Repair of O6-methylguanine, O6-ethylguanine, O6-isopropylguanine, and O4-methylthymine in synthetic oligodeoxynucleotides by Escherichia coli ada gene O6-alkylguanine-DNA-alkyltransferase

Abstract

Self-complementary oligodeoxynucleotides have been synthesized containing O⁶-methylguanine (O⁶meG), O⁶-ethylguanine (O⁶etG), O⁶-isopropylguanine (O⁶iprG) and O⁴-methylthymine (O⁴meT). They anneal in solution to give double-stranded DNA. These double helices have been used as substrates for the DNA repair protein O⁶-alkyl-guanine-DNA-alkyltransferase coded for by the ada gene of Escherichia coli. The repair followed second-order chemical kinetics. O⁶meG was repaired by the 19-kd transferase at a rate of 2.54×10⁷ M⁻¹ s⁻¹ which is close to the theoretical limit for a diffusion-controlled reaction; O⁶etG and O⁴meT are repaired 1000 and 10000 times more slowly. The 39-kd alkyltransferase (which is precursor to the 19-kd form) and the 19-kd transferase repaired O⁶etG at similar rates. O⁶iprG was not repaired. The repair of oligomers containing O⁶meG was only slightly inhibited by the presence of non-alkylated oligomers. Oligomers containing O⁶etG were only slightly more effective as inhibitors of repair than the non-alkylated oligomers, indicating that the transferase does not bind selectively to alkylated DNA. Parallel structural studies have shown that O⁶-alkylguanine:C and O⁴-alkylthymine:A base pairs have a similar geometry with the alkylated base displaced into the major groove of the DNA in contrast to O⁶-alkylguanine:T and O⁴-alkylthymine:G base pairs which retain the Watson-Crick alignment with N1 of the purine juxtaposed to N3 of the pyrimidine. Measurement of the rate of repair of these different base pairs suggests that pairs with the alkyl group exposed in the major groove may be repaired more rapidly than those with the alkyl group more deeply buried in the helix.