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| Status |
Public on Jan 01, 2023 |
| Title |
Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
The chromatin at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how the chromatin composition controls the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we used site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the histone modification landscape and identify the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to the known origin interactors, we find novel origin-associated factors, such as the kinetochore-associated Ask1/DASH complex. Strikingly, we show that Ask1 regulates the replication timing control of specific origins in yeast. Thus, our unbiased approach identifies functionally-relevant proteomes at single-copy loci and would be widely applicable to provide an in-depth quantitative characterization of histone modification and protein interaction networks of chromatin at any genomic locus of interest.
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| Overall design |
DNA sequencing based copy number analysis of origin activity in HU-arrested and synchronously released S. cerevisiae cells after targeted degradation of Ask1 or targeted recruitment of Ask1 at specific replication origins ARS305, ARS313 or ARS316.
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| Contributor(s) |
Weiß M, Chanou A, Schauer T, Hamperl S |
| Citation(s) |
36701236 |
| Submission date |
Sep 08, 2022 |
| Last update date |
Apr 03, 2023 |
| Contact name |
Tamas Schauer |
| E-mail(s) |
tamas.schauer@helmholtz-munich.de
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| Organization name |
Helmholtz Zentrum München
|
| Department |
Institute of Epigenetics and Stem Cells
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| Street address |
Feodor-Lynen-Straße 21
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| City |
Munich |
| ZIP/Postal code |
81377 |
| Country |
Germany |
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| Platforms (1) |
| GPL27812 |
Illumina NovaSeq 6000 (Saccharomyces cerevisiae) |
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| Samples (72)
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| Relations |
| BioProject |
PRJNA878541 |
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