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.

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.

Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase.

Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine. When expressed at high levels in E. coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine. The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E. coli from toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.

Reaction of O6-benzylguanine-resistant mutants of human O6-alkylguanine-DNA alkyltransferase with O6-benzylguanine in oligodeoxyribonucleotides.

Inactivation of the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), by O6-benzylguanine renders tumor cells susceptible to killing by alkylating agents. AGT mutants resistant to O6-benzylguanine can be made by converting Pro140 to an alanine (P140A) or Gly156 to an alanine (G156A). These mutations had a much smaller effect on the reaction with O6-benzylguanine when it was incorporated into a short single-stranded oligodeoxyribonucleotide. Such oligodeoxyribonucleotides could form the basis for the design of improved AGT inhibitors. AGT and mutants P140A and G156A preferentially reacted with O6-benzylguanine when incubated with a mixture of two 16-mer oligodeoxyribonucleotides, one containing O6-benzylguanine and the other, O6-methylguanine. When the 6 amino acids located in positions 159-164 in AGT were replaced by the equivalent sequence from the Escherichia coli Ada-C protein (mutant AGT/6ada) the preference for benzyl repair was eliminated. Further mutation incorporating the P140A change into AGT/6ada giving mutant P140A/6ada led to a protein that resembled Ada-C in preference for the repair of methyl groups, but P140A/6ada did not differ from P140A in reaction with the free base O6-benzylguanine. Changes in the AGT active site pocket can therefore affect the preference for repair of O6-benzyl or -methyl groups when present in an oligodeoxyribonucleotide without altering the reaction with free O6-benzylguanine.

Repair of O6-benzylguanine by the Escherichia coli Ada and Ogt and the human O6-alkylguanine-DNA alkyltransferases.

O6-Methylguanine is removed from DNA via the transfer of the methyl group to a cysteine acceptor site present in the DNA repair protein O6-alkylguanine-DNA alkyltransferase. The human alkyltransferase is inactivated by the free base O6-benzylguanine, raising the possibility that substantially larger alkyl groups could also be accepted as substrates. However, the Escherichia coli alkyltransferase, Ada-C, is not inactivated by O6-benzylguanine. The Ada-C protein was rendered capable of reaction by the incorporation of two site-directed mutations converting Ala316 to a proline (A316P) and Trp336 to alanine (W336A) or glycine (W336G). These changes increase the space at the active site of the protein where Cys321 is buried and thus permit access of the O6-benzylguanine inhibitor. Reaction of the mutant A316P/W336A-Ada-C with O6-benzylguanine was greatly stimulated by the presence of DNA, providing strong support for the concept that binding of DNA to the Ada-C protein activates the protein. The Ada-C protein was able to repair O6-benzylguanine in a 16-mer oligodeoxyribonucleotide. However, the rate of repair was very slow, whereas the E. coli Ogt, the human alkyltransferase, and the mutant A316P/W336A-Ada-C alkyltransferases reacted very rapidly with this 16-mer substrate and preferentially repaired it when incubated with a mixture of the methylated and benzylated 16-mers. These results show that benzyl groups are better substrates than methyl groups for alkyltransferases provided that steric factors do not prevent binding of the substrate in the correct orientation for alkyl group transfer.

Resistance of the human O6-alkylguanine-DNA alkyltransferase containing arginine at codon 160 to inactivation by O6-benzylguanine.

Inactivation of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine renders tumor cells more sensitive to killing by methylating and chloroethylating agents, and O6-benzylguanine is currently undergoing clinical trials for development as an agent to enhance chemotherapy. It has been reported recently that a polymorphism in the human O6-alkylguanine-DNA alkyltransferase gene exists, with about 15% of the population studied having arginine at codon 160 instead of glycine (Y. Imai et al., Carcinogenesis (Lond.), 16: 2441-2445, 1995). We have studied the effects of mutations of this glycine to arginine, tryptophan, or alanine on the interaction of human alkyltransferase with O6-benzylguanine using direct determination of the amount of activity remaining after incubation with various concentrations of the inhibitor and measurement of the rate of production of [8-3H]guanine from O6-benzyl[8-3H]guanine as assays. These mutations had little effect on the alkyltransferase activity in repairing O6-methylguanine in methylated DNA. Alteration of glycine 160 to tryptophan or alanine slightly increased the sensitivity to O6-benzylguanine (by up to 4-fold). However, alteration of glycine 160 to arginine drastically reduced the inactivation by O6-benzylguanine with at least a 20-fold increase in the ED50 value and a similar reduction in the production of guanine whether inactivation was carried out in the absence or presence of DNA. These results raise the possibility that a subpopulation of patients may be resistant to O6-benzylguanine and that higher doses or additional alkyltransferase inhibitors capable of inactivating this form of the alkyltransferase will be necessary.

The role of tyrosine-158 in O6-alkylguanine-DNA alkyltransferase activity.

