Construction of an Escherichia coli vector containing the major DNA adduct of activated benzo(a)pyrene at a defined site
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-N/sup 2/-Gua) is formed when the C(10)-position of BPDE reacts with the N/sup 2/-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 than contains BP-N/sup 2/-Gua located in the unique PstI restriction endonuclease recognition site at nucleotide position 6249 in the (-)-strand is described (designated, BP-N/sup 2/-Gua-M13mp19). First, the oligonucleotide 5'-TGCA-3' was reacted with BPDE and a product (5'-T(BP-N/sup 2/)GCA-3') was isolated by HPLC than, when enzymatically digested to deoxynucleosides, yielded an adduct that comigrated on HPLC with an authentic BP-N/sup 2/-Gua deoxynucleoside standard. Second, the 5'-hydroxyl group of 5'-T(BP-N/sup 2/)GCA-3' was phosphorylated with ATP and T4 polynucleotide kinase, and the product (5'-pT(BP-N/sup 2/)GCA-3') was purified by HPLC. This product is stable when heated at 80/sup 0/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-N/sup 2/)GCA-3' was ligated into this gap with T4DNA ligase and ATP. The product of this reaction (BP-N/sup 2/-Gua-M13mp19) was shown to be insensitive to clevage by PstI, which suggests that a modification is located in the PstI recognition site. The most likely modification is the adduct of BP-N/sup 2/-Gua.
- Research Organization:
- Boston Univ., MA (USA)
- OSTI ID:
- 6655469
- Journal Information:
- Chem. Res. Toxicol.; (United States), Vol. 1:3
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
BENZOPYRENE
TOXICITY
DNA ADDUCTS
BIOSYNTHESIS
ATP
BACTERIOPHAGES
CARCINOGENESIS
ESCHERICHIA COLI
LIQUID COLUMN CHROMATOGRAPHY
MUTAGENESIS
PHOSPHOTRANSFERASES
RECOMBINANT DNA
ADDUCTS
AROMATICS
BACTERIA
CHROMATOGRAPHY
CONDENSED AROMATICS
DNA
ENZYMES
HYDROCARBONS
MICROORGANISMS
NUCLEIC ACIDS
NUCLEOTIDES
ORGANIC COMPOUNDS
PARASITES
PATHOGENESIS
PHOSPHORUS-GROUP TRANSFERASES
SEPARATION PROCESSES
SYNTHESIS
TRANSFERASES
VIRUSES
560300* - Chemicals Metabolism & Toxicology