A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci
A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci
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DOI：
10.4161/cc.26640
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发表时间：
2013-12-15
期刊：
影响因子：
4.3
通讯作者：
Canman, Christine E.
中科院分区：
文献类型：
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作者：
Helchowski, Corey M.;Skow, Laura F.;Canman, Christine E.
DNA 双链断裂 (DSB) 周围染色质的快速泛素化驱动了称为电离辐射诱导灶 (IRIF) 的大型结构的形成，其中包含许多 DNA 损伤反应 (DDR) 蛋白。该过程由 RNF8 和 RNF168 泛素连接酶调节，被认为对于 DNA 修复和调节细胞周期检查点涉及的信号通路激活是必需的。在这里，我们证明，通过表达含有与赖氨酸 63 连接的多聚泛素链结合的泛素结合域 (UBD) 的蛋白质，可以干扰 DDR 因子的泛素依赖性招募。 E3 泛素连接酶 RAD18 的表达阻止了 53BP1 在 DSB 处的染色质扩散，这种现象取决于 RAD18 UBD 的完整性。分离的 RAD18 UBD 干扰 53BP1 染色质扩散以及其他重要的 DDR 介质，包括 RAP80 和 BRCA1 肿瘤抑制蛋白，这与 RAD18 UBD 阻止蛋白质接近泛素化染色质的模型一致。使用 RAD18 UBD 作为阻碍 53BP1 和 BRCA1 定位以修复病灶的工具，我们发现 DDR 信号传导、DNA DSB 修复和放射敏感性不受影响。我们确实发现，除了上调的同源重组修复之外，在 DNA 修复灶中未检测到激活的 ATM (S1981P) 和磷酸化的 SMC1（ATM 的特定靶标），揭示了依赖于 DSB 上某些 DDR 因子的染色质扩散的 2 个 DDR 反应。这些数据表明，选择含有靶向基序的 UBD 可能是确定蛋白质-泛素相互作用的生物学意义的有用探针。
The rapid ubiquitination of chromatin surrounding DNA double-stranded breaks (DSB) drives the formation of large structures called ionizing radiation-induced foci (IRIF), comprising many DNA damage response (DDR) proteins. this process is regulated by RNF8 and RNF168 ubiquitin ligases and is thought to be necessary for DNA repair and activation of signaling pathways involved in regulating cell cycle checkpoints. Here we demonstrate that it is possible to interfere with ubiquitin-dependent recruitment of DDR factors by expressing proteins containing ubiquitin binding domains (UBDs) that bind to lysine 63-linked polyubiquitin chains. expression of the E3 ubiquitin ligase RAD18 prevented chromatin spreading of 53BP1 at DSBs, and this phenomenon was dependent upon the integrity of the RAD18 UBD. An isolated RAD18 UBD interfered with 53BP1 chromatin spreading, as well as other important DDR mediators, including RAP80 and the BRCA1 tumor suppressor protein, consistent with the model that the RAD18 UBD is blocking access of proteins to ubiquitinated chromatin. Using the RAD18 UBD as a tool to impede localization of 53BP1 and BRCA1 to repair foci, we found that DDR signaling, DNA DSB repair, and radiosensitivity were unaffected. We did find that activated ATM (S1981P) and phosphorylated SMC1 (a specific target of ATM) were not detectable in DNA repair foci, in addition to upregulated homologous recombination repair, revealing 2 DDR responses that are dependent upon chromatin spreading of certain DDR factors at DSBs. these data demonstrate that select UBDs containing targeting motifs may be useful probes in determining the biological significance of protein-ubiquitin interactions.