Expression of the inactive C145A mutant human O6-alkylguanine-DNA alkyltransferase in E.coli increases cell killing and mutations by N-methyl-N'-nitro-N-nitrosoguanidine
Suvarchala Edara,
Sreenivas Kanugula and
Anthony E. Pegg1
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
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Abstract
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Human O6-alkylguanine-DNA alkyltransferase (AGT) counteracts the mutagenic and toxic effects of methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by removing the methyl group from O6-methylguanine lesions in DNA. The methyl group is transferred to a cysteine acceptor residue in the AGT protein, which is located at residue 145. The C145A mutant of AGT in which this cysteine is converted to an alanine residue is therefore inactive. When this C145A mutant was expressed in an Escherichia coli strain lacking endogenous alkyltransferase activity, the number of G:C
A:T mutations actually increased and the toxicity of the MNNG treatment was enhanced. These effects were not seen when an E.coli strain also lacking nucleotide excision repair (NER) was used. The enhancement of mutagenesis and toxicity of MNNG produced by the C145A mutant AGT was not seen with another inactive mutant Y114E that contains a mutation preventing DNA binding, and the double mutant C145A/Y114E was also ineffective. These results suggest that the C145A mutant AGT binds to O6-methylguanine lesions in DNA and prevents their repair by NER. The inactive C145A mutant AGT also increased the number of A:T
G:C transition mutations in MNNG-treated cells. These mutations are likely to arise from the minor methylation product, O4-methylthymine. However, expression of wild-type AGT also increased the incidence of these mutations. These results support the hypothesis that mammalian AGTs bind to O4-methylthymine but repair the lesion so slowly that they effectively shield it from more efficient repair by NER.
Abbreviations: AGT, O6-alkylguanine-DNA alkyltransferase; IPTG, isopropyl ß-D-thiogalactopyranoside; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; NER, nucleotide excision repair.
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Introduction
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A major part of the toxicity of methylating agents is due to the formation of O6-methylguanine in DNA (13). This adduct causes the guanine to be copied incorrectly by DNA polymerase inserting a thymine residue and thus producing G:C
A:T mutations (4,5). O6-methylguanine in DNA can also cause cell death, which occurs by apoptosis (69). Such killing is mediated via the mis-match repair system (10,11). This system recognizes the O6-methylguanine:thymine/cytosine pair as a mis-match and causes a section of DNA strand containing the thymine/cytosine to be degraded. When this section is filled in by DNA polymerase, the same error is re-inserted and a futile cycle of DNA synthesis and degradation that leads to cell death is set up. Although most of the work in which the toxic effects of methylating agents has been studied was carried out with compounds such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea, these agents generate a similar methylating species to therapeutic methylating agents such as procarbazine, dacarbazine and temozolomide. Repair of O6-methylguanine may be a major factor in resistance to these drugs but is also likely to reduce their genotoxicity that leads to the incidence of secondary tumors (1214).
O6-alkylguanine-DNA alkyltransferases are a ubiquitous family of DNA repair proteins that play an important role in counteracting the toxic effects of methylating agents (1518). They act by transferring the methyl group from the O6-position of guanine to a cysteine acceptor residue in the human O6-alkylguanine-DNA alkyltransferase (AGT) protein. This restores the DNA structure in a single step and very efficiently protects against mutagenesis and killing by methylating agents provided that adequate AGT is available. AGT, the human member of this family, contains 207 amino acids and has the acceptor site located at cysteine 145 (17,18).
The reaction between AGT and its substrate is thought to be brought about by the generation of a thiolate anion at a cysteine located in the active site of the protein. This facilitates transfer of the alkyl group in an SN2 reaction from the DNA to the cysteine acceptor in the AGT sequence leading to the formation of S-methylcysteine (12,13,19). This reaction is irreversible and each molecule of AGT can only be used once, as the S-methylcysteine is not converted back to cysteine.
