IN VITRO AND IN VIVO CHARACTERIZATION OF MAMMALIAN O6-METHYLGUANINE-DNA METHYLTRANSFERASE

Abstract

The potential hazard of the promutagenic lesion O6-alkylguanine in prokaryotes and eukaryotes is neutralized by a ubiquitous repair process entailing the direct transfer of the alkyl moiety. In mammalian cells, this activity is mediated by a 22kDa O6-methylguanine-DNA methyltransferase (MGMT) which contains a conserved active site with a unique cysteine residue acting as the alkyl acceptor. Despite extensive homology in sequences surrounding the active site, the N-termini of mammalian methyltransferases differ significantly but arc nonetheless essential for repair activity since deletion of the first 7 Lo 48 amino acids from the rat and human protein resulted in severe or complete loss of activity. Despite being characterized as a nuclear protein, the heterogeneous staining of MGMT in several cell lines and the lack of orthodox nuclear localization signal prompted us to undertake an extensive study on the intracellular localization of MGMT. The putative nuclear localization signal is highly basic and well-conserved in mammalian MGMTS. Nuclear staining of human MGMT was disruptcd when Lys- 125 and Arg-128 were replaced by leucines. Nevertheless, peptides containing each of the basic domains were ineffective in nuclear targeting, suggesting that the entire protein is required for nuclear translocation. Progrcssive deletion from either termini of human MGMT resulted in cytoplasmic staining of MGMT-lacZ fusion proteins, implying that nuclear translocation of MGMT is not directed by any signal peptide but depends on the wild type conformation. Moreover, in vitro nuclear import assay showed that nuclear accumulation of MGMT required cytosolic proteins but not inhibited by low temperature, thus deviating from NLS-mediated nuclear transport pathway. The fact that the Kl25L and Rl28L mutants were repair-proficient implies that intracellular localization and DNA repair of MGMT require separate domains. We therefore postulate that MGMT enters the nucleus by a novel active process and is stabilized by interaction with DNA or nuclear matrix. It is now evident that cellular protection of alkylation-induced carcinogenesis can be regulated by either post-transcriptional events or spatial distribution of the repair protein.