ABSTRACT

CST (CTC1-STN1-TEN1) is a heterotrimeric, RPA-like complex that binds to single-stranded DNA (ssDNA) and functions in the replication of telomeric and non-telomeric DNA. Previous studies demonstrated that deletion of CTC1 results in decreased cell proliferation and telomere DNA damage signaling. However, a detailed analysis of the consequences of conditional CTC1 knockout (KO) has not been fully elucidated. Here, we investigated the effects of CTC1 KO on cell cycle progression, genome-wide replication and activation of the DNA damage response. Consistent with previous findings, we demonstrate that CTC1 KO results in decreased cell proliferation, G2 arrest and RPA-bound telomeric ssDNA. However, despite the increased levels of telomeric RPA-ssDNA, global ATR-dependent CHK1 and p53 phosphorylation was not detected in CTC1 KO cells. Nevertheless, we show that RPA-ssDNA does activate ATR, leading to the phosphorylation of RPA and autophosphorylation of ATR. Further analysis determined that inactivation of ATR, but not CHK1 or ATM, suppressed the accumulation of G2 arrested cells and phosphorylated RPA following CTC1 removal. These results suggest that ATR is localized and active at telomeres but is unable to elicit a global checkpoint response through CHK1. Furthermore, CTC1 KO inhibited CHK1 phosphorylation following hydroxyurea-induced replication stress. Additional studies revealed that this suppression of CHK1 phosphorylation, following replication stress, is caused by decreased levels of the ATR activator TopBP1. Overall, our results identify CST as a novel regulator of the ATR-CHK1 pathway.

 抽象的

CST (CTC1-STN1-TEN1) 是一种异三聚体、RPA 样复合物，可与单链 DNA (ssDNA) 结合，并在端粒和非端粒 DNA 的复制中发挥作用。先前的研究表明，CTC1 的缺失会导致细胞增殖和端粒 DNA 损伤信号传导减少。然而，对条件性 CTC1 敲除 (KO) 后果的详细分析尚未完全阐明。在这里，我们研究了 CTC1 KO 对细胞周期进程、全基因组复制和 DNA 损伤反应激活的影响。与之前的研究结果一致，我们证明 CTC1 KO 会导致细胞增殖、G2 停滞和 RPA 结合端粒 ssDNA 减少。然而，尽管端粒 RPA-ssDNA 水平增加，但在 CTC1 KO 细胞中并未检测到全局 ATR 依赖性 CHK1 和 p53 磷酸化。尽管如此，我们发现 RPA-ssDNA 确实激活 ATR，导致 RPA 磷酸化和 ATR 自身磷酸化。进一步的分析确定，ATR 的失活（而非 CHK1 或 ATM）抑制了 CTC1 去除后 G2 停滞细胞和磷酸化 RPA 的积累。这些结果表明，ATR 在端粒上是局部且活跃的，但无法通过 CHK1 引发全局检查点反应。此外，CTC1 KO 在羟基脲诱导的复制应激后抑制 CHK1 磷酸化。其他研究表明，复制应激后 CHK1 磷酸化的抑制是由 ATR 激活剂 TopBP1 水平降低引起的。总的来说，我们的结果确定 CST 是 ATR-CHK1 通路的新型调节剂。