Dataset Information

Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Ann-Christine König

LAB HEAD: Dr Stephan Hamperl

PROVIDER: PXD032930 | Pride | 2023-02-16

REPOSITORIES: Pride

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			Action	DRS
	92-243013_HAT13883X002__MW71_Y65_40_81964.raw	Raw
	92-243014_HAT13883X003__MW71_Y66_40_81968.raw	Raw
	92-243015_HAT13883X004__MW72_Y65_40_81971.raw	Raw
	92-243017_HAT13883X006__MW72_Y66_40_81978.raw	Raw
	92-243018_HAT13883X007__MW73_Y65_40_81983.raw	Raw

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Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control.

Weiβ Matthias M Chanou Anna A Schauer Tamas T Tvardovskiy Andrey A Meiser Stefan S König Ann-Christine AC Schmidt Tobias T Kruse Elisabeth E Ummethum Henning H Trauner Manuel M Werner Marcel M Lalonde Maxime M Hauck Stefanie M SM Scialdone Antonio A Hamperl Stephan S

Cell reports 20230125 2

The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the protein composition of native chromatin regions su ...[more]

PMID: 36701236

Similar Datasets

Project description:Background: Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation events can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, transiently disabling these mechanisms causes replication origins to re-initiate with diverse efficiencies, which do not correlate with known differences in the efficiency or timing of origin initiation during normal DNA replication. These observations suggest an additional local layer of replication control that can differentially influence how re-initiation is regulated at distinct origins. Principal Findings: We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs). We have mapped the two RIPs to ~60bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 re-associates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Conclusions/Significance: We have uncovered a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are peeled away. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some regions of the genome may be more susceptible to these alterations than others. 142 array CGH experiments are presented. Each experiment is done in experimental replicate. Cells grown as described in Richardson CD and Li JJ PLoS Genetics 2014. Ch1 samples taken from replicating (S phase) or re-replicating (M phase + 3/6 hour induced). Ch2 samples taken from M phase arrested DNA.