A Single Amino Acid Change in Human 06-Alkylguanine-DNA Alkyltransferase Decreasing Sensitivity to Inactivation by O6-Benzylguanine1

Mammalian O'-alkylguanine-DNA alkyltransferases (AGTs) are readily inactivated by incubation with the pseudosubstrate, O6-benzylguanine, but the equivalent protein from the Escherichia coli ogt gene is much less sensitive and the Saccharomyces cerevisiae and E. coli oda gene prod uct AGTs are completely resistant to this compound. We have expressed the normal human ACT and various point mutations (C145A, W100A, and P140A) in an u</u ogt~ strain of E. coli and tested these proteins against DNA substrates containing 06-methylguanine, for inactivation by 06-benzylguanine and for the ability to produce guanine from O6-benzylguanine. The C145A mutation was inactive as expected since this residue forms the methyl acceptor site. Mutants VS1(10A and P140A were fully active against methylated DNA substrates but the P140A mutant was much less sensitive to inactivation by 06-benzylguanine and failed to form significant amounts of [3H]guanine when incubated with O6-benzyl[8-3H]guanine. The proline at position 140 in mammalian AGTs is replaced by alanine in the Ada and yeast AGTs and by serine in the Ogt AGT. These results suggest that this proline residue affects the configuration of the active site allowing the 06-benzylguanine to enter and react with the mammalian AGT. The production of resistance to 06-benzylguanine by a single base change raises the possibility that such resistance may arise quite readily in cells of tumors treated therapeutically with the combina tion of 06-benzylguanine and an alkylating agent. Introduction AGT2 is a DNA repair protein that plays an important role in protecting cells from the toxic effects of monofunctional alkylating agents and chloroethylating drugs (1-4). AGT has a unique mecha nism of action in that it brings about the transfer of alkyl groups present on the O''-position of guanine in DNA to a cysteine residue located within the AGT amino acid sequence (5, 6). The resulting S-alkylcysteine in the protein is not converted back to cysteine. There fore, the AGT can act only once and the number of O6-alkylguanine residues that can be repaired is equal to the number of available AGT molecules. Tumor cells expressing high levels of AGT are resistant to killing by therapeutic methylating or chloroethylating drugs and such inherent resistance may limit the clinical effectiveness of these agents (1-4, 6). There is, therefore, considerable interest in the synthesis of compounds that would block the AGT activity and thus enhance their action. Recent work has indicated that O6-benzylguanine may be suitable for this purpose. O6-Benzylguanine was found to be a very potent time and concentration dependent inactivator of the human alkyltransferase (7). Inactivation was irreversible suggesting that O6benzylguanine acts as an alternative substrate for the protein. This mechanism has now been confirmed by the identification of S-benReceived 7/15/93; accepted 9/15/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Grants CA-18137 and CA-57725 from the National Cancer Institute. 2 The abbreviations used are: AGT, O^-alkylguanine-DNA alkyltransferase (EC 2.1. 63); MNNG, A/-methyl-A/'-nitro-7v'-nitrosoguanidine; IPTG, isopropyl-ß-o-thiogalactopyranoside; cDNA, complementary DNA. zylcysteine in the AGT and the formation of stoichiometric amounts of guanine following incubation with O6-benzylguanine.3 There is considerable similarity between the human AGT and the AGT proteins isolated from microorganisms and the sequence surrounding the cys teine acceptor site is identical (3, 4, 8). However, regardless of this similarity, the AGT proteins derived from carboxyl terminal fragment of the Escherichia coli ado. gene (9) and the Saccharomyces cerevisiae AGT3 were found to be completely resistant to inactivation by Ohbenzylguanine and the AGT derived from the Escherichia coli ogt gene was much less sensitive.3 These results suggest that there is some difference in the active site of these AGT proteins and show that the microbial proteins are not good models for the design of inactivators of the mammalian AGT. Furthermore, they raise the possibility that resistance to O6-benzylguanine might arise in mammalian cells treated with this AGT inactivator and a toxic and mutagenic alkylating agent. We have examined this question by expressing the human AGT and defined mutants of it in E. coli. We now report a single amino acid change that bestows a significant level of resistance to O6-benzylguanine in the human AGT. Materials and Methods Materials, Bacterial Cells, and Plasmids. GWR109 cells (10) were ob tained from Dr. Leona Samson, Department of Molecular and Cellular Toxi cology, Harvard School of Public Health, Boston, MA. DH5a MCR cells were purchased from Bethesda Research Laboratories (Gaithersburg, MD). BamHI was purchased from GIBCO BRL (Gaithersburg, MD). EcoRI and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). Ampicillin, kanamycin, MNNG, and IPTG were purchased from Sigma Chemical Com pany (St. Louis, MO). CJ236 cells, plasmid pGem-3Zf( + ), and helper phage M13K07 were purchased from Promega Corporation (Madison WI). Plasmid pINAGT, which expresses the human AGT in E. coli, was produced by insert ing the human cDNA sequence (8) into the E. coli expression vector pINIIIA3(lppp"5) (11) using the £coRIand BamH\ sites in the vector and polymerase chain reaction to generate the appropriate sites in the cDNA. The means of construction changes the amino terminal sequence by the addition of 5 amino acids giving a sequence MKGGILin place of M-. Site-directed Mutagenesis. pINAGT was digested with £coRIand BamHI and the resulting piece which contains the human AGT amino acid coding sequence was inserted into pGem-3Zf(+) which was used for site directed mutagenesis using the following oligodeoxynucleotides and the Oligonucleotide-directed Mutagenesis System 2.1 kit (Amersham, Arlington Heights, IL), according to manufacturer's instructions. Oligodeoxynucleotides were synthe sized in the Macromolecular Core Facility, Hershey Medical Center, by using a Milligen 7500 DNA synthesizer. The following sense strands were synthe sized, mismatches underlined, to produce the amino acid changes of tryptophan 100 to alanine, proline 140 to alanine, and cysteine 145 to alanine, respec tively: 5'-CCAGACAGGTGTTAGCAAAGCTGCTGAAG-3'; 5'-GGCAATCCTGTCGCCATCCTCATCCCG-3'; and 5'-CCATCCTCATCCCGGCCCACAGAGTGGTC-3'. After screening to identify the desired mutants, the inserts were transferred back into the pIN expression vector. All mutants were verified by sequencing the entire AGT amino acid coding region. 3 A. E. Pegg, M. Boosalis, L. Samson, R. C. Moschel, T. L. Byers, K. Swenn, and M. E. Dolan, submitted for publication.