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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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Mechanisms for generating UV-induced recombinogenic DSBs.At the top part of the figure, chromosomal DNA molecules are depicted as unreplicated double-stranded DNA molecules. Newly-synthesized DNA is depicted as gray dashed lines. UV-induced pyrimidine dimers are shown as triangles, and centromeres of replicated chromosomes are shown as ovals. A. Excision of a dimer results in a small gap and replication produces one broken and one unbroken sister chromatid. B. During replication of a DNA molecule with an unexcised dimer, a DSB occurs in one of the two sister chromatids. C. Excision of two closely-opposed dimers results in a short (<6 bp) unstable double-stranded region between the excision tracts. The resulting broken chromosome is replicated to form two broken sister chromatids. D. As in Figure 9C, two closely-opposed dimers are excised. One of the resulting short gaps is expanded by the 5′ to 3′ Exo1p nuclease (shown in green) to generate a broken chromosome. Replication of this chromosome results in two broken sister chromatids. E. The tract resulting form excision of a single dimer is expanded, leaving a large single-stranded DNA gap. An endonuclease cleaves this single-stranded region, resulting in two broken sister chromatids.
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pgen-1003894-g009: Mechanisms for generating UV-induced recombinogenic DSBs.At the top part of the figure, chromosomal DNA molecules are depicted as unreplicated double-stranded DNA molecules. Newly-synthesized DNA is depicted as gray dashed lines. UV-induced pyrimidine dimers are shown as triangles, and centromeres of replicated chromosomes are shown as ovals. A. Excision of a dimer results in a small gap and replication produces one broken and one unbroken sister chromatid. B. During replication of a DNA molecule with an unexcised dimer, a DSB occurs in one of the two sister chromatids. C. Excision of two closely-opposed dimers results in a short (<6 bp) unstable double-stranded region between the excision tracts. The resulting broken chromosome is replicated to form two broken sister chromatids. D. As in Figure 9C, two closely-opposed dimers are excised. One of the resulting short gaps is expanded by the 5′ to 3′ Exo1p nuclease (shown in green) to generate a broken chromosome. Replication of this chromosome results in two broken sister chromatids. E. The tract resulting form excision of a single dimer is expanded, leaving a large single-stranded DNA gap. An endonuclease cleaves this single-stranded region, resulting in two broken sister chromatids.

Mentions: A central issue is the nature of the recombinogenic DNA damage generated by UV. Based on the mechanism of NER and on the observation that unrepaired pyrimidine dimers block replication, there are two obvious potential sources of DSBs [40]. First, if a DNA molecule with an unrepaired gap resulting from NER is replicated before filling-in of the gap and ligation, the net result would be a pair of sister chromatids with a single DSB (Figure 9A). Alternatively, if a replication fork encounters an unrepaired UV-induced lesion, breakage of the fork could also result in a single broken chromatid (Figure 9B). Based on the observation that UV treatment of G1- or G2-synchronized cells was not recombinogenic unless cells were allowed to divide, Galli and Schiestl (1999) [20] suggested that cell division was required to convert DNA lesions to recombinogenic lesions, consistent with both of the possibilities described above; their assay detected only intrachromatid deletions. Kadyk and Hartwell (1993) [21] found that unrepaired UV lesions stimulate gene conversion events between homologs, but have little effect on mitotic crossovers. This conclusion may be affected by the use of the rad1 mutation to prevent dimer excision, since rad1 strains have reduced frequencies of crossovers in some assays [41].


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

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

Mechanisms for generating UV-induced recombinogenic DSBs.At the top part of the figure, chromosomal DNA molecules are depicted as unreplicated double-stranded DNA molecules. Newly-synthesized DNA is depicted as gray dashed lines. UV-induced pyrimidine dimers are shown as triangles, and centromeres of replicated chromosomes are shown as ovals. A. Excision of a dimer results in a small gap and replication produces one broken and one unbroken sister chromatid. B. During replication of a DNA molecule with an unexcised dimer, a DSB occurs in one of the two sister chromatids. C. Excision of two closely-opposed dimers results in a short (<6 bp) unstable double-stranded region between the excision tracts. The resulting broken chromosome is replicated to form two broken sister chromatids. D. As in Figure 9C, two closely-opposed dimers are excised. One of the resulting short gaps is expanded by the 5′ to 3′ Exo1p nuclease (shown in green) to generate a broken chromosome. Replication of this chromosome results in two broken sister chromatids. E. The tract resulting form excision of a single dimer is expanded, leaving a large single-stranded DNA gap. An endonuclease cleaves this single-stranded region, resulting in two broken sister chromatids.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003894-g009: Mechanisms for generating UV-induced recombinogenic DSBs.At the top part of the figure, chromosomal DNA molecules are depicted as unreplicated double-stranded DNA molecules. Newly-synthesized DNA is depicted as gray dashed lines. UV-induced pyrimidine dimers are shown as triangles, and centromeres of replicated chromosomes are shown as ovals. A. Excision of a dimer results in a small gap and replication produces one broken and one unbroken sister chromatid. B. During replication of a DNA molecule with an unexcised dimer, a DSB occurs in one of the two sister chromatids. C. Excision of two closely-opposed dimers results in a short (<6 bp) unstable double-stranded region between the excision tracts. The resulting broken chromosome is replicated to form two broken sister chromatids. D. As in Figure 9C, two closely-opposed dimers are excised. One of the resulting short gaps is expanded by the 5′ to 3′ Exo1p nuclease (shown in green) to generate a broken chromosome. Replication of this chromosome results in two broken sister chromatids. E. The tract resulting form excision of a single dimer is expanded, leaving a large single-stranded DNA gap. An endonuclease cleaves this single-stranded region, resulting in two broken sister chromatids.
Mentions: A central issue is the nature of the recombinogenic DNA damage generated by UV. Based on the mechanism of NER and on the observation that unrepaired pyrimidine dimers block replication, there are two obvious potential sources of DSBs [40]. First, if a DNA molecule with an unrepaired gap resulting from NER is replicated before filling-in of the gap and ligation, the net result would be a pair of sister chromatids with a single DSB (Figure 9A). Alternatively, if a replication fork encounters an unrepaired UV-induced lesion, breakage of the fork could also result in a single broken chromatid (Figure 9B). Based on the observation that UV treatment of G1- or G2-synchronized cells was not recombinogenic unless cells were allowed to divide, Galli and Schiestl (1999) [20] suggested that cell division was required to convert DNA lesions to recombinogenic lesions, consistent with both of the possibilities described above; their assay detected only intrachromatid deletions. Kadyk and Hartwell (1993) [21] found that unrepaired UV lesions stimulate gene conversion events between homologs, but have little effect on mitotic crossovers. This conclusion may be affected by the use of the rad1 mutation to prevent dimer excision, since rad1 strains have reduced frequencies of crossovers in some assays [41].

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