Construction of an Escherichia coli vector containing the major DNA adduct of activated benzo[a]pyrene at a defined site

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Abstract
The mutagenic and carcinogenic substance benzo[.alpha.]pyrene reacts with DNA following activation to its corresponding 7,8-diol 9,10-epoxide (BPDE), and the major DNA adduct (BP-N2-Gua) is formed when the C(10)-position of BPDE reacts with the N2-position of guanine. It is unknown if this adduct is a premutagenic lesion in vivo. Herein, the construction and characterization of an M13mp19-based, E. coli vector that contains BP-N2-Gua located in the unique PstI restriction endonuclease cognition site at nucleotide position 6249 in the (.sbd.)-strand is described (designated, BP-N2-Gua-M13mp19). First, the oligonucleotide 5''-TGCA-3'' was reacted with BPDE and a product (5''-T(BP-N2)GCA-3'') was isolated by HPLC that, when enzymatically digested to deoxynucleosides, yielded an adduct that comigrated on HPLC with an authentic BP-N2-Gua deoxynucleoside standard. Second, the 5''-hydroxyl group of 5''-T-(BP-N2)GCA-3'' was phosphorylated with ATP and T4 polynucleotide kinase, and the product (5''-pT(BP-N2)GCA-3'') was purified by HPLC. This product is stable when heated at 80.degree. C at both neutral and alkaline pH. Third, M13mp19 was manipulated such that the sequence 5''-pTGCA-3'' was selectively removed from the (.sbd.)-strand in its unique PstI recognition site, and 5''-pT(BP-N2)GCA-3'' was ligated into this gap with T4 DNA ligase and ATP. The product of this reaction (BP-N2-Gua-M13mp19) was shown to be insensitive to cleavage by PstI, which suggests that a modification is located in the PstI recognition site. The mostly likely modification is the adduct BP-N2-Gua.

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