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

Pathways of DSB repair by homologous recombination. In this figure, we show accepted models of DSB repair by homologous recombination. DNA strands from two different homologs are shown in red and blue; light red and blue lines indicate newly synthesized DNA. Regions of the duplex that have strands of different colors represent heteroduplexes. These pathways are described in detail in the text. (A) Synthesis-dependent strand annealing (SDSA) pathway. Following processing of the DSB, the 3′ end of the left end of the broken DNA molecule invades the other duplex. Following DNA synthesis, the invading strand is displaced and hybridizes to the right end of the broken chromosome. This pathway results in conversion events unassociated with crossovers. (B) Double-strand break repair (DSBR) pathway. In this pathway, a double Holliday junction (dHJ) is formed. Resolution of these junctions by resolvase cleavage can result in two different crossover products (middle) and two different noncrossover products (bottom right). These products have two regions of heteroduplex located in trans. Alternatively, the dHJ can be dissolved by the action of topoisomerases/helicases, resulting in a noncrossover product with heteroduplexes located in cis. (C) Break-induced replication (BIR) pathway. One of the broken ends invades the homologous chromosome and duplicates sequences from the point of invasion to the telomere. The net result of BIR events is an apparent long terminal gene conversion event.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3316642&req=5

fig1: Pathways of DSB repair by homologous recombination. In this figure, we show accepted models of DSB repair by homologous recombination. DNA strands from two different homologs are shown in red and blue; light red and blue lines indicate newly synthesized DNA. Regions of the duplex that have strands of different colors represent heteroduplexes. These pathways are described in detail in the text. (A) Synthesis-dependent strand annealing (SDSA) pathway. Following processing of the DSB, the 3′ end of the left end of the broken DNA molecule invades the other duplex. Following DNA synthesis, the invading strand is displaced and hybridizes to the right end of the broken chromosome. This pathway results in conversion events unassociated with crossovers. (B) Double-strand break repair (DSBR) pathway. In this pathway, a double Holliday junction (dHJ) is formed. Resolution of these junctions by resolvase cleavage can result in two different crossover products (middle) and two different noncrossover products (bottom right). These products have two regions of heteroduplex located in trans. Alternatively, the dHJ can be dissolved by the action of topoisomerases/helicases, resulting in a noncrossover product with heteroduplexes located in cis. (C) Break-induced replication (BIR) pathway. One of the broken ends invades the homologous chromosome and duplicates sequences from the point of invasion to the telomere. The net result of BIR events is an apparent long terminal gene conversion event.

Mentions: DSBs can be repaired by several different HR pathways (Heyer et al. 2010). The repair of a DSB by gene conversion unassociated with a crossover is shown in Figure 1A. This process involves the nonreciprocal transfer of sequences from the intact donor molecule to the broken chromosome in several steps: (1) invasion of one broken end into the intact template molecule, followed by DNA synthesis primed by the invading 3′ strand; (2) removal of the invading end and reannealing of this end back to the other broken end, forming a heteroduplex with mismatches; and (3) repair of the mismatches. This mechanism [synthesis-dependent strand-annealing (SDSA)] was first suggested to explain some features of meiotic recombination in yeast (Allers and Lichten 2001). In the second pathway (Figure 1B), gene conversion may be associated with crossovers. In this pathway, a double Holliday junction (dHJ) is formed that can be resolved to yield a crossover or noncrossover. In this pathway, heteroduplexes flank the original position of the DSB. Although the heteroduplex regions have the same size in Figure 1B, in both meiosis (Merker et al. 2003; Jessop et al. 2005) and mitosis (Mitchel et al. 2010; Tang et al. 2011), the conversion tracts flanking the DSB are often of different lengths. The dHJ can also be dissolved without nucleolytic cleavage of DNA strands to yield noncrossover products with heteroduplexes located in cis on one of the two interacting chromosomes (Heyer et al. 2010). In the third pathway (Figure 1C), one part of the broken DNA molecule is lost and a complete chromosome is then reconstructed by break-induced replication (BIR). In this mechanism, one of the broken ends invades the intact template molecule, and a replication fork is set up that duplicates the template from the site of invasion to the telomere.


