DNA-PKcs-FACT信号通路参与DNA损伤诱导的染色质重塑

The repair of DNA double-strand breaks (DSBs),which are the most severe DNA damage induced by irradiation, involves recruitment of repair and checkpoint signaling machineries to DSB site. However, nucleosomes and chromatin architecture surrounding the DSB limit the ability of DNA-damage response to access and repair breaks. Therefore, recruitment of chromatin modifying factor to promote the formation of open, relaxed chromatin structures at damage site is necessary for DNA DSBs repair. How the DNA DSBs sensor couple the chromatin-remodeling signal has not been fully elucidated. DNA-PKcs is well known as a critical component involving the nonhomologous end joining pathway (NHEJ) of DNA DSBs repair. Here, our preliminary data demonstrate that DNA-PKcs can directly interact with histone chaperone FACT complex via its T2609 phosphorylation cluster. Moreover, DNA-PKcs phosphorylates both components of the FACT complex, SSRP1 and Spt16, in vitro. Recent work revealed that FACT complex participates in H2A-H2B exchange process around UV light-induced DNA damage site. On the basis of these results and our preliminary data, we will further explore the function of DNA-PKcs-FACT signal pathway in irradiation induced chromatin remodeling, including: 1) Whether DNA-PKcs play an important role in DNA damage-induced chromatin remodeling through regulating FACT-mediated histone exchange; FACT also regulate homologous recombination via its chromatin remodeling function, 2) therefore, we will further identify if the phosphorylation of FACT,which mediated by DNA-PKcs, affects the DSBs repair pathway choice; 3) Whether DNA-PKcs-FACT pathway affects irradiation-induced large scale heterochromatin relaxation. This investigation may be of value in the development of radioprotective compounds against DNA-PKcs-FACT signal pathway.

真核生物高度包装的染色质结构限制了DNA损伤反应蛋白与DNA接近。DNA 双链断裂损伤感受器与染色质重塑信号偶联机制尚不清楚。前期工作显示DNA-PKcs与FACT直接相互作用，并可在体外磷酸化FACT复合物。在此工作基础上，本课题将进一步探讨DNA-PKcs-FACT信号通路在辐射诱发染色质重塑中的功能，具体内容包括：1）DNA-PKcs是否通过调控FACT介导的组蛋白交换调节辐射诱发的染色质动态变化；由于FACT对染色质调控作用影响HR修复途径，因此，另一内容为：2）DNA-PKcs介导的FACT磷酸化是否影响DNA 双链断裂修复途径选择；3）DNA-PKcs-FACT通路是否影响照射诱发的大尺度异染色质松弛。本研究将为以DNA-PKcs-FACT通路为靶的抗放射药物研究提供依据。

本研究发现FACT复合物在DNA损伤修复中的重要作用，揭示了DNA-PKcs与FACT复合物间的互作关系。.(1) 在本研究中我们发现FACT复合物缺失细胞对电离辐射和化疗药物敏感性升高，FACT复合物基因在结直肠癌和食管鳞状细胞癌中表达水平升高，更为重要的是FACT复合物表达在上述肿瘤中与DNA-PKcs基因表达呈正相关。.（2）本课题研究基础中发现DNA-PKcs Thr2609磷酸化集簇区段与FACT复合物存在相互作用，DNA-PKcs Thr2609磷酸化集簇对于DNA损伤修复和组织功能完整性至关重要。DNA-PKcs3A/3A小鼠造血干细胞死亡，进一步研究发现DNA-PKcs3A/3A小鼠MEFs细胞具有较高的恶性化，与对照细胞相比DNA-PKcs3A/3A MEFs细胞HR修复能力降低、端粒融合比率增高，结果显示DNA-PKcs Thr2609集簇区段对于HR修复、端粒稳定性、基因组稳定性和抑制肿瘤发生非常重要。.（3）本研究发现小分子物质VND3207通过激活NDA-PKcs和抑制p53-Noxa介导的隐窝细胞凋亡缓解电离辐射诱发的肠损伤。.