As expected for a DNA repair protein, alkyltransferase binds to DNA although the mechanism of binding is not well understood and two different models have been put forward (20,21). Both of these are based on the crystal structure for the Ada-C alkyltransferase from Escherichia coli (20,22). This structure was of the protein alone and did not contain a bound substrate. The cysteine acceptor site is not readily accessible in this structure suggesting that a change in the protein conformation upon binding DNA is necessary in order to permit access of the target O6-alkylguanine. Conformational changes in the protein as a result of binding to DNA have been detected by using CD and fluorescent anisotropy (2325). Alterations in DNA conformation after AGT binding consistent with local melting have been detected by near-UV CD spectral changes (26). Other studies of the repair of double-stranded oligodeoxyribonucleotides containing O6-methylguanine analogs suggested that the duplex has to open up in order for the reaction to take place (27). These results are consistent with a base flipping mechanism in which the O6-alkylguanine is flipped out of the DNA helix to permit the reaction to occur. Such base flipping is now well established for DNA repair enzymes and has been suggested for AGT (21,2831).
AGT binding to DNA with and without O6-methylguanine adducts has been detected by gel shift experiments, footprinting (32) and sedimentation analysis (33). In such experiments, when oligodeoxynucleotides containing O6-methylguanine are used, it is very hard to prevent the reaction of wild-type AGT with the substrate. Therefore, the C145A mutant AGT has been used as a surrogate (32). This mutant in which the normal methyl acceptor site is converted to an alanine has been shown to have no DNA repair activity in in vitro assays (34,35).
In the course of studies on the ability of wild-type AGT and mutants to protect cells from the toxic effects of alkylating agents in which C145A AGT was used as a negative control, we have now observed that this C145A mutant AGT actually increases the toxic effects of MNNG. The present paper gives a detailed description of this phenomenon and discusses the underlying biochemical mechanism responsible for it.
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Materials and methods
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Materials
All restriction enzymes were purchased from New England Biolabs (Beverly, MA) and Gibco BRL (Gaithersburg, MD). Isopropyl ß-D-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranose and phenyl-ß-D-galactoside, MNNG, ampicillin, kanamycin, chloramphenicol and all electrophoresis and media reagents were purchased from Sigma (St Louis, MO). Taq I DNA polymerase was purchased from Amersham (Arlington Heights, IL). Nitrocellulose was purchased from Schleicher and Schuell (Keene, NH). Tween-20 was purchased from Bio-Rad (Hercules, CA). GENECLEAN DNA isolation kit was purchased from BIO101 (Vista, CA). DH5
MCR cells were purchased from Bethesda Research Laboratories (Gaithersburg, MD). All oligodeoxynucleotides were made in the Macromolecular Core Facility, Hershey Medical Center, using a Milligen 7500 DNA synthesizer.
Bacterial strains
The bacterial strains GWR109 (36), FC218, FC326 (37) and CJM2 (38) were a generous gift from Dr L.Samson (Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA).
Plasmids
All AGT mutants were expressed in E.coli using the pINIII-A3(lppP5) expression vector (39). Preparation of this vector plasmid containing sequences for expression of the E11Q mutant of S-adenosylmethionine decarboxylase (40), wild-type human AGT (41), C145A mutant AGT (35) and Y114E mutant AGT (42) have been described. The pIN plasmid containing the Y114E/C145A double mutant was made by three rounds of PCR with conditions as described (43). The first round was carried out using pINAGT as template and primers 5'-CCCATCCTCATCCCGGCCCACAGAGTGGTCTGC-3' (mismatch underlined) to introduce the C145A mutation and primer A (5'-TTTAGCAGCCTGAACGTCGG-3') matching a nucleotide sequence downstream to the AGT stop codon. Second-round PCR was then carried out using pIN-Y114E plasmid DNA as template using a primer B (5'-CAGCTATGACCATGATTACGGATTC-3') matching a nucleotide sequence upstream to AGT start codon and the 265 bp first-round PCR product excised from 1% agarose gels. The product from the second-round PCR was then used as a template in the third round PCR using primers A and B. The third-round PCR product was ethanol precipitated, digested with EcoRI and BamHI enzymes and ligated into pIN vector digested with the same enzymes to form pIN-Y114E/C145A. The entire AGT protein coding sequence of this plasmid was verified by sequencing and no additional mutations were found. The plasmids were transferred into the bacterial strains by electroporation.
Determination of AGT protein expression
The amount of recombinant human AGT or its mutants expressed in GWR109 cells was determined by western blotting after separation by SDSPAGE on 16% gels as described (44) using antibody MAP-1, which was raised to peptide KRTTLDSPLGKLE corresponding to residues 820 of the human AGT amino acid sequence (45).
