Detection of the DNA primary structure modifications induced by the base analog 6-n-hydroxylaminopurine in the alpha-test in yeast saccharomyces cerevisiae

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Abstract

Background. The alpha-test allows to detect inherited genetic changes of different types, as well as phenotypic expression of primary DNA lesions before the lesions are fixed by repair. Here we investigate ability of the alpha-test to detect base modifications induced by 6-N-hydroxylaminopurine (HAP) and determine frequency of inherited and non-inherited genetic changes in yeast strains treated with HAP.

Materials and methods. The alpha-test is based on mating type regulation and detects cell type switch from α to a in heterothallic yeast Saccharomyces cerevisiae. The frequency of mating type switching reflects level of both spontaneous and induced by a mutagen DNA instability. The alpha-test may be performed in two variants: “illegitimate” hybridization and cytoduction. Conducting both complementary tests and analysis of phenotypes of the “illegitimate” hybrids and cytoductants allows to detect the full spectrum of genetic events that lead to mating type switching, such as chromosome III loss and chromosome III arm loss, mutations and temporary lesions, recombination and conversion.

Results. HAP increases the frequency of illegitimate hybridization by 5-fold, and illegitimate cytoduction by 10-fold. A large proportion of the primary lesions induced by HAP causes temporary mating type switch and the remainder parts are converted into inherited point mutations.

Conclusion. The alpha-test can detect HAP-induced base modifications and may be used to investigate the ratio between correct and error-prone processing of such primary DNA lesions. Like other genetic toxicology tests the alpha-test has limitations, which are discussed.

About the authors

Anna S. Zhuk

ITMO University

Author for correspondence.
Email: ania.zhuk@gmail.com
ORCID iD: 0000-0001-8926-8238
SPIN-code: 2223-5306
Scopus Author ID: 54953157500
ResearcherId: N-5270-2015

PhD, Researcher, Laboratory of Genomic Diversity, International Laboratory of Computer Technologies

Russian Federation, Saint Petersburg

Elena I. Stepchenkova

ITMO University; Vavilov Institute of General Genetics Russian Academy of Science

Email: stepchenkova@gmail.com
ORCID iD: 0000-0002-5854-8701
SPIN-code: 9121-7483
Scopus Author ID: 8862552900
ResearcherId: F-9931-2014

PhD, Head of Laboratory of Mutagenesis and Genetic Toxicology; Assistant, Department of Genetics and Biotechnology

Russian Federation, Saint Petersburg

Sergey G. Inge-Vechtomov

ITMO University; Vavilov Institute of General Genetics Russian Academy of Science

Email: ingevechtomov@gmail.com
SPIN-code: 3743-7626
Scopus Author ID: 23473232500

Doctor of Science, Director; Professor, Department of Genetics and Biotechnology

Russian Federation, Saint Petersburg

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2. Figure: 1. Genetic events detected in the alpha test in the systems of "illegal" hybridization and cytoduction.

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3. Figure: 2. The incorporation of 6-N-hydroxylaminopurine (HAP) into DNA during replication and the mechanism of gene mutations (transitions): a - GC → AT substitutions; b - replace AT → GC

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4. Figure: 3. Distribution of classes of genetic events detected in the test for "illegal" hybridization, arising spontaneously and under the influence of HAP, in percentage ratio (a) and the frequency of spontaneous and induced HAP mutations and transient damages in the test for "illegal" hybridization (b). The graph shows the median for frequencies and its confidence interval. HR — loss of chromosome III; PPH - loss of the right shoulder of chromosome III; MEP - mutations and transient damage at the MATα locus; Rec - reciprocal recombination between the MATα locus and the HMRa cassette; Conv. - conversion of the HMRa cassette to the MATα locus. * The values ​​are statistically significantly different from the frequency of the same events occurring spontaneously, according to the Mann-Whitney test (p <0.0001). The underlining indicates a statistically significant change in the proportion of the corresponding class of genetic events after HAP treatment in comparison with the spontaneous level according to the Z-test.

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5. Figure: 4. Distribution of classes of genetic events detected in the test for "illegal" cytoduction, in percentage, arising spontaneously and under the influence of HAP (a) and the frequency of spontaneous and induced by HAPs inherited and non-inherited changes in genetic material in the test for "illegal" cytoduction (b ). VP - temporary damage at the MATα locus (simultaneously in MATα1 and MATα2, or in a two-sided promoter); Conv. - conversion of the HMRa cassette to the MATα locus; Mut a * - mutations simultaneously in MATα1 and MATα2, or in a two-sided promoter, deletion of MATα); Mut. n / m - mutations in MATα1 or MATα2; * - the values ​​are statistically significantly different from the frequency of the same events occurring spontaneously, according to the Mann-Whitney test (p <0.0001). The underlining indicates a statistically significant change in the proportion of the class of genetic events after HAP treatment in comparison with the corresponding proportion of spontaneous events according to the Z-test.

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Copyright (c) 2020 Zhuk A.S., Stepchenkova E.I., Inge-Vechtomov S.G.

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