Chapter 1 Measuring Spontaneous Mutation Rates in Yeast

Borstel, R. C. von

doi:10.1016/s0091-679x(08)62005-1

Cited by 77 publications

(51 citation statements)

References 19 publications

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“…Mutation rates were determined by using the method of the median (26). Briefly, 20 5-ml cultures were first grown to saturation, as described above, in CM-leu plus galactose.…”

Section: Methodsmentioning

confidence: 99%

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Mutagenic specificity of endogenously generated abasic sites in Saccharomyces cerevisiae chromosomal DNA

Auerbach

Bennett

Bailey

et al. 2005

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

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Abasic [apurinic/apyrimidinic (AP)] sites are common, noncoding DNA lesions. Despite extensive investigation, the mutational pattern they provoke in eukaryotic cells remains unresolved. We constructed Saccharomyces cerevisiae strains in which chromosomal AP sites were generated during normal cell growth by altered human uracil-DNA glycosylases that remove undamaged cytosines or thymines. The mutation target was the URA3 gene inserted near the ARS309 origin to allow defined replication polarity. Expression of the altered glycosylases caused a 7- to 18-fold mutator effect in AP endonuclease-deficient (Δ apn1 ) yeast, which depended highly on the known translesion synthesis enzymes Rev1 and DNA polymerase ζ. For the C-glycosylase, GC>CG transversions were the predominant mutations, followed by GC>AT transitions. AT>CG transversions predominated for the T-glycosylase. These results support a major role for Rev1-dependent dCMP insertion across from AP sites and a lesser role for dAMP insertion. Unexpectedly, there was also a significant proportion of dTMP insertions that suggest another mutational pathway at AP sites. Although replication polarity did not strongly influence mutagenesis at AP sites, for certain mutation types, there was a surprisingly strong difference between the transcribed and non-transcribed strands of URA3 . The basis for this strand discrimination requires further exploration.

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“…Mutation rates were determined by using the method of the median (26). Briefly, 20 5-ml cultures were first grown to saturation, as described above, in CM-leu plus galactose.…”

Section: Methodsmentioning

confidence: 99%

“…The mean number of colonies on the YPD plates was divided into the corresponding total number of 5-FOA R colonies to give a mutation frequency for each culture. The median value of the 20 mutation frequencies was then used to estimate the mutation rate from a statistical table (26).…”

Section: Methodsmentioning

confidence: 99%

Mutagenic specificity of endogenously generated abasic sites in Saccharomyces cerevisiae chromosomal DNA

Auerbach

Bennett

Bailey

et al. 2005

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

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“…This assay is a measure of spontaneous mutation rates in cells during the log phase of growth and has the advantage of avoiding the "jackpot effect." Mutation rates were performed using a modification of the Von Borstel 1,000-well assay (29). Cultures were grown overnight in 5 ml of liquid YPAD, washed in water, and diluted to 2.8 ϫ 10 3 cells/ml in 40 ml of SCϪArg liquid medium.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Respiratory Electron Carriers Are Involved in Oxidative Stress during Heat Stress in Saccharomyces cerevisiae

Davidson

Schiestl

2001

Molecular and Cellular Biology

206

120

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In the present study we sought to determine the source of heat-induced oxidative stress. We investigated the involvement of mitochondrial respiratory electron transport in post-diauxic-phase cells under conditions of lethal heat shock. Petite cells were thermosensitive, had increased nuclear mutation frequencies, and experienced elevated levels of oxidation of an intracellular probe following exposure to a temperature of 50°C. Cells with a deletion in COQ7 leading to a deficiency in coenzyme Q had a much more severe thermosensitivity phenotype for these oxidative endpoints following heat stress compared to that of petite cells. In contrast, deletion of the external NADH dehydrogenases NDE1 and NDE2, which feed electrons from NADH into the electron transport chain, abrogated the levels of heat-induced intracellular fluorescence and nuclear mutation frequency. Mitochondria isolated from COQ7-deficient cells secreted more than 30 times as much H 2 O 2 at 42 as at 30°C, while mitochondria isolated from cells simultaneously deficient in NDE1 and NDE2 secreted no H 2 O 2 . We conclude that heat stress causes nuclear mutations via oxidative stress originating from the respiratory electron transport chains of mitochondria.

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“…Spontaneous Trp ϩ reversion rates of DBY747 derivatives were measured by a modified Luria and Delbruck fluctuation test as described (34). An overnight yeast culture was used to inoculate five tubes, each containing 10 ml of fresh YPD, to a final titer of 20 cells per ml.…”

Section: Methodsmentioning

confidence: 99%

MMS2 , encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway

Broomfield

Chow

Xiao

1998

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

249

222

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Among the three Saccharomyces cerevisiae DNA repair epistasis groups, the RAD6 group is the most complicated and least characterized, primarily because it consists of two separate repair pathways: an error-free postreplication repair pathway, and a mutagenesis pathway. The rad6 and rad18 mutants are defective in both pathways, and the rev3 mutant affects only the mutagenesis pathway, but a yeast gene that is involved only in error-free postreplication repair has not been reported. We cloned the MMS2 gene from Genetic analyses indicate that the mms2 mutation is hypostatic to rad6 and rad18 but is synergistic with the rev3 mutation, and the mms2 mutant is proficient in UV-induced mutagenesis. These phenotypes are reminiscent of a pol30-46 mutant known to be impaired in postreplication repair. The mms2 mutant also displayed a REV3-dependent mutator phenotype, strongly suggesting that the MMS2 gene functions in the error-free postreplication repair pathway, parallel to the REV3 mutagenesis pathway. Furthermore, with respect to UV sensitivity, mms2 was found to be hypostatic to the rad6 ⌬1-9 mutation, which results in the absence of the first nine amino acids of Rad6. On the basis of these collective results, we propose that the mms2 null mutation and two other allele-specific mutations, rad6 ⌬1-9 and pol30-46, define the error-free mode of DNA postreplication repair, and that these mutations may enhance both spontaneous and DNA damage-induced mutagenesis.

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Chapter 1 Measuring Spontaneous Mutation Rates in Yeast

Cited by 77 publications

References 19 publications

Mutagenic specificity of endogenously generated abasic sites in Saccharomyces cerevisiae chromosomal DNA

Mutagenic specificity of endogenously generated abasic sites in Saccharomyces cerevisiae chromosomal DNA

Mitochondrial Respiratory Electron Carriers Are Involved in Oxidative Stress during Heat Stress in Saccharomyces cerevisiae

MMS2 , encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway

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