Chem. Res. Toxicol. 2003, 16, 1405-1409
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O6-Alkylguanine-DNA Alkyltransferases Repair O6-Methylguanine in DNA with Michaelis-Menten-like Kinetics Aviva S. Meyer,† Melodie D. McCain,† Qingming Fang,‡ Anthony E. Pegg,‡ and Thomas E. Spratt*,† Institute for Cancer Prevention, American Health Foundation Cancer Center, One Dana Road, Valhalla, New York 10595, and Pennsylvania State University Milton Hershey Medical Center, Hershey, Pennsylvania 17033 Received June 23, 2003
O6-Alkylguanine-DNA alkyltransferase (AGT) repairs O6-methylguanine (O6mG) by transferring the methyl group from the DNA to a cysteine residue on the protein. The kinetics of this reaction was examined by reacting an excess of AGT (0-300 nM) with [5′-32P]-labeled oligodeoxynucleotides (0.5 nM) of the sequence 5′-CGT GGC GCT YZA GGC GTG AGC-3′ in which Y or Z was G or O6mG, annealed to its complementary strand. The reactions, conducted at 25 °C, were quenched by the addition of 0.1 N NaOH at various times, and the extents of reaction were monitored by ion exchange HPLC with radiochemical detection. The time courses followed first-order kinetics. The first-order rate constants were plotted against the initial concentration of AGT and fitted to the hyperbolic equation kobs ) kinact[AGT]0/(KS + [AGT]0). The KS values for hAGT of 81-91 nM are 10-fold lower than the dissociation constants of hAGT (C145S) to unmodified and O6mG-containing DNA obtained by EMSA and indicate that AGT has a preference for binding to O6mG in DNA. The proteins reacted with DNA in which Y ) O6mG and Z ) G faster than Y ) G and Z ) O6mG due to an approximately 10-fold increase in kinact. These results suggest that the sequence specificity in the repair of O6mG is manifested in the methyl transfer not in the O6mG recognition step.
Introduction AGT1 is a crucial protein that defends cells against the toxic and mutagenic effects of various endogenous and exogenous alkylating agents (1-4). AGT is a small monomeric protein found in archaea (5), bacteria (6-9), and eukarya (10, 11). A principle target of AGT is O6mG, which if not repaired leads to G to A transition mutations (12, 13). AGT also repairs bulkier adducts such as O6-(2-chloroethyl)guanine (14), O6-benzylguanine (15), and O6-[4-oxo-4-(3-pyridyl)butyl]guanine (16). AGT repairs DNA by transferring the alkyl group from the O6position of guanine to a cysteine residue on the protein (17, 18). The original guanine is restored, but the free cysteine on AGT is never regenerated; consequently, the protein can act only once. AGT and O6mG in DNA have been shown to react with second-order kinetics (19-23), consistent with a simple bimolecular reaction. However, other evidence suggests that there should be an initial binding of the DNA to the protein prior to methyl transfer (Scheme 1), a mechanism that should exhibit saturation kinetics. For example, EMSAs and fluorescence anisotropy experiments have * To whom correspondence should be addressed. Tel: 914-789-7289. Fax: 914-729-3344. E-mail:
[email protected]. † American Health Foundation Cancer Center. ‡ Pennsylvania State University Milton Hershey Medical Center. 1Abbreviations: Ada, AGT expressed by the ada gene of E. coli.; Ada-C, the C-terminal domain of Ada; AGT, O6-alkylguanine-DNA alkyltransferase; BSA, bovine serum albumin; EMSA, electrophoretic mobility shift assay; C145S, hAGT with the serine substitution at residue 145; hAGT, human O6-alkylguanine-DNA alkyltransferase; O6mG, O6-methylguanine; Ogt, AGT expressed by the ogt gene of E. coli.
