The Wistar Institute, Philadelphia, PA 19104, USA.
J Virol. 2010 May;84(10):4979-87. doi: 10.1128/JVI.01300-09. Epub 2010 Mar 3.
The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is required for episome stability during latent infection. Telomere repeat factor 2 (TRF2) binds directly to OriP and facilitates DNA replication and plasmid maintenance. Recent studies have found that TRF2 interacts with the DNA damage checkpoint protein Chk2. We show here that Chk2 plays an important role in regulating OriP plasmid stability, chromatin modifications, and replication timing. The depletion of Chk2 by small interfering RNA (siRNA) leads to a reduction in DNA replication efficiency and a loss of OriP-dependent plasmid maintenance. This corresponds to a change in OriP replication timing and an increase in constitutive histone H3 acetylation. We show that Chk2 interacts with TRF2 in the early G(1)/S phase of the cell cycle. We also show that Chk2 can phosphorylate TRF2 in vitro at a consensus acceptor site in the amino-terminal basic domain of TRF2. TRF2 mutants with a serine-to-aspartic acid phosphomimetic substitution mutation were reduced in their ability to recruit the origin recognition complex (ORC) and stimulate OriP replication. We suggest that the Chk2 phosphorylation of TRF2 is important for coordinating ORC binding with chromatin remodeling during the early S phase and that a failure to execute these events leads to replication defects and plasmid instability.
Epstein-Barr 病毒（EBV）的质粒复制原点（OriP）对于潜伏感染期间的 episome 稳定性是必需的。端粒重复因子 2（TRF2）直接与 OriP 结合，促进 DNA 复制和质粒维持。最近的研究发现，TRF2 与 DNA 损伤检查点蛋白 Chk2 相互作用。我们在这里表明，Chk2 在调节 OriP 质粒稳定性、染色质修饰和复制时间方面起着重要作用。通过小干扰 RNA（siRNA）耗尽 Chk2 会导致 DNA 复制效率降低和 OriP 依赖性质粒维持丧失。这对应于 OriP 复制时间的改变和组成型组蛋白 H3 乙酰化的增加。我们表明 Chk2 在细胞周期的早期 G1/S 期与 TRF2 相互作用。我们还表明，Chk2 可以在体外 TRF2 的氨基末端碱性结构域的一个公认的受体位点上磷酸化 TRF2。具有丝氨酸到天冬氨酸磷酸模拟突变的 TRF2 突变体在招募起始识别复合物（ORC）和刺激 OriP 复制的能力方面降低。我们认为 Chk2 对 TRF2 的磷酸化对于协调 ORC 在早期 S 期与染色质重塑的结合是重要的，而未能执行这些事件会导致复制缺陷和质粒不稳定。
