The nuclear targeting and nuclear retention properties of a human DNA repair protein O6-methylguanine-DNA methyltransferase are both required for its nuclear localization: the possible implications.

Human O6-methylguanine-DNA methyltransferase (MGMT) protects human cells from the mutagenic effects of alkylating agents by repairing the O6-alkylguanine residues formed by these agents in the nuclear DNA. We report here a study showing a possible two-step model for the nuclear localization of the 21 kDa human protein. The first step is the translocation of the protein from the cytosol to the nucleus. This appears to require the nuclear targeting property associated with the holoprotein in combination with a cellular factor(s) to effect the nuclear translocation of MGMT. The second step involves the nuclear retention of MGMT (to prevent its export from the nucleus). This requires a basic region (PKAAR, codons 124-128) that can bind to the non-diffusible DNA elements in the nucleus. Supporting data for such mechanisms are: (i) the holoprotein can target the cytosolic 110 kDa beta-galactosidase into the nucleus; (ii) purified recombinant MGMT requires a cellular factor for transport across the nuclear membrane; (iii) nuclear MGMT can be removed selectively by DNase I; (iv) the repair-positive K125L mutant, which alters the PKAAR motif, remains in the cytosol and fails to bind DNA in vitro; and (v) polypeptide containing the PKAAR motif has no nuclear targeting property. Interestingly, mutants in another basic region, KLLKVVK (codons 101-107) are DNA binding and repair deficient but entirely nuclear. As these substitutions affect the functional properties of human MGMT, they are potential targets for genetic screening of individuals for risk assessment to alkylating agents.