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Dephosphorylation of the pre-initiation complex is critical for origin firing

Fiona Jenkinson1, Kang Wei Tan2, Barbara Schöpf2

  • 1Department of Biochemistry, 80 Tennis Court Road, University of Cambridge, Cambridge CB2 1GA, UK; Wellcome Trust, Cancer Research UK Gurdon Institute and Department of Biochemistry, The Henry Wellcome Building of Cancer and Developmental Biology, University of Cambridge, Cambridge CB2 1QN, UK.

Molecular Cell
|December 21, 2022
PubMed

Insights

Phosphatases PP2A and PP4 rapidly reverse cyclin-dependent kinase (CDK) phosphorylation of Sld3 and Sld2 during S phase. Targeted dephosphorylation of Sld3 by PP2ARts1 is critical for DNA replication initiation and cell viability.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Cyclin-dependent kinases (CDKs) regulate eukaryotic DNA replication, ensuring genome duplication occurs only once.
  • CDK activity is crucial for activating DNA replication origins by phosphorylating key substrates like Sld2 and Sld3, forming the pre-initiation complex (pre-IC).

Purpose of the Study:

  • To investigate the role of phosphatases in regulating CDK-mediated phosphorylation of Sld3 and Sld2 during DNA replication in Saccharomyces cerevisiae.
  • To determine the specific phosphatases involved and their mechanisms of action in controlling replication initiation.

Main Methods:

  • Utilized budding yeast (Saccharomyces cerevisiae) as a model organism.
  • Employed biochemical assays and genetic approaches to study protein phosphorylation and dephosphorylation events.
  • Investigated the interaction between PP2ARts1 and Sld3 using an Rts1-interaction motif.

Main Results:

  • Demonstrated that PP2A and PP4 phosphatases rapidly reverse CDK phosphorylations of Sld3 and Sld2 during S phase.
  • Showed that PP2ARts1 specifically targets Sld3 for dephosphorylation via an Rts1-interaction motif.
  • Found that targeted Sld3 dephosphorylation is essential for genome-wide origin firing, pre-IC formation, Sld3 dephosphorylation in G1 phase, and cell viability.
  • Observed that PP2ARts1 promotes DNA replication in vitro.

Conclusions:

  • Phosphatases, in addition to kinases, are critical regulators of DNA replication initiation.
  • Targeted dephosphorylation by phosphatases enforces the correct order of replication factor phosphorylation, ensuring proper cell cycle progression.
  • PP2ARts1-mediated dephosphorylation of Sld3 is a key mechanism controlling replication initiation and is vital for cell survival.
Keywords:
DNA replicationcell cyclegenome stabilityorigin firingphosphatasesyeast

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