Ability of AGT mutant proteins to affect survival and mutagenesis in cells treated with MNNG
GWR109 cells containing AGT plasmids were grown in 10 ml of M9 media supplemented with 50 µg/ml ampicillin, 50 µg/ml kanamycin and 0.3 mM IPTG. Cultures were grown in a 37°C water bath shaken at 220 r.p.m. to an A600 of 0.7. The cultures were pelleted and resuspended in 5 ml M9 media and treated with 04 µg/ml MNNG (dissolved in 100 mM sodium acetate buffer, pH 5.0) at 25°C for 1 h. The cells were centrifuged at 3000 r.p.m., suspended in 1 ml of M9 salts. Dilutions ranging from 1:100 to 1:10 000 were made and plated on M9 plates supplemented with 0.2% glucose, histidine (40 µg/ml), ampicillin (50 µg/ml) and kanamycin (50 µg/ml) and incubated at 37°C for 12 days to estimate the number of surviving colonies. In order to assess his+ revertants, cells were plated on M9 media plates lacking histidine. The mutation frequency was calculated as the number of his+ revertants/108 survivors. The incidence of Lac+ revertants in response to MNNG in the strains FC218, FC326 and CJM2 were measured under the same conditions using growth on galactose as described (37,46).
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Results
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When the inactive human AGT mutant C145A was expressed in the his ada ogt E.coli strain GWR109 (36), there was a clear decrease in survival and an increase in mutations leading to a reversion to the ability to grow without histidine after treatment with MNNG. In order to confirm that this difference was due to the C145A protein, the cells expressing mutant C145A were compared with cells expressing a similar amount of an irrelevant protein SAMDC, an inactive E11Q mutant of the enzyme, S-adenosylmethionine decarboxylase (40), or to cells expressing another inactive AGT mutant Y114E (Figure 1
). As shown in Figure 1a
, the cells expressing either SAMDC or Y114E gave a similar number of MNNG-induced mutations to the control cells that contained the pIN vector plasmid without an insert but C145A gave an ~2-fold increase in mutations. As expected, the expression of the wild-type AGT almost completely protected the cells from these mutations (Figure 1a
) or from killing (Figure 1b
). The presence of the C145A mutant increased the killing of GWR109 cells by MNNG when compared with either Y114E or SAMDC (Figure 1b
).

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Fig. 1. Effect of MNNG on mutations and killing in GWR109 cells expressing AGT mutant C145A. Results are shown for mutations to revertants to ability to grow without histidine (a) and for survival (b) in cells expressing no AGT protein (closed triangles) or wild-type AGT (closed inverted triangles), C145A mutant AGT (open circles), Y114E mutant AGT (closed circles), SAMDC (closed squares) and the double mutant Y114E/C145A AGT (open triangles) were treated with different concentrations of MNNG as shown. The values that were statistically different (P < 0.01) from the SAMDC used as a control are marked with asterisks. Results are means ± SD for at least four estimations.
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The C145A mutant AGT is known to bind to DNA containing O6-methylguanine (32) whereas the Y114E mutant is deficient in DNA binding (42). The results can therefore be explained if the C145A binds to the methylated DNA produced by MNNG and prevents repair by other pathways. In order to test this hypothesis, a double mutant Y114E/C145A to disrupt the DNA binding region of the C145A mutant was made. When this protein was expressed in GWR109 cells, the level of AGT protein formed was the same as wild-type and the C145A (Figure 2
). However, the cells expressing the Y114E/C145A mutant AGT protein did not show the increase in mutations reverting to the his+ phenotype or the increase in killing brought about by the C145A single mutation (Figure 1
). These results suggest that the binding of the C145A mutant to methylated DNA is responsible for the increase in MNNG-mediated toxicity.

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Fig. 2. Expression of mutant AGT proteins in GWR109 cells. Extracts from GWR109 cells expressing human wild-type AGT (lanes 1 and 2) or its mutants C145A (lanes 3 and 4) and Y114E/C145A lanes 5 and 6) and control GWR109 cells (lanes 7 and 8 ) were resolved by SDSPAGE, transferred to nitrocellulose and developed using antibodies to a peptide corresponding to amino acids 820 of the human AGT. In lanes 1, 3, 5 and 7, 5 µg of protein were loaded and in lanes 2, 4, 6 and 8, 10 µg of protein were used.
