). The apparent rate constants were approximately 0.44 min–1 for N/O compared with 0.42 min–1 for T/I (Fig. 5). Excess of enzyme (E:S = 10:1) increased the cleavage of the N/O substrates to ~30%. Cleavage of T/I substrate approached completion in a 60-min incubation period (Fig. 5C), in keeping with a previous analysis (28).



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  		FIG. 5.
Cleavage and binding of Oxa-containing and T/I substrates by hAAG. Cleavage assays were performed as described under "Experimental Procedures," with 1 nM {Delta}54 wild type hAAG ({blacktriangleup}) (E:S = 1:10), 10 nM {Delta}54 wild type hAAG ()(E:S = 1:1), and 100 nM {Delta}54 wild type hAAG ({blacksquare}) (E:S = 10:1). Reactions were stopped on ice at indicated time points and followed by adding 0.5 µl of 1 N NaOH. Gel mobility shift analysis was performed as described under "Experimental Procedures." Lane 1, 0 nM {Delta}54-E125Q; lane 2, 10 nM; lane 3, 50 nM; lane 4, 100 nM; lane 5, 250 nM; lane 6, 500 nM. Data are based on at least two independent experiments. A, cleavage of C/O substrate. B, binding of C/O substrate. C, cleavage of T/I substrate. D, binding of T/I substrate. E, cleavage of single-stranded Oxa substrate. F, binding of single-stranded Oxa substrate.

 
We then compared the binding affinity of hAAG to the Oxa-containing substrates. Unlike the weak binding to the C/I base pair (Ref. 28 and Fig. S1 in Supplemental Data), binding to the C/O substrate was strong (Fig. 5B). Because the substrate-containing DNA was long (62-mer), hAAG forms a primary complex (ES) at low enzyme concentrations and secondary complexes (EnS) at higher enzyme concentrations. The apparent Kd values for the primary complex ranged from 115 nM for C/O and 160 nM for T/O to 180 nM for A/O and G/O. Under the same assay conditions, the apparent Kd value for T/I was approximately 460 nM (Fig. 5D). Evidently, there is no significant difference in binding affinity among the four Oxa-containing substrates.

Based on the observation that hAAG does not show significant preference in cleavage and binding to double-stranded Oxa-containing DNA, we determined the cleavage and binding to single-stranded Oxa-containing DNA. The cleavage patterns for the single-stranded Oxa substrate did not deviate much compared with the double-stranded Oxa substrates (Figs. 5E). The apparent rate constant for the single-stranded Oxa substrate assayed under the same conditions was approximately 0.42 min–1, a value very similar to the double-stranded Oxa substrates. Likewise, hAAG showed comparable binding affinity to the single-stranded Oxa substrate, with an apparent Kd value of 130 nM under the same assay conditions (Fig. 5F). On the other hand, the cleavage and binding to single-stranded inosine-containing DNA were barely detectable, even at high enzyme concentrations (Fig. S1 in Supplemental Data). This represents a significant biochemical difference between the two deaminated bases.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Oxanine is a newly discovered stable base lesion derived from deamination of guanine. This work investigates the mutagenicity and potential oxanine DNA glycosylase activities in mammalian systems. The potential of oxanine to mispair is addressed by in vitro studies as reported previously using Klenow fragment (17) and, in this study, using human polymerase {beta} (Fig. 2). Oxanine nucleotide triphosphate appears to be misinserted opposite thymine in DNA by a Klenow fragment, which would result in A/T to G/C mutations (17). The Klenow fragment also incorporates nucleotides to mispair with oxanine in DNA,3 suggesting that oxanine is promutagenic in either nucleotide or DNA. Human polymerase {beta} appears to preferentially insert dOTP to pair with Cyt in DNA (Fig. 2). However, oxanine in the DNA template is more mutagenic, as manifested by incorporation of Cyt, Thy, Ade, and, to a lesser extent, Gua opposite Oxa in DNA by human polymerase {beta} (Fig. 2). Thus, the mispairing property of oxanine in the DNA template appears broader than the natural universal base, hypoxanthine, which pairs primarily with Cyt and Ade (38).

Mammalian AAGs are broad substrate enzymes that act on alkylated or cyclic alkylated and deaminated bases (39). In addition to 3-methyladenine, 7-methylguanine, 1,N6-ethenoadenine ({epsilon}A), and hypoxanthine, xanthine is also found as a substrate for hAAG (9). Removal of 1,N2-ethenoguanine ({epsilon}G), however, appears to require full-length AAG (40). Crystal structures of {Delta}79-AAG complexed with the abasic pyrrolidine inhibitor or with {epsilon}A provide important insights on base discrimination and catalytic mechanisms (23, 29). Interestingly, the active site appears to act more to discriminate against the normal purines than to specifically recognize the damaged purines. One key feature of this discrimination in the active site is the potential steric clash between Asn-169 of AAG and the N2-amino group of guanine (24, 33). The damaged adenine derivatives 3-methylA, {epsilon}A, and hypoxanthine lack the N2-amino group. Discrimination against adenine is accomplished by the main chain amide of His-136 (29). However, how guanine-derived substrates xanthine, oxanine, 7-methylG, and {epsilon}G occupy the AAG active site is not understood. A subtle conformational adjustment appears to be needed to accommodate the N2-amino group of xanthine, oxanine and 7-methylG without clashing with Asn-169, while maintaining strong discrimination against guanine. A more substantial conformational change, which involves the nonconserved N-terminal region, may be needed to prevent a steric clash caused by the bulkier {epsilon}G.

