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Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus.

ABSTRACT: AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. The AGT genes from two extremely thermophilic organisms, the bacterium Aquifex aeolicus and the archaeon Archaeoglobus fulgidus were PCR-derived and cloned into an expression vector. The nucleotide sequence of the Aq. aeolicus AGT encodes a 201-amino-acid protein with a molecular mass of 23000 Da and Ar. fulgidus AGT codes for a 147-amino-acid protein with a molecular mass of 16718 Da. The Aq. aeolicus and Ar. fulgidus AGTs were expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allowed single-step isolation and purification by metal-affinity chromatography. Both AGTs formed inclusion bodies and were not soluble under native purification conditions. Therefore AGT isolation was performed under protein-denaturation conditions in the presence of 8.0 M urea. Soluble AGT was obtained by refolding the AGT in the presence of calf thymus DNA. Both AGTs were active in repairing O6-methylguanine and, at a lower rate, O4-methylthymine in DNA. They exhibited thermostability and optimum activity at high temperature. The thermostable AGTs, particularly that from Aq. aeolicus, were readily inactivated by the low-molecular-mass inhibitor O6-benzylguanine, which is currently in clinical trials to enhance cancer chemotherapy.

SUBMITTER: Kanugula S

PROVIDER: S-EPMC1223701 | biostudies-other | 2003 Oct

REPOSITORIES: biostudies-other

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Project description:1,2,3,4-Diepoxybutane (DEB) is a key carcinogenic metabolite of the important industrial chemical 1,3-butadiene. DEB is a bifunctional alkylating agent capable of reacting with DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can react with another nucleophilic site to form cross-linked adducts. A recent report revealed a strong correlation between cellular expression of the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) and the cytotoxic and mutagenic activity of DEB, suggesting that DEB induces AGT-DNA cross-links (Valadez, J. G., et al. (2004) Activation of bis-electrophiles to mutagenic conjugates by human O6-alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 17, 972-982). The purpose of our study was to analyze the formation and structures of DEB-induced AGT-DNA conjugates and to identify specific amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of hAGT that had been treated with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the protein to form either one or two butanediol-dG cross-links, corresponding to mass shifts of +353 and +706 Da, respectively. HPLC-ESI+ -MS/MS sequencing of the tryptic peptides obtained from dG monoepoxide-treated protein indicated that the two cross-linking sites were the alkyl acceptor site, Cys145, and a neighboring active site residue, Cys150. The same two amino acid residues of hAGT became covalently cross-linked to DNA following DEB treatment. Modification of Cys145 was further confirmed by HPLC-ESI+ -MS/MS analysis of dG monoepoxide-treated synthetic peptide GNPVPILIPCHR which represents the active site tryptic fragment of hAGT (C = Cys145). The replacement of the catalytic cysteine residue with alanine in the C145A hAGT mutant abolished DEB-induced cross-linking at this site, while the formation of conjugates via neighboring Cys150 was retained. The exact chemical structure of the cross-linked lesion was established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI+ -MS/MS analysis of the amino acids resulting from the total digestion of modified proteins analyzed in parallel with an authentic standard. AGT-DNA cross-linking is a likely mechanism of DEB-mediated cytotoxicity in cells expressing this important repair protein.

Project description:O(6)-POB-dG (O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O(6)-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O(6)-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O(6)-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O(6)-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI(+)-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O(6)-POB-dG adducts. In our approach, synthetic DNA duplexes containing O(6)-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D(4)-O(6)-POB-dG internal standard and mild acid hydrolysis to release O(6)-POB-guanine (O(6)-POB-G) and D(4)-O(6)-POB-guanine (D(4)-O(6)-POB-G), samples are purified by solid phase extraction (SPE), and O(6)-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI(+)-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O(6)-POB-G-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O(6)-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI(+)-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O(6)-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5'-AATAGTATCT[O(6)-POB-G]GAGCC-3') opposite either C or T. Faster rates of alkyl transfer were observed when O(6)-POB-dG was paired with T rather than with C (k = 1.74 × 10(6) M(-1) s(-1) vs 1.17 × 10(6) M(-1) s(-1)).

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Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus.

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