Do replication forks control late origin firing in Saccharomyces cerevisiae?

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Eukaryotic DNA replication timing profiles suggest a universal shape for the rate of origin firing, I(t), crucial for reproducible replication completion.
• Population-averaged measurements of I(t) may be biased by cell synchrony and cell-to-cell variability.
• Understanding the true shape of I(t) is essential for comprehending DNA replication dynamics.

Purpose of the Study:

• To measure the population-averaged I(t) profile in Saccharomyces cerevisiae using DNA combing.
• To extract the single-cell I(t) profile via numerical deconvolution.
• To investigate the relationship between single-cell I(t), fork density, and the role of Clb5p in DNA replication.

Main Methods:

• Synchronization of Saccharomyces cerevisiae cells.
• DNA combing to measure population-averaged replication timing profiles.
• Numerical deconvolution to extract single-cell I(t) profiles.
• Analysis of wild-type and clb5Δ mutant cells.

Main Results:

• Single-cell I(t) profiles closely resemble population-averaged profiles, indicating genome-scale replication process invariance.
• Cell-to-cell variability in replication timing does not explain the observed I(t) shape.
• Single-cell I(t) correlates with fork density in wild-type cells, with a notable loosening in clb5Δ mutants.
• A modified numerical model accurately describes I(t) in both wild-type and clb5Δ cells, incorporating CDK activity and fork density dependencies.

Conclusions:

• The universal shape of the DNA replication origin firing rate, I(t), is maintained at the single-cell level.
• Replication forks from early-firing origins appear to promote origin firing in later-replicating regions.
• The clb5Δ mutant data highlights the role of Clb5p in regulating replication fork density and origin firing coordination.