Protein phosphatase 2A regulates binding of Cdc45 to the prereplication complex

Area of Science:

• Molecular Biology
• Cell Cycle Regulation
• DNA Replication

Background:

• Eukaryotic DNA replication initiation involves ordered steps, including prereplication complex (pre-RC) assembly during G1 phase.
• At the G1/S transition, MCM10 and kinases recruit Cdc45 to the pre-RC, initiating origin unwinding and DNA polymerase recruitment.

Purpose of the Study:

• To investigate the role of Protein Phosphatase 2A (PP2A) in the initiation of eukaryotic DNA replication.
• To elucidate the mechanism by which PP2A regulates Cdc45 loading to the prereplication complex.

Main Methods:

• Utilized a soluble DNA replication system from Xenopus egg extracts.
• Employed immunodepletion of PP2A and inhibition of PP2A activity using okadaic acid.
• Assessed the impact on the loading of key replication factors, including Cdc45, MCM10, RPA, and DNA polymerase alpha.

Main Results:

• PP2A depletion or inhibition abolished Cdc45 loading to the pre-RC, consequently inhibiting origin unwinding, RPA, and DNA polymerase alpha loading.
• PP2A activity inhibition did not affect MCM10 loading or the activity of Cdc7-Dbf4 and Cdk2 kinases.
• The PP2A substrate is not a pre-RC component or Cdc45, but a soluble factor essential for Cdc45 recruitment.

Conclusions:

• PP2A is essential for eukaryotic DNA replication initiation by regulating Cdc45 loading.
• PP2A likely functions by dephosphorylating and activating a soluble factor, counteracting an inhibitory kinase.
• Eukaryotic DNA replication initiation is fine-tuned by a balance of stimulatory and inhibitory phosphorylation events at the Cdc45 loading step.