Repair of Oligodeoxyribonucleotides by O6-Alkylguanine-DNA Alkyltransferase

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Abstract
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.

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