DNA sequence analysis of the mutational specificity of u.v. light in the <i>SUP4</i>-o gene of yeast

Kunz, Bernard A.; Pierce, M K; Mis, Joseph R.A.; Giroux, Craig N.

doi:10.1093/mutage/2.6.445

Cited by 48 publications

(16 citation statements)

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“…Therefore, we conclude that in human cells G C-*A T transitions result from both unexcised Py-Py dimers and 6-4 photoproducts. This could explain why such transitions are the most common mutations induced by UV radiation in a variety of organisms (5,8,10,21,24).…”

Section: Discussionmentioning

confidence: 99%

Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

McGregor¹,

Chen²,

Лукаш³

et al. 1991

Mol. Cell. Biol.

140

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To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G, phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which [HPRT]) is significantly higher than in cells irradiated in early G1 phase (9). This difference in frequency cannot be attributed to differences in the physical state of the DNA, since no such difference in mutation frequency is found when diploid xeroderma pigmentosum cells (XP12BE, complementation group A), which are virtually devoid of nucleotide excision repair (18), are irradiated in early S phase or in early G1 phase (9). These data suggest that S-phase replication is centrally involved in the conversion of potentially mutagenic DNA damage into mutations (fixation) and that excision repair prior to the onset of S phase decreases the frequency of mutants by eliminating such lesions.Of the two major classes of potentially mutagenic UV photoproducts, i.e., cyclobutane pyrimidine dimers (Py-Py dimers) and 6-4 pyrimidine-pyrimidone (6-4 Py-Py) lesions, the latter have been shown to be removed from human genomic DNA more rapidly than the former (15). It has also been shown that Py-Py dimers are excised more rapidly from an actively transcribed gene of human cells, the dihydrofolate reductase gene (DHFR), than from bulk DNA (1, 12). Moreover, Mellon et al. (14) demonstrated that there is a strand bias in the rate of removal of Py-Py dimers from the * Corresponding author. DHFR gene in human cells. The majority of such lesions are removed within 24 h, but dimers in the transcribed strand are excised much faster than those in the nontranscribed strand. Information on strand-specific excision repair of UV-induced damage from the HPRT gene of human cells is not yet available, but if such repair occurs, HPRT mutations induced by UV in excision repair-proficient human cells irradiated at the onset of S phase should arise from 6-4 Py-Py lesions and Py-Py dimers located in either strand of DNA. In contrast, the lesions responsible for the mutations induced in such cells allowed 6 h for repair before the onset of S phase should arise mainly from Py-Py dimers, and these should be located primarily in the nontranscribed strand. This switch in strands should not occur in XP12BE cells.There are no published data on the kinds and locations (i.e., the spectrum) of UV-induced mutations in an endogenous gene of human cells, nor are there data showing whether excision repair in such cells alters the spectrum. This study was designed to examine the spectrum of mutations induced by UV in the HPRT gene of diploid human cells and to determine, at the DNA ...

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Section: Discussionmentioning

confidence: 99%

Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

McGregor¹,

Chen²,

Лукаш³

et al. 1991

Mol. Cell. Biol.

140

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“…The haploid, repair-proficient yeast strain MKP-op (MATa, canl-100, ade2-1, lys2-1, ura3-52, leu2-3,112, his3-A200, trpl-A901, YCpMP2) (25) and isogenic derivatives deleted either for APNI (apnl-Al:: HIS3) (DRY373-p) (12) (34), transformed into JF1754 by using calcium chloride (25), and isolated from JF1754 by an alkaline extraction procedure (35). SUP4-o alleles were sequenced on double-stranded YCpMP2 molecules (36). x2 tests with Yates' correction for continuity (37) were used to assess differences in several parameters.…”

Section: Methodsmentioning

confidence: 99%

Specificity of the mutator caused by deletion of the yeast structural gene (APN1) for the major apurinic endonuclease.

