Graduate School of Life Science, University of Hyogo, Kouto 3-2-1, Kamigori, Ako-gun, Hyogo 678-1297, Japan.
J Biol Chem. 2010 Dec 31;285(53):41993-2000. doi: 10.1074/jbc.M110.161661. Epub 2010 Oct 7.
The licensing factor Cdt1 is degraded by CRL4(Cdt2) ubiquitin ligase dependent on proliferating cell nuclear antigen (PCNA) during S phase and when DNA damage is induced in G(1) phase. Association of both Cdt2 and PCNA with chromatin was observed in S phase and after UV irradiation. Here we used a micropore UV irradiation assay to examine Cdt2 accumulation at cyclobutane pyrimidine dimer-containing DNA-damaged sites in the process of Cdt1 degradation in HeLa cells. Cdt2, present in the nucleus throughout the cell cycle, accumulated rapidly at damaged DNA sites during G(1) phase. The recruitment of Cdt2 is dependent on prior PCNA chromatin binding because Cdt2 association was prevented when PCNA was silenced. Cdt1 was also recruited to damaged sites soon after UV irradiation through its PIP-box. As Cdt1 was degraded, the Cdt2 signal at damaged sites was reduced, but PCNA, cyclobutane pyrimidine dimer, and XPA (xeroderma pigmentosum, complementation group A) signals remained at the same levels. These findings suggest that Cdt1 degradation following UV irradiation occurs rapidly at damaged sites due to PCNA chromatin loading and the recruitment of Cdt1 and CRL4(Cdt2), before DNA damage repair is completed.
在 S 期，Cdt1 被 CRL4(Cdt2)泛素连接酶依赖于增殖细胞核抗原 (PCNA) 降解；在 G1 期，当 DNA 受到损伤时，Cdt1 也会被降解。在 S 期和紫外线照射后，观察到 Cdt2 和 PCNA 与染色质的结合。在这里，我们使用微孔紫外线照射测定法，在 HeLa 细胞中 Cdt1 降解过程中，检查 Cdt2 在含有环丁烷嘧啶二聚体的 DNA 损伤部位的积累。Cdt2 在整个细胞周期中存在于核内，在 G1 期迅速积累在受损的 DNA 部位。Cdt2 的募集依赖于 PCNA 染色质结合，因为沉默 PCNA 会阻止 Cdt2 的结合。紫外线照射后，Cdt1 通过其 PIP 盒也很快被募集到受损部位。随着 Cdt1 的降解，受损部位的 Cdt2 信号减少，但 PCNA、环丁烷嘧啶二聚体和 XPA（着色性干皮病，互补组 A）信号仍保持在相同水平。这些发现表明，在 DNA 损伤修复完成之前，由于 PCNA 染色质加载和 Cdt1 和 CRL4(Cdt2)的募集，紫外线照射后 Cdt1 降解迅速发生在受损部位。
