Enhanced in vivo repair of O4-methylthymine by a mutant human DNA alkyltransferase
Lance P. Encell and
Lawrence A. Loeb1
The Joseph Gottstein Memorial Cancer Research Laboratory, Departments of Pathology and Biochemistry, University of Washington School of Medicine, Seattle, WA 98195-7705 USA
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Abstract
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The repair of O6-methylguanine (m6G) by human O6-alkylguanine-DNA alkyltransferase (hAGT) is ~5000-fold greater than that for O4-methylthymine (m4T). To evaluate each adduct's contribution to mutagenesis, we previously created a mutant hAGT with increased specificity for m4T in vitro. The mutant and wild-type (WT) hAGT have now been expressed in bacterial strains that allow for the specific detection of A:T
G:C and G:C
A:T mutations induced by m4T and m6G, respectively. After exposure to the mutagenic methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine, A:T
G:C substitutions were reduced >4-fold in cells expressing the mutant hAGT compared with 1.1-fold for WT hAGT. G:C
A:T substitutions were decreased >2.5-fold in cells expressing the mutant hAGT, whereas WT hAGT totally prevented G:C
A:T mutations. These results demonstrate that the altered substrate specificity of hAGT observed in vitro also occurs in vivo, and that it is responsible for the observed differences in mutations.
Abbreviations: BG, O6-benzylguanine; hAGT, human O6-alkylguanine-DNA alkyltransferase; m6G, O6-methylguanine; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; m4T, O4-methylthymine; WT, wild-type.
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Introduction
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Alkylating agents react extensively with DNA to produce a multitude of different adducts (1). This diversity makes it difficult to determine the biological significance of individual lesions. In vitro and in vivo studies using site-specific DNA adducts suggest that certain adducts, e.g. 3-methyladenine can block polymerases during replication (2,3), whereas other lesions, e.g. O6-methylguanine (m6G) and O4-methylthymine (m4T), allow bypass, and miscode during polymerization to produce mutations (46). m4T is formed less frequently than m6G by alkylating agents; however, it is a more potent source of mutations when examined in both prokaryotic and eukaryotic cells (712). Both adducts are repaired in human cells by O6-alkylguanine-DNA alkyltransferase (hAGT, EC 2.1.1.63), a protein that transfers alkyl groups from DNA to a cysteine residue within the active site (13,14). hAGT is a suicide protein because each protein carries out a single reaction before being hydrolyzed by cellular proteases (15,16).
We have previously used directed evolution to create and identify a hAGT that contains eight amino acid substitutions near the active site (C150Y, S152R, A154S, V155G, N157T, V164M, E166Q, A170T) (17). The mutant, referred to as 56-8, was selected for its ability to protect bacteria from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced killing in the presence of the potent hAGT inhibitor, O6-benzylguanine (BG). In addition to being highly resistant to BG, mutant 56-8 exhibits enhanced in vitro repair of m4T accompanied by a decrease in the repair of m6G (18). To determine if the change in substrate specificity is indicative of DNA repair in vivo we have utilized strains of Escherichia coli that allow for the specific detection of A:T
G:C or G:C
A:T transitions, the predominant base substitutions induced by m4T and m6G, respectively. Our results indicate that altered substrate specificity in vitro is predictive of repair in vivo.
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Materials and methods
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Materials
All reagents were purchased from Sigma (St Louis, MO) unless noted otherwise.
Plasmids
Construction of pUC118-based vectors expressing mutant 56-8 (pLE56-8), WT hAGT (pFC6) and inactive hAGT (pFC9) has been described previously (17,19). pFC9 contains a 1.7 kb stuffer fragment that replaces a 141 base fragment from the WT hAGT cDNA which includes the codon for the active cysteine. pUCogt (20) expressing the bacterial alkyltransferase, Ogt, was a generous gift from L.Samson (Harvard School of Public Health, Boston, MA).
