CONSTRUCTION OF ESCHERICHIA-COLI VECTORS CONTAINING DEOXYADENOSINE AND DEOXYGUANOSINE ADDUCTS FROM (-ANTI-DIBENZ[A,J]ANTHRACENE DIOL EPOXIDE AT A DEFINED SITE())

Dibenz[a,j]anthracene (DB[a,j]A) is a carcinogenic Polycyclic aromatic hydrocarbon, which is metabolically activated through the formation o f bay region diol epoxides. Site-specifically modified M13mp19-based v ectors containing a single (+)-anti-dibenz[a,j]anthracene diol epoxide {(+)-anti-DB[a,j]A-DE}-deoxyguanosine (dGuo) or -deoxyadenosine (dAdo ) adduct were constructed. Four-base oligonucleotides, 5'-(HO)TGCA-3' and 5'-(HO)CATG-3', corresponding to the central four base pairs in th e PstI and SphI restriction endonuclease sites, respectively, in the m ultiple cloning region of M13mp19, were reacted in solution with (+/-) -anti-DB[a,j]A-DE. The resulting adducted oligonucleotides were separa ted and purified using reverse-phase HPLC. Several different singly ad ducted oligonucleotides were isolated, consisting of the various cis a nd trans addition products of the (+) and (-) enantiomers of the diol epoxide bound to dGuo or dAdo in the oligonucleotides. 5'-(HO)TGCA-3' containing the (+)-anti-DB[a,j]A-trans-N2-dGuo adduct [T(DB[a,j]A-N2)G CA] and 5'-(HO)CATG-3' containing the (+)-anti-DB[a,j]A-trans-N6-dAdo adduct [C(DB[a,j]A-N6)ATG) were selected for subsequent ligation into M13mp19 vectors that had been constructed with a corresponding four ba se gap in the minus strand. Both unmodified and adducted oligonucleoti des were successfully ligated into the M13mp19 vectors, [yields: unmod ified -TGCA-M13mp19 (approximately 32%) and -CATG- M13mp19 (approximat ely 42%); adducted T(DB[a,j]A-N2)GCA-M13mp19 (approximately 13%) and C (DB[a,j]A-N6)ATG-M13mp19 (approximately 12%)]. The dAdo adduct-contain ing vector was characterized. The presence of a dAdo-DNA adduct at the recognition site of SphI inhibited restriction by SphI. The dAdo addu ct-containing vector was cleaved with HindIII/XbaI to release the addu ct in a 24-mer, which migrated slower than the corresponding 24-mer re leased from the unmodified vector. The dGuo adduct-containing vector w as characterized similarly. While the results were more complicated, a pproximately 25% of the vectors contained the dGuo adduct. These site- specifically modified vectors will be used in future studies in vitro and in vivo to compare the effects of dAdo vs dGuo adducts on replicat ion, repair, and mutagenesis.