TY - JOUR
T1 - In vitro replication by prokaryotic and eukaryotic polymerases on DNA templates containing site-specific and stereospecific benzo [a]pyrene-7, 8-dihydrodiol-9, 10-epoxide adducts
AU - Chary, Parvathi
AU - Lioyd, Stephen
N1 - Funding Information:
We are grateful to Drs T. M. Harris and C. M. Harris for synthesis of the BPDE-adducted oligodeoxynucleotides, to Dr S. H. Wilson for his generous gift of rat polymerase fj, to Drs B. M. Alberts and R. D. Schrock for providing T4 DNA polymerase holoenzyme, and Drs M. F. Goodman and L. Bloom for their gift of exo~ KF. We are especially thankful to G. J. Latham and A. G. McNees for helpful comments during the course of this study. We are thankful to the personnel in the Recombinant DNA Laboratory, University of Texas Medical Branch, for providing the non-adducted templates, and to J. A. Daniels for the typing of this manuscript. This work was supported by United States Public Health Service grants ES05355 and ACS FRA 381.
PY - 1995/4/25
Y1 - 1995/4/25
N2 - DNA adducts of the environmental carcinogen benzo[a]pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE) interact stereospecifically with prokaryotic and eukaryotic polymerases in vitro. Toward understanding the capacity to replicate past different diastereomers of BPDE at specific sites in DNA, six deoxyoligonucleotides, each 33 baseslong, were constructed with stereochemically defined BPDE adducts on adenine N6 at position two of the human N-ras codon 61. Four polymerases that were studied under single en counters with the template-primer complex terminated synthesis one base 3' to the lesion with all the adducted templates. When multiple encounters between polymerase and substrate were permitted, each of the polymerases analyzed revealed a unique pattern for a given adducted template. The general replication pattern was encompassed under two categories, reflecting the significance of the A and S configurations of C10 of the pyrenyl ring attached to the single-stranded DNA template. Furthermore, within each of these categories, every polymerase demon strated distinct quantitative differences in product accumulation at a given site, for the various adducted templates. Among the polymerases utilized in this study, exonuclease-deficient Klenow fragment of polymerase I (exo-KF) exhibited the most efficient translesion synthesis resulting in ∼1 6% full-length products with the modified templates bearing adducts with C10-S configuration. In contrast, chain elongatlon with bacteriophage T4 DNA polymerase bearing an active 3'→5' exonucleolytic activity was most strongly inhibited by all six BPDE-adducted templates. Misincorporation of A opposite the adduct occurred in all the templates when polymerized with Sequenase, whereas exo- KF preferentially incorporated C opposite the C10-R BPDE adducts and A opposite the C10- BPDE adducts.
AB - DNA adducts of the environmental carcinogen benzo[a]pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE) interact stereospecifically with prokaryotic and eukaryotic polymerases in vitro. Toward understanding the capacity to replicate past different diastereomers of BPDE at specific sites in DNA, six deoxyoligonucleotides, each 33 baseslong, were constructed with stereochemically defined BPDE adducts on adenine N6 at position two of the human N-ras codon 61. Four polymerases that were studied under single en counters with the template-primer complex terminated synthesis one base 3' to the lesion with all the adducted templates. When multiple encounters between polymerase and substrate were permitted, each of the polymerases analyzed revealed a unique pattern for a given adducted template. The general replication pattern was encompassed under two categories, reflecting the significance of the A and S configurations of C10 of the pyrenyl ring attached to the single-stranded DNA template. Furthermore, within each of these categories, every polymerase demon strated distinct quantitative differences in product accumulation at a given site, for the various adducted templates. Among the polymerases utilized in this study, exonuclease-deficient Klenow fragment of polymerase I (exo-KF) exhibited the most efficient translesion synthesis resulting in ∼1 6% full-length products with the modified templates bearing adducts with C10-S configuration. In contrast, chain elongatlon with bacteriophage T4 DNA polymerase bearing an active 3'→5' exonucleolytic activity was most strongly inhibited by all six BPDE-adducted templates. Misincorporation of A opposite the adduct occurred in all the templates when polymerized with Sequenase, whereas exo- KF preferentially incorporated C opposite the C10-R BPDE adducts and A opposite the C10- BPDE adducts.
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U2 - 10.1093/nar/23.8.1398
DO - 10.1093/nar/23.8.1398
M3 - Article
C2 - 7753632
AN - SCOPUS:0029014360
SN - 0305-1048
VL - 23
SP - 1398
EP - 1405
JO - Nucleic acids research
JF - Nucleic acids research
IS - 8
ER -