Essential roles of Xenopus TRF2 in telomere end protection and replication
Essential roles of Xenopus TRF2 in telomere end protection and replication
复制标题
非洲爪蟾 TRF2 在端粒末端保护和复制中的重要作用
DOI：
10.1111/j.1365-2443.2011.01520.x
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发表时间：
2011
期刊：
影响因子：
2.1
通讯作者：
K.
中科院分区：
文献类型：
--
作者：
Muraki;K.
TRF1 和 TRF2 是双链 (ds) 端粒 DNA 结合蛋白，也是庇护蛋白的核心成员，庇护蛋白是一种为端粒功能提供结构和功能基础的复合物。我们已经报道，与组成性结合染色质的哺乳动物 TRF1 不同，非洲爪蟾 TRF1 (xTRF1) 与有丝分裂染色质结合，但与非洲爪蟾提取物中重建的间期染色质分离。这一发现提出了 xTRF1 和 XenopusTRF2 (xTRF2) 以不同于哺乳动物 TRF1 和 TRF2 的方式对端粒功能做出贡献的可能性。在这里，我们重点关注 xTRF2 的作用。我们制备了在免疫耗竭 xTRF2 的鸡蛋提取物中重建的染色质。与模拟耗尽的细胞核相比，端粒处的 DNA 损伤反应被激活，并且大量 DNA 在 xTRF2 耗尽的细胞核中复制很差。通过添加抗 ATR 中和抗体灭活 ATR 可以挽救复制缺陷，这表明 ATR 在缺陷中发挥了作用。有趣的是，通过用重组 xTRF2 (rTRF2) 补充 xTRF2 耗尽的提取物，可以挽救大量 DNA 复制缺陷，但不能挽救端粒处的 DNA 损伤反应。我们认为 xTRF2 是大量 DNA 的有效复制和防止 DNA 损伤检查点通路激活所必需的，并且这两个功能在机制上是可分离的。
TRF1 and TRF2 are double‐stranded (ds) telomere DNA‐binding proteins and the core members of shelterin, a complex that provides the structural and functional basis of telomere functions. We have reported that unlike mammalian TRF1 that constitutively binds to chromatin,XenopusTRF1 (xTRF1) associates with mitotic chromatin but dissociates from interphase chromatin reconstituted inXenopusegg extracts. This finding raised the possibility that xTRF1 andXenopusTRF2 (xTRF2) contribute to telomere functions in a manner different from mammalian TRF1 and TRF2. Here, we focused on the role of xTRF2. We prepared chromatin reconstituted in egg extracts immunodepleted for xTRF2. Compared to mock‐depleted nuclei, DNA damage response at telomeres was activated, and bulk DNAs were poorly replicated in xTRF2‐depleted nuclei. The replication defect was rescued by inactivating ATR through the addition of anti‐ATR neutralizing antibody, suggesting that ATR plays a role in the defect. Interestingly, the bulk DNA replication defect, but not the DNA damage response at telomeres, was rescued by supplementing the xTRF2‐depleted extracts with recombinant xTRF2 (rTRF2). We propose that xTRF2 is required for both efficient replication of bulk DNA and protection from the activation of the DNA damage checkpoints pathway, and that those two functions are mechanistically separable.