發(fā)布時(shí)間：2018-11-20 18:22

【摘要】： Mrell復(fù)合物(MRN復(fù)合物)由Mrell、Nbs 1及Rad50組成，Mrell復(fù)合物在DNA雙鏈損傷修復(fù)，細(xì)胞周期檢驗(yàn)點(diǎn)，維持端粒長度，維持基因組穩(wěn)定性等方面起著重要的作用。目前有一些證據(jù)表明Mrell復(fù)合物在S期中也起到了作用。 本研究主要采用了RNA干擾技術(shù)研究Mrell復(fù)合物在S期的作用。我們發(fā)現(xiàn)Mrell表達(dá)下調(diào)的細(xì)胞具有ATLD病人細(xì)胞的表型之一，即對(duì)離子照射的敏感性增加。Mrell表達(dá)下調(diào)的細(xì)胞對(duì)DNA合成抑制藥物，，如Mitomycin(MMC)、Hydroxyurea(Hu)和Aphidicolin的敏感性增加，相同劑量的藥物導(dǎo)致Mrell表達(dá)下調(diào)的細(xì)胞存活率降低，在使用pMrellrescue質(zhì)�；謴�(fù)了Mrell在細(xì)胞中的表達(dá)后，細(xì)胞對(duì)藥物的敏感性也基本恢復(fù)正常。Mrell缺陷的細(xì)胞在不加任何藥物處理的情況下，其S期的自然進(jìn)程減慢，用pMrellrescue回復(fù)Mrell的表達(dá)后，細(xì)胞的S期進(jìn)程恢復(fù)正常。我們進(jìn)一步探究了Mrell下調(diào)細(xì)胞對(duì)S期藥物敏感性增加和S期自然進(jìn)程減慢的原因，我們通過免疫印跡和免疫熒光的方法檢測(cè)到Mrell表達(dá)下調(diào)的細(xì)胞中的DNA雙鏈損傷(DSBs)的標(biāo)志物γ-H2AX ser139磷酸化水平增高，并在細(xì)胞核中成點(diǎn)狀聚集。而且用LM-PCR進(jìn)一步證明了Mrell下調(diào)的細(xì)胞在復(fù)制的過程中，在遇到發(fā)卡結(jié)構(gòu)時(shí)累積了更多的DSBs，細(xì)胞也因此激活了細(xì)胞周期檢驗(yàn)點(diǎn)蛋白Chk1。此外，目前關(guān)于Mrell核酸酶的作用存在著爭(zhēng)議，有的學(xué)者認(rèn)為其核酸酶活性是冗余的，而另外一些學(xué)者認(rèn)為其核酸酶活性是必須的。我們構(gòu)建了Mrell核酸酶結(jié)構(gòu)域Ⅲ缺陷的突變體，在實(shí)驗(yàn)中我們觀察到轉(zhuǎn)染了突變體的細(xì)胞不但不能回復(fù)細(xì)胞的正常表型，還使細(xì)胞表型異常更加明顯。轉(zhuǎn)染了突變體的細(xì)胞對(duì)S期抑制藥物的敏感性進(jìn)一步增加，S期進(jìn)程更加緩慢，γ-H2AX ser139磷酸化水平增高，并在細(xì)胞核內(nèi)呈更大的點(diǎn)狀聚集，另外在遇到發(fā)卡結(jié)構(gòu)時(shí)同樣累積了大量未修復(fù)的DSBs。 綜上所述，我們證明了Mrell在DNA復(fù)制期中發(fā)揮了作用，Mrell可以修復(fù)DNA復(fù)制過程中產(chǎn)生的DSBs。另外，Mrell的核酸酶活性在這一過程中也發(fā)揮了重要作用，核酸酶活性并非冗余的。我們的研究結(jié)果對(duì)于闡述ATLD及腫瘤細(xì)胞發(fā)生的自發(fā)染色體不穩(wěn)定性的機(jī)理具有一定的理論意義。
[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 緯-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

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