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| Status |
Public on Feb 17, 2023 |
| Title |
Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Genome variation profiling by genome tiling array
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| Summary |
Ribosomal DNA (rDNA) is organized as large arrays of tandem repeats that vary in copy number from a few dozen to hundreds. In the budding yeast Saccharomyces cerevisiae, each rDNA repeat includes a potential origin of replication. Previous work has led to the model that the rDNA replication origins compete for limiting replication initiation factors with origins in the rest of the genome, suggesting that reduction in rDNA copy number would reduce competition for these limiting factors and therefore promote origin usage in the rest of the genome. To test this hypothesis, we compared genome-wide replication in strains with either wild type rDNA copy number of ~180 (“180 rDNA”) or just ~35 copies (“35 rDNA”) by performing dense-to-light isotope transfer experiments to physically separate replicated, hybrid-density (HL or heavy-light) DNA from unreplicated, HH (heavy-heavy) DNA in cell samples collected at different times in S phase. Contrary to our expectations, we find that although there are no apparent differences in non-rDNA origin activity between the two strains, the 35 rDNA strain shows a genome-wide delay in progression through S phase compared to the 180 rDNA strain.
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| Overall design |
To profile genome-wide replication, we grew cells in isotopically dense (13C-glucose, 15N-ammonium sulfate) for ≥7 population doublings. We then arrested the cells in G1 using a-factor, filtered and resuspended the cells in isotopically light (12C, 14N) medium, then released them into S phase by addition of Pronase. We collected samples at intervals through the ensuing S phase (30,35,40,45,50 min for 35 rDNA strain; 25,30,35,40,45,55 min for 180 rDNA strain). We extracted the DNA, and digested it with a restriction enzyme. Unreplicated (HH) DNA was separated from replicated (HL) DNA by ultracentrifugation in cesium chloride gradients, which were subsequently drip-fractionated. Small aliquots of each fraction were hybridized on a slot blot with a [32P]-labeled whole-genome probe, both to identify fractions with HH and HL DNA and to quantify the percent replication in each S phase sample. For each sample, we then pooled all the HH fractions and labeled the DNA with Cy3, likewise labeled the HL DNA with Cy5, and co-hybridized the pair to a microarray. After extraction of hybridization intensities, we used the known % replication values from the slot blot analysis to normalize the overall % replication in each sample, with an added correction for the percentage of cells in the population that successfully completed S phase (obtained from the maximum % replication seen at the end of S phase from the slot blot analysis). The normalized % replication values were loess-smoothed with a window of 22 kb and plotted as a function of chromosome coordinate with a step size of 500 bp. For a detailed description of this protocol, see Peng et al. (2014), Methods Mol Biol 1170:477-99 (PMCID: PMC4338859).
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| Contributor(s) |
Alvino GM, Kwan EX, Raghuraman MK, Brewer BJ |
| Citation(s) |
36842087 |
| Submission date |
May 30, 2022 |
| Last update date |
May 12, 2023 |
| Contact name |
Mosur K Raghuraman |
| E-mail(s) |
raghu@u.washington.edu
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| Organization name |
University of Washington
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| Department |
Genome Sciences Box 355065
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| Lab |
Brewer/Raghuraman
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| Street address |
Foege Bldg, 3720 15th Ave NE
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platforms (1) |
| GPL10930 |
Agilent-014810 Yeast Whole Genome ChIP-on-Chip Microarray 4x44K (G4493A) [Probe Name version] |
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| Samples (11)
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| Relations |
| BioProject |
PRJNA843612 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE205068_RAW.tar |
50.7 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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