SYNTHESIS AND STRUCTURAL CHARACTERIZATION OF THE N(2)G-MITOMYCIN C-N(2)G INTERSTRAND CROSS-LINK IN A MODEL SYNTHETIC 23 BASE-PAIR OLIGONUCLEOTIDE DNA DUPLEX

Mitomycin C (MMC) is a genotoxic cancer chemotherapeutic agent that re acts principally at the N-2 position of guanine to form one of two pre dominant monoadducts, or a G-G interstrand cross-link at CpG sites, or a G-G intrastrand cross-link at GpG sites. Previous studies of MMC ad duction have principally used very short duplex oligonucleotides (5-15 bp) or very long native duplex DNAs. We examined the formation and st ructural features of the MMC CpG interstrand cross-link on a model 23 bp synthetic oligonucleotide duplex having the (upper strand) sequence (5')-ATAAATACGTATTTATTTATAAA-(3'). MMC was reacted with the duplex ol igonucleotide in the presence of sodium dithionite at ratios of 5 mM d ithionite:1.5 mM MMC:0.03 mM duplex. The yield of cross-link in the re action was determined to be approximately 4.8% by denaturing gel. elec trophoresis, which represented approximately 75% ofthe total bound MMC . The crosslinked DNA was isolated to greater than 97% purity in a sin gle step by high temperature size exclusion column chromatography. Cha racterization of the purified product confirmed that the complex conta ined exclusively the N(2)G-MMC-N(2)G cross-link at the single central CpG site. CD spectroscopy demonstrated a negative band at approximatel y 290-320 nm which has previously been shown to be characteristic ofth e MMC cross-link. The relative intensity of this band compared to thos e reported for shorter duplexes suggested that the majority of the dup lex is in a normal B-DNA helical configuration. Base-specific chemical footprinting techniques also indicated that there were subtle but dis tinct structural perturbations principally within the central four to six base pairs containing and adjacent to the cross-link.