Localization of X-ray cross complementing gene 1 protein in the nuclear matrix is controlled by casein kinase II-dependent phosphorylation in response to oxidative damage
Localization of X-ray cross complementing gene 1 protein in the nuclear matrix is controlled by casein kinase II-dependent phosphorylation in response to oxidative damage
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DOI：
10.1016/j.dnarep.2009.06.003
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发表时间：
2009-08-06
期刊：
影响因子：
3.8
通讯作者：
Horiuchi, Saburo
中科院分区：
文献类型：
--
作者：
Kubota, Yoshiko;Takanami, Takako;Horiuchi, Saburo
DNA损伤的碱基切除修复/单链断裂修复（BER/SSBR）是一种高效的修复过程。X射线交叉互补蛋白1（XRCC 1）是BER/SSBR因子的关键支架蛋白。最近的研究表明，XRCC 1以依赖于酪蛋白激酶II（CK 2）磷酸化的方式在DNA损伤部位形成致密灶。为了研究病灶形成的机制，我们通过HeLa细胞蛋白的生化分离分析了修复过程中XRCC 1的亚核定位和磷酸化状态。固定细胞的原位提取也验证了定位。在未受攻击的细胞中，XRCC 1主要以高度磷酸化的形式存在于染色质组分中，此外，在核基质（NM）中存在少量（10-15%）不磷酸化或边缘磷酸化的XRCC 1。病灶形成和XRCC 1分布的变化可以通过CK 2的敲低、XRCC 1的非磷酸化版本的表达或损伤位点处的聚ADP核糖基化的抑制来消除。其他BER因子，如DNA聚合酶β，也被发现在过氧化氢诱导的DNA损伤后在NM中积累，尽管它与NM的关联似乎相对较弱。我们的研究结果表明，XRCC 1在染色质中的组成性磷酸化及其DNA损伤诱导的NM的招聘是至关重要的焦点形成，BER/SSBR的核心反应可能发生在NM。(C)2009 Elsevier B. V.保留所有权利。
Base excision repair/single strand break repair (BER/SSBR) of damaged DNA is a highly efficient process. X-ray cross complementing protein 1 (XRCC1) functions as a key scaffold protein for BER/SSBR factors. Recent work has shown that XRCC1 forms dense foci at sites of DNA damage in a manner dependent on casein kinase II (CK2) phosphorylation. To investigate the mechanism underlying foci formation, we analyzed the subnuclear localization and phosphorylation status of XRCC1 during the repair process by biochemical fractionation of HeLa cellular proteins. The localization was also verified by in situ extraction of the fixed cells. In unchallenged cells, XRCC1 was primarily found in the chromatin fraction in a highly phosphorylated form; in addition, a minor population (10-15%) existed in the nuclear matrix (NM) with no or marginal phosphorylation.After hydrogen peroxide treatment, hyperphosphorylated XRCC1 appeared in the NM and accordingly, those in the chromatin fraction decreased. Foci formation and changes in XRCC1 distribution could be abolished by the knockdown of CK2, the expression of a non-phosphorylatable version of XRCC1, or the inhibition of poly-ADP ribosylation at the damage sites. Other BER factors, like DNA polymerase beta, were also found to accumulate in the NM after hydrogen peroxide-induced DNA damage, although its association with the NM seemed relatively weak. Our results suggest that the constitutive phosphorylation of XRCC1 in the chromatin and its DNA damage-induced recruitment to the NM are critical for foci formation, and that the core reactions of BER/SSBR may occur in the NM. (C) 2009 Elsevier B.V. All rights reserved.