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Since the majority of the mutations produced by MNNG are due to O6-methylguanine, it would be expected that the C145A mutant AGT increases G:C
A:T transitions. This was confirmed using the strain FC218, which has a point mutation at the Glu-461 codon that renders it Lac but can be reverted to the Lac+ phenotype by a G:C
A:T transition (37,46). As shown in Figure 3a
, the C145A mutant AGT increased the frequency of MNNG-induced Lac+ revertants in this strain supporting the hypothesis that these mutations are due to an increased persistence of O6-methylguanine in the DNA. The expression of wild-type AGT greatly reduced the production of Lac+ revertants by MNNG in this strain (Figure 3a
).
It is known that O6-methylguanine is a substrate for nucleotide excision repair (NER) in E.coli (47,48). As previously reported (38), strain CJM2, which is similar to FC218 but also has a deficiency in NER, was more sensitive to both killing (Figure 3c and d
) and reversion to the Lac+ phenotype (Figure 3a and b
) when treated with MNNG. The expression of the wild-type AGT decreased the incidence of mutations and the killing of CJM2 cells by MNNG. However, the expression of C145A mutant AGT did not increase the toxicity and mutagenicity of MNNG in this strain (Figure 3b and d
). These results suggest that the binding of the C145A mutant to DNA containing O6-methylguanine prevent its repair by NER.
Another DNA methylation product that has been reported to be a substrate for AGT is O4-methylthymine (15,19,4951). O4-Methylthymine is a very minor component of the total methylation damage (52) but is highly mutagenic causing A:T
G:C transition mutations (5,53).These mutations can be assayed by reversion to the Lac+ phenotype using strain FC326 (37). However, as shown in Figure 4
, we were able to confirm the report by Samson et al. (38) that the presence of wild-type AGT does not reduce the frequency of the A:T
G:C transition mutations produced by MNNG but, instead, actually increases this frequency. The C145A mutant also increased the mutation frequency. This increase was slightly less than that produced by wild-type AGT but the difference between wild-type and C145A mutant AGT was not statistically significant.

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Fig. 4. Effect of C145A AGT and wild-type AGT protein on effects of MNNG on A:T G:C transition mutations. Mutations were measured in strain FC326 using cells expressing wild-type AGT (inverted triangles), C145A mutant AGT (circles) and SAMDC (squares). The values that were statistically different (P < 0.01) from the cells expressing SAMDC used as a control are marked with asterisks. Results are means ± SD for at least four estimations.
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Discussion
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The most plausible explanation for the increase in MNNG-mediated mutations and cell killing produced by the presence of the C145A mutant AGT is that this protein binds to O6-methylguanine sufficiently tightly to prevent its repair by other DNA pathways. This interpretation is supported by several different results: (i) the increase in mutations was clearly due to a greater persistence of O6-methylguanine in DNA since mutations were seen in the FC218 strain which requires a G:C
A:T transition to revert to Lac+; (ii) the effect was not seen with the Y114E mutant of AGT, which is also inactive but fails to bind to DNA (42,54), and incorporation of the Y114E mutation into the C145A mutant AGT abolished its ability to increase MNNG toxicity; and (iii) the effect of the C145A mutant AGT was not seen in the CJM2 strain lacking NER.
These results provide further support for experiments showing that NER is able to recognize O6-methylguanine despite the minimal distortion of the DNA structure produced by this small lesion (47,48,55). Although alkyltransferase-catalyzed repair is the predominant route for removal of this lesion, NER provides a back-up pathway for cells with limited amounts of alkyltransferase. However, in order for NER to be efficient, any interference by inactive AGT must be prevented. After repair of O6-methylguanine by AGT, the protein is converted to an inactive form with S-methylcysteine at the active site. Our results suggest that a mechanism to ensure that this protein does not continue to bind to DNA containing O6-methylguanine must exist in order to allow repair by other pathways.
The fate of the methylated form of the AGT protein that results from repair of DNA is not fully understood but several studies have shown that the protein undergoes a conformational change after alkylation of the active site cysteine (5658) and is degraded rapidly (59,60). There is also evidence that the alkylated form of the protein becomes ubiquitinated (60,61). All of these changes could help to prevent the binding of the inactive form of the AGT to remaining lesions in the DNA and thus avoid the toxic effects that we observe with the C145A mutant AGT.
