Department of Genetics, Microbiology, and Toxicology, Stockholm University, S-10691, Stockholm, Sweden.
Nucleic Acids Res. 2012 Sep 1;40(17):8440-8. doi: 10.1093/nar/gks646. Epub 2012 Jun 29.
Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine--inhibiting ATM and ATR amongst other kinases--is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase η (Polη), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.
紫外线 (UV) 诱导的 DNA 损伤会导致延伸中的复制叉有效地停滞。研究表明，羟基脲处理后，检查点激酶 CHK1 可以稳定复制叉。因此，我们想测试非特异性激酶抑制剂咖啡因（抑制 ATM 和 ATR 等激酶）是否会导致 UV 敏感性增加，原因是否是无法激活 CHK1 激酶来稳定复制结构。为此，我们使用缺乏聚合酶 η (Polη) 的细胞进行实验，Polη 是一种跨损伤合成聚合酶，能够正确绕过 UV 诱导的顺式 - 顺式 TT 嘧啶二聚体，该二聚体阻止复制。这些细胞在 UV 暴露后会在正在进行的复制叉后面积累缺口。我们证明，咖啡因和 CHK1 抑制剂同样会在 UV 处理后延迟连续复制叉的延伸。有趣的是，我们发现在用咖啡因处理时，细胞的 UV 敏感性比用 CHK1 抑制剂处理时更为明显。此外，我们还发现，在用咖啡因处理的 UV 照射细胞中，复制结构的崩溃更为明显，而在用 CHK1 抑制剂处理的细胞中则没有。这表明 CHK1 活性不是稳定 UV 损伤 DNA 复制过程中产生的缺口所必需的。这些数据表明，在 UV 诱导的 DNA 损伤处，复制叉的延伸和稳定是两种不同的机制，而 CHK1 仅参与复制叉的延伸。
