DETERMINATION OF THE DNA CROSS-LINKING SEQUENCE SPECIFICITY OF REDUCTIVELY ACTIVATED MITOMYCIN-C AT SINGLE-NUCLEOTIDE RESOLUTION - DEOXYGUANOSINE RESIDUES AT CPG ARE CROSS-LINKED PREFERENTIALLY

Several synthetic oligodeoxyribonucleotide duplexes containing one or more 5'-CG and/or 5'-GC sites and singly radiolabeled at the 5'- or 3'-termini were subjected to interstrand cross-linking by mitomycin C activated with sodium dithionite in the absence of oxygen. Isolation of the interstrand cross-linked products by polyacrylamide gel electrophoresis (PAGE) was followed by fragmentation with Fe(II) EDTA/hydrogen peroxide/ascorbic acid and analysis of the resulting fragment sizes (by PAGE). It was demonstrated that reductively activated mitomycin C cross-links deoxyguanosine residues at duplex 5'-CG sites in strong preference to duplex 5'-GC sites and numerous A/T-containing sites. The identity of neighboring residues had a significant impact on the relative reactivity toward cross-linking of 5'-CG sites. Constrained molecular mechanics calculations on a model of the monoadduct of reductively activated mitomycin C with N2 of deoxyguanosine in a pentanucleotide duplex indicate a strong preference for the conformation required for cross-linking at 5'-CG over that for 5'-GC. © 1990, American Chemical Society. All rights reserved.