Rad18 regulates DNA polymerase κ and is required for recovery from S-phase checkpoint-mediated arrest
Rad18 regulates DNA polymerase κ and is required for recovery from S-phase checkpoint-mediated arrest
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DOI：
10.1128/mcb.26.9.3527-3540.2006
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发表时间：
2006-05-01
影响因子：
5.3
通讯作者：
Vaziri, C
中科院分区：
文献类型：
--
作者：
Bi, XH;Barkley, LR;Vaziri, C
我们研究了将跨损伤合成 (TLS) DNA 聚合酶 Pol kappa 募集至停滞的复制叉的机制。 DNA 聚合酶持续合成因子 PCNA 被单泛素化，并与用大体积加合物形成基因毒素苯并[a]芘二氢二醇环氧化物 (BPDE) 处理的细胞中的 Pol kappa 相互作用。 PCNA 的单泛素化缺陷突变形式无法与 Pol kappa 相互作用。小干扰 RNA 介导的 E3 连接酶 Rad18 下调会抑制 BPDE 诱导的 PCNA 泛素化以及 PCNA 和 Pol kappa 之间的关联。相反，过表达的 Rad18 会以 DNA 损伤无关的方式诱导 PCNA 泛素化以及 PCNA 和 Pol kappa 之间的关联。因此，Pol kappa 与 PCNA 的关联受到 Rad18 介导的 PCNA 泛素化的调节。来自 Rad18(-/-) 转基因小鼠的细胞显示出 BPDE 诱导的 S 期检查点恢复有缺陷。在 Rad18(-/-) 细胞中，BPDE 诱导检查点激酶升高且持续激活，表明由于 TLS 缺陷而导致分叉持续停滞。与野生型细胞相比，Rad18 缺陷细胞在 BPDE 攻击后表现出活力降低（但羟基脲或电离放射治疗后的存活率不受 Rad18 缺陷的影响）。抑制 RPA/ATR/Chk1 介导的 S 期检查点信号传导可部分抑制 BPDE 诱导的 PCNA 泛素化，并阻止 PCNA 和 Pol kappa 之间的相互作用。综上所述，我们的结果表明 Rad18 介导的 PCNA 单泛素化需要 ATR/Chk1 信号传导。将 Pol kappa 招募到泛素化 PCNA 中可以绕过病变并消除停滞的分叉，从而减弱 S 期检查点。
We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Pol kappa to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Pol kappa in cells treated with the bulky adduct-forming genotoxin benzo[a]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Pol kappa. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Pol kappa. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Pol kappa in a DNA damage-independent manner. Therefore, association of Pol kappa with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18(-/-) transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18(-/-) cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18-deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Pol kappa. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Pol kappa to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.