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

Self-complementary oligodeoxynucleotides have been synthesizedcontaining O⁶-methylguanine (O⁶meG), O⁶-ethylguanine (O⁶etG),O⁶-isopropylguanine (O⁶iprG) and O⁴-methylthymine (O⁴meT). Theyanneal in solution to give double-stranded DNA. These doublehelices have been used as substrates for the DNA repair proteinO⁶-alkyl-guanine-DNA-alkyltransferase coded for by the ada geneof Escherichia coli. The repair followed second-order chemicalkinetics. O⁶meG was repaired by the 19-kd transferase at a rateof 2.54x10⁷ M^–1 s^–1 which is close to the theoreticallimit for a diffusion-controlled reaction; O⁶etG and O⁴meT arerepaired 1000 and 10000 times more slowly. The 39-kd alkyltransferase(which is precursor to the 19-kd form) and the 19-kd transferaserepaired O⁶etG at similar rates. O⁶iprG was not repaired. Therepair of oligomers containing O⁶meG was only slightly inhibitedby the presence of non-alkylated oligomers. Oligomers containingO⁶etG were only slightly more effective as inhibitors of repairthan the non-alkylated oligomers, indicating that the transferasedoes not bind selectively to alkylated DNA. Parallel structuralstudies have shown that O⁶-alkylguanine:C and O⁴-alkylthymine:Abase pairs have a similar geometry with the alkylated base displacedinto the major groove of the DNA in contrast to O⁶-alkylguanine:Tand O⁴-alkylthymine:G base pairs which retain the Watson-Crickalignment with N1 of the purine juxtaposed to N3 of the pyrimidine.Measurement of the rate of repair of these different base pairssuggests that pairs with the alkyl group exposed in the majorgroove may be repaired more rapidly than those with the alkylgroup more deeply buried in the helix.