Recognition of specific DNA sequences by mitomycin C for alkylation.

Synthetic oligodeoxyribonucleotides were reacted with mitomycin C (MC) under conditions which restricted MC to monofunctional alkylating activity. The yields of monofunctional alkylation of oligonucleotides with variable sequence were determined by enzymatic digestion of the reaction mixture to unreacted nucleosides and the product of alkylation, a MC-deoxyguanosine adduct (2), followed by quantitative analysis by HPLC. The relative yields of 2 reflected relative monoalkylation reactivities. They were compared in a series of oligonucleotides having the sequence 5'-NGN' in which the 5'-base was varied while the 3'-base was kept constant as T. Under Na2S2O4 activation conditions a striking enhancement of the yield was observed at the 5'-CG sequence: 36%, compared to 2% at 5'-AG and 4.1% at 5'-TG. The 5'-GG sequence also showed enhanced reactivity although to a lesser extent (14.7%). The enhancements were specific to the duplex state of the oligonucleotides. Using NADPH:cytochrome c reductase as the reducing agent gave similar results. MC activated by acidic pH also displayed 5'-CG alkylation specificity. 10-Decarbamoyl-MC activated by Na2S2O4 showed the same 5'-CG specificity as MC. Replacement of deoxyguanosine by deoxyinosine in the opposite strand at a 5'-CG site abolished the enhancement of alkylation. Such replacement at a 5'-GG site had a similar effect. It was found that the base 3' to the guanine had only a relatively modest modulating effect on the enhanced reactivity of the G at the 5'-CG sequence. This 3'-base effect appeared to be independent of the 5'-base of the 5'-NGN' triplet. The order of reactivity is 3'-(C greater than T greater than A).(ABSTRACT TRUNCATED AT 250 WORDS)