Scheme 1. Kinetic Scheme for the Reaction between AGT and Oligodeoxynucleotides Containing O6mGa
a E represents AGT, S represents the substrates 1 or 2, ES represents the Michaelis complex, and X inactivated AGT and D the repaired DNA 3. KS ) k-1/k1.
shown that the oligodeoxynucleotides bind to wild-type AGT with a Kd in the micromolar range (23-26). Crystal structures of AGT (5, 27-29) show a potential binding cleft for the DNA that may allow AGT to scan the DNA in search of O6mG. In addition, the reaction between the free base O6mG and Ada-C exhibits saturation kinetics with a Km in the millimolar range (30). To determine whether AGT reacts with O6mG in DNA with an initial complex prior to methyl transfer, we examined the kinetics of the reaction between an excess of AGT with O6mG in oligodeoxynucleotide duplexes (21mers) in two sequence contexts. We have found that the reaction exhibited saturation kinetics with a KS from 59 to 91 nM, an indication that AGT has a 10-fold preference for O6mG over unmodified DNA. In addition, we found
10.1021/tx0341254 CCC: $25.00 © 2003 American Chemical Society Published on Web 10/24/2003
1406 Chem. Res. Toxicol., Vol. 16, No. 11, 2003
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that the sequence specificity of the reaction is derived from differences in kinact, not KS.
Materials and Methods AGT. hAGT with a C-terminal (His)6 tail was purified as described (31). The C145S mutant of hAGT with a C-terminal (His)6 tail was purified as described (32). Two alkyltransferases from Escherichia coli, Ogt (33) and the C-terminal domain of the Ada protein (Ada-C) (30), were purified as described. The activity of AGT was assayed as described previously (30, 34, 35). Oligodeoxynucleotide Substrates. Oligodeoxynucleotides of the sequences shown in Scheme 1 were obtained from Oligos Etc. (Corvallis, OR) and were purified by reverse phase HPLC on either a Prodigy 5 µm ODS(3) (4.6 mm × 250 mm) or a Luna 5 µm C18(2) (4.6 mm × 250 mm) column, which were eluted with a linear gradient of 10-40% methanol in 100 mM Et3NHOAc (pH 7.0) over 30 min. The O6mG-containing oligodeoxynucleotides (25 pmol) were incubated with 1 unit of polynucleotide T4 kinase (Promega, Madison, WI) and 50 µCi of [γ-32P]ATP (6000 Ci/mmol, Amersham) in 70 mM Tris-HCl (pH 7.6), 10 mM MgCl2, and 5 mM DTT at 37 °C for 30 min. The reaction was quenched by heating at 60 °C for 5 min, and the oligodeoxynucleotide was purified with a spin column containing 1 mL of Sephadex G-25 equilibrated with 50 mM Tris-HCl (pH 8.0) and 1 mM EDTA. For the kinetic assays, the labeled oligodeoxynucleotides were annealed to a 25% excess of the complementary strand by heating at 90 °C and slowly cooling to room temperature over approximately 2 h. For the EMSA experiments, the labeled oligodeoxynucleotides were annealed to a 100-fold excess of the complementary strand. AGT-Substrate Reactions. The reactions were performed on a RQF-3 Rapid Quench instrument (KinTek Corp., Austin, TX) in which 16.9 µL of AGT was mixed with 17.2 µL of DNA in 25 mM HEPES-NaOH (pH 7.8), 1 mM DTT, and 100 µg/mL BSA at 25 °C. The reactions were quenched by the addition of 0.1 N NaOH. The quenched solutions were kept on dry ice until analyzed. The extents of reaction were monitored by HPLC using a strong anion exchange DNA Pac PA-100 (4.6 mm × 250 mm) (Dionex, Sunnyvale, CA) column (36, 37). The column was eluted with a 0.55-0.7 M linear NaCl gradient in 10 mM NaOH containing 0.5% acetonitrile over 30 min. The substrate eluted at 21 min while the product eluted at 24 min. Kinetic Data Analysis. The extent of the reaction was determined by dividing the area under the product peak by the sum of the areas under the substrate and product peaks. The concentration of product was determined by multiplying the initial concentration of the substrate by the extent of reaction. This procedure is illustrated in eq 1 in which AP and AS are the areas under the product and substrate peaks, respectively; [S]0 is the initial substrate concentration, and [X] is the concentration of the inactivated AGT as well as the repaired DNA, at a particular time, t.
AP [X] ) [S]0 AP + AS
(1)
The formation of 1 from the reactions between AGT and 2 or 3 were fitted to the first-order kinetic equation shown in eq 2. Values for kinact and KS were determined by plotting the kobs vs initial AGT concentration using eq 3. The experimental data were fitted to the theoretical equations using the nonlinear regression module of GraphPad Prism version 4 for Windows, GraphPad Software, San Diego, CA, www.graphpad.com.