Bacterial strains and media
The bacterial strains CJM1 and CJM2 (21) were kindly provided by L.Samson. Both strains are deficient in alkyltransferase (ada, ogt) and nucleotide excision repair (uvr), and are derived from strains CC102 and CC106 that carry mutated lacZ sequences contained on an F' episome (22). The mutant lacZ alleles are Lac and do not support growth of Escherichia coli on lactose as a sole carbon source; however, the E.coli can be reverted to Lac+ via either A:T
G:C (CJM1) or G:C
A:T (CJM2) transition mutations. Cells were grown in either 2x YT or M9 minimal medium containing the appropriate antibiotics and 50 µM isopropyl-ß-D-galactopyranoside (IPTG). Minimal plates contained either 0.025% glucose or lactose, 0.025% thiamine, 40 µg/ml methionine, 1 mM MgSO4, 0.1 mM CaCl2 and antibiotics (carbenicillin, kanamycin, chloramphenicol and tetracycline) as described previously (23).
MNNG-induced mutagenicity assay
CJM1 or CJM2 cells, transformed with either pLE56-8 (hAGT mutant 56-8), pFC6 (WT hAGT), pFC9 (inactive hAGT) or pUCogt (E.coli Ogt) were grown in 2x YT medium containing the appropriate antibiotics and IPTG to OD600 = 0.8. Ten milliliter aliquots (1010 cells) were washed and resuspended in 10 ml of M9 salts, treated with varying concentrations of MNNG for 30 min at 37°C, and washed again with M9 salts. In experiments including BG, the inhibitor was present at a concentration of 100 µM both prior to and during exposure to MNNG as described previously (17). Appropriate dilutions of the cells were spread on minimal plates containing either glucose (for estimating survival) or lactose (for estimating the number of lacZ revertants). Following incubation at 37°C for 48 h, colonies were counted and mutagenicity was quantitated. Mutation frequencies are expressed as the number of induced lacZ revertants per 108 surviving cells for strain CJM2 and per 109 surviving cells for strain CJM1. Experiments were performed between three and five times each and the results were reproducible.
Sequencing of Lac+ revertants
Episomal lacZ DNA from colonies on glucose and lactose plates was isolated using a kit from 5 Prime
3 Prime, Inc. (Boulder, CO). A region of the lacZ allele containing the altered codon at position 461 was PCR-amplified using the forward primer, 5'-GCGAACGCGTAACGCG-3' (nucleotides 13131328) and the reverse primer, 5'-GCTCCGCCGCCTTCATAC-3' (nucleotides 14751458). The annealing temperature used in the reactions was 57°C. PCR products were purified and then sequenced using PE Biosytems (Foster City, CA) Big dye terminator chemistry. The primer used for sequencing was the forward PCR primer mentioned above.
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Results
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hAGT mutant 56-8 repairs m4T and m6G in vivo
We have shown previously that the hAGT mutant 56-8 repairs m4T more effectively than the WT protein in vitro (18). In order to determine if this altered specificity is exhibited in vivo, we measured cell survival and mutations produced by unrepaired m4T and m6G in cells expressing the mutant and WT hAGTs. We made use of a series of reporter strains established by J.Miller to detect the induction of specific types of mutations. These strains are also deficient in nucleotide excision repair (uvr), a pathway that could contribute to the repair of m4T and m6G (10,21,24). Plasmids encoding alkyltransferases were introduced into alkyltransferase-deficient E.coli strains CJM1 and CJM2. In vivo repair was measured by the mutant's ability to prevent A:T
G:C mutations (induced by m4T) and G:C
A:T mutations (induced by m6G) in CJM1 and CJM2 cells, respectively. The reporter strains each contain specific nucleotide substitutions in their episomal lacZ gene, rendering the cells unable to grow on lactose. The lacZ mutations can revert from Lac to Lac+ by specific point mutations that are induced by either m4T (CJM1) or m6G (CJM2). Mutations are scored by colony formation on plates containing minimal medium and lactose as a sole source of carbon. We compared the efficiency of DNA repair of m4T and m6G in vivo by three DNA alkyltransferases: mutant 56-8 hAGT, WT hAGT and the bacterial alkyltransferase, Ogt. Ogt was used as a positive control because it repairs both adducts very effectively (14,21). Bacteria harboring these genes were incubated with MNNG, a methylating agent that produces both m4T and m6G (1). Each of the three constructs protected the m4T reporter cells (CJM1) against killing (Figure 1A
). In the absence of active hAGT, survival was reduced proportionately by increasing amounts of MNNG. A plot of mutations per survivor is presented in Figure 1B
. In the absence of hAGT there was a linear increase in mutagenesis as a function of increasing amounts of MNNG. Mutations at the highest dose of MNNG (1 µg/ml) were reduced by 10% in cells transformed with WT hAGT. In contrast, mutagenesis at this dose was reduced 76% in cells expressing mutant 56-8 hAGT. Cells expressing Ogt failed to produce detectable A:T
G:C mutations at these doses of MNNG. This is in accord with the known ability of Ogt to repair m4T in vivo very effectively (21).
