RAD18 mediates DNA double-strand break-induced ubiquitination of chromatin protein
RAD18 mediates DNA double-strand break-induced ubiquitination of chromatin protein
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DOI：
10.1093/jb/mvab010
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发表时间：
2021-01-28
影响因子：
2.7
通讯作者：
Tateishi, Satoshi
中科院分区：
文献类型：
--
作者：
Mustofa, Md Kawsar;Tanoue, Yuki;Tateishi, Satoshi
E3泛素连接酶RAD18对增殖细胞核抗原（PCNA）进行单泛素化修饰，以促进复制叉绕过导致其停滞的DNA损伤。另一方面，RAD18也参与DNA双链断裂（DSB）修复。RAD18被招募到电离辐射（IR）诱导的双链断裂处，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，据推测它能传播DNA双链断裂信号并招募DNA修复蛋白。我们发现，纯化的与RAD6B结合的人源RAD18蛋白（RAD6B - RAD18）在体外可催化组蛋白H2A的单泛素化和多泛素化，而与RAD6B蛋白结合的UBZ结构域突变的RAD18只能催化组蛋白H2A的单泛素化，不能催化多泛素化。与野生型对照细胞相比，处于G1期同步化的人源RAD18基因敲除（RAD18(-/-)）细胞在受到电离辐射后，染色质中泛素化蛋白的信号减弱。通过导入RAD18的互补DNA（cDNA）可恢复RAD18(-/-)细胞中泛素化蛋白信号的减弱，但导入缺失UBZ结构域的RAD18的cDNA时，恢复程度较小。综上所述，这些结果表明，RAD18在G1期介导双链断裂诱导的染色质蛋白泛素化。
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.