Physicochemical studies of human O⁶‐methylguanine‐DNA methyltransferase

O⁶-Methylguanine-DNA methyltransferase, present in most organisms, removes mutagenic and carcinogenic O⁶-alkylguanine from DNA by accepting the alkyl group in a stoichiometric reaction. The protein has been partially purified from human placenta. It reacts with second-order rate constants of 2.20 x 10⁸ and 0.067 x 10⁸ lmol^-1 min^-1 at 37°C for duplex and single-stranded DNA substrates, respectively. The corresponding value for the alkylated base in synthetic poly(dC, dG, m⁶dG) is 0.02 x 10⁸ l mol^-1 min^-1. The native protein is monomeric with a molecular mass of 22-24 kDa. Methylation of the protein does not lead to a gross change in its conformation but causes a slight reduction in its isoelectric point of 6.2. Although DNA protects the protein from heat inactivation, both duplex and single-stranded DNAs inhibit its activity in a concentration-dependent manner. The transferase reaction rate is also strongly inhibited by salt with about 20% of the maximum rate observed in physiological ionic strength. This inhibition is nonspecific with respect to the ions of univalent salts.