Human RAD18 is involved in S phase-specific single-strand break repair without PCNA monoubiquitination.
Human RAD18 is involved in S phase-specific single-strand break repair without PCNA monoubiquitination.
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DOI：
10.1093/nar/gkl979
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发表时间：
2007
影响因子：
14.9
通讯作者：
Shiomi T
中科院分区：
文献类型：
--
作者：
Shiomi N;Mori M;Tsuji H;Imai T;Inoue H;Tateishi S;Yamaizumi M;Shiomi T
从复制型聚合酶转换为跨损伤聚合酶是在DNA损伤位点进一步继续复制的重要步骤。最近，RAD 18（一种泛素连接酶）被证明与RAD 6（一种泛素缀合酶）在复制停滞位点协同单泛素化增殖细胞核抗原（PCNA），引起聚合酶转换。通过分析从人HCT 116细胞中产生的RAD 18-敲除（RAD 18-/-）细胞，除了聚合酶开关之外，我们发现了RAD 18在S期特异性DNA单链断裂修复（SSBR）中的新功能。与聚合酶转换的情况不同，SSBR不需要PCNA单泛素化。与野生型HCT 116细胞相比，X射线诱导的染色体畸变修复缺陷的RAD 18 −/−细胞对X射线照射和能够诱导单链断裂的拓扑异构酶I抑制剂喜树碱（CPT）显著超敏，但对能够诱导双链断裂的拓扑异构酶II抑制剂依托泊苷不那么敏感。然而，在RAD 18 −/−细胞中观察到的这种对CPT的超敏反应仅限于S期，因为缺乏RAD 18 S期特异性功能。此外，碱性彗星试验也证实了在RAD 18 −/−细胞的S期观察到的缺陷SSBR。
Switching from a replicative to a translesion polymerase is an important step to further continue on replication at the site of DNA lesion. Recently, RAD18 (a ubiquitin ligase) was shown to monoubiquitinate proliferating cell nuclear antigen (PCNA) in cooperation with RAD6 (a ubiquitin-conjugating enzyme) at the replication-stalled sites, causing the polymerase switch. Analyzing RAD18-knockout (RAD18−/−) cells generated from human HCT116 cells, in addition to the polymerase switch, we found a new function of RAD18 for S phase-specific DNA single-strand break repair (SSBR). Unlike the case with polymerase switching, PCNA monoubiquitination was not necessary for the SSBR. When compared with wild-type HCT116 cells, RAD18−/− cells, defective in the repair of X-ray-induced chromosomal aberrations, were significantly hypersensitive to X-ray-irradiation and also to the topoisomerase I inhibitor camptothecin (CPT) capable of inducing single-strand breaks but were not so sensitive to the topoisomerase II inhibitor etoposide capable of inducing double-strand breaks. However, such hypersensitivity to CPT observed with RAD18−/− cells was limited to only the S phase due to the absence of the RAD18 S phase-specific function. Furthermore, the defective SSBR observed in S phase of RAD18−/− cells was also demonstrated by alkaline comet assay.