Anomalous cross-linking by mechlorethamine of DNA duplexes containing C-C mismatch pairs

Nitrogen mustards such as mechlorethamine have previously been shown to cov alently crosslink DNA through the N7 position of the two guanine bases of a d[GXC].d[GYC] duplex sequence, a so-called 1,3 G-G-cross-link, when X-Y = C-G or T-A. Here, we report the formation of a new mechlorethamine cross-li nk with the d[GXC] d[GYC] fragment when X-Y is a C-C mismatch pair. Mechlor ethamine cross-links this fragment preferentially between the two mismatche d cytosine bases, rather than between the guanine bases. The cross-link als o forms when one or both of the guanine bases of the d[GCC].d[GCC] fragment are replaced by N7-deazaguanine, and, more generally, forms with any C-C m ismatch, regardless of the flanking base pairs. Piperidine cleavage of the cross-link species containing the d[GCC].d[GCC] sequence gives DNA fragment s consistent with alkylation at the mismatched cytosine bases. We also prov ide evidence that the cross-link reaction occurs between the N3 atoms of th e two cytosine bases by showing that the formation of the C-C cross-link is pH dependent for both mechlorethamine and chlorambucil. Dimethyl sulfate ( DMS) probing of the cross-linked d[GCC].d[GCC] fragment showed that the maj or groove of the guanine adjacent to the C-C mismatch is still accessible t o DMS. In contrast, the known minor groove binder Hoechst 33258 inhibits th e cross-link formation with a C-C mismatch pair flanked by A-T base pairs. These results suggest that the C-C mismatch is cross-linked by mechloretham ine in the minor groove. Since C-C pairs may be involved in unusual seconda ry structures formed by the trinucleotide repeat sequence d[CCG](n), and as sociated with triplet repeat expansion diseases, mechlorethamine may serve as a useful probe for these structures.