果蝇 S2 细胞中 DNA 复制对 MCM 蛋白的差异需求。
Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells.
机构信息
Basic Medical Sciences, St. George's University of London, London, United Kingdom.
出版信息
PLoS One. 2007 Sep 5;2(9):e833. doi: 10.1371/journal.pone.0000833.
BACKGROUND
The MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they are significantly more abundant than other preRC components, we were interested in determining whether the entire cellular content was necessary for DNA replication in vivo.
METHODOLOGY/PRINCIPLE FINDINGS: We performed a systematic depletion of the MCM proteins in Drosophila S2 cells using dsRNA-interference. Reducing MCM2-6 levels by >95-99% had no significant effect on cell cycle distribution or viability. Depletion of MCM7 however caused an S-phase arrest. MCM2-7 depletion produced no change in the number of replication forks as measured by PCNA loading. We also depleted MCM8. This caused a 30% reduction in fork number, but no significant effect on cell cycle distribution or viability. No additive effects were observed by co-depleting MCM8 and MCM5.
CONCLUSIONS/SIGNIFICANCE: These studies suggest that, in agreement with what has previously been observed for Xenopus in vitro, not all of the cellular content of MCM2-6 proteins is needed for normal cell cycling. They also reveal an unexpected unique role for MCM7. Finally they suggest that MCM8 has a role in DNA replication in S2 cells.
背景
MCM2-7 蛋白是真核生物复制前复合物（preRC）的关键组成部分。由于它们的含量明显高于其他 preRC 成分，我们有兴趣确定细胞内的全部 MCM 蛋白是否对体内的 DNA 复制是必需的。
方法/原理发现：我们使用 dsRNA 干扰在果蝇 S2 细胞中进行了 MCM 蛋白的系统性耗竭。将 MCM2-6 水平降低>95-99%对细胞周期分布或活力没有显著影响。然而，MCM7 的耗竭会导致 S 期停滞。MCM2-7 的耗竭不会改变 PCNA 加载所测量的复制叉数量。我们还耗尽了 MCM8。这导致叉数减少 30%，但对细胞周期分布或活力没有显著影响。当同时耗尽 MCM8 和 MCM5 时，没有观察到相加效应。
结论/意义：这些研究表明，与之前在 Xenopus 体外观察到的结果一致，并非所有细胞内的 MCM2-6 蛋白都需要正常的细胞循环。它们还揭示了 MCM7 的一个意外独特作用。最后，它们表明 MCM8 在 S2 细胞的 DNA 复制中具有作用。
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