Limits...
High-resolution genome-wide analysis of irradiated (UV and γ-rays) diploid yeast cells reveals a high frequency of genomic loss of heterozygosity (LOH) events.

St Charles J, Hazkani-Covo E, Yin Y, Andersen SL, Dietrich FS, Greenwell PW, Malc E, Mieczkowski P, Petes TD - Genetics (2012)

Bottom Line: Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected.Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV.To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.

ABSTRACT
In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.

Show MeSH

Related in: MedlinePlus

Genetic system used to select mitotic crossovers and associated conversions on the left arm of chromosome V. The starting diploid strain PG311 has the ochre-suppressible can1-100 on one copy of chromosome V (shown in red) and the SUP4-o gene (encoding an ochre suppressor tRNA gene) at an allelic position on the other homolog (shown in black). The strain is homozygous for the ochre-suppressible ade2-1 allele. Strains with an unsuppressed ade2-1 mutation form red colonies. The starting diploid strain is canavanine-sensitive and forms pink colonies. (A) Reciprocal crossover without an associated gene conversion initiated by a single DSB in G2. This type of event produces a canavanine-resistant red/white sectored colony (Barbera and Petes 2006). The transition from heterozygous markers to LOH is identical in the two sectors. (B) Reciprocal crossover with an associated conversion event initiated by a single DSB in G2. If a DSB forms on one of the black chromatids, a conversion associated with the crossover may occur. This event will also result in a canavanine-resistant red/white sectored colony in which the transitions between heterozygous markers and LOH are different in the two sectors. The region of conversion is indicated by the blue rectangle. (C) Reciprocal crossover and conversion resulting from a DSB formed in G1. A black chromosome with a DSB is replicated to form two sister chromatids that are broken at the same place. One chromatid is repaired to yield a reciprocal crossover and an associated conversion; the second is repaired to yield a conversion without a crossover. The resulting red and black sectors will have a 4:0 conversion event, a region in which both sectors are homozygous for SNPs derived from the red chromatid (included within the blue rectangle).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3316642&req=5

fig2: Genetic system used to select mitotic crossovers and associated conversions on the left arm of chromosome V. The starting diploid strain PG311 has the ochre-suppressible can1-100 on one copy of chromosome V (shown in red) and the SUP4-o gene (encoding an ochre suppressor tRNA gene) at an allelic position on the other homolog (shown in black). The strain is homozygous for the ochre-suppressible ade2-1 allele. Strains with an unsuppressed ade2-1 mutation form red colonies. The starting diploid strain is canavanine-sensitive and forms pink colonies. (A) Reciprocal crossover without an associated gene conversion initiated by a single DSB in G2. This type of event produces a canavanine-resistant red/white sectored colony (Barbera and Petes 2006). The transition from heterozygous markers to LOH is identical in the two sectors. (B) Reciprocal crossover with an associated conversion event initiated by a single DSB in G2. If a DSB forms on one of the black chromatids, a conversion associated with the crossover may occur. This event will also result in a canavanine-resistant red/white sectored colony in which the transitions between heterozygous markers and LOH are different in the two sectors. The region of conversion is indicated by the blue rectangle. (C) Reciprocal crossover and conversion resulting from a DSB formed in G1. A black chromosome with a DSB is replicated to form two sister chromatids that are broken at the same place. One chromatid is repaired to yield a reciprocal crossover and an associated conversion; the second is repaired to yield a conversion without a crossover. The resulting red and black sectors will have a 4:0 conversion event, a region in which both sectors are homozygous for SNPs derived from the red chromatid (included within the blue rectangle).

Mentions: One problem with studying spontaneous mitotic recombination is that most analytic systems do not allow the selection of both daughter cells that contain the recombinant chromosomes. Several years ago, we developed a method of selecting reciprocal crossovers on chromosome V that surmounts this difficulty (Barbera and Petes 2006; Lee et al. 2009). One copy of chromosome V has the can1-100 allele (an ochre mutation) and, in the other copy, the CAN1 gene is replaced by SUP4-o, a gene encoding an ochre-suppressing transfer RNA (tRNA) (Figure 2A). In the absence of the suppressor, strains with the can1-100 allele are resistant to canavanine, but because of the suppressor, the diploid used in our experiments is canavanine-sensitive. In addition, the diploid is homozygous for the ade2-1 mutation (an ochre mutation). Strains with this mutation, in the absence of the SUP4-o gene, form red colonies, but form pink colonies if one copy of the SUP4-o gene is present. Thus, the diploid strain is canavanine-sensitive and forms pink colonies. If a crossover occurs between the centromere of chromosome V and the can1-100/SUP4-o markers (a distance of ∼120 kb), a canavanine-resistant red/white colony is formed (Figure 2A).


