Accumulation of Werner protein at DNA double-strand breaks in human cells
Accumulation of Werner protein at DNA double-strand breaks in human cells
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DOI：
10.1242/jcs.02544
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发表时间：
2005-09-15
影响因子：
4
通讯作者：
Yasui, A
中科院分区：
文献类型：
--
作者：
Lan, L;Nakajima, S;Yasui, A
Werner综合征是由WRN基因缺陷引起的常染色体隐性加速衰老疾病，WRN基因编码具有核酸外切酶活性的RecQ DNA解旋酶家族成员。体外实验表明，WRN在几个DNA修复过程中发挥作用，但WRN在活细胞中的实际功能仍然未知。在这里，我们分析了WRN蛋白的核内动员的动力学，以响应各种类型的DNA损伤局部产生的人细胞核。在激光诱导的双链断裂处观察到WRN的显著积累，但在单链断裂或氧化性碱基损伤处没有观察到WRN的显著积累。WRN在双链断裂处的积累是快速的，持续数小时，并且在不存在几种已知的相互作用蛋白质的情况下发生，包括聚合酶P、聚（ADP-核糖）聚合酶1（PARP 1）、Ku 80、DNA依赖性蛋白激酶（DNA-PKcs）、NBS 1和组蛋白H2 AX。取消解旋酶活性或删除的核酸外切酶结构域的积累没有影响，而HRDC（解旋酶和RNaseD C-末端）结构域的存在是必要的，足以积累。我们的数据表明，WRN的功能主要是在DNA双链断裂和结构类似的双链断裂在活细胞中，并通过HRDC域的自主积累是WRN的双链断裂的初始响应。
Werner syndrome is an autosomal recessive accelerated-aging disorder caused by a defect in the WRN gene, which encodes a member of the RecQ family of DNA helicases with an exonuclease activity. In vitro experiments have suggested that WRN functions in several DNA repair processes, but the actual functions of WRN in living cells remain unknown. Here, we analyzed the kinetics of the intranuclear mobilization of WRN protein in response to a variety of types of DNA damage produced locally in the nucleus of human cells. A striking accumulation of WRN was observed at laser-induced double-strand breaks, but not at single-strand breaks or oxidative base damage. The accumulation of WRN at double-strand breaks was rapid, persisted for many hours, and occurred in the absence of several known interacting proteins including polymerase P, poly(ADP-ribose) polymerase 1 (PARP1), Ku80, DNA-dependent protein kinase (DNA-PKcs), NBS1 and histone H2AX. Abolition of helicase activity or deletion of the exonuclease domain had no effect on accumulation, whereas the presence of the HRDC (helicase and RNaseD C-terminal) domain was necessary and sufficient for the accumulation. Our data suggest that WRN functions mainly at DNA double-strand breaks and structures resembling double-strand breaks in living cells, and that an autonomous accumulation through the HRDC domain is the initial response of WRN to the double-strand breaks.