The cleavage and binding of Oxa-containing double-stranded DNA by AAG are essentially independent of the opposite base. In fact, the ODG activity of AAG does not require a complementary strand at all, as evidenced by similar cleavage and binding of single-stranded DNA (Fig. 5, A, B, E, and F). Cleavage and binding of {epsilon}A in double-stranded DNA are also independent of opposite pyrimidine bases (28, 33, 37). We subsequently tested the excision of {epsilon}A from single-stranded DNA. Indeed, AAG can excise {epsilon}A from single-stranded DNA, albeit with compromised efficiency compared with {epsilon}A from double-stranded DNA (Fig. S2 in Supplemental Data). These observations are in stark contrast with hypoxanthine-containing DNA. Removal of hypoxanthine is highly dependent on the opposite base. The T/I base pair is a very good substrate for AAG, but C/I is a poor one (Refs. 28 and 35–37 and Fig. S1 in Supplemental Data). At least part of the poor activity of C/I can be attributed to weak binding by AAG (Ref. 28 and Fig. S1 in Supplemental Data). There is little cleavage of single-stranded inosine-containing DNA by AAG (Ref. 41 and Fig. S1 in Supplemental Data), suggesting that the interactions with the complementary strand are important for the cleavage of the inosine-containing substrates. Human AAG preferentially removes 7-methylG opposite Thy over Cyt (33) and shows very poor activity on single-stranded 7-methylG-containing DNA.4 It appears that AAG exhibits two modes of interaction with its alkylated and deaminated base substrates, as classified by its ability to remove lesions in single-stranded DNA. The first mode of interaction can be represented by oxanine and {epsilon}A. AAG can recognize these lesions in single-stranded DNA. Furthermore, the identity of the DNA base opposing these lesions does not severely affect excision of the lesion. The second mode of interaction can be exemplified by hypoxanthine and 7-methylguanine. Recognition and excision of these lesions are highly dependent on the double-stranded structure and highly sensitive to the identity of the base opposite of the lesion.

ODG activities dealing with Oxa-containing lesions appear to be ubiquitous in nature. AlkA, similar to hAAG, is a broad substrate DNA glycosylase that can remove alkylated and deaminated bases. However, the deaminated base DNA glycosylase activities of E. coli AlkA seem low compared with its alkylbase DNA glycosylase activities (18, 41). Endonuclease VIII is a DNA glycosylase that removes oxidative base lesions (42, 43). Compared with other lesions, the ODG activities of both AlkA and endo VIII are quite low (18). Enzyme in excess (30-fold) is required to detect cleavage products (18). It is reported (18) that both AlkA and endo VIII preferentially remove oxanine from A/O, C/O, and G/O base pairs. Both enzymes show even poorer activities for the T/O base pair. This study demonstrates that the ODG activity of hAAG is more robust than E. coli AlkA, in keeping with the previous observation of higher hypoxanthine DNA glycosylase activity in hAAG (41). Another distinct feature of human AAG is that it shows no preference to Oxa-containing base pairs. It has been suggested that AAG evolved from a hypoxanthine-specific DNA glycosylase to a broader substrate enzyme (33, 44). The novel ODG activities in mammalian AAG underscore the intriguing adaptation of the active site to all three deaminated purine and several alkylated purine lesions.


   FOOTNOTES
 
* This work was supported in part by Cooperative State Research, Education, and Extension Service, U. S. Department of Agriculture, Grant SC-1700153, technical contribution No. 4994, National Institutes of Health Grant GM 067744, and the Concern Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Figs. 1S and 2S. Back

Supported by NIEHS, National Institutes of Health Grant K22 ES00333. Back

** To whom correspondence should be addressed: Dept. of Genetics, Biochemistry & Life Science Studies, SC Experiment Station, Clemson University, Rm. 219, Biosystems Research Complex, 51 New Cherry St., Clemson, SC 29634. Tel.: 864-656-4176; Fax: 864-656-0393; E-mail: wgc{at}clemson.edu.

1 The abbreviations used are: 5-MeC, 5-methyl cytosine; A, adenine; G, guanine; C, cytosine; I, inosine or hypoxanthine; X, xanthine; U, uracil; T, thymine; O, oxanine; ODG, oxanine DNA glycosylase; AAG, alkyladenine glycosylase; hAAG, human AAG; RP-HPLC, reverse phase high performance liquid chromatography; DTT, dithiothreitol; hOGG1, human 8-oxo-guanine DNA glycosylase 1; hNEIL, human Neilike; KO, knock-out (tissue); {epsilon}A, 1, N6-ethenoadenine; {epsilon}G, 1,N2-ethenoguanine; EcoPol buffer, 10 mM Tris-HCl, 5 mM MgCl2, 7.5 mM DTT, pH 7.5, at 25 °C. Back

2 L. Dong and W. Cao, unpublished data. Back

3 Hitchcock, T. M., Gao, H., and Cao, W. (2004) Nucl. Acids Res. 32, 4071–4080. Back

4 P. J. O'Brien and T. Ellenberger, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Tom Ellenberger, Patrick O'Brien, Peter Dedon, and members of the Cao laboratory for stimulating discussions. We also thank Sam Wilson for providing human DNA polymerase {beta}, Tom Ellenberger and Patrick O'Brien for {Delta}79-hAAG proteins and clones, Sankar Mitra and Tapas K. Hazra for hOGG1, hNEIL1, and hNEIL2, and Jonathan Campbell at Clemson University Meat Laboratory for mammalian tissues.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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