Because MNNG induces more m6G than m4T, lower doses of methylating agent are required to produce detectable G:C
A:T mutations in m6G reporter cells (CJM2). MNNG was not lethal to CJM2 cells expressing any of the active alkyltransferases tested (56-8, WT or Ogt), and only marginally toxic to cells expressing inactive protein (Figure 2A
). Results of mutagenesis in the m6G reporter strain are presented in Figure 2B
. In cells transfected with the inactive construct there was a linear increase in mutations with increasing doses of MNNG. Mutagenesis was reduced 63% in cells expressing the mutant at the highest dose of MNNG. An even greater reduction was observed in cells harboring the WT hAGT or Ogt constructs, as no mutations above background were detected with either alkyltransferase. Thus, mutant 56-8 hAGT is more effective in diminishing mutagenesis in the m4T reporter strain than in the m6G reporter strain. In contrast, WT hAGT prevents mutations in the m6G reporter strain better than in the m4T reporter strain.
Verification of point mutations responsible for lacZ reversions in CJM1 and CJM2 cells
To verify the nature of the Lac+ phenotype acquired by the mutant clones following exposure to MNNG, we sequenced a region of the F' episomal DNA surrounding codon 461 of the lacZ gene from untreated Lac and MNNG-induced Lac+ cells harboring either mutant 56-8 or WT hAGT. With each strain, five Lac and 15 Lac+ colonies were used to prepare episomal DNA for sequencing. Table I
summarizes these results. For every revertant examined, the predicted mutation had occurred to restore the WT GAG codon at position 461.
The substrate specificity of mutant 56-8 hAGT is unaltered by BG
It has been shown previously that the hAGT inhibitor, BG can render bacteria harboring WT hAGT sensitive to cytotoxicity induced by MNNG (25). In contrast, cells expressing mutant 56-8 hAGT are resistant (17). To examine the effect of BG on mutagenesis, m4T or m6G reporter cells (CJM1 and CJM2, respectively) harboring either the mutant or WT hAGT were treated with increasing concentrations of MNNG in the presence of 100 µM BG. In the case of CJM1 cells harboring the mutant (Figure 3A
), BG did not reduce cell survival, and survival was similar to that shown in Figures 1 and 2
. In the case of the cells expressing the WT hAGT, BG increased MNNG-induced killing. At the highest dose of MNNG used (1 µg/ml), the survival of cells harboring WT hAGT was ~10-fold greater than cells expressing an inactive protein. In the presence of BG this protection was reduced to ~2-fold. BG had no effect on the onset of A:T
G:C mutations in m4T reporter cells (CJM1) harboring either mutant 56-8 or WT hAGT (Figure 3B
). Presumably, even though BG inactivates WT hAGT, the WT protein is not rate limiting for the repair of m4T.
In the case of m6G reporter cells (CJM2) there was no reduction in survival in bacteria harboring the mutant or WT hAGT as a function of increasing amounts of MNNG in the presence of BG (Figure 4A
). However, mutagenesis was a more sensitive indicator of DNA damage. BG did have a significant enhancing effect on mutagenesis (G:C
A:T) in CJM2 cells expressing WT hAGT, with the greatest effect observed at the higher doses of MNNG. The finding that at 0.3 µg/ml MNNG, cells harboring WT hAGT accumulated no detectable mutations in the absence of BG suggests that the WT protein is able to completely repair the m6G lesions induced by MNNG. However, when it was inactivated by BG, these cells develop nearly 5000 mutations per 108 surviving cells, which is >1000 (~33%) more than were observed in cells harboring the mutant hAGT. Sensitivity to BG was dependent on an active alkyltransferase; it should be noted that the inhibitor did not have an effect on the survival or mutagenicity of cells harboring an inactive protein (data not shown).
