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Regulation of Epstein-Barr virus origin of plasmid replication (OriP) by the S-phase checkpoint kinase Chk2

Jing Zhou1, Zhong Deng, Julie Norseen

  • 1The Wistar Institute, Philadelphia, PA 19104, USA.

Journal of Virology
|March 5, 2010
PubMed

Insights

The DNA damage protein Chk2 phosphorylates TRF2, crucial for Epstein-Barr virus (EBV) OriP plasmid replication and stability. This phosphorylation coordinates DNA replication and chromatin changes, preventing instability.

Area of Science:

  • Molecular Biology
  • Virology
  • Cell Cycle Regulation

Background:

  • Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is essential for episome stability during latent infection.
  • Telomere repeat factor 2 (TRF2) binds OriP, aiding DNA replication and plasmid maintenance.
  • TRF2 has been observed to interact with the DNA damage checkpoint protein Chk2.

Purpose of the Study:

  • To investigate the role of Chk2 in regulating OriP plasmid stability, chromatin modifications, and replication timing.
  • To elucidate the interaction between Chk2 and TRF2 during the cell cycle.
  • To determine if Chk2-mediated phosphorylation of TRF2 impacts OriP replication and plasmid maintenance.

Main Methods:

  • Small interfering RNA (siRNA) was used to deplete Chk2.
  • Analysis of DNA replication efficiency, OriP plasmid maintenance, and replication timing.
  • Investigation of histone H3 acetylation.
  • Cell cycle analysis to determine Chk2-TRF2 interaction timing.
  • In vitro kinase assays to assess Chk2 phosphorylation of TRF2.
  • Site-directed mutagenesis to create phosphomimetic TRF2 mutants.
  • Assays to measure origin recognition complex (ORC) recruitment and OriP replication stimulation.

Main Results:

  • Chk2 depletion reduced DNA replication efficiency and led to loss of OriP plasmid maintenance.
  • Chk2 depletion altered OriP replication timing and increased histone H3 acetylation.
  • Chk2 and TRF2 interact in early G(1)/S phase.
  • Chk2 phosphorylates TRF2 in vitro at a specific site.
  • TRF2 phosphomimetic mutants showed reduced ORC recruitment and OriP replication stimulation.

Conclusions:

  • Chk2 plays a critical role in OriP plasmid stability, chromatin modification, and replication timing.
  • Chk2-mediated phosphorylation of TRF2 is important for coordinating ORC binding and chromatin remodeling in early S phase.
  • Disruption of these events by impaired Chk2 phosphorylation leads to replication defects and plasmid instability.

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