Function of domains of human O⁶-alkylguanine-DNA alkyltransferase

0⁶-Alkylguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that protects from alkylating agents by converting O ⁶-alkylguanine to guanine forming 5-methylcysteine in the AGT protein. The crystal structure of human AGT shows clearly the presence of two domains. The N-terminal domain contains a bound zinc atom, and zinc binding confers a mechanistic enhancement to repair activity, but this domain has no known function. The C-terminal domain contains all residues so far implicated in alkyl transfer including the cysteine acceptor site (Cys145), the O ⁶-alkylguanine binding pocket, and a DNA binding domain. We have expressed and purified the two domains of human AGT separately. The C-terminal domain was totally inactive in vitro, but good activity forming 5-alkylcysteine at Cys145 was obtained after recombination with the N-terminal domain via a freeze-thawing procedure. This suggests that the N-terminal domain plays a critical structural role in maintaining an active configuration of the C-terminal domain. However, this C-terminal domain alone had activity in protecting against the cytotoxic and mutagenic activity of the methylating agent, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) when expressed in Escherichia coli cells lacking endogenous AGT, suggesting that other proteins can fulfill this function. Remarkably, the free N-terminal domain of hAGT was able to repair O⁶-alkylguanine in vitro via alkyl transfer provided that zinc ions were present. The N-terminal domain was also able to produce moderate protection from MNNG when expressed in E. coli. This cryptic Zn ²⁺-dependent DNA repair activity may be relevant to the evolution and function of AGTs.