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Discussion
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Although hAGT repairs both m4T and m6G, it has a preference for m6G. This is of interest because evidence suggests both adducts contribute to mutagenesis by alkylating agents (2629). Although formed less frequently by alkylating agents, m4T is a more persistent adduct (30,31). Presumably this is due to inefficient repair. Using random mutagenesis and genetic selection we have evolved an alkyltransferase that repairs m4T with increased efficiency compared with the WT protein (18). In vitro biochemical analysis of this mutant indicated it repairs m4T >10-fold more rapidly than the WT protein; the relative m4T specificity [k(m4T)/k(m6G)] for the mutant is increased 75-fold. In order to determine the biological effects of this altered specificity we have now further characterized the mutant by examining its repair capacity in vivo.
Using two strains of E.coli (CJM1 and CJM2) that detect specific mutations resulting from either unrepaired m4T or m6G, mutant 56-8 and WT hAGT were compared for their abilities to reduce mutagenesis following exposure to MNNG. We hypothesized that mutant 56-8 hAGT would be more efficacious than WT hAGT at preventing the accumulation of A:T
G:C transitions based on in vitro data indicating enhanced repair of m4T. This was indeed the case, as the mutant offered the cells >3.5-fold more protection than WT hAGT using a m4T reporter strain. WT hAGT offered very little protection against these mutations, in agreement with a previous report by Samson et al. (21). In contrast to its improved ability to prevent A:T
G:C mutations, mutant 56-8 did not protect the bacteria as well as WT hAGT against G:C
A:T mutations, suggesting that the mutant protein is less effective at repairing m6G than the WT. Together these findings verify our previous biochemical characterization of the mutant as having a reversed substrate specificity for m4T and m6G compared with WT hAGT. More importantly these data suggest that in vitro repair by alkyltransferases can be predictive of repair in vivo.
Mutant 56-8 hAGT is unusual in that it contains eight amino acid substitutions adjacent to the active site and yet maintains WT-like activity for protecting cells against MNNG-induced cytotoxicity. The number of substitutions within a contiguous sequence in this mutant is greater than any other enzyme so far generated by selection from random sequences. In addition to having an altered substrate specificity for m4T, mutant 56-8 also confers resistance to BG in bacteria (17) and in human cells (B.M.Davis, L.P.Encell, S.P.Zielske, F.C.Christians, L.Liu, S.E.Friebert, L.A.Loeb and S.L.Gerson, manuscript in preparation). In contrast, WT hAGT is highly sensitive to inhibition by BG, an inhibitor that is being used in clinical trials for sensitizing tumors to chemotherapeutic alkylating agents whose effectiveness is diminished by repair involving hAGT (32). In order to determine the effect of BG on mutagenesis, the same strains (CJM1, CJM2) were used to measure mutant 56-8's ability to prevent mutations following exposure to a combination of MNNG and BG. The inhibitor had no effect on the induction of either type of mutation (A:T
G:C or G:C
A:T) by MNNG in cells harboring mutant 56-8 hAGT, in accord with the high BG resistance observed previously for this mutant (17). BG also had no effect on A:T
G:C mutations in cells expressing the WT protein, indicating that WT hAGT does not participate significantly in the repair of m4T. In contrast, BG highly sensitized CJM2 cells harboring WT hAGT to an accumulation of G:C
A:T mutations, presumably resulting from unrepaired m6G. Cells harboring WT hAGT developed more G:C
A:T mutations (30%) than cells expressing the mutant. Thus, in the presence of BG, the mutant is more efficient than the WT hAGT at repairing both m4T and m6G. It should be pointed out that the effects of BG on cells harboring WT hAGT were greatest when higher concentrations of MNNG were used. The WT protein offers some protection against killing at the lower doses of MNNG even in the presence of BG. One explanation for this is that residual hAGT that is not depleted by BG is ample for the efficient repair of the relatively low levels of alkylation damage resulting from these concentrations of MNNG. If this is true, it suggests that at the higher doses of MNNG (in the presence of BG), the residual level of the WT hAGT is inadequate for protecting cells against killing and that cells harboring mutant 56-8 might have a selective growth advantage compared with cells harboring the WT hAGT.
