Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Eukaryotic chromosome replication initiates from multiple origins during S-phase.
• Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) activities are essential throughout S-phase for replication initiation.

Purpose of the Study:

• To investigate the regulatory mechanisms controlling the temporal program of replication origin firing.
• To understand why replication origins do not initiate simultaneously at the beginning of S-phase.

Main Methods:

• Analysis of the abundance of CDK and DDK pathway components in Saccharomyces cerevisiae.
• Genetic manipulation, including over-expression of Sld3, Sld2, Dpb11, and Dbf4, and deletion of the RPD3 gene.
• Assessment of replication initiation timing and checkpoint activation.

Main Results:

• Key CDK and DDK pathway factors (Sld3, Sld2, Dpb11, Dbf4) are found in low abundance.
• Over-expression of these factors promotes early firing of late origins and activation of heterochromatic, dormant origins.
• The normal origin firing program prevents aberrant checkpoint activation and regulates S-phase duration.

Conclusions:

• Competition for limiting DDK kinase and CDK targets at replication origins governs initiation kinetics.
• This study elucidates a system for investigating the biological significance of temporal origin firing programs.