The repair of O4-methylthymine in DNA by mammalian AGTs has been the subject of some controversy. Although it has been well established for some time that certain microbial alkyltransferases can repair O4-methylthymine (15,19,49), repair of this lesion by extracts from mammalian cells could not be demonstrated in several laboratories (62). However, work carried out with recombinant AGTs, which in some cases were present in great excess over the amount of substrate, showed that even human AGT could bring about this repair (50). Detailed studies have indicated that the efficiency with which O4-methylthymine is repaired by AGTs varies considerably according to the source of the repair protein and repair by the human AGT is very slow, in particular when compared with certain microbial AGTs (18,49,51). Although this work is of considerable interest with respect to the information provided concerning the active site of alkyltransferases, it does not answer the question of whether human AGT is effective in repairing this lesion in vivo. Our results using strain FC326 to measure A:T
G:C transition mutations arising from O4-methylthymine confirm the report of Samson et al. that the presence of mammalian AGTs in E.coli actually increases the incidence of such mutations in response to MNNG (38). Therefore, it appears unlikely that AGT plays a significant role in removing the minor methylation product O4-methylthymine from DNA in human cells. It is noteworthy that recent studies have shown that even in E.coli, mutations arising from plasmids containing O4-methylthymine were not reduced when measured in alkyltransferase deficient strains (5).
In the case of O4-methylthymine-mediated mutations, both the wild-type and the C145A mutant human AGT increase the number of mutations (Figure 4
). This is consistent with the proposal that wild-type AGT repairs O4-methylthymine so slowly that it interferes with repair of the lesion by NER (38). Our results are in agreement with this hypothesis and show that, when a totally inactive AGT protein is used, the O6-methylguanine-induced G:C
A:T transition mutations and cell killing by AGT are also increased. The small difference in the A:T
G:C transition mutation frequency between the cells expressing wild-type AGT and the cells expressing C145A (Figure 4
) was not statistically significant but could indicate that the C145A mutant does not bind quite so tightly to O4-methylthymine lesions as the wild-type AGT.
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Notes
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1 To whom correspondence should be addressed Email: aep1{at}psu.edu 
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Acknowledgments
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We are most grateful to Dr L.Samson for the gifts of the bacterial strains used in this work. This work was supported by the National Cancer Institute with grants CA-18137, CA-71976 and CA-57725.
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References
|
---|
- Lawley,P.D. (1976) Carcinogenesis by alkylating agents. In Searle,C.E. (ed.) Chemical Carcinogens. ACS Symposium Series no. 173. ACS, Washington, DC, pp. 83244.
- Pegg,A.E. (1977) Formation and metabolism of alkylated nucleosides: possible role in carcinogenesis by nitroso compounds and alkylating agents. Adv. Cancer Res., 25, 195269.[Medline]
- Pegg,A.E. and Singer,B. (1984) Is O6-alkylguanine necessary for initiation of carcinogenesis by alkylating agents? Cancer Invest., 2, 221238.[ISI]
