Human RAD18 Interacts with Ubiquitylated Chromatin Components and Facilitates RAD9 Recruitment to DNA Double Strand Breaks

RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2.

RAD18是一种泛素连接酶（ubiquitin ligase），参与复制损伤旁路（replicative damage bypass）及DNA双链断裂（DNA double-strand break, DSB）的修复过程。我们发现，细胞周期中RAD18定位的动态模式，以及RAD18在γ射线辐照诱导的DNA损伤位点的富集，均依赖于复制蛋白A（Replication Protein A, RPA）。此外，在整个细胞周期及辐照后，RAD18均可与染色质结合的缀合泛素（chromatin-associated conjugated ubiquitin）及泛素化组蛋白H2A（ubiquitylated H2A）共定位。该定位模式依赖于完整的泛素结合锌指结构域（Zinc finger domain）的存在。通过生化实验方法（biochemical approach），我们证实RAD18可直接结合泛素化组蛋白H2A及其他多种未知的泛素化染色质组分。该相互作用同样依赖于RAD18的锌指结构域，且在γ射线辐照诱导DSB形成后会显著增强。值得注意的是，RAD18并非总能与H2A泛素化水平升高的区域共定位。在人类女性原代成纤维细胞中，两条X染色体之一会发生失活以平衡雌雄细胞间的X染色体基因表达水平，该失活X染色体富含泛素化组蛋白H2A，但仅极少富集RAD18。这表明RAD18与泛素化组蛋白H2A的结合具有情境依赖性。关于RAD18在DSB位点定位的功能相关性，我们发现RAD18对于9-1-1检查点复合物（9-1-1 checkpoint complex）的组分之一RAD9向此类损伤位点的招募是必需的。RAD9的招募依赖于RAD18的环指结构域（RING domain）与锌指结构域的功能。综上，本研究数据表明，RAD18通过泛素化组蛋白H2A及其他泛素化染色质组分与DSB位点结合，可介导RAD9的招募，这一过程可能直接参与DSB修复，且不依赖于下游检查点激酶CHK1与CHK2的激活。