DNA strand-specific repair of ( ± )-3 a , 4 , 8-dihydroxy-1 a , 2 a-epoxy-1 , 2 , 3 , 4-tetrahydrobenzo [ c ] phenanthrene adducts in the hamster dihydrofolate reductase gene

We evaluated the formation and removal of (+)-3a,48-dihydroxy-la,2a-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPHDE)-DNA adducts in two Chinese hamster ovary (CHO) cell lines. One line of repair-proficient cells (MK42) carries a stable 150-fold amplification of the dihydrofolate reductase (DHFR) locus. The other line ofrepairdeficient cells (UV-5) is diploid for this gene and is defective in excision of bulky DNA lesions. Two methods were used to quantitate adduct levels in treated cells: Eseherichia coUl UvrABC excision nuclease cleavage and 32P-postlabeling. DNA repair was examined in the actively transcribedDHFR gene, in an inactive region located 25 kilobases downstream, and in the overall genome. Between 8 and 24 hr after BcPHDE exposure, preferential repair of the DHFR gene compared to the noncoding region was apparent in MK42 cells. This gene-specific repair was associated with adduct removal from the DHFR transcribed strand. However, UV-5 cells showed no lesion reduction from this strand of the gene. By both quantitation methods, regions accessible to repair in MK42 cells showed a 2-fold reduction in DNA adduct levels by 24 hr. That the decline in adducts reflects genomic repair was demonstrated by the constant damage level remaining in UV-5 cells. Since BcPHDE-induced mutations in DHFR apparently arise from adducted purines on the nontranscribed strand, results from the present study support the idea that a consequence of strand-specific repair is strand-biased mutations. We have been studying the mechanism of strand-biased mutations induced by polycyclic aromatic hydrocarbons (PAHs) in a model mammalian gene. Greater than 90% of mutations induced by (+)-7/,38a-dihydroxy-9f3,10,3-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (+)-3a,4f3dihydroxy-la,2a-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPHDE) in the dihydrofolate reductase gene (DHFR) of Chinese hamster ovary (CHO) cells affected purine targets on the nontranscribed coding strand of DNA (1, 2). A similar strand bias for BPDE-induced mutations in the hypoxanthine phosphoribosyl transferase gene (HPRT) of human fibroblasts was described where 77% of the premutagenic guanine bases were also on this strand (3). Assuming that carcinogen modification ofDNA in vivo occurs with similar frequency on both strands, several possible explanations for the apparent strand selectivity of induced mutations can be considered and tested. One explanation for the strand bias of mutations is that the selection for dihydrofolate reductase-deficient mutants is very stringent, causing mutations to occur on one strand preferentially (4). We recently devised a reversion assay to address this possibility (5). BcPHDE-induced forward mutations were transversions favored at triple purine sequences where the 5' base was altered (2). The assay used DHFRnonsense mutants that carry this target sequence on both strands at each of two stop codons, and all purine thymine mutations yield amino acid substitutions compatible with dihydrofolate reductase activity. Randomly induced mutations at these sites would produce a 2:1 ratio of substitutions on the nontranscribed versus the transcribed strand. Sequence analysis of 66 independent BcPHDE-induced revertants showed that the combined ratio of mutations affecting purines on the nontranscribed strand was 15:1. Hence, the strand bias here was independent of phenotypic selection. Analysis of induced mutations in rodent HPRT led to the suggestion that strand-biased mutations relate to the polarity of the marker gene with respect to DNA replication (6). Since strand-biased UV-induced mutations were found in the absence ofDNA repair on the transcribed strand ofHPRT (7), the misinsertion rate differences for replicative polymerases a and 6 may also contribute to the strand-bias phenomenon. Indeed, the overall fidelity of polymerase a appears significantly poorer for base substitutions compared with other polymerases tested (8). A third explanation for strand-biased mutations is that DNA repair of carcinogen adducts occurs preferentially in the DHFR gene and specifically removes adducts from the transcribed strand. This model predicts that mutations will occur on the nontranscribed strand because adducts persist there. Repair of UV-induced damage was demonstrated to occur rapidly in the CHO DHFR gene but inefficiently (15% in 24 hr) in the genome overall (9). This intragenomic DNA repair heterogeneity is thought to reflect aspects ofchromatin structure, and repair in DHFR was shown to be maximally efficient in the hypomethylated 5' end of the gene (10, 11). Gene-specific repair of UV-induced pyrimidine dimers correlated with selective repair of the transcribed strand in active loci of eukaryotes and prokaryotes (11, 12). Facilitation of damage recognition on the transcribed strand is mediated by a transcription-repair coupling factor in Escherichia coli (13). The human coupling factor is presumably encoded by ERCC6, a gene mutated in Cockayne syndrome (14). In the work presented here, we quantitated the adduct levels inDNA from cells treated with BcPHDE and evaluated whether there was gene-specific and/or strand-specific repair ofthese lesions. In addition, we examined the overall genome repair of BcPHDE-induced DNA damage. For these experAbbreviations: BPDE, (+-)-7,8,8a-dihydroxy-9/,108-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene; BcPHDE, (-+-)-3a,4f8-dihydroxyla,2a-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene; PAH, polycyclic aromatic hydrocarbon. 11925 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 11926 Biochemistry: Carothers et al. iments, we used CHO cells that are repair-proficient (MK42), as well as cells that are defective in repair of bulky DNA adducts (UV-5) (15, 16). MK42 cells were derived from the hamster K-1 line and carry DHFR amplified 150 times. The level ofDHFR mRNA is proportional to the relative number of amplified gene copies (17). Since these cells are highly resistant to methotrexate (0.2 mM) and have a 200-fold increase in DHFR activity over wild-type parental cells (15), it is reasonable to assume that most of the gene copies are transcribed normally. In both strains, BcPHDE-DNA adducts formed to about maximal levels within 1 hr after treatment. These adducts in MK42 but not UV-5 cells were repaired on the transcribed strand of DHFR. Thus, the results are consistent with the model that the induced mutational strand bias is a consequence of transcription-coupled