Selective recognition of the m5CpG dinucleotide sequence in DNA by mitomycin C for alkylation and cross-linking

The clinically used natural antitumor agent mitomycin C (MC) is known to alkylate DNA monofunctionally and bifunctionally, resulting in the cross-linking of DNA. These reactions occur selectively with guanines at the CpG sequence. We show, confirming a previous report (Millard, J. T.; Beachy, T. M. Biochemistry 1993, 32, 12850) that cross-linking in oligonucleotides is further enhanced when the cytosines in CpG.CpG are 5-methylated to m5CpG.m5CpG. It is shown, furthermore, that guanines in m5CpG are monoalkylated two- to three-times faster than in CpG indicating that the m5C-induced rate enhancement occurs at the first, monoalkylation step of the two-step cross-linking process. The same MC-DNA adducts are formed in methylated as in non-methylated DNA. The basepaired but not the 5'-flanking, m5C residue is responsible for the enhanced alkylation of guanine. Enzymatically activated or Na2S2O4-activated MC shows identical rate-enhancement of alkylation at m5CpG. pBR322 DNA methylated by CpG-methylase was cross-linked two- to three-times more efficiently by MC than non-methylated DNA, indicating that the m5C effect is not an artifact of oligonucleotides. An electronic effect of the 5-methyl group of cytosine transmitted via G.C H-bonding to N2 of guanine is suggested as responsible for increased reactivity with MC. CpG is severely depleted in mammalian DNA and it is speculated that this factor attenuates MC cytotoxicity in human cells.