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.

Carothers, Adelaide M.; Mucha, Jitka; Grünberger, Dezider

doi:10.1073/pnas.88.13.5749

Cited by 24 publications

(13 citation statements)

References 38 publications

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“…In several studies of mutation in cultured mammalian cells, it has been observed that the nontranscribed strand is much more prone to mutation by agents that produce bulky adducts or pyrimidine dimers (8,16,77,81). Our data are also consistent with this finding.…”

Section: Resultssupporting

confidence: 82%

“…Mutations at 5 of the 7 less-well-conserved positions were found; more often than not, the phenotype was severe, with little or no correctly spliced transcripts detected (NS79, 42,8,91, and 90 at -3, -2, -1, +3, and +4, respectively). No mutants were isolated that affected positions +5 or +6.…”

Section: Resultsmentioning

confidence: 99%

“…Its sequence, AGG/GUUUGU, has a modest fit to the consensus, 69.8 compared to 86.3 for the wild-type sequence (scoring system of Shapiro and Senapathy [65]), which may explain its low use. What is puzzling is why this cryptic site is activated by four donor site mutations (Table 1, NS7, 82, 60, and 90) but not by seven others (NS1, 79,42,92,8,5, and 91). This dichotomy extends to mutations at the same position; a G-to-A change at + 1 activates the cryptic splice, whereas a G-to-U change does not.…”

Section: Resultsmentioning

confidence: 99%

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Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

Chen¹,

Chasin²

1993

Mol. Cell. Biol.

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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[clphenanthrene 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 transcriptionpolymerase 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.The development of cell-free splicing systems has led to rapid and continuing progress in understanding biochemical mechanisms involved in intron removal and in the definition of the spliceosomal machinery. However, the exact requirements for recognition of splices sites and the rules governing splice site selection remain unclear. Analysis of consensus sequences provided the first definition of splice sites (49). Most efforts to move beyond that initial step have focused on site-directed mutagenesis studies (for reviews, see references 4 and 31). In these experiments, the effects of splice site mutations are typically assayed by using cell-free splicing extracts (in. vitro) or after transient transfection of cultured mammalian cells or injection of RNA directly into frog oocytes (in vivo).Conflicting results between these in vitro and in vivo assays for the effect of mutations on splicing have frequently been reported (2,38,39,45,83), raising the possibility that artifacts are being introduced in one system or the other. Moreover, recent experiments suggesting that the splicing machinery may be compartmentalized within t...

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

Chen¹,

Chasin²

1993

Mol. Cell. Biol.

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“…Whatever the origin of this unexpected bias, it could explain why the lack of CPD repair in the nontranscribed strand in XP129 cells does not greatly increase the frequency of 6-thioguanine resistance. In contrast to the results for repair-deficient mutants, strand-specific mutational analysis in repair-proficient hamster and human cells reveals a strong bias for mutations resulting from lesions in the nontranscribed strand (4,15,18,32). These results suggest that preferential repair of the transcribed strand exerts a substantial influence on UV-induced mutagenesis.…”

Section: Discussioncontrasting

confidence: 48%

Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene.

Lommel¹,

Hanawalt²

1993

Mol. Cell. Biol.

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A UV-resistant revertant (XP129) of a xeroderma pigmentosum group A cell line has been reported to be totally deficient in repair of cyclobutane pyrimidine dimers (CPDs) but proficient in repair of 6-4 photoproducts. This finding has been interpreted to mean that CPDs play no role in cell killing by UV. We have analyzed the fine structure of repair of CPDs in the dihydrofolate reductase gene in the revertant. In this essential, active gene, we observe that repair of the transcribed strand is as efficient as that in normal, repair-proficient human cells, but repair of the nontranscribed strand is not. Within 4 h after UV at 7.5 J/m2, over 50% of the CPDs were removed, and by 8 h, 80%o of the CPDs were removed. In contrast, there was essentially no removal from the nontranscribed strand even by 24 h. Our results demonstrate that overall repair measurements can be misleading, and they support the hypothesis that removal of CPDs from the transcribed strands of expressed genes is essential for UV resistance.

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“…This mutational preference re¯ects the propensity of BPhDE to react primarily with G and A in DNA, as well as the tendency of these adducts to mispair with adenine during replication (14). A strand bias in mutations was also observed for racemic anti-BPhDE, with the non-transcribed strand giving rise to most of the mutations (15).…”

Section: Introductionmentioning

confidence: 89%

Human RNA polymerase II is partially blocked by DNA adducts derived from tumorigenic benzo[c]phenanthrene diol epoxides: relating biological consequences to conformational preferences

Schinecker¹

2003

Nucleic Acids Research

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Environmental polycyclic aromatic hydrocarbons (PAHs) are metabolically activated to diol epoxides that can react with DNA, resulting in covalent modi®cations to the bases. The (+)-and (±)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene (anti-BPhDE) isomers are diol epoxide metabolites of the PAH benzo[c]phenanthrene (BPh). These enantiomers readily react with DNA at the N 6 position of adenine, forming bulky (+)-1R-or (±)-1S-trans-anti-[BPh]-N 6 -dA adducts. Transcription-coupled nucleotide excision repair clears such bulky adducts from cellular DNA, presumably in response to RNA polymerase transcription complexes that stall at the bulky lesions. Little is known about the effects of [BPh]-N 6 -dA lesions on RNA polymerase II, hence, the behavior of human RNA polymerase II was examined at these adducts. A site-speci®c, stereochemically pure [BPh]-N 6 -dA adduct was positioned on the transcribed or non-transcribed strand of a DNA template with a suitable promoter for RNA polymerase II located upstream from the lesion. Transcription reactions were then carried out with HeLa nuclear extract. Each [BPh]-dA isomer strongly impeded human RNA polymerase II progression when it was located on the transcribed strand; however, a small but signi®cant degree of lesion bypass occurred, and the extent of polymerase blockage and bypass was dependent on the stereochemistry of the adduct. Molecular modeling of the lesions supports the idea that each adduct can exist in two orientations within the polymerase active site, one that permits nucleotide incorporation and another that blocks the RNA polymerase nucleotide entry channel, thus preventing base incorporation and causing the polymerase to stall or arrest.

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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.

Cited by 24 publications

References 38 publications

Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene.

Human RNA polymerase II is partially blocked by DNA adducts derived from tumorigenic benzo[c]phenanthrene diol epoxides: relating biological consequences to conformational preferences

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