Mitomycin C-induced distortions of DNA at minor alkylation sites.

Reductively-activated mitomycin C (MC) presents a high specificity to the 5'-CG site and to a lesser extent the 5'-GG site. However, its affinity is different for each 5'-CG site. This was evidenced by using the 3'-5' exonuclease activity of T4 DNA polymerase on a short DNA fragment exposed to MC, which was gradually activated by several Na2S2O4 additions. The time-delayed appearance of some exonuclease digestion stop sites (corresponding to MC-monofunctional adducts) suggests that MC discriminates between very fine structural variations. The feature of the stop sites suggests a good fit of MC in the DNA groove, in the case of the major alkylation sites, but not in the case of a minor 5'-TG alkylation site. Furthermore, it is evidenced by the use of the chemical probe hydroxylamine (HA) that MC-monoalkylation of 5'-CG (or 5'-GG) does not induce notable local structural disturbance of the DNA double helix, as opposed to alkylation of the 5'-TG site of minor specificity, which leads to significant local DNA distortion. This suggests that the 'in vivo' effect of MC is related, not only to amount of alkylated sites (essentially 5'-CG sites), but also to possible local DNA deformations (at minor alkylation sites).