- 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. USA, 81, 62716275.[Abstract]
- Pauly,G.T., Hughes,S.H. and Moschel,R.C. (1998) Comparison of mutagenesis by O6-methyl- and O6-ethylguanine and O4-methylthymine in Escherichia coli using double-stranded and gapped plasmids. Carcinogenesis, 19, 457461.[Abstract]
- Kaina,B., Ziouta,A., Ochs,K. and Coquerelle,T. (1997) Chromosomal instability, reproductive cell death and apoptosis induced by O6-methylguanine in Mex, Mex+ and methylation-tolerant mismatch repair compromised cells: facts and models. Mutat. Res., 381, 227241.[ISI][Medline]
- Tominaga,Y., Tsuzuki,T., Shiraishi,A., Kawate,H. and Sekiguchi,M. (1997) Alkylation-induced apoptosis of embryonic stem cells in which the gene for DNA-repair, methyltransferase, had been disrupted by gene targeting. Carcinogenesis, 18, 889896.[Abstract]
- Tentori,L., Orlando,L., Lacal,P.M., Benincasa,E., Faraoni,I., Bonmassar,E., D'Atri,S. and Graziani,G. (1997) Inhibition of O6-alkylguanine DNA-alkyltransferase or poly(ADP-ribose) polymerase increases susceptibility of leukemic cells to apoptosis induced by temozolomide. Mol. Pharmacol., 52, 249258.[Abstract/Free Full Text]
- Meikrantz,W., Bergom,M.A., Memisoglu,A. and Samson,L. (1998) O6-alkylguanine DNA lesions trigger apoptosis. Carcinogenesis, 19, 369372.[Abstract]
- Karran,P. and Bignami,M. (1996) Drug-related killings: a case of mistaken identity. Chem. Biol., 3, 875879.[ISI][Medline]
- Karran,P. and Hampson,R. (1996) Genomic instability and tolerance to alkylating agents. Cancer Surv., 28, 6985.[ISI][Medline]
- Pegg,A.E. and Byers,T.L. (1992) Repair of DNA containing O6-alkylguanine. FASEB J., 6, 23022310.[Abstract/Free Full Text]
- Pegg,A.E., Swenn,K., Dolan,M.E. and Moschel,R.C. (1995) Increased killing of prostate, breast, colon and lung tumor cells by the combination of inactivators of O6-alkylguanine-DNA alkyltransferase and N,N-bis(2-chloroethyl)-N-nitrosourea. Biochem. Pharmacol., 50, 11411148.[ISI][Medline]
- Dolan,M.E. (1997) Inhibition of DNA repair as a means of increasing the antitumor activity of DNA reactive agents. Adv. Drug Delivery Rev., 26, 105118.[ISI][Medline]
- Lindahl,T., Sedgwick,B., Sekiguchi,M. and Nakabeppu,Y. (1988) Regulation and expression of the adaptive response to alkylating agents. Annu. Rev. Biochem., 57, 133157.[ISI][Medline]
- Samson,L. (1992) The suicidal DNA repair methyltransferases of microbes. Mol. Microbiol., 6, 825831.[ISI][Medline]
- Mitra,S. and Kaina,B. (1993) Regulation of repair of alkylation damage in mammalian genomes. Progr. Nucleic Acid Res. Mol. Biol., 44, 109142.[ISI][Medline]
- Pegg,A.E., Dolan,M.E. and Moschel,R.C. (1995) Structure, function and inhibition of O6-alkylguanine-DNA alkyltransferase. Progr. Nucleic Acid Res. Mol. Biol., 51, 167223.[ISI][Medline]
- Demple,B. (1990) Self-methylation by suicide DNA repair enzymes. In Paik,W.K. and Kim,S. (eds) Protein Methylation. CRC Press, Boca Raton, FL, pp. 285304.
- Moore,M.H., Gulbus,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, 14951501.[Abstract]
- Vora,R., Pegg,A.E. and Ealick,S.E. (1998) A new model for how O6-methylguanine-DNA methyltransferase binds DNA. Prot. Struc. Function Genet., 32, 36.
- Moody,P.C.E. and Moore,M.E. (1995) Crystal structure of E.coli O6-methylguanine-DNA methyltransferase. In Zeller,W.J., D'Incalci,M. and Newell,D.R. (eds) Novel Approaches in Anticancer Drug Design Molecular ModellingNew Treatment Strategies, vol. 49. Karger, Basel, pp. 1624.
