Ray Alo, Blevins Chessica, Wani Gulzar, Wani Altaf A
Department of Radiology, The Ohio State University, Columbus, Ohio, 43210, United States of America.
Department of Radiology, Department of Molecular and Cellular Biochemistry, James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, Ohio, 43210, United States of America.
PLoS One. 2016 Jul 21;11(7):e0159344. doi: 10.1371/journal.pone.0159344. eCollection 2016.
Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.
细胞周期检查点由ATR和ATM激酶介导，作为对多种DNA损伤的快速早期反应，并最终形成高度协调的信号转导级联反应。此前，我们通过ATR和ATM磷酸化以及募集到紫外线（UVR）诱导的损伤位点，定义了核苷酸切除修复（NER）因子DDB2和XPC在检查点以及ATR/ATM依赖性修复途径中的调节作用。在此，我们剖析了UVR照射后DDB2和XPC介导的ATR和ATM募集及激活的分子机制。我们表明，ATR和ATM的激活以及向UVR诱导损伤的积累不仅依赖于DDB2和XPC，还依赖于NER蛋白XPA，这表明活性NER复合物的组装对于ATR和ATM的募集至关重要。在异步以及G1期停滞的细胞中，损伤位点的ATR和ATM定位以及H2AX磷酸化早在十分钟时就会出现，这表明由ATR和ATM介导的修复和检查点在紫外线照射后早期就开始了。此外，我们的结果表明，ATR和ATM的募集以及H2AX磷酸化在G1期依赖于NER蛋白，但在S期则不然。我们推测，在G1期，UVR诱导的单链DNA缺口或加工后的单链DNA以及结合的NER复合物促进了ATR和ATM的募集。在S期，当UV损伤导致带有长单链DNA的复制叉停滞时，ATR和ATM向这些位点的募集由不同的蛋白质组调节。综上所述，这些结果提供了证据，表明由于存在不同类型的DNA损伤，UVR诱导的ATR和ATM募集及激活在G1期和S期有所不同，这些损伤促进了参与DNA修复和检查点激活过程的不同蛋白质的组装。