The tyrosine residue present at position 158 in the human O6-alkylguanine-DNA alkyltransferase is one of 22 amino acid residues that are conserved in all known alkyltransferase protein sequences. The importance of this amino acid in the reactions brought about by the alkyltransferase was studied by changing this residue to alanine or to phenylalanine. The control and mutant alkyltransferase proteins were expressed in an Escherichia coli strain lacking alkyltransferase activity and the proteins purified to near homogeneity and their activities measured using both methylated DNA and O6-benzylguanine (BG) as substrates. The alteration to alanine led to a very large decrease in activity towards both substrates but removal of O6-methylguanine from DNA and the conversion of BG to guanine could still be detected when large amounts of the protein were used. The activity of the Y158A mutant was at least 800 times less than that of the control alkyltransferase. The change of tyrosine-158 to phenylalanine reduced the rate of reaction with methylated DNA only slightly (to about one-third). The conversion of BG to guanine by the Y158F mutant was also reduced to about one-third when assayed in the absence of DNA and by about one-half in the presence of DNA. These results suggest that the presence of tyrosine at position 158 plays an important but not absolutely essential role in the reactions brought about by the alkyltransferase. This role is likely to involve the stabilization of the bound substrate by interaction with the aromatic ring of the tyrosine. The hydrogen bond formed by the hydroxyl group from tyrosine-158 may also facilitate the reaction but the contribution from this interaction is relatively small.

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.

O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes the promutagenic O6-methylguanine lesion from DNA. In order to obtain more information about the mechanism of action of AGT, two conserved residues in a putative DNA binding domain were changed by site-directed mutagenesis, and the abilities of the mutant proteins to bind to DNA, to repair methylated DNA, and to convert O6-benzylguanine to guanine were examined. The alteration of arginine-128 to alanine (R128A) reduced the AGT activity toward methylated DNA substrates by a factor of more than 1000 but did not decrease the rate of reaction with O6-benzylguanine. The change of residue tyrosine-114 to glutamic acid (Y114E) completely abolished the ability to repair O6-methylguanine in DNA in the assays used showing that this was reduced by > 15,000-fold, but the ability to convert O6-benzylguanine to guanine was reduced by only 60-fold. Alteration of this residue to alanine (Y114A) reduced activity toward methylated DNA by > 1000-fold and toward O6-benzylguanine by about 80-fold. Neither the R128A nor the Y114E mutant AGT were able to compete with the control AGT for the repair of methylated DNA whereas the inactive mutant, C145A, in which the cysteine acceptor site is changed to alanine, competed effectively in this assay. These results suggest that the residues arginine-128 and tyrosine-114 are involved in the DNA binding properties of the AGT. The ability of the AGT proteins to form stable complexes with DNA was therefore examined by measuring the retardation of DNA during electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in human O6-alkylguanine-DNA alkyltransferase imparting resistance to O6-benzylguanine.

O6-Benzylguanine is an inactivator of O6-alkylguanine-DNA alkyltransferases (AGT) which is currently entering clinical trials as an agent improving the cancer chemotherapeutic activity of chloroethylnitrosoureas and other alkylating agents. O6-Benzylguanine acts by virtue of its ability to serve as a substrate for the AGT forming S-benzylcysteine at the cysteine acceptor site. The effects of a number of mutations in the human AGT sequence on the reaction with O6-benzylguanine were investigated by two methods: (a) by measuring the loss of the ability of the AGT to repair a methylated DNA substrate after preincubation with O6-benzylguanine; and (b) by measuring the production of guanine from O6-benzylguanine by the AGT proteins. Both assays gave similar results and showed that mutations of the proline residues at positions 138 and 140 and of the glycine residue at position 156 significantly reduced the ability to react with O6-benzylguanine. The combination of these mutations gave even greater resistance. Thus, the 50% effective dose for O6-benzylguanine was increased from 0.25 microM in the control AGT to 29 microM by mutations P138K/P140A, to 60 microM by mutation G156A and to > 300 microM by mutations P140A/G156A. Truncation of the AGT at the carboxyl end, removing either 31 or 23 amino acids did not affect the activity or the ability to react with O6-benzylguanine, but removal of the 36 carboxyl terminal amino acids, which includes a highly conserved glutamic acid residue, led to the loss of all activity. The rate of the reaction between the AGT and O6-benzylguanine was increased when DNA was present. This increase amounted to about 6-fold with the control AGT and the carboxyl-truncated mutants but was reduced to only 2-fold with G156A mutant and increased to 11-18-fold with the mutations of proline residues at 138 and 140. These results indicate that several residues in the AGT sequence affect the access of the active site to O6-benzylguanine and that these residues are located in at least two regions on either side of the active site cysteine, which is located at residue 145. Mutations in these regions may occur during therapy with alkylating agents and O6-benzylguanine. The development of other AGT inactivators which are still able to inactivate the resistant mutants may be necessary to maximize the potential of AGT inhibition for cancer chemotherapy.