High-resolution genome-wide analysis of irradiated (UV and γ-rays) diploid yeast cells reveals a high frequency of genomic loss of heterozygosity (LOH) events.

St Charles J, Hazkani-Covo E, Yin Y, Andersen SL, Dietrich FS, Greenwell PW, Malc E, Mieczkowski P, Petes TD - Genetics (2012)

Genetic system used to select mitotic crossovers and associated conversions on the left arm of chromosome V. The starting diploid strain PG311 has the ochre-suppressible can1-100 on one copy of chromosome V (shown in red) and the SUP4-o gene (encoding an ochre suppressor tRNA gene) at an allelic position on the other homolog (shown in black). The strain is homozygous for the ochre-suppressible ade2-1 allele. Strains with an unsuppressed ade2-1 mutation form red colonies. The starting diploid strain is canavanine-sensitive and forms pink colonies. (A) Reciprocal crossover without an associated gene conversion initiated by a single DSB in G2. This type of event produces a canavanine-resistant red/white sectored colony (Barbera and Petes 2006). The transition from heterozygous markers to LOH is identical in the two sectors. (B) Reciprocal crossover with an associated conversion event initiated by a single DSB in G2. If a DSB forms on one of the black chromatids, a conversion associated with the crossover may occur. This event will also result in a canavanine-resistant red/white sectored colony in which the transitions between heterozygous markers and LOH are different in the two sectors. The region of conversion is indicated by the blue rectangle. (C) Reciprocal crossover and conversion resulting from a DSB formed in G1. A black chromosome with a DSB is replicated to form two sister chromatids that are broken at the same place. One chromatid is repaired to yield a reciprocal crossover and an associated conversion; the second is repaired to yield a conversion without a crossover. The resulting red and black sectors will have a 4:0 conversion event, a region in which both sectors are homozygous for SNPs derived from the red chromatid (included within the blue rectangle).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316642&req=5

fig2: Genetic system used to select mitotic crossovers and associated conversions on the left arm of chromosome V. The starting diploid strain PG311 has the ochre-suppressible can1-100 on one copy of chromosome V (shown in red) and the SUP4-o gene (encoding an ochre suppressor tRNA gene) at an allelic position on the other homolog (shown in black). The strain is homozygous for the ochre-suppressible ade2-1 allele. Strains with an unsuppressed ade2-1 mutation form red colonies. The starting diploid strain is canavanine-sensitive and forms pink colonies. (A) Reciprocal crossover without an associated gene conversion initiated by a single DSB in G2. This type of event produces a canavanine-resistant red/white sectored colony (Barbera and Petes 2006). The transition from heterozygous markers to LOH is identical in the two sectors. (B) Reciprocal crossover with an associated conversion event initiated by a single DSB in G2. If a DSB forms on one of the black chromatids, a conversion associated with the crossover may occur. This event will also result in a canavanine-resistant red/white sectored colony in which the transitions between heterozygous markers and LOH are different in the two sectors. The region of conversion is indicated by the blue rectangle. (C) Reciprocal crossover and conversion resulting from a DSB formed in G1. A black chromosome with a DSB is replicated to form two sister chromatids that are broken at the same place. One chromatid is repaired to yield a reciprocal crossover and an associated conversion; the second is repaired to yield a conversion without a crossover. The resulting red and black sectors will have a 4:0 conversion event, a region in which both sectors are homozygous for SNPs derived from the red chromatid (included within the blue rectangle).
Mentions: One problem with studying spontaneous mitotic recombination is that most analytic systems do not allow the selection of both daughter cells that contain the recombinant chromosomes. Several years ago, we developed a method of selecting reciprocal crossovers on chromosome V that surmounts this difficulty (Barbera and Petes 2006; Lee et al. 2009). One copy of chromosome V has the can1-100 allele (an ochre mutation) and, in the other copy, the CAN1 gene is replaced by SUP4-o, a gene encoding an ochre-suppressing transfer RNA (tRNA) (Figure 2A). In the absence of the suppressor, strains with the can1-100 allele are resistant to canavanine, but because of the suppressor, the diploid used in our experiments is canavanine-sensitive. In addition, the diploid is homozygous for the ade2-1 mutation (an ochre mutation). Strains with this mutation, in the absence of the SUP4-o gene, form red colonies, but form pink colonies if one copy of the SUP4-o gene is present. Thus, the diploid strain is canavanine-sensitive and forms pink colonies. If a crossover occurs between the centromere of chromosome V and the can1-100/SUP4-o markers (a distance of ∼120 kb), a canavanine-resistant red/white colony is formed (Figure 2A).

Bottom Line: Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected.Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV.To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.

ABSTRACT
In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.

Show MeSH
Related in: MedlinePlus