[X] ) [S]0(1 - e-kobst) kobs )
kinact[E]0 KS + [E]0
(2) (3)
EMSA. Radiolabeled oligodeoxynucleotide (10 nM) was incubated for 30 min with C145S (0-30 µM) in 25 mM Hepes, pH 7.8, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, and 10% glycerol at 25 °C in a total volume of 10 µL. The solution was then loaded on a 6% polyacrylamide gel (20 cm × 16 cm × 0.1 cm) in 45 mM Tris, 45 mM boric acid, and 1 mM EDTA (pH 8.3) (0.5 × TBE) and run at 200 V for 1 h. The gel was visualized with a BioRad G250 Molecular Imager. The binding of n molecules of AGT (E) to one DNA duplex (D) may be represented by nE + D h EnD. The dissociation constant K ) [E]n[D]/[EnD] can be rearranged to eq 4, in which the ratio of the free DNA (D) to total DNA (D + EnD) can be fit to the hyperbolic equation dependent on [E]n. The value of n was confirmed by plotting the natural logarithm of the ratio of bound to free DNA vs the natural logarithm of the concentration of AGT according to eq 5.
[D] 1 ) [D] + [EnD] [E]n +1 K ln
( ) [EnD] [D]
) nln[E] - lnK
(4)
(5)
Results AGT was reacted with oligodeoxynucleotide duplexes 2 or 3 (Scheme 1), which contain the 9th to 15th codons of the H-ras gene. Georgiadis et al. (20) found that Ada repairs O6mG at the first G of the 12th codon 20-fold more rapidly than when placed at the second G. These results suggested that this difference in repair rate might be part of the reason that the 12th codon is mutated at the first G but not the second G in mammary and skin tumors induced by N-methyl-N-nitrosourea. We chose to study the kinetics of repair of this sequence to examine the mechanisms underlying sequence specific repair. AGT had previously been found to react with O6mG in DNA with second-order kinetics in which the rate of reaction is proportional to the concentrations of the substrate and protein (19-23). This type of kinetics is typical of the reaction of two small molecules. However, as in the case of an enzyme-catalyzed reaction, we would expect to observe saturation of the rate of repair as the substrate concentration was increased to fully fill the active site of AGT. However, because AGT is not an enzyme, if the substrate concentration was increased too far above the AGT concentration, the percent conversion of O6mG to G would be insignificant and therefore difficult to measure. Consequently, we examined this reaction by varying the concentration of AGT. We analyzed the kinetics of the reaction with the simple enzymelike reaction mechanism illustrated in Scheme 1. Under the conditions in which the concentration of AGT is much greater than substrate ([E]0 . [S]0), the free AGT concentration will be approximately equal to the total AGT concentration and lead to the steady state approximation in eq 6. Rearrangement of eq 6 to eq 7 allows the derivation of rate eqs 2 and 3.
d[E] ) 0 ) k-1[ES] - k1[E][S] dt Ks )
k-1 [E][S] ) k1 [ES]
(6) (7)
As illustrated in Figure 1A, an excess of hAGT reacted with 2 in a time-dependent manner that could be fit to a
Communications
Figure 1. Time course of the reaction between hAGT and 1. (A) The reaction between 25 nM hAGT and 0.5 nM 2 was fit to eq 2. The Y-axis in the insert is ln((Pi - P∞)/(P0 - P∞)), where Pi is the concentration of product at ti. (B) The time courses of the reaction between 5 nM 2 and 2 (9), 5 (4), 10 (1), 15, (]), 25 (b), 50 (0), 100 (2), and 150 (O) nM AGT. Additional, longer time points for 5 and 10 nM reactions are not shown. The solid lines are the best fit of all of the data to eq 2.
Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1407
Figure 2. Dependence of kobs on the concentration of AGT. Plots of kobs vs [hAGT] (9), Ada-C (2), and Ogt (b) with 0.5 nM 2 (A) or 3 (B). The error bars are standard errors obtained from the analyses of eq 2. The lines (hAGT (s), Ada-C (‚ ‚ ‚), and Ogt (- - -)) are the best fits of the data to eq 3. Table 1. Kinetic Constants for the Repair and Binding of Oligodeoxynucleotides by AGTa protein
first-order equation. We also found that as the hAGT concentration increased, the rate of reaction increased and then leveled off (Figure 1B). The first-order rate constants for the reactions of 2 or 3 with hAGT, Ogt, and Ada-C were fitted to eq 3 as illustrated in Figure 2, and the kinetic parameters are summarized in Table 1. The three variants of AGT repaired 3 (Figure 2B) more slowly than 2 (Figure 2A) due to an approximately 10-fold decrease in kinact, while the KS values remained unaffected by the difference in substrate. Ada-C and Ogt reacted with 2 and 3 two to three times faster than hAGT due to increased kinact values. The AGT-DNA binding affinity was also measured by EMSA as previously described (38, 39). The inactive C145S mutant of AGT was used so that we could measure the association with 2 and 3, which contain O6mG. Although C145S cannot react with O6mG because the alkyl acceptor cysteine-145 is replaced with a serine residue, the protein retains affinity for DNA (24). Stoichiometries and dissociation constants were determined for 1-3 with the inactive C145S mutant of hAGT by fitting the fraction of free DNA ([D]/([D] + [EnD])) vs C145S concentration with eq 4 as illustrated in Figure 3. The data were consistent with three molecules of C145S bound to one DNA duplex. Previously, it was found that up to four AGT molecules could bind to a 16mer duplex (31, 38, 39). The resulting dissociation constants are equal to the product of the three individual dissociation constants K ) K1K2K3 and have units of M3. The cubed roots of this value, which we have referred to
hAGT hAGT Ogt Ogt Ada Ada C145S C145S C145S
substrate (Y,Z) (O6mG,G)
2 3 (G,O6mG) 2 (O6mG,G) 3 (G,O6mG) 2 (O6mG,G) 3 (G,O6mG) 1 (G,G) 2 (O6mG,G) 3 (G,O6mG)
kinact (s-1)
KS or Kd (nM)
0.39 ( 0.03 0.035 ( 0.002 1.1 ( 0.1 0.13 ( 0.02 1.33 ( 0.22 0.12 ( 0.02
81 ( 11 91 ( 12 67 ( 15 68 ( 14 59 ( 29 80 ( 30 760 ( 80 380 ( 50 240 ( 40
a Reaction performed in 25 mM HEPES-NaOH (pH 7.8), 1 mM DTT, 1 mM EDTA, and 100 µg/mL BSA at 25 °C with 0.5 nM DNA substrate and 0-300 nM AGT. EMSA experiments performed with AGT (0-30 µM) and oligodeoxynucleotide duplex (50 nM modified strand and 500 nM complementary strand) in 25 mM HEPESNaOH (pH 7.8), 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, and 10% glycerol.
as the monomer equivalent dissociation constant (Kd), are also the concentrations of AGT at which equal amounts of DNA are free and bound to C145S, and they are presented in Table 1. The Kd for 1 of 760 nM is 7-fold less than that obtained for oligodeoxynucleotides containing the central 15 base pairs of 1 to wild-type or C145A hAGT as reported previously (23) but is consistent with recent studies (31, 38). The increased affinity of the protein for the DNA may be due to the use of C145S, which better maintains the hydrogen-bonding network at the active site (27), in place of C145A, and/or by the improved zinc content (31). The Kd values for 2 and 3 are 2-3-fold less than for 1, suggesting a preference for binding to O6mG over unmodified DNA. Quantifying this
1408 Chem. Res. Toxicol., Vol. 16, No. 11, 2003
Figure 3. Analysis of binding of DNA to C145S by EMSA. The fraction of unbound DNA ([D]/[D] + [EnD]) is plotted against the concentration of C145S ([E]) for 1 (O, - - -), 2 (9, ;), and 3 (b, ‚ ‚ ‚). The lines are the nonlinear best fits of the data to eq 4 in which n ) 3. The error bars are the SDs of at least three independent determinations.
preference is difficult because the Kd values represent the binding to the entire duplex and not just to the O6mG site.
