Alteration in the choice of DNA repair pathway with increasing sequence selective DNA alkylation in the minor groove

Many conventional DNA alkylating anticancer drugs form adducts in the major groove of DNA. These are known to be chiefly repaired by both nucleotide (NER) and base (BER) excision repair in eukaryotic cells. Much less is known about the repair pathways acting on sequence specific minor groove purine adducts, which result from a promising new class of anti-tumour agents.

Benzoic acid mustards (BAMs) tethering 1-3 pyrrole units (compounds 1, 2 and 3) show increasing DNA sequence selectivity for alkylation from BAM and 1, alkylating primarily at guanine-N7 in the major groove, to 3 which is selective for alkylation in the minor groove at purine-N3 in the sequence 5'-TTTTGPu (Pu=guanine or adenine). This increasing sequence selectivity is reflected in increased toxicity in human cells. In the yeast Saccharomyces cerevisiae, the repair of untargeted DNA adducts produced by BAM, 1 and 2 depends upon both the NER and BER pathways. In contrast, the repair of the sequence specific minor groove adducts of 3 does not involve known BER or NER activities. In addition, neither recombination nor mismatch repair are involved. Two disruptants from the RAD6 mutagenesis defective epistasis group (rad6 and rad18), however, showed increased sensitivity to 3. In particular, the rad18 mutant was over three orders of magnitude more sensitive to 3 compared to its isogenic parent, and 3 was highly mutagenic in the absence of RAD18. Elimination of the sequence specific DNA adducts formed by 3 was observed in the wild type strain, but these lesions persisted in the rad18 mutant.

We have demonstrated that the repair of DNA adducts produced by the highly sequence specific minor groove alkylating agent 3 involves an error free adduct elimination pathway dependent on the Rad18 protein. This represents the first systematic analysis of the cellular pathways which modulate sensitivity to this new class of DNA sequence specific drugs, and indicates that the enhanced cytotoxicity of certain sequence specific minor groove adducts in DNA is the result of evasion of the common excision repair pathways.