Synthesis of oligodeoxyribonucleotides containing a tricyclic thio analogue of O6-methylguanine and their recognition by MGMT and Atl1

Promutagenic O6-alkylguanine adducts in DNA are repaired in humans by O6-methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of O6-methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1. Examination of the active sites of both MGMT and Atl1 suggest large steric clashes hindering binding of the analogue. Such analogues, if mutagenic, are likely to be highly toxic

Atl1 Regulates Choice between Global Genome and Transcription-Coupled Repair of O6-Alkylguanines

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O6-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O6-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O6-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER

Mass spectrometric analysis of the active site tryptic peptide of recombinant O6-methylguanine-DNA methyltransferase following incubation with human colorectal DNA reveals the presence of an O6-alkylguanine adductome

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O6-methylguanine O6-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O6-alkylguanines (O6-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O6-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O6-alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S-methyl, S-ethyl, S-propyl, S-hydroxyethyl, S-carboxymethyl, S-benzyl, and S-pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O6-alkGs. The LOQ of ASPs containing S-methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S-methylcysteine at levels that correlated with those of O6-MeG determined previously by HPLC-radioimmunoassay (r2 = 0.74; p = 0.014). O6-CMG, a putative O6-hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O6-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O6-alkG adductome and, given the mutagenic potential of O6-alkGs, can provide mechanistic information about cancer pathogenesis

Personal exposure to inhalable dust and the specific latex-aeroallergen, Hev b6.02, in latex glove manufacturing in Thailand.

OBJECTIVES: Latex product manufacturing is an important industry in south-east Asia but has the potential for considerable occupational exposure of workers to latex allergens. Although exposure to latex allergens can result in adverse health reactions, few studies to characterize this exposure have been conducted to date. This study therefore aimed to characterize current airborne inhalable dust and the specific allergen, Hev b 6.02, exposures in this industry in Thailand. METHODS: Workers were recruited from three factories in the southern part of Thailand. Full-shift inhalable dust personal air sampling was conducted using IOM sampling heads equipped with polytetrafluoroethylene filters at a 2.0 l min(−1) flowrate. After weighing to determine inhalable dust levels, filters were extracted and analysed for Hev b 6.02 using an enzyme immunometric assay. RESULTS: Two hundred and seventy-five workers agreed to participate, resulting in a total of 292 measurements. Geometric mean (GM) personal exposure to inhalable dust was 0.88mg m(–3), but individual exposures up to 12.34mg m(–3) were measured. The pattern of exposure was similar across factories, with highest exposures in the stripping (GM 2.08–4.05mg m(–3) for the 3 factories) and tumbling departments (1.11–2.17mg m(–3)). Within-worker (day-to-day) variability contributed 92% to total variability. The Hev b 6.02 exposure pattern was similar with time-weighted average GM exposure levels in the oldest factory ranging from 8.7mg m(–3) in the laboratory to 30.2mg m(–3) in the stripping department. In contrast to inhalable dust exposure, total exposure variability was primary driven by variability between workers (67%). CONCLUSIONS: Workers in these latex product factories get routinely exposed to measurable Hev b 6.02 levels, which may give rise to increased incidence of allergic symptoms and occupational asthma. Also, in this measurement campaign a 10mg m(–3), but not 15mg m(–3), occupational exposure limit for inhalable dust was occasionally exceeded. Highest Hev b 6.02 exposures were found in the stripping and tumbling departments, which would be natural targets for interventions aimed at reducing exposure