Discussion We have shown that the reaction between O6mG in an oligodeoxynucleotide duplex and AGT exhibits saturation kinetics. These results are consistent with the mechanism illustrated in Scheme 1 in which the methyl transfer is preceded by the formation of the Michaelis complex. The KS was found to be between 59 and 91 nM for the substrates and proteins tested. The kinact varied from 0.035 to 1.33 s-1 depending on protein and the O6mG sequence context. By EMSA, we have found that AGT binds to O6mG more tightly than unmodified DNA. This difference in affinity is difficult to quantify because Kd values from the EMSA experiments represent binding to an entire oligodeoxynucleotide duplex and not to the O6mG/G site. However, by analyzing the kinetics of the methyl transfer, we obtain KS, the dissociation constant of the Michaelis complex, which is presumably O6mG, bound to the active site of the protein. The difference between the KS for hAGT and the Kd for unmodified DNA with hAGT (C145S) by EMSA is approximately 10-fold. Thus, we conclude that hAGT binds to O6mG approximately 10 times more tightly than unmodified DNA. Differences in rates of repair of many types of DNA damage are influenced by the local DNA environment (40, 41). The sequence specificity of AGT has been investigated in few studies (20, 42-44), and the results have not yet reached a point where we understand the mechanisms underlying the sequence specificity. Differences in rate of 25-fold have been observed between purified Ada-C and different sequences containing O6mG (20). We examined the repair of O6mG in 21-mers, in which the central 15 base pairs were the sequences used by Georgiadis et al. (20). We found that 2 was repaired approximately 10-fold faster than 3 by Ada-C, Ogt, and hAGT, and the differences in rate were due to differences in kinact and not KS. Thus, AGT recognizes O6mG in different sequences equally but repairs them with different rates. We analyzed the kinetics of the AGT reaction according to the simple reaction mechanism in Scheme 1, but the
Communications
mechanism of action of AGT is undoubtedly more complex. Crystal structures of AGT show that the active site cysteine is not on the exterior of the protein but buried in a hydrophobic pocket, and there is a groove into which the DNA can potentially fit (5, 27-29). These structures have lead to the hypothesis that AGT flips O6mG out of the helix in order to displace the methyl group (27, 29). The possibility that AGT binds to an extrahelical O6mG suggests a multistep mechanism in which (i) AGT first binds nonspecifically to DNA with a Kd of 0.8 µM, (ii) AGT binds to and flips O6mG out of the helix into the active site pocket, and (iii) AGT transfers the methyl group to a cysteine residue on the protein. In this scenario, kinact would represent the rate of methyl transfer while KS would represent the binding of the extrahelical O6mG to AGT. Consequently, the sequence surrounding O6mG would affect the rate of methyl transfer. Our kinetic data, however, cannot rule out a second mechanism, in which KS represents the binding of AGT to the O6mG:C base pair while kinact is the rate of base flipping, which is followed by rapid methyl transfer. In this model, the rate of base flipping would be dependent on the surrounding sequence.
Acknowledgment. This work was supported by NCI grants CA75074 (T.E.S.) and CA18137 (A.E.P.). Supporting Information Available: Tritium assay to determine concentration of active AGT, example of analysis of extent of reaction using HPLC, EMSA to determine binding of hAGT to DNA, and mathematical derivation of rate equation. This material is available free of charge at http://pubs.acs.org.
References (1) Samson, L. (1992) The suicidal DNA repair methyltransferases of microbes. Mol. Microbiol. 6, 825-831. (2) Sekiguchi, M., Nakabeppu, Y., Sakumi, K., and Tuzuki, T. (1996) DNA-repair methyltransferase as a molecular device for preventing mutation and cancer. J. Cancer Res Clin. Oncol. 122, 199206. (3) Pegg, A. E. (2000) Repair of O6-alkylguanine by alkyltransferases. Mutat. Res. 462, 83-100. (4) Margison, G. P., and Santibanez-Koref, M. F. (2002) O6-alkylguanine-DNA alkyltransferase: role in carcinogenesis and chemotherapy. BioEssays 24, 255-266. (5) Hashimoto, H., Inoue, T., Nishioka, M., Fujiwara, S., Takagi, M., Imanaka, T., and Kai, Y. (1999) Hyperthermostable protein structure maintained by intra and inter-helix ion-pairs in archaeal O6-methylguanine-DNA methyltransferase. J. Mol. Biol. 292, 707-716. (6) Samson, L., and Cairns, J. (1977) A new pathway for DNA repair in Escherichia coli. Nature 267, 281-282. (7) Demple, B., Jacobsson, A., Olsson, M., Robins, P., and Lindahl, T. (1982) Repair of alkylated DNA in Escherichia coli. J. Biol. Chem. 257, 13776-13780. (8) Potter, P. M., Wilkinson, M. C., Fitton, J., Carr, F. J., Brennand, J., Cooper, D. P., and Margison, G. P. (1987) Characterization and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E. coli. Nucleic Acids Res. 15, 9177-9193. (9) Margison, G. P., Cooper, D. P., and Potter, P. M. (1990) The E. coli ogt gene. Mutat. Res. 233, 15-21. (10) Pegg, A. E., and Dolan, M. E. (1987) Properties and assay of mammalian O6-alkylguanine-DNA alkyltransferase. Pharmacol. Ther. 34, 167-179. (11) Hakura, A., Morimoto, K., Sofuni, T., and Nohmi, T. (1991) Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase. J. Bacteriol. 173, 3663-3672. (12) Loechler, E. L., Green, C. L., and Essigmann, J. M. (1984) In vivo mutagenesis by O6-methylguanine built into a unique site in a viral genome. Proc. Natl. Acad. Sci. U.S.A. 81, 6271-6275. (13) Pauly, G. T., and Moschel, R. C. (2001) Mutagenesis by O6methyl-, O6-ethyl-, and O6-benzylguanine and O4-methylthymine in human cells: effects of O6-alkylguanine-DNA alkyltransferase and mismatch repair. Chem. Res. Toxicol. 14, 894-900.
