The National Laboratory of Protein Engineering and Plant Genetic Engineering, The College of Life Sciences, Peking University, Beijing, China.
Cell. 2012 Jun 8;149(6):1221-32. doi: 10.1016/j.cell.2012.04.030.
When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ∼10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.
当复制叉在受损碱基或核苷酸耗尽时停滞，S 期内检验点确保它们稳定并能够重新启动。在 S 期内检验点缺陷的芽殖酵母中，停滞的叉会崩溃，并且大约 10%形成致病性的鸡脚结构，导致不完全复制和细胞死亡（Lopes 等人，2001；Sogo 等人，2002；Tercero 和 Diffley，2001）。我们使用裂殖酵母报告说，Cds1（Chk2）效应激酶在 S220 上靶向 Dna2，以调节体内和体外染色质中停滞的复制叉与 Dna2 的结合。我们证明 Dna2-S220 磷酸化和 Dna2 的核酸酶活性对于防止叉反转是必需的。与此一致，Dna2 可以有效地切割叉回归的必需前体-回归的领头或滞后链-在模型复制叉上。我们提出 Dna2 对回归的新生链的切割防止叉反转，从而稳定停滞的叉，以在复制应激期间维持基因组稳定性。
