Positional and directional repair of O?-alkylguanine lesions by human O?-alkylguanine-DNA alkyltransferase

by 1975- Luu, Kieu X.

iii Human O 6-alkylguanine-DNA alkyltranserase (AGT) is a widely expressed DNA repair protein present in prokaryotes and eukaryotes. AGT repairs O 6-alkylguanine adducts in DNA via a stoichiometric reaction that transfers the alkyl group onto an internal cysteine residue of the protein. This irreversible reaction inactivates AGT and de novo protein synthesis is required to produce more active protein. AGT protects normal cells from the mutagenic and carcinogenic effects of alkylation, but overexpression of AGT by cancer cells confers resistant to chemotherapeutic agents. The development of AGT inhibitors may prove useful in enhancing cancer chemotherapy. O 6-Benzylguanine (b 6G) is a potent inactivator of AGT. We showed that a singlestranded (ss) 7-mer oligodeoxyribonucleotide (oligo) containing multiple b6G can inhibit AGT activity in vitro. The protein is more efficient at reacting with b6G residues positioned near the 5'-end compared to those positioned near the 3'-end. In vivo, oligos containing single or multiple b6G can also sensitize HT29 cells to killing by BCNU. The position of b6G in an oligo may confer some protection from serum nucleases as oligos with b 6G near the 5'- or 3'-end tend to have a longer half-life. Studies done with 16-mer oligos containing O 6-methylguanine (m 6 G) at different positions also showed a trend in which there is an increase in the ED50 values for AGT inactivation with methylated lesions located towards the extreme 3'-end of the oligo. The ED50 values for the 16-mers with m 6G positioned at the second, sixth, and eleventh nucleotide from the 5'-end were between 9-13 nM for wild type AGT. However, the ED50 values for the 16-mers with m 6G positioned at the fourteenth and fifteenth nucleotide from the 5'-end were 57 nM and > 50 µM, respectively. AGT repaired adducts at all iv positions equally well in duplex 16-mer oligos except those at positions 2 and 15. These results suggest that AGT recognizes the polarity in DNA and requires a minimum of four nucleotides 3' of an m 6 G lesion in order to productively bind and efficiently repair that lesion. The preference for AGT to repair 5'-end lesions in short oligos suggested that AGT may bind DNA in an oriented manner and scans directionally (3' to 5') to identify and repair O 6-alkylguanine lesions. Using a single-stranded 70-mer oligo with an m 6G lesion near either end, we confirmed that AGT preferentially (3.5 fold) repaired the 5'end m 6G lesion than the 3'-end lesion. However, this preference was not apparent in double-stranded oligos. A streptavidin/biotin-dT block was inserted between the two m 6G lesions in the 70-mer oligo to impede the action of AGT. Compared to the appropriate controls, a streptavidin/biotin-dT block proximal to the 5'-end m 6 G in a single-stranded oligo decreased the repair of that lesion by about 2.6 fold while the repair of the 3'-end m 6G was unaffected. A block distal to the 5'-end m 6G decreased the repair of the 5'-end m 6 G to a lesser extent (1.7 fold) than the proximal block, while the repair of the 3'-end m 6G was decreased by 1.6 fold. Similar results were obtained using double-stranded substrates. In summary, AGT preferentially repaired 5'-end m 6G lesions and a streptavidin/biotin-dT block affected the repair of the 5'-end m 6G more than the 3'-end lesion, supporting a model for AGT to move 3' to 5' along DNA. These findings provide new insights into how AGT repairs O 6-alkylguanine lesions in an oligo context for the purpose of rational drug design of novel AGT inhibitors.