- Chan,C.Z.W., Ciardelli,T., Eastman,A. and Bresnick,E. (1993) Kinetic and DNA-binding properties of recombinant human O6-methylguanine-DNA methyltransferase. Arch. Biochem. Biophys., 300, 193200.[ISI][Medline]
- Federwisch,M., Hassiepen,U., Bender,K., Dewor,M., Rajewsky,M.F. and Wollmer,A. (1997) Recombinant human O6-alkylguanine-DNA alkyltransferase (AGT), Cys145-alkylated AGT and Cys145 to Met145 mutant AGT: comparison by isoelectric focusing, CD and time-resolved fluorescence spectroscopy. Biochem. J., 324, 321328.[ISI][Medline]
- Takahashi,M., Sakumi,K. and Sekiguchi,M. (1990) Interaction of Ada protein with DNA examined by fluorescence anisotropy of the protein. Biochemistry, 29, 34313436.[ISI][Medline]
- Federwisch,M., Hassiepen,U., Bender,K., Rajewsky,M.F. and Wollmer,A. (1997) Recombinant human O6-alkylguanine-DNA alkyltransferase induces conformational change in bound DNA. FEBS Lett., 407, 333336.[ISI][Medline]
- Spratt,T.E. and Campbell,C.R. (1994) Synthesis of oligonucleotides containing analogs of O6-methylguanine and reaction with O6-alkylguanine-DNA alkyltransferase. Biochemistry, 33, 1136411371.[ISI][Medline]
- Demple,B. (1995) DNA repair flips out. Curr. Biol., 5, 719721.[ISI][Medline]
- Roberts,R.J. (1995) On base flipping. Cell, 82, 912.[ISI][Medline]
- Pearl,L.H. and Savva,R. (1995) DNA repair in three dimensions. Trends Biochem. Sci., 20, 421426.[ISI][Medline]
- Verdine,G.L. (1994) The flip side of DNA methylation. Cell, 76, 197200.[ISI][Medline]
- 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, 57695776.[ISI][Medline]
- 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.[ISI][Medline]
- Harris,L.C., Potter,P.M. and Margison,G.P. (1992) Site directed mutagenesis of two cysteine residues in the E.coli ogt O6-alkylguanine DNA alkyltransferase protein. Biochem. Biophys. Res. Commun., 187, 425431.[ISI][Medline]
- Crone,T.M. and Pegg,A.E. (1993) A single amino acid change in human O6-alkylguanine-DNA alkyltransferase decreasing sensitivity to inactivation by O6-benzylguanine. Cancer Res., 53, 47504753.[Abstract]
- Rebeck,G.W. and Samson,L. (1991) Increased spontaneous mutation and alkylation sensitivity of Escherichia coli strains lacking the ogt O6-methylguanine DNA repair methyltransferase. J. Bacteriol., 173, 20682076.[ISI][Medline]
- Mackay,W.J., Han,S. and Samson,L.D. (1994) DNA alkylation repair limits spontaneous base substitution mutations in Escherichia coli. J. Bacteriol., 176, 32243230.[Abstract]
- Samson,L., Han,S., Marquis,J.C. and Rasmussen,L.J. (1997) Mammalian DNA repair methyltransferases shield O4MeT from nucleotide excision repair. Carcinogenesis, 18, 919924.[Abstract]
- Duffaud,G.D., March,P.E. and Inouye,M. (1987) Expression and secretion of foreign proteins in Escherichia coli. Methods Enzymol., 153, 492507.[ISI][Medline]
- Stanley,B.A., Shantz,L.M. and Pegg,A.E. (1994) Expression of mammalian S-adenosylmethionine decarboxylase in E.coli. J. Biol. Chem., 269, 79017907.[Abstract/Free Full Text]
- 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, 1199812006.[ISI][Medline]
- Kanugula,S., Goodtzova,K., Edara,S. and Pegg,A.E. (1995) Alteration of arginine-128 to alanine abolishes the ability of human O6-alkylguanine-DNA alkyltransferase to repair methylated DNA but has no effect on its reaction with O6-benzylguanine. Biochemistry, 34, 71137119.[ISI][Medline]
- Edara,S., Kanugula,S., Goodtzova,K. and Pegg,A.E. (1996) Resistance of the human O6-alkylguanine-DNA alkyltransferase containing arginine at codon 160 to inactivation by O6-benzylguanine. Cancer Res., 56, 55715575.[Abstract]
- Crone,T.M., Goodtzova,K. and Pegg,A.E. (1996) Amino acid residues affecting the activity and stability of human O6-alkylguanine-DNA alkyltransferase. Mutat. Res., 363, 1525.[ISI][Medline]
- Pegg,A.E., Wiest,L., Mummert,C. and Dolan,M.E. (1991) Production of antibodies to peptide sequences present in human O6-alkylguanine-DNA alkyltransferase and their use to detect this protein in cell extracts. Carcinogenesis, 12, 16711677.[Abstract]
