Ewald Brett, Sampath Deepa, Plunkett William
Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston Graduate School of Biomedical Sciences, Houston, Texas 77030, USA.
Cancer Res. 2008 Oct 1;68(19):7947-55. doi: 10.1158/0008-5472.CAN-08-0971.
The Mre11-Rad50-Nbs1 complex and autophosphorylated Ser(1981)-ATM are involved in recognizing and repairing DNA damage, such as double-strand breaks (DSB). However, the role of these factors in response to stalled replication forks is not clear. Nucleoside analogues are agents that are incorporated into DNA during replication, which cause stalling of replication forks. The molecular mechanisms that sense these events may signal for DNA repair and contribute to survival but are poorly understood. Cellular responses to both DSBs and stalled replication forks are marked by H2AX phosphorylation on Ser(139) (gamma-H2AX), which forms nuclear foci at sites of DNA damage. Here, concentrations of the nucleoside analogues 1-beta-d-arabinofuranosylcytosine (cytarabine; ara-C), gemcitabine, and troxacitabine, which inhibited DNA synthesis by 90% within 2 hours, were determined for each agent. Using gamma-H2AX as a marker for changes in chromatin structure, we show that Mre11, Rad50, Nbs1, and phosphorylated ATM respond to nucleoside analogue-induced stalled replication forks by forming nuclear foci that colocalize with gamma-H2AX within 2 hours. Because neither DSBs nor single-strand breaks were detectable after nucleoside analogue exposure, we conclude that this molecular response is not due to the presence of DNA breaks. Deficiencies in ATM, Mre11, or Rad50 led to a 2- to 5-fold increase in clonogenic sensitization to gemcitabine, whereas Nbs1 and H2AX deficiency did not affect reproductive growth. Taken together, these results suggest that ATM, Mre11, and Rad50 are required for survival after replication fork stalling, whereas Nbs1 and H2AX are inconsequential.
Mre11-Rad50-Nbs1复合物和自磷酸化的Ser(1981)-ATM参与识别和修复DNA损伤，如双链断裂（DSB）。然而，这些因子在应对停滞的复制叉时所起的作用尚不清楚。核苷类似物是在复制过程中掺入DNA的试剂，可导致复制叉停滞。感知这些事件的分子机制可能会发出DNA修复信号并有助于细胞存活，但目前了解甚少。细胞对DSB和停滞的复制叉的反应均以Ser(139)位点的H2AX磷酸化（γ-H2AX）为特征，γ-H2AX在DNA损伤位点形成核灶。在此，测定了每种试剂的核苷类似物1-β-D-阿拉伯呋喃糖基胞嘧啶（阿糖胞苷；ara-C）、吉西他滨和曲西他滨的浓度，这些试剂在2小时内可抑制DNA合成达90%。使用γ-H2AX作为染色质结构变化的标志物，我们发现Mre11、Rad50、Nbs1和磷酸化的ATM通过在2小时内形成与γ-H2AX共定位的核灶来响应核苷类似物诱导的停滞复制叉。由于在核苷类似物暴露后未检测到DSB或单链断裂，我们得出结论，这种分子反应不是由于DNA断裂的存在。ATM、Mre11或Rad50的缺陷导致对吉西他滨的克隆形成敏感性增加2至5倍，而Nbs1和H2AX缺陷不影响生殖生长。综上所述，这些结果表明，ATM、Mre11和Rad50是复制叉停滞后细胞存活所必需的，而Nbs1和H2AX则无关紧要。