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)

Pathways of DSB repair by homologous recombination. In this figure, we show accepted models of DSB repair by homologous recombination. DNA strands from two different homologs are shown in red and blue; light red and blue lines indicate newly synthesized DNA. Regions of the duplex that have strands of different colors represent heteroduplexes. These pathways are described in detail in the text. (A) Synthesis-dependent strand annealing (SDSA) pathway. Following processing of the DSB, the 3′ end of the left end of the broken DNA molecule invades the other duplex. Following DNA synthesis, the invading strand is displaced and hybridizes to the right end of the broken chromosome. This pathway results in conversion events unassociated with crossovers. (B) Double-strand break repair (DSBR) pathway. In this pathway, a double Holliday junction (dHJ) is formed. Resolution of these junctions by resolvase cleavage can result in two different crossover products (middle) and two different noncrossover products (bottom right). These products have two regions of heteroduplex located in trans. Alternatively, the dHJ can be dissolved by the action of topoisomerases/helicases, resulting in a noncrossover product with heteroduplexes located in cis. (C) Break-induced replication (BIR) pathway. One of the broken ends invades the homologous chromosome and duplicates sequences from the point of invasion to the telomere. The net result of BIR events is an apparent long terminal gene conversion event.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Pathways of DSB repair by homologous recombination. In this figure, we show accepted models of DSB repair by homologous recombination. DNA strands from two different homologs are shown in red and blue; light red and blue lines indicate newly synthesized DNA. Regions of the duplex that have strands of different colors represent heteroduplexes. These pathways are described in detail in the text. (A) Synthesis-dependent strand annealing (SDSA) pathway. Following processing of the DSB, the 3′ end of the left end of the broken DNA molecule invades the other duplex. Following DNA synthesis, the invading strand is displaced and hybridizes to the right end of the broken chromosome. This pathway results in conversion events unassociated with crossovers. (B) Double-strand break repair (DSBR) pathway. In this pathway, a double Holliday junction (dHJ) is formed. Resolution of these junctions by resolvase cleavage can result in two different crossover products (middle) and two different noncrossover products (bottom right). These products have two regions of heteroduplex located in trans. Alternatively, the dHJ can be dissolved by the action of topoisomerases/helicases, resulting in a noncrossover product with heteroduplexes located in cis. (C) Break-induced replication (BIR) pathway. One of the broken ends invades the homologous chromosome and duplicates sequences from the point of invasion to the telomere. The net result of BIR events is an apparent long terminal gene conversion event.
Mentions: DSBs can be repaired by several different HR pathways (Heyer et al. 2010). The repair of a DSB by gene conversion unassociated with a crossover is shown in Figure 1A. This process involves the nonreciprocal transfer of sequences from the intact donor molecule to the broken chromosome in several steps: (1) invasion of one broken end into the intact template molecule, followed by DNA synthesis primed by the invading 3′ strand; (2) removal of the invading end and reannealing of this end back to the other broken end, forming a heteroduplex with mismatches; and (3) repair of the mismatches. This mechanism [synthesis-dependent strand-annealing (SDSA)] was first suggested to explain some features of meiotic recombination in yeast (Allers and Lichten 2001). In the second pathway (Figure 1B), gene conversion may be associated with crossovers. In this pathway, a double Holliday junction (dHJ) is formed that can be resolved to yield a crossover or noncrossover. In this pathway, heteroduplexes flank the original position of the DSB. Although the heteroduplex regions have the same size in Figure 1B, in both meiosis (Merker et al. 2003; Jessop et al. 2005) and mitosis (Mitchel et al. 2010; Tang et al. 2011), the conversion tracts flanking the DSB are often of different lengths. The dHJ can also be dissolved without nucleolytic cleavage of DNA strands to yield noncrossover products with heteroduplexes located in cis on one of the two interacting chromosomes (Heyer et al. 2010). In the third pathway (Figure 1C), one part of the broken DNA molecule is lost and a complete chromosome is then reconstructed by break-induced replication (BIR). In this mechanism, one of the broken ends invades the intact template molecule, and a replication fork is set up that duplicates the template from the site of invasion to the telomere.

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