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| Status |
Public on Dec 31, 2007 |
| Title |
Meiotic DNA double strand breaks in the yeast Saccaromyces cerevisiae |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Genome binding/occupancy profiling by genome tiling array
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| Summary |
DNA double-strand breaks (DSBs) initiate meiotic recombination. Past DSB-mapping studies have used rad50S or sae2? mutants, which are defective in break processing, to accumulate DSBs, and report large (= 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2? mutants. We therefore developed novel methods that detect DSBs using ssDNA enrichment and microarray hybridization, and that use background-based normalization to allow cross-comparison between array datasets, to map genome-wide the DSBs that accumulate in processing-capable, repair-defective dmc1î and dmc1î rad51î mutants. DSBs were observed at known hotspots, but also in most previously-identified “DSB-cold” regions, including near centromeres and telomeres. While about 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1? shows that most of these regions have significant DSB activity. Thus, DSBs are distributed much more uniformly than was previously believed. Southern-blot assays of DSBs in selected regions in dmc1?, rad50S and wild-type cells confirm these findings. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as the primary strand transfer activity genome-wide, and Spo11-induced lesions as initiating all meiotic recombination.
Keywords: DSB mapping, ChIP-chip, single strand DNA , BND cellulose
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| Overall design |
We use two different strategies to map the genome-wide distribution of meiotic DSBs in the yeast Saccharomyces cerevisiae. The first is a chromatin immunoprecipitation (ChIP) based approach that targets the Spo11p protein, which remains covalently attached to DSB ends in the rad50S mutant background. The second approach involves BND cellulose enrichment of the single strand DNA (ssDNA) recombination intermediate formed by end-resection at DSB sites following Spo11p removal. We use dmc1 and dmc1 rad51 mutants that accumulates meiotic single strand DNA intermediates
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| Contributor(s) |
Buhler C, Borde V, Lichten M |
| Citation(s) |
18076285 |
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| Submission date |
Sep 07, 2007 |
| Last update date |
Mar 17, 2012 |
| Contact name |
Michael Lichten |
| E-mail(s) |
[email protected]
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| Phone |
301 496 9760
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| Fax |
301 402 3095
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| Organization name |
National Cancer Institute
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| Department |
LBMB
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| Street address |
37 Convent Dr
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892-4260 |
| Country |
USA |
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| Platforms (1) |
| GPL3737 |
Agilent-012713 Yeast Whole Genome ChIP-on-chip Microarray (G4486A) |
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| Samples (20)
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| Relations |
| BioProject |
PRJNA102441 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE8981_RAW.tar |
125.5 Mb |
(http)(custom) |
TAR (of GPR) |
| Processed data included within Sample table |
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