Equipe Labellisée La Ligue Contre le Cancer 2013, INSERM UMR 1037, CNRS ERL 505294, CRCT (Cancer Research Center of Toulouse), Toulouse, France.
EMBO J. 2013 Jul 31;32(15):2172-85. doi: 10.1038/emboj.2013.148. Epub 2013 Jun 25.
Formation of primed single-stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR-mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA-mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y-family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9-1-1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9-1-1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells.
在停滞的复制叉处形成引发的单链 DNA 会触发复制检查点信号级联的激活，导致 ATR 介导的 Chk1 蛋白激酶磷酸化，从而防止基因组不稳定性。通过在人细胞中使用 siRNA 介导的耗尽以及在非洲爪蟾卵提取物中的免疫耗竭和重建实验，我们报告说 Y 家族跨损伤（TLS）DNA 聚合酶 κ（Pol κ）有助于复制检查点反应，并且在复制应激后恢复是必需的。我们发现 Pol κ 参与在停滞的叉处合成短的 DNA 中间体，促进 9-1-1 检查点夹的募集。此外，我们表明 Pol κ 与 9-1-1 复合物的 Rad9 亚基相互作用。最后，我们表明 Pol κ 的这种新的检查点功能对于未受应激的人细胞中的基因组稳定性和细胞增殖的维持是必需的。