- Cupples,C.G. and Miller,J.H. (1989) A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc. Natl Acad. Sci. USA, 86, 53455349.[Abstract]
- Samson,L., Thomale,J. and Rajewsky,M.F. (1988) Alternative pathways for the in vivo repair of O6-alkylguanine and O4-alkylthymine in Escherichia coli: the adaptive response and nucleotide excision repair. EMBO J., 7, 22612267.[Abstract]
- Voigt,J.M., Van Houten,B., Sancar,A. and Topal,M.D. (1989) Repair of O6-methylguanine by ABC excinuclease of Escherichia coli in vitro. J. Biol. Chem., 264, 51725176.[Abstract/Free Full Text]
- Sassanfar,M., Dosanjh,M.K., Essigmann,J.M. and Samson,L. (1991) Relative efficiencies of the bacterial, yeast, and human DNA methyltransferases for the repair of O6-methylguanine and O4-methylthymine. J. Biol. Chem., 266, 27672771.[Abstract/Free Full Text]
- Koike,G., Maki,H., Takeya,H., Hayakawa,H. and Sekiguchi,M. (1990) Purification, structure and biochemical properties of human O6-methylguanine-DNA methyltransferase. J. Biol. Chem., 265, 1475414762.[Abstract/Free Full Text]
- Zak,P., Kleibl,K. and Laval,F. (1994) Repair of O6-methylguanine and O4-methylthymine by the human and rat O6-methylguanine-DNA methyltransferase. J. Biol. Chem., 269, 730733.[Abstract/Free Full Text]
- Singer,B. and Kusmierek,J.T. (1982) Chemical mutagenesis. Annu. Rev. Biochem., 52, 655693.[ISI]
- Preston,B.D., Singer,B. and Loeb,L.A. (1986) Mutagenic potential of O4-methylthymine in vivo determined by an enzymatic approach to site specific mutagenesis. Proc. Natl Acad. Sci. USA, 83, 85018505.[Abstract]
- Goodtzova,K., Kanugula,S., Edara,S. and Pegg,A.E. (1998) Investigation of the role of tyrosine-114 in O6-alkylguanine-DNA alkyltransferase. Biochemistry, 37, 1248912495.[ISI][Medline]
- Sancar,A. (1996) DNA excision repair. Annu. Rev. Biochem., 65, 4381.[ISI][Medline]
- Ayi,T.C., Oh,H.K., Lee,T.K.Y. and Li,B.F.L. (1994) A method for simultaneous identification of human active and active-site alkylated O6-methylguanine-DNA methyltransferase and its possible application for monitoring human exposure to alkylating carcinogens. Cancer Res., 54, 37263731.[Abstract]
- Oh,H.-K., Teo,A.K.-C., Ali,R.B., Lim,A., Ayi,T.-C., Yarosh,D.B. and Li,B.F.-L. (1996) Conformational change in human DNA repair enzyme O6-methylguanine-DNA methyltransferase upon alkylation of its active site by SN1 (indirect-acting) and SN2 (direct acting) alkylating agents: breaking a `salt-link'? Biochemistry, 35, 1225912266.[ISI][Medline]
- Kanugula,S., Goodtzova,K. and Pegg,A.E. (1998) Probing of conformational changes in human O6-akylguanine-DNA alkyltransferase protein in its alkylated and DNA bound states by limited proteolysis. Biochem. J., 329, 545550.[ISI][Medline]
- Pegg,A.E., Wiest,L., Mummert,C., Stine,L., Moschel,R.C. and Dolan,M.E. (1991) Use of antibodies to human O6-alkylguanine-DNA alkyltransferase to study the content of this protein in cells treated with O6-benzylguanine or N-methyl-N'-nitro-N-nitrosoguanidine. Carcinogenesis, 12, 16791683.[Abstract]
- Srivenugopal,K.S., Yuan,X.H., Bigner,D.D., Friedman,H.S. and Ali-Osman,F. (1996) Ubiquitination of O6-alkylguanine-DNA alkyltransferase in human tumor cells following inactivation with O6-benzylguanine or 1,3 bis(2-chloroethyl)-1-nitrosourea. Biochemistry, 35, 13281334.[ISI][Medline]
- Major,G.J., Brady,M., Notarianni,G.B., Collier,J.D. and Douglas,M.S. (1997) Evidence for ubiquitin-mediated degradation of the DNA repair enzyme for O6-methylguanine in non-tumour derived human cell and tissue extracts. Biochem. Soc. Trans., 25, 3595.
- Brent,T.P., Dolan,M.E., Fraenkel-Conrat,H. et al. (1988) Repair of O-alkylpyrimidines in mammalian cells: a present consensus. Proc. Natl Acad. Sci. USA, 85, 17591762.[Abstract]
Received July 22, 1998;
revised September 2, 1998;
accepted September 3, 1998.