Dataset_A96193B - samples
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ABSTRACT:
PROVIDER: EGAD00001005347 | EGA |
REPOSITORIES: EGA
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Project description:Eukaryotic genome replication is stochastic with each cell using a different cohort of replication origins. Interpreting high-resolution genome replication profiles with a mathematical model allowed us to quantify the stochastic nature of genome replication. This approach included estimation of the activity of every replication origin and the genome-wide location of replication termination events. Single-cell measurements verified the inferred values for stochastic origin replication time. Strains in which multiple origins had been inactivated, confirmed that the location of termination events is primarily dictated by the stochastic activation time of origins. Cell-to-cell variability in origin activity ensures that termination events are widely distributed across virtually the whole genome. We propose that the heterogeneity in origin usage contributes to genome stability by limiting potentially deleterious events from accumulating at particular loci. Measurement of genome replication time for two S. cerevisiae strains. For each strain six S phase samples werecompared with a non-replicating sample.
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Project description:Eukaryotic genome replication is stochastic with each cell using a different cohort of replication origins. Interpreting high-resolution genome replication profiles with a mathematical model allowed us to quantify the stochastic nature of genome replication. This approach included estimation of the activity of every replication origin and the genome-wide location of replication termination events. Single-cell measurements verified the inferred values for stochastic origin replication time. Strains in which multiple origins had been inactivated, confirmed that the location of termination events is primarily dictated by the stochastic activation time of origins. Cell-to-cell variability in origin activity ensures that termination events are widely distributed across virtually the whole genome. We propose that the heterogeneity in origin usage contributes to genome stability by limiting potentially deleterious events from accumulating at particular loci.
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Project description:Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of specific genomic loci after targeted purification in their native chromatin context. Applying MATAC-Seq to selected early-efficient (EE) and late-inefficient (LI) budding yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an optimal 100-115bp nucleosome-free region in combination with surrounding well-positioned nucleosomes and open +2 linker region is a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during DNA replication of eukaryotic cells. Consequently, our single-molecule assay for chromatin accessibility will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.
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