Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo[a]pyrene, is a mutagenic in both bacterial and mammalian systems. Toward understanding the mutagenic effects of different stereoisomers of BPDE at specific sites in DNA, six stereochemically defined BPDE adducts were constructed on adenine N6 at position 2 of the human N-ras 61 codon within an 11-base oligonucleotide fragment. Both the nonadducted and BPDE-adducted N-ras 61 11-mers were inserted into a unique EcoRI site in single-stranded M13mp7L2 DNA and utilized for in vivo studies. The ligation efficiencies of BPDE-adducted 11-mers into the single-stranded vector were determined by Southern hybridization and confirmed by electron microscopy. Repair-deficient AB2480 E. coli cells were transformed with adducted and non-adducted DNA samples. The resultant plaque-forming abilities were used to evaluate the replication competence of the various BPDE adducts with respect to the nonadducted 11-mer. Point mutations due to aberrant replication at the adducted site were identified by the technique of differential DNA hybridization. All of the six BPDE adducts examined were mutagenic in vivo, generating exclusively A-->G mutations at frequencies ranging from 0.26 to 1.20%. In vitro replication studies using these BPDE-adducted 11-mers involved primer extension assays with Klenow fragment. All of the BPDE-modified templates demonstrated distinct blockage at the adducted site and/or 1 base 3' to the adducted site, allowing essentially no translesion synthesis to form fully extended polymerization products in vitro.
Deoxyadenosine 3‘-phosphates bearing cis and
trans
N
6 adducts of
(7R) and (7S)-anti
7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrenes
have been prepared in good yield by
reaction of 6-fluoropurinyl 2‘-deoxyriboside
3‘-(bis(2-[4-nitrophenyl]ethyl )phosphate with the
(±)-(7β,8α,9α,10β)- and
(±)-(7β,8α,9α,10α)-10-amino-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8,9-triols.
The
protected phosphates are easily prepared as diasteromeric mixtures,
readily resolved by reversed
phase HPLC, and efficiently deprotected with DBU to give the adducted
3‘-phosphates. These
nucleotides are of value as standards for the
32P-postlabeling procedure of Randerath for
determination of benzo[a]pyrene adducts in DNA
(Reddy et al. Carcinogenesis
1984,
5, 231).
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