TY - JOUR
T1 - In vivo and in vitro replication consequences of stereoisomeric benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide adducts on adenine N6 at the second position of N-ras codon 61
AU - Chary, Parvathi
AU - Latham, Gary J.
AU - Robberson, Donald L.
AU - Kim, Seong J.
AU - Han, Shin
AU - Harris, Constance M.
AU - Harris, Thomas M.
AU - Lloyd, R. Stephen
PY - 1995/3/10
Y1 - 1995/3/10
N2 - Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo[a]pyrene, is 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 nonadducted 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.
AB - Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo[a]pyrene, is 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 nonadducted 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.
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U2 - 10.1074/jbc.270.10.4990
DO - 10.1074/jbc.270.10.4990
M3 - Article
C2 - 7890605
AN - SCOPUS:0028959993
SN - 0021-9258
VL - 270
SP - 4990
EP - 5000
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
ER -