Kunz

Henson²,

Roche³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The loss of bases from cellular DNA occurs via both spontaneous and mutagen-induced reactions. The resulting apurinic/apyrimidinic (AP) sites are cytotoxic and mutagenic but are counteracted by repair initiated by AP endonucleases. Previously, in vitro and bacterial transfection studies suggested that AP sites often prompt insertion of dAMP residues during replication, the A-rule. Dissimilar results have been obtained by transfecting DNA into eukaryotic cells. It seemed possible that these differences might be due to idiosyncrasies of transfection or aberrant replication of the transfecting DNA. The observation that AP endonuclease-deficient strains of the yeast Saccharomyces cerevisiae have elevated spontaneous mutation rates allowed us to determine the mutational specificity of endogenously generated AP sites in nuclear DNA. With the yeast SUP4-o gene as a mutational target, we found that a deficiency in the major yeast AP endonuclease, Apnl, provoked mainly single base-pair substitution; the rate of transposon Ty insertion was also enhanced. The rate of transversion to a G-C pair was increased 10-fold in Apnl-deficient yeast, including a 59-fold increase in the rate of AFT -+ CG events. In contrast, the rate of transversion to an AFT pair was increased by only 3-fold. A deficiency in N3-methyladenine glycosylase offset these substitution rate increases, indicating that they are due primarily to AP sites resulting from glycosylase action. Thus, the A-rule does not seem to apply to the mutagenic processing of endogenous abasic sites in S. cereviswae. Other results presented here show that AP endonuclease-deficient Escherichia coi exhibit a mutator phenotype consistent with the A-rule.The loss of DNA bases to form apurinic/apyrimidinic (AP) sites occurs constantly. Such abasic lesions result from the inherent chemical instability of DNA, as well as the incessant assault of endogenous and environmental agents (1). AP sites are formed directly through the slow hydrolysis of the N-glycosyl bonds that anchor bases to DNA, but pyrimidines are lost at a much slower rate than purines (2). Spontaneous and mutagen-induced DNA damages include modified bases that have unstable glycosyl bonds or are substrates for DNA glycosylases, enzymes that remove the damaged bases to generate AP sites (3, 4). AP sites in DNA provide blocks to DNA synthesis and are substrates for mutagenesis in both prokaryotes and eukaryotes (3).The onslaught of AP sites is opposed primarily by the class of repair activities called AP endonucleases (5-8). The predominant forms of these enzymes hydrolyze the phosphodiester bond on the immediate 5' side of an AP site in duplex DNA; the nicked molecule is then further processed by nucleases that remove the dangling 5' AP site, followed by DNA repair synthesis and ligation (4). AP endonucleases and the genes that encode them have been isolated from bacteria, the yeast Saccharomyces cerevisiae, Drosophila melanogaster, and mammalian cells (5-11). In Escherichia coli, exonuclease III and endonuclease...

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“…Bacterial cells were transformed by a modification of the CaCl2 method (39). Mutant SUP4-o genes were sequenced by dideoxynucleotide-mediated chain termination on linearized double-stranded YCpMP2 molecules (23).…”

Section: Methodsmentioning

confidence: 99%

Specificity of the mutator effect caused by disruption of the RAD1 excision repair gene of Saccharomyces cerevisiae

et al. 1990

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The product of the RADI gene of the yeast Saccharomyces cerevisiae is believed to function at the incision step of excision repair of DNA damage (9). Thus, it is not surprising that defects in this gene increase sensitivity to a variety of DNA-damaging agents, including UV, the UV-mimetic chemical 4-nitroquinoline-1-oxide, mono-and bifunctional alkylating agents,.and photoactivated psoralens (6,8,9,40,41,49). As well, the RADI gene product is required for the repair of NM-methyladenine (13). Not only do RADI deficiencies sensitize cells to the lethal effects of certain genotoxic agents, but also they enhance UV and 4-nitroquinoline-1-oxide-induced mutagenesis (27, 40) and UV-induced mitotic interchromosomal recombination (45). Recent findings suggest that the RADI gene product also plays a role in mitotic intrachromosomal recombination and in integration of linear DNA molecules into homologous genomic sequences (2,17,44). In addition to these various properties, defects in RADl confer a mutator phenotype (35,43,47). Both enhanced locus reversion to prototrophy and forward mutation to suppression and canavanine resistance have been reported, but neither the precise mutational changes involved nor the specificity of the mutator effect has been elucidated.To account for the association between repair defects and enhanced spontaneous mutagenesis in yeast cells, von Borstel and colleagues (12, 42) have invoked the repair channeling hypothesis (5, 10). According to this hypothesis, impairment of a specific repair pathway results in channeling of damage normally repaired by that pathway along other, competitive pathways. In a similar fashion, shunting of spontaneous DNA lesions through mutagenic repair pathways might account for elevated spontaneous tnutation in repair-deficient strains. However, the nature of these errorprone pathways has remained obscure, and generally the magnitudes of the mutator effects in yeast cells are relatively small. Consequently, it has been suggested that repair defects might have a more indirect influence on spontaneous * Corresponding author. mutation, perhaps by somehow reducing the fidelity. of DNA replication (43) or the efficiency of correcting replication errors (26). Alternatively, the products of some repair genes might play a regulatory role (7,16) so that modulation of processes other than DNA repair could be responsible for the mutator phenotypes of certain repair-deficient mutants.Characterization of the locations and types of DNA sequence alteration occurring spontaneously within a single gene in a radl background would provide valuable infoi-mation about the specificity of the radl mutator effect. In turn, this could yield important clues about the mechanism(s) of enhanced spontaneous mutagenesis in excision repair-deficient strains. To examine mutational specificity, we previously developed a system for the DNA sequence analysis of forward mutations in the yeast tRNA suppressor gene . In this system, all types of base pair substitution as well as deletions, duplications, in...

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DNA sequence analysis of the mutational specificity of u.v. light in the SUP4-o gene of yeast

Cited by 48 publications

References 0 publications

Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

Specificity of the mutator caused by deletion of the yeast structural gene (APN1) for the major apurinic endonuclease.

Specificity of the mutator effect caused by disruption of the RAD1 excision repair gene of Saccharomyces cerevisiae

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