Information on the DNA-bonding and sequence specificity for mitomycin C (la) and select derivatives under limiting reducing conditions has been gleaned by using the X exonuclease stop assay and DNA restriction fragments. This procedure demonstrated that covalent modification of DNA by mitomycin C occurred preferentially at guanine residues within 5'CG and 5'GG sequences. The observed selectivity was shown to proceed at the monoalkylation stage and was independent of the second (cross-linking) drug-bonding event. The bonding properties of mitomycin C have been compared with anthramycin, an antineoplastic agent of comparable size. The results of this investigation are discussed in light of findings previously reported for the bonding specificity of mitomycin C to DNA. Potential explanations are offered for the observed guanine specificity as well as the preference for 5'CG and 5'GG sequences within the duplex DNA.

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The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity.

The structural requirements for mitomycin C-1 bonding have been investigated by comparing the bonding specificity of mitomycin C (1) with selectively modified porfiromycins (N-methylmitomycin C) at the C-10 position under reductive conditions and then comparing N-methyl-7-methoxyaziridinomitosene (10) with 7-methoxy-10-noraziridinomitosene (11) under nonreductive conditions. Enzymatic and chemical reductive activation of mitomycin C in the presence of the 129-bp fragment from pBR322 led to exclusive guanine (G*) modification with drug bonding that occurred preferentially at 5‘CG* sites, while C-10 chloro (8) and C-10 bromo (9) deoxycarbamoylporfiromycins modified DNA at guanines but with significantly diminished 5‘CG* sequence selectivity. A similar set of bonding profiles were observed with 10 and 11 upon incubation with DNA. Mitosene 10 selectively modified 5‘CG* sites in DNA, while 11 did not. These studies provided support for the hypothesis that a hydrogen bond between the C-10 oxygen in the activated mitomycin species and the guanine N(2)-amino proton on the nonbonding DNA strand in the precovalent complex permits selective modification of 5‘CG* sites in in vitro transformations. We have also found that the kinetics of UVRABC incision of N-methyl-7-methoxyaziridinomitosene (10)−DNA adducts at different sequences are identical. This finding leads us to conclude that drug modification-induced UVRABC incision at different sites represents the sequence selectivity of drug−DNA bonding.