Fundación Instituto Leloir - Instituto de Investigaciones Bioquímicas de Buenos Aires, Consejo de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
Equipe «Labellisée LA LIGUE CONTRE LE CANCER», Laboratoire d'Excellence Toulouse Cancer LABEX TOUCAN - Cancer Research Center of Toulouse, Inserm U1037, CNRS ERL5294, University Paul Sabatier, Toulouse, France.
EMBO J. 2019 Aug 15;38(16):e101284. doi: 10.15252/embj.2018101284. Epub 2019 Jul 11.
The effectiveness of checkpoint kinase 1 (Chk1) inhibitors at killing cancer cells is considered to be fully dependent on their effect on DNA replication initiation. Chk1 inhibition boosts origin firing, presumably limiting the availability of nucleotides and in turn provoking the slowdown and subsequent collapse of forks, thus decreasing cell viability. Here we show that slow fork progression in Chk1-inhibited cells is not an indirect effect of excess new origin firing. Instead, fork slowdown results from the accumulation of replication barriers, whose bypass is impeded by CDK-dependent phosphorylation of the specialized DNA polymerase eta (Polη). Also in contrast to the linear model, the accumulation of DNA damage in Chk1-deficient cells depends on origin density but is largely independent of fork speed. Notwithstanding this, origin dysregulation contributes only mildly to the poor proliferation rates of Chk1-depleted cells. Moreover, elimination of replication barriers by downregulation of helicase components, but not their bypass by Polη, improves cell survival. Our results thus shed light on the molecular basis of the sensitivity of tumors to Chk1 inhibition.
细胞周期检查点激酶 1（Chk1）抑制剂杀死癌细胞的效果被认为完全取决于其对 DNA 复制起始的影响。Chk1 抑制作用会促进起始原点的激活，可能会限制核苷酸的可用性，并反过来引发叉的减速和随后的崩溃，从而降低细胞活力。在这里，我们表明 Chk1 抑制剂处理的细胞中叉的缓慢进展并不是由于过度新的起始原点激活的间接影响。相反，叉减速是由于复制障碍的积累，其被 CDK 依赖性磷酸化的特殊 DNA 聚合酶 eta（Polη）所阻碍。与线性模型相反，Chk1 缺陷细胞中的 DNA 损伤积累取决于起始原点的密度，但在很大程度上与叉的速度无关。尽管如此，起始原点的失调对 Chk1 耗竭细胞的增殖率不良的贡献很小。此外，通过下调解旋酶成分消除复制障碍，但不是通过 Polη 绕过它们，可提高细胞存活率。因此，我们的研究结果阐明了肿瘤对 Chk1 抑制敏感的分子基础。
