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

• Published in: Chemical research in toxicology Vol. 1; no. 3; pp. 160 - 168
• Main Authors: Benasutti, Matt, Ezzedine, Z. Diala, Loechler, Edward L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.05.1988
• Subjects: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Base Sequence; Benzo(a)pyrene - metabolism; Biotransformation; DNA; DNA Adducts; Escherichia coli - genetics; Genetic Vectors; Genome, Bacterial; Oligodeoxyribonucleotides - chemical synthesis
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx00003a006

The mutagenic and carcinogenic substance benzo[a]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 recognition site at nucleotide position 6249 in the (-)-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 degrees C at both neutral and alkaline pH. Third, M13mp19 was manipulated such that the sequence 5'-pTGCA-3' was selectively removed from the (-)-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 most likely modification is the adduct BP-N2-Gua.