Dataset Information

Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA.

ABSTRACT: The cDNA encoding Chinese hamster O6-alkylguanine-DNA-alkyltransferase (ATase) has been isolated from a library prepared from RNA isolated from V79 lung fibroblasts which had an upregulated level of this repair activity following stepwise selection with a chloroethylating agent (1, 2). Expression of the cDNA in E. coli produced functionally active ATase at levels of 2.5% of total cellular protein as determined by in vitro assay. The recombinant hamster protein has a molecular weight of 28 kDa as estimated by SDS-PAGE and fluorography and this was identical to that in the upregulated cells. The characteristic PCHRV pentapeptide of the alkyl acceptor site has been identified and there is a 68 amino acid residue region which is 90% conserved across all the mammalian proteins so far analysed: in contrast, the N- and C-terminal domains diverge by as much as 50% between species. Polyclonal antibodies to the human and rat ATases hybridised to the hamster protein on western analysis suggesting at least one common epitope shared across species. However, in antibody inhibition experiments neither of the antisera cross reacted with the hamster ATase in a way which interfered with functional activity whereas the anti-human antibodies inhibited the human ATase and the anti-rat antibodies inhibited the rat and mouse ATases. There may therefore be significant tertiary structural differences between the hamster protein and the other mammalian ATases.

SUBMITTER: Rafferty JA

PROVIDER: S-EPMC312303 | biostudies-other | 1992 Apr

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA.

Rafferty J A JA Elder R H RH Watson A J AJ Cawkwell L L Potter P M PM Margison G P GP

Nucleic acids research 19920401 8

The cDNA encoding Chinese hamster O6-alkylguanine-DNA-alkyltransferase (ATase) has been isolated from a library prepared from RNA isolated from V79 lung fibroblasts which had an upregulated level of this repair activity following stepwise selection with a chloroethylating agent (1, 2). Expression of the cDNA in E. coli produced functionally active ATase at levels of 2.5% of total cellular protein as determined by in vitro assay. The recombinant hamster protein has a molecular weight of 28 kDa as ...[more]

PMID: 1579490

Similar Datasets

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)).

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.

Dataset Information

Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA.

Publications

Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets