Quantitation of O6-methylguanine-DNA methyltransferase in HeLa cells |
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| Affiliation: | 1. Life Cycle Thinking Group, Department of Graphic Design and Engineering Projects. University of the Basque Country (UPV/EHU), Plaza Ingeniero Torres Quevedo 1, 48013 Bilbao, Biscay, Spain;2. Novus Educare Research Group, Minuto de Dios University Corporation (UNIMINUTO), Barrio Chicalá, Calle 87 #20-98, Ibagué 730003, Tolima, Colombia;3. Ekopol: Transition Pathways Research Group, University of the Basque Country (UPV/EHU), Barrio Sarriena, s/n, 48940 Leioa, Biscay, Spain |
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| Abstract: | A synthetic DNA polymer containing [8-3H]O6-methylguanine m6G) was used as a substrate to assay the in situ demethylation of the alkylated base by an activity in HeLa cell extracts. The repair activity appears to be similar to the O6-methylguanine-DNA methyltransferase of E. coli and to be inactivated by reaction with the substrate. Extracts of a methylation-repair proficient (Mer+) cell strain, HeLa CCL2, were found to contain m6G repair activity equivalent to approx. 100 000 molecules of methyltransferase per cell, assuming that each molecule can demethylate one m6G residue. No activity could be detected in the extract of a repair deficient (Mer−) cell strain, HeLa S3, and there is no evidence of an inhibitor of repair activity in this strain. |
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