Biotech Research and Innovation Centre, University of Copenhagen, 2200 Copenhagen N, Denmark.
J Cell Biol. 2010 Mar 8;188(5):629-38. doi: 10.1083/jcb.200905059. Epub 2010 Mar 1.
Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication.
基因组完整性的维持对细胞至关重要。为了鉴定基因组完整性的关键调控因子，我们筛选了一个带有激酶组小干扰 RNA 文库的人类细胞系。有丝分裂进入的主要调节因子 WEE1 和 CHK1 是鉴定出的基因之一。这两种激酶都是 CDK1 和 CDK2 的重要负调控因子。令人惊讶的是，WEE1 的耗竭会迅速诱导新复制 DNA 中 S 期的 DNA 损伤，同时伴随着单链 DNA 的明显增加。这种 DNA 损伤依赖于 CDK1 和 CDK2 以及复制蛋白 MCM2 和 CDT1，但不依赖于 CDC25A。相反，CHK1 抑制后的 DNA 损伤高度依赖于 CDC25A。此外，CHK1 耗竭细胞的增殖能力较差，通过共耗竭 CDC25A 可得到显著改善。我们得出结论，有丝分裂激酶 WEE1 和 CHK1 共同维持正常 DNA 复制过程中 Cdk 活性的细胞平衡控制，这对于防止复制过程中产生有害的 DNA 损伤至关重要。
