Characterization of O⁶-methylguanine-DNA-methyltransferase (O⁶-MGMT) activity in Xiphophorus fishes

We utilized a custom-synthesized double-strand oligonucleotide containing a single O⁶-methylguanine (O⁶-MG) residue within a restriction endonuclease recognition site to determine O⁶-methylguanine-DNA-methyltransferase (O⁶-MGMT) activity in various tissue extracts prepared from Xiphophorus fish. The results suggest Xiphophorus fish O⁶-MGMT activity has many of the same characteristics as Escherichia coli and mammalian O⁶-MGMT's including rapid reaction kinetics consistent with stoichiometric removal of methyl groups, but exhibits a temperature optimum of 23°C. Results from protein extract activity assays indicate O⁶-MGMT activity patterns among four Xiphophorus tissues followed the order: brain ≥ testes > gill ≥ liver. In mammals, O⁶-MGMT activity is high in liver, while activity in brain is minimal (i.e. ≈9% of liver); however, we report that in the Xiphophorus fishes examined, brain tissue extracts exhibited much higher (≈six-fold) O⁶-MGMT activity levels than liver. Comparison of O⁶-MGMT activity between Xiphophorus species employed in tumor induction experiments did not indicate significant differences in ability to clear the pre-mutagenic O⁶-MG from the oligonucleotide substrate.