Checkpoint kinase interaction with DNA polymerase alpha regulates replication progression during stress

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Eukaryotic DNA replication relies on a robust checkpoint response to manage replication stress.
• Checkpoint kinases are crucial for stabilizing the replisome, but their specific targets within the replisome are not fully elucidated.
• Understanding these interactions is key to comprehending genome duplication fidelity.

Purpose of the Study:

• To identify novel targets of checkpoint kinases within the replisome during replication stress.
• To investigate the role of Rad53 kinase in regulating replisome components.
• To uncover mechanisms coordinating leading and lagging strand replication machinery.

Main Methods:

• Employed an unbiased biotin proximity-ligation assay in Saccharomyces cerevisiae.
• Screened for novel Rad53 interactors and substrates in vivo.
• Utilized genetic and biochemical approaches to validate findings.

Main Results:

• Identified Sld7, a replication initiation factor, as a Rad53 substrate.
• Identified Pol1, the catalytic subunit of DNA polymerase a, as a Rad53 interactor.
• Demonstrated that CDK phosphorylation of Pol1 mediates its interaction with Rad53, crucial for viability and replisome progression under stress.

Conclusions:

• Elucidated how Rad53's interactions with the replisome are modulated by replication stress and cell cycle.
• Highlighted the significance of the Rad53-Pol1 interaction for maintaining genome stability.
• Provided insights into coordinating leading and lagging strand replication machineries during stress.