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Genome-wide high-resolution mapping of UV-induced mitotic recombination events in Saccharomyces cerevisiae.

Yin Y, Petes TD - PLoS Genet. (2013)

Bottom Line: Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH).UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells.Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology and University Program in Genetics and Genomics, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

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A system for detecting mitotic crossovers by a colony sectoring assay.G1-synchronized diploid cells were treated with UV and immediately plated on solid medium. The diploid is homozygous for the ade2-1 mutation, an ochre mutation that when unsuppressed results in a red colony. The diploid has one copy of the ochre suppressor gene SUP4-o inserted near the telomere of chromosome IV on the black homolog. Strains with zero, one, and two copies of SUP4-o form red, pink, and white colonies, respectively. A. Crossover in G2 of the first division following irradiation. A DSB in one chromatid repaired during G2 will generate a red/white sectored colony, the white sector derived from daughter cell 1 (D1) and the red sector derived from daughter cell 2 (D2). B. Crossover delayed to G2 of the second division. If DNA damage induced in G1 is not repaired during the first division, a pink/white/red sectored colony would be generated. The abbreviation “GD” indicates the granddaughter of the irradiated cell.
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pgen-1003894-g003: A system for detecting mitotic crossovers by a colony sectoring assay.G1-synchronized diploid cells were treated with UV and immediately plated on solid medium. The diploid is homozygous for the ade2-1 mutation, an ochre mutation that when unsuppressed results in a red colony. The diploid has one copy of the ochre suppressor gene SUP4-o inserted near the telomere of chromosome IV on the black homolog. Strains with zero, one, and two copies of SUP4-o form red, pink, and white colonies, respectively. A. Crossover in G2 of the first division following irradiation. A DSB in one chromatid repaired during G2 will generate a red/white sectored colony, the white sector derived from daughter cell 1 (D1) and the red sector derived from daughter cell 2 (D2). B. Crossover delayed to G2 of the second division. If DNA damage induced in G1 is not repaired during the first division, a pink/white/red sectored colony would be generated. The abbreviation “GD” indicates the granddaughter of the irradiated cell.

Mentions: In order to determine different types of mitotic recombination and to determine whether the conversion events are of the 3∶1 or 4∶0 configuration, we used a method of identifying recombination events that allows the recovery of both daughter cells with the recombinant chromosomes. The system used in the present study (Figure 3) is similar to that employed previously [2], [28]. Near the telomere of chromosome V, one homolog (shown in black in Figure 3A) has an insertion of SUP4-o, an ochre-suppressing tRNA gene. The diploid is also homozygous for the ade2-1 ochre mutation. Diploids homozygous for the ade2-1 mutation and zero, one or two copies of SUP4-o form colonies that are red, pink, and white, respectively [28].


Genome-wide high-resolution mapping of UV-induced mitotic recombination events in Saccharomyces cerevisiae.

Yin Y, Petes TD - PLoS Genet. (2013)

A system for detecting mitotic crossovers by a colony sectoring assay.G1-synchronized diploid cells were treated with UV and immediately plated on solid medium. The diploid is homozygous for the ade2-1 mutation, an ochre mutation that when unsuppressed results in a red colony. The diploid has one copy of the ochre suppressor gene SUP4-o inserted near the telomere of chromosome IV on the black homolog. Strains with zero, one, and two copies of SUP4-o form red, pink, and white colonies, respectively. A. Crossover in G2 of the first division following irradiation. A DSB in one chromatid repaired during G2 will generate a red/white sectored colony, the white sector derived from daughter cell 1 (D1) and the red sector derived from daughter cell 2 (D2). B. Crossover delayed to G2 of the second division. If DNA damage induced in G1 is not repaired during the first division, a pink/white/red sectored colony would be generated. The abbreviation “GD” indicates the granddaughter of the irradiated cell.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814309&req=5

pgen-1003894-g003: A system for detecting mitotic crossovers by a colony sectoring assay.G1-synchronized diploid cells were treated with UV and immediately plated on solid medium. The diploid is homozygous for the ade2-1 mutation, an ochre mutation that when unsuppressed results in a red colony. The diploid has one copy of the ochre suppressor gene SUP4-o inserted near the telomere of chromosome IV on the black homolog. Strains with zero, one, and two copies of SUP4-o form red, pink, and white colonies, respectively. A. Crossover in G2 of the first division following irradiation. A DSB in one chromatid repaired during G2 will generate a red/white sectored colony, the white sector derived from daughter cell 1 (D1) and the red sector derived from daughter cell 2 (D2). B. Crossover delayed to G2 of the second division. If DNA damage induced in G1 is not repaired during the first division, a pink/white/red sectored colony would be generated. The abbreviation “GD” indicates the granddaughter of the irradiated cell.
Mentions: In order to determine different types of mitotic recombination and to determine whether the conversion events are of the 3∶1 or 4∶0 configuration, we used a method of identifying recombination events that allows the recovery of both daughter cells with the recombinant chromosomes. The system used in the present study (Figure 3) is similar to that employed previously [2], [28]. Near the telomere of chromosome V, one homolog (shown in black in Figure 3A) has an insertion of SUP4-o, an ochre-suppressing tRNA gene. The diploid is also homozygous for the ade2-1 ochre mutation. Diploids homozygous for the ade2-1 mutation and zero, one or two copies of SUP4-o form colonies that are red, pink, and white, respectively [28].

Bottom Line: Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH).UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells.Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology and University Program in Genetics and Genomics, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

Show MeSH
Related in: MedlinePlus