||Benzo(a)pyrene Diol Epoxide I Binds to DNA at Replication Forks (Journal Version).
Paules, R. S. ;
Cordeiro-Stone, M. ;
Mass, M. J. ;
Poirier, M. C. ;
Yuspa, S. H. ;
||Health Effects Research Lab., Research Triangle Park, NC. ;North Carolina Univ. at Chapel Hill. ;National Cancer Inst., Bethesda, MD.
Electron microscopy ;
Benzopyrene diol epoxide ;
DNA replication ;
Neoplastic cell transformation ;
DNA damage ;
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The distribution in replication forks of DNA lesions caused by the treatment of S phase calls with benzo(a)pyrene-diol-epoxide-1 (BPDE-1) was studied in synchronized C3H10T1/2 cells. Sites of carcinogen modification of DNA were identified by polyclonal rabbit antibodies that were specific in their recognition of the deoxyguanosine adduct formed in DNA upon exposure to BPDE-1. The total DNA from S phase calls treated with 0.5 microgram/ml BPDE-1 for 10 min contained an average of 19 and 30 fmoles of BPDE-1 adduct for microgram of DNA, respectively. Under these conditions, overall rates of DNA synthesis measured by the uptake of (3H)thymidine decreased to 32% and 13% of control levels, 60 min after the carcinogen treatment. DNA replication fork structures with antibodies bound to BPDE-1 adducts were analyzed by EM. The highest carcinogen binding was found in close proximity to the fork junction. The proportion of replication forks that were apparently blocked at the site of the DNA damage was found to increase with the amount of replication allowed after carcinogen exposure. The results support the evidence that BPDE-1 adducts block the displacement of replication forks during DNA synthesis in intact cells.