Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena.
Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena.
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DOI：
10.1371/journal.pgen.1005405
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发表时间：
2015-07
期刊：
影响因子：
4.5
通讯作者：
Kapler GM
中科院分区：
文献类型：
--
作者：
Sandoval PY;Lee PH;Meng X;Kapler GM
后生动物和酵母菌的S期内检查点激酶ATR/MEC1通过磷酸化复制解旋酶的亚基MCM2-7来保护染色体免受DNA损伤和复制应激。在这里，我们描述了在嗜热四膜虫中一种前所未有的ATR依赖的途径，在羟基脲处理的S期细胞中，必不可少的复制前复合蛋白Orc1p、Orc2p和Mcm6p被降解。在HU停滞期间，染色体经历了全局性的变化，包括组蛋白H2A.X的磷酸化，组蛋白H3的脱乙酰化，以及DNA含量的明显减少，这可以被脱乙酰酶抑制剂丁酸钠所阻断。最值得注意的是，去除羟基脲后，细胞周期迅速恢复，整个基因组在补充ORC和MCM之前复制。虽然停滞的复制叉子在这些条件下会被拉长，但DNA纤维成像显示，大多数复制分子是由新的启动事件产生的。此外，核糖体DNA微染色体的唯一来源是不活跃的，复制似乎是在rRNA启动子附近开始的。这些收集的数据提出了在从HU诱导的复制应激恢复过程中通过ORC无关的机制启动复制的可能性。DNA损伤和复制应激激活细胞周期检查点反应，保护真核染色体的完整性。一个保守的反应涉及复制解旋酶MCM2-7的可逆磷酸化，它与起源识别复合体(ORC)一起决定了复制在染色体上开始的时间和地点。ORC和MCM在DNA复制中的核心作用体现在以下事实上：从酵母到人类，这些复制前复合体(Pre-RC)成分丰度的微小变化很难耐受。在这里，我们描述了在早期分枝真核生物四膜虫中一种前所未有的复制应激检查点反应，这种反应是由羟基脲(HU)耗尽dNTP池所触发的。在HU处理的四膜虫中，ORC和MCM蛋白被物理降解，而不是瞬时磷酸化MCM亚基。出乎意料的是，当HU被移除时，基因组在补充ORC和MCM组件之前被完全和毫不费力地复制。使用DNA纤维成像和2D凝胶电泳，我们表明在恢复阶段绕过了ORC依赖的机制，在整个基因组中产生了双向复制叉子。我们的发现表明，四膜虫采用了一种替代的复制启动机制，并且潜在的过程可以在基因组范围内进行操作。
The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress. DNA damage and replication stress activate cell cycle checkpoint responses that protect the integrity of eukaryotic chromosomes. A well-conserved response involves the reversible phosphorylation of the replicative helicase, MCM2-7, which together with the origin recognition complex (ORC) dictates when and where replication initiates in chromosomes. The central role of ORC and MCMs in DNA replication is illustrated by the fact that small changes in abundance of these pre-replicative complex (pre-RC) components are poorly tolerated from yeast to humans. Here we describe an unprecedented replication stress checkpoint response in the early branching eukaryote, Tetrahymena thermophila, that is triggered by the depletion of dNTP pools with hydroxyurea (HU). Instead of transiently phosphorylating MCM subunits, ORC and MCM proteins are physically degraded in HU-treated Tetrahymena. Unexpectedly, upon HU removal the genome is completely and effortlessly replicated prior to replenishment of ORC and MCM components. Using DNA fiber imaging and 2D gel electrophoresis, we show that ORC-dependent mechanisms are bypassed during the recovery phase to produce bidirectional replication forks throughout the genome. Our findings suggest that Tetrahymena enlists an alternative mechanism for replication initiation, and that the underlying process can operate on a genome-wide scale.