Mre11及其核酸酶结构域Ⅲ在DNA复制中的作用
发布时间：2018-11-20 18:22
【摘要】： Mrell复合物(MRN复合物)由Mrell、Nbs 1及Rad50组成，Mrell复合物在DNA双链损伤修复，细胞周期检验点，维持端粒长度，维持基因组稳定性等方面起着重要的作用。目前有一些证据表明Mrell复合物在S期中也起到了作用。 本研究主要采用了RNA干扰技术研究Mrell复合物在S期的作用。我们发现Mrell表达下调的细胞具有ATLD病人细胞的表型之一，即对离子照射的敏感性增加。Mrell表达下调的细胞对DNA合成抑制药物，，如Mitomycin(MMC)、Hydroxyurea(Hu)和Aphidicolin的敏感性增加，相同剂量的药物导致Mrell表达下调的细胞存活率降低，在使用pMrellrescue质粒恢复了Mrell在细胞中的表达后，细胞对药物的敏感性也基本恢复正常。Mrell缺陷的细胞在不加任何药物处理的情况下，其S期的自然进程减慢，用pMrellrescue回复Mrell的表达后，细胞的S期进程恢复正常。我们进一步探究了Mrell下调细胞对S期药物敏感性增加和S期自然进程减慢的原因，我们通过免疫印迹和免疫荧光的方法检测到Mrell表达下调的细胞中的DNA双链损伤(DSBs)的标志物γ-H2AX ser139磷酸化水平增高，并在细胞核中成点状聚集。而且用LM-PCR进一步证明了Mrell下调的细胞在复制的过程中，在遇到发卡结构时累积了更多的DSBs，细胞也因此激活了细胞周期检验点蛋白Chk1。此外，目前关于Mrell核酸酶的作用存在着争议，有的学者认为其核酸酶活性是冗余的，而另外一些学者认为其核酸酶活性是必须的。我们构建了Mrell核酸酶结构域Ⅲ缺陷的突变体，在实验中我们观察到转染了突变体的细胞不但不能回复细胞的正常表型，还使细胞表型异常更加明显。转染了突变体的细胞对S期抑制药物的敏感性进一步增加，S期进程更加缓慢，γ-H2AX ser139磷酸化水平增高，并在细胞核内呈更大的点状聚集，另外在遇到发卡结构时同样累积了大量未修复的DSBs。 综上所述，我们证明了Mrell在DNA复制期中发挥了作用，Mrell可以修复DNA复制过程中产生的DSBs。另外，Mrell的核酸酶活性在这一过程中也发挥了重要作用，核酸酶活性并非冗余的。我们的研究结果对于阐述ATLD及肿瘤细胞发生的自发染色体不稳定性的机理具有一定的理论意义。
[Abstract]:Mrell complex (MRN complex) is composed of Mrell,Nbs 1 and Rad50. Mrell complex plays an important role in the repair of DNA double strand damage, cell cycle detection point, maintenance of telomere length, and maintenance of genomic stability. There is now some evidence that Mrell complexes also play a role in S-term. In this study, RNA interference technique was used to study the role of Mrell complex in S phase. We found that cells with down-regulated Mrell expression had one of the phenotypes of ATLD patients, that is, increased sensitivity to ion irradiation. Cells with down-regulated Mrell expression were more sensitive to DNA synthesis inhibitors such as Mitomycin (MMC), Hydroxyurea (Hu) and Aphidicolin. The same dose of drugs reduced the survival rate of the cells with down-regulated Mrell expression, and the expression of Mrell in the cells was restored by using the pMrellrescue plasmid. The natural process of S phase of Mrell deficient cells slowed down without any drug treatment, and the S phase process of cells returned to normal when pMrellrescue was used to restore the expression of Mrell. We further explored the reasons for the increased sensitivity of Mrell down-regulated cells to S phase drugs and the slowing down of natural processes in S phase. We detected the increased phosphorylation of 纬-H2AX ser139, a marker of DNA double strand (DSBs) damage, in cells with down-regulated Mrell expression by Western blotting and immunofluorescence. Moreover, LM-PCR further demonstrated that Mrell down-regulated cells accumulated more DSBs, cells in the process of replication when they encountered the hairpin structure, thus activating the cell cycle test point protein Chk1.. In addition, the role of Mrell nuclease is controversial. Some scholars think that its nuclease activity is redundant, while others think that its nuclease activity is necessary. We constructed Mrell nuclease domain 鈪
本文编号：2345635
[Abstract]:Mrell complex (MRN complex) is composed of Mrell,Nbs 1 and Rad50. Mrell complex plays an important role in the repair of DNA double strand damage, cell cycle detection point, maintenance of telomere length, and maintenance of genomic stability. There is now some evidence that Mrell complexes also play a role in S-term. In this study, RNA interference technique was used to study the role of Mrell complex in S phase. We found that cells with down-regulated Mrell expression had one of the phenotypes of ATLD patients, that is, increased sensitivity to ion irradiation. Cells with down-regulated Mrell expression were more sensitive to DNA synthesis inhibitors such as Mitomycin (MMC), Hydroxyurea (Hu) and Aphidicolin. The same dose of drugs reduced the survival rate of the cells with down-regulated Mrell expression, and the expression of Mrell in the cells was restored by using the pMrellrescue plasmid. The natural process of S phase of Mrell deficient cells slowed down without any drug treatment, and the S phase process of cells returned to normal when pMrellrescue was used to restore the expression of Mrell. We further explored the reasons for the increased sensitivity of Mrell down-regulated cells to S phase drugs and the slowing down of natural processes in S phase. We detected the increased phosphorylation of 纬-H2AX ser139, a marker of DNA double strand (DSBs) damage, in cells with down-regulated Mrell expression by Western blotting and immunofluorescence. Moreover, LM-PCR further demonstrated that Mrell down-regulated cells accumulated more DSBs, cells in the process of replication when they encountered the hairpin structure, thus activating the cell cycle test point protein Chk1.. In addition, the role of Mrell nuclease is controversial. Some scholars think that its nuclease activity is redundant, while others think that its nuclease activity is necessary. We constructed Mrell nuclease domain 鈪
本文编号：2345635
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2345635.html
最近更新
教材专著