Communications (14) Brent, T. P., Remack, J. S., and Smith, D. G. (1987) Characterization of a novel reaction by human O6-alkylguanine-DNA alkyltransferase with 1,3-bis(2-chloroethyl)-1-nitrosourea-treated DNA. Cancer Res. 47, 6158-6188. (15) Pegg, A. E., Boosalis, M., Samson, L., Moschel, R. C., Byers, T. L., Swenn, K., and Dolan, M. E. (1993) Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine. Biochemistry 32, 11998-12006. (16) Wang, L., Spratt, T. E., Liu, X. K., Hecht, S. S., Pegg, A. E., and Peterson, L. A. (1997) Pyridyloxobutyl adduct (O6-[4-oxo-4-(3pyridyl)butyl]guanine, is present in 4-(acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanone-treated DNA and is a substrate for O6alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 10, 562567. (17) Demple, B., Sedgwick, B., Robind, P., Totty, N., Waterfield, M. D., and Lindahl, T. (1985) Active site and complete sequence of the suicidal methyltransferase that counters alkylation mutagenesis. Proc. Natl. Acad. Sci. U.S.A. 82, 2688-2692. (18) Major, G. N., Gardner, E. J., Carne, A. F., and Lawley, P. D. (1990) Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine. Nucleic Acids Res. 18, 1351-1359. (19) Dolan, M. E., Oplinger, M., and Pegg, A. E. (1988) Sequence specificity of guanine alkylation and repair. Carcinogenesis 9, 2139-2143. (20) Georgiadis, P., Smith, C. A., and Swann, P. F. (1991) Nitrosamineinduced cancer: selective repair and conformational differences between O6-methylguanine residues in different positions in and around codon 12 of rat H-ras. Cancer Res. 51, 5843-5850. (21) Graves, R. J., Li, B. F. L., and Swann, P. F. (1989) Repair of O6methylguanine, O6-ethylguanine, O6-isopropylguanine and O4methylthymine in synthetic oligodeoxynucleotides by Escherichia coli ada gene O6-alkylguanine-DNA-alkyltransferase. Carcinogenesis 10, 661-666. (22) Scicchitano, D., and Pegg, A. E. (1982) Kinetics of repair of O6methylguanine in DNA by O6-methylguanine-DNA methyltransferase in vitro and in vivo. Biochem. Biophys. Res. Commun. 109, 995-1001. (23) Spratt, T. E., Wu, J. D., Levy, D. E., Kanugula, S., and Pegg, A. E. (1999) Reaction and binding of oligodeoxynucleotides containing analogues of O6-methylguanine with wild-type and mutant human O6-alkylguanine-DNA alkyltransferase. Biochemistry 38, 6801-6806. (24) Hazra, T. K., Roy, R., Biswas, T., Grabowski, D. T., Pegg, A. E., and Mitra, S. (1997) Specific recognition of O6-methylguanine in DNA by active site mutants of human O6-methylguanine-DNA methyltransferase. Biochemistry 36, 5769-5776. (25) Chan, C. L., Wu, Z., Ciardelli, T., Eastman, A., and Bresnick, E. (1993) Kinetic and DNA binding properties of recombinant human O6-methylguanine-DNA methyltransferase. Arch. Biochem. Biophys. 300, 193-200. (26) Takahashi, M., Sakumi, K., and Sekiguchi, M. (1990) Interaction of Ada protein with DNA examined by fluorescence anistropy of the protein. Biochemistry 29, 3431-3436. (27) Daniels, D. S., Mol, C. D., Arvai, A. S., Kanugula, S., Pegg, A. E., and Tainer, J. A. (2000) Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding. EMBO J. 19, 1719-1730. (28) Moore, M. H., Gulbis, J. M., Dodson, E. J., Demple, B., and Moody, P. C. E. (1994) Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli. EMBO J. 13, 1495-1501.
Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1409 (29) Wibley, J. A. E., Pegg, A. E., and Moody, P. C. E. (1999) Crystal structure of the human O6-alkylguanine-DNA alkyltransferase. Nucleic Acids Res. 28, 393-401. (30) Spratt, T. E., and de los Santos, H. (1992) Reaction of O6alkylguanine-DNA alkyltransferase with O6-methylguanine analogues: evidence that oxygen of O6-methylguanine is protonated by the protein to effect methyl transfer. Biochemistry 31, 36883694. (31) Rasimas, J. J., Kanugula, S., Dalessio, P. M., Ropson, I. J., Fried, M. G., and Pegg, A. E. (2003) Effects of zinc occupancy on human O6-alkylguanine-DNA alkyltransferase. Biochemistry 42, 980990. (32) Liu, L., Xu-Welliver, M., Kanugula, S., and Pegg, A. E. (2002) Inactivation and degradation of O6-alkylguanine-DNA alkyltransferase after reaction with nitric oxide. Cancer Res. 62, 3037-3043. (33) Goodtzova, K., Kanugula, S., Edara, S., Pauly, G. T., Moschel, R. C., and Pegg, A. E. (1997) Repair of O6-benzylguanine by the Escherichia coli Ada and Ogt and the human O6-alkylguanineDNA alkyltransferases. J. Biol. Chem. 272, 8332. (34) Karran, P., Lindahl, T., and Griffin, B. (1979) Adaptive response to alkylating agents involves alteration in situ of O6-methylguanine residues in DNA. Nature 280, 76-77. (35) Spratt, T. E., Zydowsky, T. M., and Floss, H. G. (1997) Stereochemistry of the in vitro and in vivo methylation of DNA by (R)and (S)-N-[2H1,3H]methyl-N-nitrosourea and (R)- and (S)-Nnitroso-N-[2H1,3H]methyl-N-methylamine. Chem. Res. Toxicol. 10, 1412-1419. (36) Xu, Y. Z., and Swann, P. F. (1992) Chromatographic separation of oligodeoxynucleotides with identical length: application to purification of oligomers containing a modified base. Anal. Biochem. 204, 185-189. (37) Spratt, T. E., and Campbell, C. R. (1994) Synthesis of oligodeoxynucleotides containing analogues of O6-methylguanine and reaction with O6-alkylguanine-DNA alkyltransferase. Biochemistry 33, 11364-11371. (38) Rasimas, J. J., Pegg, A. E., and Fried, M. G. (2003) DNA-binding mechanism of O6-alkylguanine-DNA alkyltransferase. Effects of protein and DNA alkylation on complex stability. J. Biol. Chem. 278, 7973-7980. (39) Fried, M. G., Kanugula, S., Bromberg, J. L., and Pegg, A. E. (1996) DNA binding mechanism of O6-alkylguanine-DNA alkyltransferase: stoichiometry and effects of DNA base composition and secondary structure on complex stability. Biochemistry 35, 1529515301. (40) Singer, B., and Hang, B. (1997) What structural features determine repair enzyme specificity and mechanism in chemically modified DNA? Chem. Res. Toxicol. 10, 713-732. (41) Singer, B., and Hang, B. (2000) Nucleic acid sequence and repair: role of adduct, neighbor bases and enzyme specificity. Carcinogenesis 21, 1071-1078. (42) Delaney, J. C., and Essigmann, J. M. (1999) Context-dependent mutagenesis by DNA lesions. Chem. Biol. 6, 743-753. (43) Delaney, J. C., and Essigmann, J. M. (2001) Effect of sequence context on O6-methylguanine repair and replication in vivo. Biochemistry 40, 14968-14975. (44) Bender, K., Federwisch, M., Loggen, U., Nehls, P., and Rajewsky, M. F. (1996) Binding and repair of O6-ethylguanine in doublestranded oligodeoxynucleotides by recombinant human O6-alkylguanine-DNA alkyltransferase do not exhibit significant dependence on sequence content. Nucleic Acids Res. 24, 2087-2094.
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