Active-site alkylation destabilizes human O⁶-alkylguanine DNA alkyltransferase

O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs pro-mutagenic O⁶-alkylguanine and O⁴-alkylthymine lesions in DNA. The alkylated form of the protein is not reactivated; instead, it is rapidly ubiquitinated and degraded. Here, we show that alkylation destabilizes the native fold of the protein by 0.5-1.2 kcal/mole and the DNA-binding function by 0.8-1.4 kcal/mole. On this basis, we propose that destabilization of the native conformational ensemble acts as a signal for ubiquitination.