Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We evaluated the formation and removal of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-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 of repair-deficient 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: Escherichia coli UvrABC excision nuclease cleavage and 32P-postlabeling. DNA repair was examined in the actively transcribed DHFR 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.
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PMID:DNA strand-specific repair of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene adducts in the hamster dihydrofolate reductase gene. 146 20

We previously showed that the preferred mutation induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy- 1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPHDE) in the dihydrofolate reductase gene in Chinese hamster ovary cells was a purine to thymine transversion on the nontranscribed strand at the sequence 5'-RRR-3' (R is a purine and the mutated base is underlined). To determine whether the observed mutational strand specificity was due to bias in the phenotypic selection, we designed a nonsense-codon reversion assay in which a triple purine target was present on both strands and all R----T transversion mutations yielded amino acid substitutions that were compatible with dihydrofolate reductase enzyme activity. From the size of the targets, a 2:1 ratio of mutations at the purines on the nontranscribed strand was expected if the DNA strands were mutationally equivalent. We isolated a total of 66 BcPHDE-induced revertants of two mutants that carry point mutations at either the 5' or the 3' end of the gene. All reversions at the 5' end arose by substitution on the nontranscribed strand; those at the 3' end showed a strand bias that favored this strand by 7:1. For both mutants, R----T transversions accounted for 88% of all the induced base changes. Thus, in this system, mutational strand bias is independent of the selection for phenotype. The results are consistent with the model of preferential repair of the transcribed strand as proposed by others. The involvement of RNA polymerase in the selective repair recruitment is discussed.
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PMID:DNA strand-specific mutations induced by (+/-)-3 alpha,4 beta-dihydroxy- 1 alpha,2 alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene in the dihydrofolate reductase gene. 206 55

Cultured Chinese hamster ovary (CHO) cells were treated with the polycyclic aromatic hydrocarbon racemic 3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene. Mutants deficient in dihydrofolate reductase activity were isolated. A carcinogen treatment at 0.1 microM yielded at 46% survival of the treated population and an induced frequency of mutation of 1.7 x 10(-4), 10(3)-fold greater than the spontaneous rate. By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 38 mutants. Base substitutions accounted for 78% (30/38) of the mutations. We obtained, in addition, four frameshift and four complex mutations. The preferred type of mutation was transversion (A.T----T.A and G.C----T.A) occurring in 69% of the analyzed mutants. A purine was on the 3' side of the putative adduct site in every mutant. Mutations were favored at sequences AGG, CAG, and AAG (the underlined base is the target). Surprisingly, 42% of the mutations created mRNA splicing defects (16/38), especially at splice acceptor sites for each of the five introns. Thus, this chemical carcinogen may recognize some aspect of DNA structure in regions corresponding to pre-mRNA splice sites.
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PMID:Splicing mutations in the CHO DHFR gene preferentially induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene. 237 Dec 81

Polycyclic aromatic hydrocarbons (PAHs) with sterically hindered fjord region diol epoxides are interesting with respect to their potency as carcinogens, interactions with DNA and mutagenic specificities. Unlike the bay region PAH derivative, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydroxybenzo[a]pyrene (BPDE), reactive metabolites of two fjord region PAH, trans-3,4-dihydroxy-anti-1, 2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene [(+/-)-anti-BcPHDE] and trans-11,12-dihydroxy-syn-BgCDE], react with DNA to yield high levels of adenine adducts. We previously found that forward mutations induced by (+/-)-anti-BcPHDE in the dihydrofolate reductase (dhfr) gene of Chinese hamster ovary (CHO) cells preferentially targeted mRNA splice acceptor sites. (+/-)-anti-BcPHDE and (+/-)-syn-BgCDE are structurally similar; they differ only by the presence of an additional benzene ring. Thus we used (+/-)-syn-BgCDE to learn if the mutational target bias reflects aspects of the mutagen structure or its capacity to efficiently modify deoxyadenosine (dA) in vivo. dhfr(-) mutants were induced after treatment of hemizygous UA21 cells with a 0.75 microM dose of (+/-)-syn-BgCDE. Cell survival after carcinogen exposure was 40%. The induced mutation frequency was 9 x 10(-6), nearly 10-fold higher than the spontaneous one, but approximately 19-fold lower than formerly observed using (+/-)-anti-BcPHDE. In the 26 confirmed null dhfr(-) mutants 27 mutations were identified by DNA sequencing. The types of (+/-)-syn-BgCDE-induced mutations were very similar to those formerly induced by (+/-)-anti-BcPHDE. Consistent with the binding specificity, both chemicals induced transversion base substitution at purines (R-->T). The most prevalent type of mutation was A-->T, which represented 59% of the induced changes, compared with 42% for (+/-)-anti-BcPHDE. (+/-)-syn-BgCDE mutated mostly novel targets in the dhfr gene, sites not found mutated with any of the several other mutagens we have used in former studies. Whereas the 25 kg dhfr gene contains six coding exons, the majority (16/27) of (+/-)-syn-BgCDE-induced mutations were located in a single one (exon 4). A random distribution of mutations affecting splice acceptor sites (22%) was induced by (+/-)-syn-BgCDE. Hence, preferential mutation of these sites by (+/-)-anti-BcPHDE may reflect aspects of chromatin structure in vivo which make these sequences better targets for modification. Alternatively, the sequence context of these sites may dictate an adduct conformation which is poorer for damage recognition and/or efficient repair.
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PMID:Mutagenic specificity of syn-benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide in the dihydrofolate reductase gene of Chinese hamster ovary cells. 758 12

A Chinese hamster cell line containing an extra exon 2 (50 bp) inserted into a single intron of a dihydrofolate reductase (dhfr) minigene was constructed. The extra exon 2 was efficiently spliced into the RNA, resulting in an mRNA that is incapable of coding for the DHFR enzyme. Mutations that decreased splicing of this extra exon 2 caused it to be skipped and so produced normal dhfr mRNA. In contrast to the parental cell line, the splicing mutants display a DHFR-positive growth phenotype. Splicing mutants were isolated from this cell line after treatment with four different mutagens (racemic benzo[c]phenanthrene diol epoxide, ethyl methanesulfonate, ethyl nitrosourea, and UV irradiation). By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 66 mutants. Each of the mutagens generated highly specific base changes. All mutations were single-base substitutions and comprised 24 different changes distributed over 16 positions. Most of the mutations were within the consensus sequences at the exon 2 splice donor, acceptor, and branch sites. The RNA splicing patterns in the mutants were analyzed by quantitative reverse transcription-polymerase chain reaction. The recruitment of cryptic sites was rarely seen; simple exon skipping was the predominant mutant phenotype. The wide variety of mutations that produced exon skipping suggests that this phenotype is the typical consequence of splice site damage and supports the exon definition model of splice site selection. A few mutations were located outside the consensus sequences, in the exon or between the branch point and the polypyrimidine tract, identifying additional positions that play a role in splice site definition. That most of these 66 mutations fell within consensus sequences in this near-saturation mutagenesis suggests that splicing signals beyond the consensus may consist of robust RNA structures.
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PMID:Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene. 841 32