本文選題：非經(jīng)典末端連接 + 抗體類別轉(zhuǎn)換�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：保持基因組的完整性和穩(wěn)定性對于生物體的存活是至關(guān)重要的。DNA雙鏈斷裂(DSB)是所有DNA損傷中最為嚴(yán)重的一種,可以通過外界因素(如電離輻射和化學(xué)誘變劑)和內(nèi)源因素(如內(nèi)源性DNA氧化以及DNA復(fù)制壓力)產(chǎn)生。哺乳動物體內(nèi)的DSB修復(fù)方式主要有兩種:依賴于同源序列的同源重組(HR)和不依賴于同源序列的非同源末端連接(NHEJ)。近幾年的研究發(fā)現(xiàn),在經(jīng)典的非同源末端連接(C-NHEJ)缺失的情況下,DSB末端仍然可以被有效地修復(fù),預(yù)示著非經(jīng)典末端連接(A-EJ)修復(fù)通路的存在。A-EJ活性已經(jīng)在多種系統(tǒng)中得到證實,特別是在C-NHEJ核心因子(如DNA連接酶IV)缺陷的B細胞中,A-EJ介導(dǎo)的IgH基因座位置的抗體類別轉(zhuǎn)換(CSR)效率是野生型細胞的50%。大量研究已經(jīng)證實A-EJ的顯著特點是對微同源序列(MH)的偏好性,以及在染色體轉(zhuǎn)位過程中的重要作用。雖然目前已經(jīng)有多個蛋白因子被報道參與A-EJ過程,但是其結(jié)果往往存在爭議,特別是對參與A-EJ修復(fù)的DNA連接酶的研究還很不清楚。本研究利用CRISPR/Cas9系統(tǒng),在Lig4缺陷的小鼠B細胞系CH12F3中完全敲除Lig1或者細胞核內(nèi)的Lig3,建立只含有單個DNA連接酶(分別為Lig3和Lig1)的CH12F3細胞系。令人驚訝的是,只含有Lig1或Lig3的CH12F3細胞仍然具有A-EJ活性,可以有效地催化IgH基因座的CSR過程以及同一條染色體上由CRISPR/Cas9產(chǎn)生的DSB之間的染色體缺失;并且其活性與Lig1/Lig3同時存在的細胞沒有明顯差別。然而,在催化染色體轉(zhuǎn)位方面,含有Lig1和Lig3的復(fù)合物顯示出不同的特性。完全敲除細胞核中的Lig3,Lig4-/-細胞的染色體轉(zhuǎn)位明顯減少,而在完全敲除Lig1的細胞中則沒有這種變化。另外,染色體轉(zhuǎn)位連接處微同源序列的分析揭示了不同DNA連接酶催化活性的特異性。這些數(shù)據(jù)表明存在多種含DNA連接酶的復(fù)合物用以催化A-EJ過程。另外,本研究在小鼠B細胞中建立了 CRISPR/Cas9高通量篩選方法,在野生型和Lig4缺陷的細胞中篩選參與CSR過程中DSB修復(fù)的基因。首先建立了 Cas9穩(wěn)定表達的CH12F3細胞系(野生型和Lig4缺陷型),并對sgRNA篩選的整個過程進行了優(yōu)化。利用優(yōu)化的實驗條件,在Cas9穩(wěn)定表達的WT和Lig4-/-CH12F3細胞中進行sgRNA文庫的高通量篩選。對不同細胞群中的sgRNA進行高通量測序,通過MAGeCK分析方法,篩選IgA陽性細胞群中顯著減少和IgA/IgM雙陰性細胞群中顯著增加的基因。在WT細胞中,篩選得到了 DNA-PKcs和Lig4等核心C-NHEJ蛋白因子,以及一些已知的參與細胞因子介導(dǎo)的CSR過程的基因。對其它未知功能的候選基因進行檢測,發(fā)現(xiàn)Brd9和Fbxo28缺失嚴(yán)重影響細胞因子介導(dǎo)的CSR。目前正在對這一結(jié)果進行進一步驗證。同時,基于Cas9/sgRNA介導(dǎo)的CSR模型的高通量篩選也正在進行。
[Abstract]:Maintaining the integrity and stability of the genome is essential to the survival of organisms. DSBs are the most serious of all DNA damage.It can be produced by external factors (such as ionizing radiation and chemical mutagens) and endogenous factors (such as endogenous DNA oxidation and DNA replication pressure).There are two main ways of DSB repair in mammals: homologous recombination of DSB which is dependent on the homologous sequence and non-homologous terminal ligation of DSB that does not depend on the homologous sequence.In recent years, it has been found that the end of DSB can still be repaired effectively in the absence of classical non-homologous terminal junctions (C-NHEJEJB), indicating that the existence of the repair pathway of non-classical terminal junctions (EJA-EJA-EJB) has been confirmed in many systems.Especially in B cells with C-NHEJ core factors (such as DNA ligase IV-deficient), the efficiency of A-EJ mediated antibody class conversion of IgH locus is 50% of wild-type cells.A large number of studies have confirmed that A-EJ is characterized by its preference for microhomologous sequences and its important role in chromosome translocation.Although several protein factors have been reported to be involved in the A-EJ process, the results are often controversial, especially the study of the DNA ligase involved in A-EJ repair is still unclear.In this study, CRISPR/Cas9 system was used to completely knockout Lig1 or Lig3 in Lig4 deficient mouse B cell line CH12F3, and to establish CH12F3 cell line containing only a single DNA ligase (Lig3 and Lig1, respectively).Surprisingly, CH12F3 cells containing only Lig1 or Lig3 still have A-EJ activity, which can effectively catalyze the CSR process of IgH locus and the deletion of DSB between DSB produced by CRISPR/Cas9 on the same chromosome.And its activity is not significantly different from that of Lig1/Lig3 cells.However, complexes containing Lig1 and Lig3 exhibit different characteristics in catalytic chromosomal translocation.The chromosomal translocation of Lig3 + Lig4-r- cells in the complete knockout nucleus was significantly reduced, but there was no such change in the cells with complete knockout of Lig1.In addition, the specific catalytic activity of different DNA ligases was revealed by the analysis of the microhomologous sequences at the chromosomal translocation junctions.These data indicate that there are many complexes containing DNA ligase to catalyze the A-EJ process.In addition, a high-throughput CRISPR/Cas9 screening method was established in mouse B cells to screen genes involved in DSB repair in wild type and Lig4 deficient cells.Firstly, CH12F3 cell lines (wild type and Lig4 deficient type) expressing Cas9 stably were established, and the whole process of sgRNA screening was optimized.High throughput screening of sgRNA library was carried out in WT and Lig4-/-CH12F3 cells stably expressed by Cas9 using optimized experimental conditions.The high throughput sequencing of sgRNA in different cell groups was carried out. The genes in IgA positive cell group and IgA/IgM double negative cell group were significantly decreased and significantly increased by MAGeCK analysis.In WT cells, core C-NHEJ protein factors such as DNA-PKcs and Lig4, as well as some known genes involved in the cytokine mediated CSR process, were screened.Candidate genes of other unknown functions were detected and the deletion of Brd9 and Fbxo28 seriously affected cytokine mediated CSRs.This result is currently being further validated.At the same time, high throughput screening of CSR model based on Cas9/sgRNA is also underway.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R730.5

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