Repair of oligodeoxyribonucleotides by O⁶-alkylguanine-DNA alkyltransferase

Activity of the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT) is an important source of tumor cell resistance to alkylating agents. AGT inhibitors may prove useful in enhancing chemotherapy. AGT is inactivated by reacting stoichiometrically with O⁶-benzylguanine (b⁶G), which is currently in clinical trials for this purpose. Short oligodeoxyribonucleotides containing a central b⁶G are more potent inactivators of AGT than b⁶G. We examined whether human AGT could react with oligodeoxyribonucleotides containing multiple b⁶G residues. The single-stranded 7-mer 5′-d[T(b⁶G)₅G]-3′ was an excellent AGT substrate with all five b⁶G adducts repaired although one adduct was repaired much more slowly. The highly b⁶G-resistant Y158H and P140K AGT mutants were also inactivated by 5′-d[T(b⁶G)₅G]-3′. Studies with 7-mers containing a single b⁶G adduct showed that 5′-d[TGGGG(b⁶G)G]-3′ was more poorly repaired by wild-type AGT than 5′-d[T(b⁶G)GGGGG]-3′ and 5′-d[TGG(b⁶G)GGG]-3′ and was even less repairable by mutants Y158H and P140K. This positional effect was unaffected by interchanging the terminal 5′- or 3′-nucleotides and was also observed with single-stranded 16-mer oligodeoxyribonucleotides containing O⁶-methylguanine, where a minimum of four nucleotides 3′ to the lesion was required for the most efficient repair. Annealing with the reverse complementary strands to produce double-stranded substrates increased the ability of AGT to repair adducts at all positions except at positions 2 and 15. Our results suggest that AGT recognizes the polarity of single-stranded DNA, with the best substrates having an adduct adjacent to the 5′-terminal residue. These findings will aid in designing novel AGT inhibitors that incorporate O⁶-alkylguanine adducts in oligodeoxyribonucleotide contexts.