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Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast.

Stepchenkova EI, Kozmin SG, Alenin VV, Pavlov YI - BMC Genet. (2005)

Bottom Line: We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA.We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP.Three of them also protect from AHA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Sankt-Petersburg State University, Sankt-Petersburg, 199034, Russia. stepchenkova@yahoo.com <stepchenkova@yahoo.com>

ABSTRACT

Background: N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA), are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast.

Results: We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism.

Conclusion: We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA.

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Results of the screening of the yeast deletion library for elevated mutagenesis and sensitivity in micro-titer plates. Left panel – Mutagenesis on selective plates with canavanine. Right panel – The estimation of the number of colony-forming units on YPD medium.
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Figure 3: Results of the screening of the yeast deletion library for elevated mutagenesis and sensitivity in micro-titer plates. Left panel – Mutagenesis on selective plates with canavanine. Right panel – The estimation of the number of colony-forming units on YPD medium.

Mentions: The results are presented in Fig. 3. The left panel of Fig. 3 shows the induction of canavanine-resistant mutants by HAP and AHA; and the right panel represents the survival of the tested strains on YPD plates in the presence of base analogs. In the wild-type strain, as in the ham1 and aah1 mutants, 1–3 spontaneous canavanine-resistant colonies per spot arise in the absence of mutagen (Fig. 3A and 3B, left panel). In our experimental conditions, 1 μg/ml of HAP did not induce Canr mutants in the wild-type strain. A moderate induction of Canr clones (fewer than ten per spot) was observed at 10 μg/ml of HAP and a very strong HAP-induced mutagenesis was observed in the wild-type strain at 100 μg/ml of HAP (Fig. 3A, left panel). For comparison, 100 μg/ml of AHA were only moderately mutagenic (compare Fig. 3A and Fig. 3E, left panel). Furthermore, both HAP and AHA did not affect the viability of the wild-type strain, even at the maximal concentration of 100 μg/ml (Fig. 3A and 3E, right panel).


Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast.

Stepchenkova EI, Kozmin SG, Alenin VV, Pavlov YI - BMC Genet. (2005)

Results of the screening of the yeast deletion library for elevated mutagenesis and sensitivity in micro-titer plates. Left panel – Mutagenesis on selective plates with canavanine. Right panel – The estimation of the number of colony-forming units on YPD medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1173102&req=5

Figure 3: Results of the screening of the yeast deletion library for elevated mutagenesis and sensitivity in micro-titer plates. Left panel – Mutagenesis on selective plates with canavanine. Right panel – The estimation of the number of colony-forming units on YPD medium.
Mentions: The results are presented in Fig. 3. The left panel of Fig. 3 shows the induction of canavanine-resistant mutants by HAP and AHA; and the right panel represents the survival of the tested strains on YPD plates in the presence of base analogs. In the wild-type strain, as in the ham1 and aah1 mutants, 1–3 spontaneous canavanine-resistant colonies per spot arise in the absence of mutagen (Fig. 3A and 3B, left panel). In our experimental conditions, 1 μg/ml of HAP did not induce Canr mutants in the wild-type strain. A moderate induction of Canr clones (fewer than ten per spot) was observed at 10 μg/ml of HAP and a very strong HAP-induced mutagenesis was observed in the wild-type strain at 100 μg/ml of HAP (Fig. 3A, left panel). For comparison, 100 μg/ml of AHA were only moderately mutagenic (compare Fig. 3A and Fig. 3E, left panel). Furthermore, both HAP and AHA did not affect the viability of the wild-type strain, even at the maximal concentration of 100 μg/ml (Fig. 3A and 3E, right panel).

Bottom Line: We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA.We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP.Three of them also protect from AHA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Sankt-Petersburg State University, Sankt-Petersburg, 199034, Russia. stepchenkova@yahoo.com <stepchenkova@yahoo.com>

ABSTRACT

Background: N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA), are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast.

Results: We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism.

Conclusion: We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA.

Show MeSH
Related in: MedlinePlus