Benzo[a]pyrene diol epoxide I binds to DNA at replication forks

Abstract

The distribution of lesions in DNA caused by (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyren e (B[a]P diol epoxide-I) was studied in synchronized C3H/10T 1/2 cells treated in S phase. Sites of carcinogen modification of DNA were identified by polyclonal rabbit antibodies elicited against DNA modified with B[a]P diol epoxide-1 in vitro. This antigenic DNA contained trans-(7R)-N2-[10-(7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo[a] pyrene)-yl]-deoxyguanosine; other adducts were not detected by liquid chromatography. In this study, DNA replication forks with antibodies bound to B[a]P diol epoxide-I adducts were detected by electron microscopy. The frequency of replication forks containing carcinogen adducts associated with the fork junction was found to be 8-fold higher than expected for an average distribution. The proportion of replication forks that were apparently blocked at the site of the DNA damage increased when replication was allowed to occur after carcinogen exposure. These results support the conclusions that the fork junction is particularly vulnerable to adduction by B[a]P diol epoxide-I and that B[a]P diol epoxide-I adducts block the displacement of replication forks during DNA synthesis in intact cells.