Disruption of Telomere Maintenance by Depletion of the MRE11/RAD50/NBS1 Complex in Cells That Use Alternative Lengthening of Telomeres*
Disruption of Telomere Maintenance by Depletion of the MRE11/RAD50/NBS1 Complex in Cells That Use Alternative Lengthening of Telomeres*
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DOI：
10.1074/jbc.m701413200
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发表时间：
2007-10
影响因子：
4.8
通讯作者：
Ze-Huai Zhong;Wei‐Qin Jiang;Anthony J. Cesare;A. Neumann;R. Wadhwa;R. Reddel
中科院分区：
文献类型：
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作者：
Ze-Huai Zhong;Wei‐Qin Jiang;Anthony J. Cesare;A. Neumann;R. Wadhwa;R. Reddel
永生化的人类细胞能够通过端粒酶或重组介导的 DNA 复制机制（称为端粒选择性延长 (ALT)）来维持其端粒。我们之前表明，Sp100 蛋白的过度表达可以抑制 ALT，这与 Sp100 对 MRE11/RAD50/NBS1 (MRN) 重组蛋白复合物的隔离有关。在本研究中，我们确定了 ALT 活性是否需要 MRN 蛋白。通过小发夹 RNA 介导的敲低，ALT 细胞中的 MRN 蛋白被耗尽，这种敲低可维持多达 100 次群体倍增。 NBS1 的敲低对 RAD50 或 MRE11 的水平没有影响，但 RAD50 的敲低也会耗尽细胞中的 NBS1，而 MRE11 的敲低则会耗尽细胞中的所有三种 MRN 蛋白。 NBS1 的耗尽，无论是否耗尽复合体的其他成员，都会导致 ALT 介导的端粒维持受到抑制，这一点可以通过 ALT 相关的早幼粒细胞白血病小体数量减少和端粒长度缩短来证明。在一些克隆中，端粒长度有一个快速缩短的初始阶段，随后端粒长度稳定，而在其他克隆中，在缺乏端粒维持机制的正常人类体细胞的报告范围内，持续缩短。相比之下，端粒酶阳性细胞中NBS1的消耗不会导致端粒缩短。最近的一项研究表明，NBS1 是染色体外端粒环形成所必需的（Compton, S. A.、Choi, J. H.、Cesare, A. J.、Ozgur, S. 和 Griffith, J. D. (2007) Cancer Res. 67, 1513-1519），也是 ALT 的标记。我们得出结论，MRN 复合体，尤其是 NBS1，是 ALT 机制所必需的。
Immortalized human cells are able to maintain their telomeres by telomerase or by a recombination-mediated DNA replication mechanism known as alternative lengthening of telomeres (ALT). We showed previously that overexpression of Sp100 protein can suppress ALT and that this was associated with sequestration of the MRE11/RAD50/NBS1 (MRN) recombination protein complex by Sp100. In the present study, we determined whether MRN proteins are required for ALT activity. ALT cells were depleted of MRN proteins by small hairpin RNA-mediated knockdown, which was maintained for up to 100 population doublings. Knockdown of NBS1 had no effect on the level of RAD50 or MRE11, but knockdown of RAD50 also depleted cells of NBS1, and knockdown of MRE11 depleted cells of all three MRN proteins. Depletion of NBS1, with or without depletion of other members of the complex, resulted in inhibition of ALT-mediated telomere maintenance, as evidenced by decreased numbers of ALT-associated promyelocytic leukemia bodies and decreased telomere length. In some clones there was an initial period of rapid shortening followed by stabilization of telomere length, whereas in others there was continuous shortening at a rate within the reported range for normal human somatic cells lacking a telomere maintenance mechanism. In contrast, depletion of NBS1 in telomerase-positive cells did not result in telomere shortening. A recent study showed that NBS1 was required for the formation of extrachromosomal telomeric circles (Compton, S. A., Choi, J. H., Cesare, A. J., Ozgur, S., and Griffith, J. D. (2007) Cancer Res. 67, 1513-1519), also a marker for ALT. We conclude that the MRN complex, and especially NBS1, is required for the ALT mechanism.