It is difficult to predict what the structural changes are in mutant 56-8 hAGT that produce its altered substrate specificity. It is likely that all eight of the substitutions are not required for the interesting phenotype that has been achieved; however, it would be difficult to identify the critical substitution(s) among the eight. It should be pointed out that seven of the eight mutations are at sites where WT hAGT differs from the E.coli alkyltransferase, Ogt. The similarity in the positions mutated between Ogt and mutant 56-8 hAGT is depicted in the sequence alignment shown in Figure 5
. Of the alkyltransferases examined, Ogt was clearly the most efficient at repairing m4T in vivo, as there was no accumulation of either type of mutation assayed following the treatment of cells harboring this alkyltransferase with MNNG. This is in agreement with a previous report describing the extremely efficient in vivo repair of both m4T and m6G by Ogt (21). In addition to effective repair of these lesions, Ogt is also resistant to BG (33). Taken together this information suggests that the evolved mutant, 56-8, is a human alkyltransferase with increased functional homology to Ogt.

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Fig. 5. Alignment of WT hAGT, bacterial Ogt and mutant 56-8 hAGT. Bold residues indicate sites in Ogt and mutant 56-8 hAGT that differ from WT hAGT. The numbers to the left of the sequences refer to the first amino acid in the homologous segment.
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This type of mutant could be used as a tool to improve our understanding of the adducts most responsible for the biological effects elicited by alkylating agents. In the absence of agents that form m4T or m6G exclusively, it is difficult to know which adduct is more critical for mutagenesis. This can now be addressed by modifying the cellular machinery for repairing specific adducts. Ideally, one would like to obtain a mutant alkyltransferase that exclusively repairs m4T, and another that repairs only m6G. After exposure of cells expressing such alkyltransferases, sensitivity to overall mutagenesis conferred by each type of mutant should implicate one of the lesions as being more biologically significant. This type of approach to studying DNA damage could be extended to other types of adducts. DNA repair enzymes with altered substrate specificities offer a new approach for elucidating the complex relationship that exists between specific DNA adducts and mutations in cancer.
Mutant 56-8 hAGT possesses two distinct changes in substrate specificity that provide cells with a unique phenotype: enhanced repair of m4T and resistance to BG. The importance of such a mutant is even more profound in the presence of BG, because under these conditions the mutant is actually superior to the WT protein at repairing both m4T and m6G. As a result, mutant 56-8 hAGT may be an attractive candidate for protecting human cells against toxicity and mutagenicity associated with chemotherapeutic alkylating agents administered in combination with BG. A potential application for this mutant is the transformation of bone marrow cells ex vivo and their reintroduction into patients prior to receiving combination treatment of alkylating agents and BG. Patients who currently undergo this treatment are limited to doses of alkylating agent that can be tolerated without toxicity to bone marrow, a tissue that expresses inherently low levels of hAGT (34). Using gene therapy it may be possible to introduce a mutant such as mutant 56-8 to bone marrow cells ex vivo, and then infuse the modified cells back into the patient. Even if only a small percentage of stem cells or hematopoeitic precursor cells are transformed by the mutant, they may have a selective advantage for repopulating the bone marrow during therapy. Increased resistance in the bone marrow may minimize toxicity and/or allow for more aggressive treatment protocols that could increase chemotherapeutic effectiveness. Another important issue concerning chemotherapeutic alkylating agents is the formation of new mutations that could potentially initiate the development of secondary tumors in patients. It is possible that due to its persistence in DNA, m4T is an important lesion in inducing secondary cancers. If this is true, than the introduction of mutant 56-8 to the bone marrow of patients would offer better protection against the initiation of secondary cancers.
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Notes
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1 To whom correspondence should be addressed Email: laloeb{at}u.washington.edu 
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Acknowledgments
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We thank L.Samson for providing the bacterial reporter strains used in these studies, and E.Glick and H.Guo for critical comments on the manuscript. This work was supported by NIH grant CA78885 (L.A.L) and NIEHS training grant T32 ES07032 (L.P.E.).
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Received December 20, 1999;
revised March 22, 2000;
accepted March 29, 2000.