Butadiene-induced intrastrand DNA cross-links: A possible role in deletionmutagenesis

To initiate studies designed to identify the mutagenic spectrum associated with butadiene diepoxide-induced N-2-N-2 guanine intrastrand cross-links, s ite specifically adducted oligodeoxynucleotides were synthesized in which t he adducted bases were centrally located within the context of the human ra s 12 codon, The two stereospecifically modified DNAs and the corresponding unmodified DNA were ligated into a single-stranded M13mp7L2 vector and tran sfected into Escherichia coil, Both stereoisomeric forms (R,R and S,S) of t he DNA cross-links resulted in very severely decreased plaque-forming abili ty, along with an increased mutagenic frequency for both single base substi tutions and deletions compared with unadducted DNAs, with the S,S stereoiso mer being the most mutagenic. Consistent with decreased plaque formation, i n vitro replication of DNA templates containing the cross-links by the thre e major E. coli polymerases revealed replication blockage by both stereoiso meric forms of the cross-links. The same DNAs that were used for replicatio n studies were also assembled into duplex DNAs and tested as substrates for the initiation of nucleotide excision repair by the E, coil UvrABC complex . UvrABC incised linear substrates containing these intrastrand cross-links with low efficiency, suggesting that these lesions may be inefficiently re paired by the nucleotide excision repair system.