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Abstract：
The E3 ubiquitin ligase RAD18 mono-ubiquitinates PCNA to promote bypass of replication fork-stalling DNA lesions. On the other hand, RAD18 also contributes to DNA double-strand break (DSB) repair. RAD18 is recruited to ionizing radiation (IR)-induced DSB and colocalizes with ubiquitinated chromatin proteins. RAD18 interacts with the ubiquitinated chromatin proteins via its ubiquitin-binding Zinc finger (UBZ) domain and is proposed to propagate DNA DSB signalling and recruit DNA repair proteins. We found that purified human RAD18 protein complexed with RAD6B (RAD6B-RAD18) catalyzes mono- and poly-ubiquitination of histone H2A in vitro while UBZ domain-mutated RAD18 complexed with RAD6B protein catalyzes mono- but not poly-ubiquitination of histone H2A. Human RAD18-/-cells synchronized at the G1 phase show a reduced signal of ubiquitinated protein in chromatin after IR when compared to that of wild-type control cells. The reduced signal of ubiquitinated protein in RAD18-/-cells is rescued by the introduction of RAD18 cDNA but to a lesser extent by the introduction of cDNA coding RAD18 lacking UBZ domain. Taken together, these results indicate that RAD18 mediates DSB-induced ubiquitination of chromatin protein during the G1 phase.
摘要：
E3泛素连接酶RAD18单泛素化PCNA以促进复制叉停滞的DNA损伤的旁路。另一方面,RAD18也有助于DNA双链断裂(DSB)修复。RAD18被募集到电离辐射（IR）诱导的DSB并与泛素化染色质蛋白共定位。RAD18通过其泛素结合锌指（UBZ）结构域与泛素化染色质蛋白相互作用，并被提议传播DNADSB信号并招募DNA修复蛋白。我们发现与RAD6B复合的纯化的人RAD18蛋白（RAD6B-RAD18）在体外催化组蛋白H2A的单泛素化和多泛素化，而与RAD6B蛋白复合的UBZ结构域突变的RAD18催化组蛋白H2A的单泛素化而非多泛素化。与野生型对照细胞相比，在G1期同步的人RAD18-/-细胞在IR后显示染色质中泛素化蛋白的信号降低。RAD18-/-细胞中泛素化蛋白的降低的信号通过引入RAD18cDNA但在较小程度上通过引入编码缺乏UBZ结构域的RAD18的cDNA来挽救。一起来看,这些结果表明，RAD18在G1期介导DSB诱导的染色质蛋白泛素化。
