NONO regulates the intra-S-phase checkpoint in response to UV radiation
NONO regulates the intra-S-phase checkpoint in response to UV radiation
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DOI：
10.1038/onc.2015.107
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发表时间：
2016-02-04
期刊：
影响因子：
8
通讯作者：
Pentimalli, F.
中科院分区：
文献类型：
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作者：
Alfano, L.;Costa, C.;Pentimalli, F.
皮肤癌的主要风险因素是紫外线(UV)暴露，它会导致DNA损伤。细胞通过激活S期内检查点来响应紫外线诱导的DNA损伤，该检查点防止复制叉崩溃、延迟起始激发并稳定脆弱部位。最近发现，54 kDa的多功能蛋白NONO参与了非同源末端连接的DNA修复过程和多聚ADP-核糖聚合酶1的激活。有趣的是，NONO在几种肿瘤类型中都发生了突变，并成为黑色素瘤发生和发展的关键因素。因此，我们开始评估NONO是否参与了对紫外线辐射的DNA损伤反应。我们建立了NONO沉默的HeLa细胞克隆，发现缺乏NONO会降低细胞的生长速度。然后，我们用紫外线照射对NONO沉默的细胞进行挑战，发现与对照细胞相比，NONO沉默的细胞继续合成DNA，未能阻止新的起源激活，并损害CHK1S345的磷酸化，显示出缺陷的检查点激活。一直以来，NONO存在于紫外线诱导的DNA损伤部位，定位于RAD9焦点。为了定位NONO在DNA损伤反应级联反应中的位置，我们分析了不同的S期检查点介质在染色质上的负载，发现NONO有助于负载作用于ATM上游的拓扑异构酶II结合蛋白1和RAD3相关的激酶活性。值得注意的是，NONO的重新表达，通过抗SH的mRNA，挽救了NONO沉默的细胞中CHK1S345的磷酸化。有趣的是，在黑色素瘤细胞系中，NONO沉默也影响细胞对紫外线辐射的反应。总体而言，我们的数据揭示了NONO在介导细胞对紫外线诱导的DNA损伤的反应中的新角色。
The main risk factor for skin cancer is ultraviolet (UV) exposure, which causes DNA damage. Cells respond to UV-induced DNA damage by activating the intra-S-phase checkpoint, which prevents replication fork collapse, late origin firing and stabilizes fragile sites. Recently, the 54-kDa multifunctional protein NONO was found to be involved in the non-homologous end-joining DNA repair process and in poly ADP-ribose polymerase 1 activation. Interestingly, NONO is mutated in several tumour types and emerged as a crucial factor underlying both melanoma development and progression. Therefore, we set out to evaluate whether NONO could be involved in the DNA-damage response to UV radiations. We generated NONO-silenced HeLa cell clones and found that lack of NONO decreased cell growth rate. Then, we challenged NONO-silenced cells with exposure to UV radiations and found that NONO-silenced cells, compared with control cells, continued to synthesize DNA, failed to block new origin firing and impaired CHK1S345 phosphorylation showing a defective checkpoint activation. Consistently, NONO is present at the sites of UV-induced DNA damage where it localizes to RAD9 foci. To position NONO in the DNA-damage response cascade, we analysed the loading onto chromatin of various intra-S-phase checkpoint mediators and found that NONO favours the loading of topoisomerase II-binding protein 1 acting upstream of the ATM and Rad3-related kinase activity. Strikingly, re-expression of NONO, through an sh-resistant mRNA, rescued CHK1S345 phosphorylation in NONO-silenced cells. Interestingly, NONO silencing affected cell response to UV radiations also in a melanoma cell line. Overall, our data uncover a new role for NONO in mediating the cellular response to UV-induced DNA damage.