Abeyta Antonio, Castella Maria, Jacquemont Celine, Taniguchi Toshiyasu
a Divisions of Human Biology and Public Health Sciences , Fred Hutchinson Cancer Research Center , Seattle , WA , USA.
b Molecular & Cellular Biology Graduate Program , University of Washington , Seattle , WA , USA.
Cell Cycle. 2017 Feb 16;16(4):335-347. doi: 10.1080/15384101.2016.1259038. Epub 2016 Nov 28.
Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.
同源重组所必需的蛋白质在DNA双链断裂修复、DNA链间交联和复制叉稳定性方面发挥着关键作用。同源重组缺陷在癌症发展以及这些癌症对化疗的敏感性方面也起着关键作用。RAD51是同源重组和复制叉保护的必需因子，在DNA损伤位点积累并形成免疫细胞化学可检测的核灶。为了鉴定可能调节RAD51定位到DNA损伤位点的激酶，我们进行了一项人类激酶组siRNA文库筛选，以DNA损伤诱导的RAD51灶形成作为读数。我们发现，NIMA家族激酶成员NEK8是高效DNA损伤诱导的RAD51灶形成所必需的。有趣的是，在小鼠胚胎成纤维细胞中敲除Nek8会导致细胞对复制抑制剂羟基脲以及对ATR激酶抑制的敏感性增加。此外，在用羟基脲使复制停滞之后，NEK8是适当的复制叉保护所必需的。在NEK8缺失的细胞和Nek8敲除的细胞中，RAD51加载到染色质上的量减少。单分子DNA纤维分析显示，在用羟基脲处理后，在没有NEK8的情况下新生DNA片段会被降解。与此一致的是，Nek8敲除的细胞在用羟基脲处理后显示出染色体断裂增加。因此，NEK8通过调节DNA修复和复制叉保护蛋白RAD51在复制叉稳定性方面发挥着关键作用。
