参与A-EJ DNA修复通路的相关蛋白的研究

论文总字数：25302字

摘 要

DNA双链断裂（double-strand break，DSB）是在外源辐射或细胞内活性氧等代谢物的作用下产生的一种DNA损伤，其直接破坏了染色体的物理连续性，如不能及时修复，可能会因为DNA的损伤而造成细胞衰老、凋亡、癌变等。DSBs的修复主要有同源重组(homologous recombination，HR）和非同源末端连接（non-homologous end joining，C-NHEJ）等修复方式。近年来，越来越多的证据表明在C-NHEJ之外仍然存在一条或多条末端连接通路，即Alternative end joining（A-EJ，替代末端连接）。A-EJ DSB修复的机制和潜在的在生理学功能仍然存在许多的疑点和空白。Poly-ADP-ribose polymerase (PARP )家族是一个已知的在单链DNA断裂修复中具有重要作用的因子。有研究发现其在Ku70或DNA-PKcs缺失的细胞中参与了DSBs的末端连接，认为其能够和Ku竞争并结合到DSBs上，进而引导DSBs的修复。但对于PARP具体的作用及机制仍然不是十分不明确。本课题通过CRISPR-Cas9技术在NHEJ缺陷的仓鼠B细胞（Lig4-null CH12）中分别敲除了PARP1和PARP2，得到了LIG4^-/-PARP1^-/- CH12细胞系和LIG4^-/-PARP2^-/- CH12细胞系。并通过CD-40，TGF-β，IL-4等细胞因子刺激促使细胞发生类别转换重组（Class Switch Recombination，CSR）。通过检测IgA阳性细胞，我们发现在LIG4缺陷的背景下分别敲除PARP1和PARP2后，细胞的CSR水平并未发生明显的改变。而且令人困惑的是，PARP1的敲除反而使得CSR水平发生了微弱的增加。这很可能是因为PARP1和PARP2功能的冗余和重叠所造成的。当其中一者缺失时，另外一者可以回补甚至代替DSB的修复活性，使得CSR活性维持在一个相对稳定的水平。这些结果表明获得PARP1和PARP2双敲除的细胞系对于进一步阐释A-EJ机理是非常重要的。

关键字：替代末端连接（A-EJ）；PARP；类型转换重组；

Exploration of PARP1 and PARP2 function in Alternative End Joining

Abstract

Double strand breaks (DSBs) is a kind of DNA lesion, which result from environmental factors such as various forms of radiation or from products of cellular metabolism, for example, reactive oxygen species. DSBs destroy the physical consistency of chromosome. If unrepaired, the DNA damage can lead to cellular aging, death, cancer and so on. There are two main pathways to repair DSBs. One is homologous recombination (HR), another is non-homologous end joining (NHEJ). In recent years, evidence for an end-joining pathway (or pathways) in addition to C-NHEJ has emerged, namely alternative end joining (A-EJ) pathway. DSBs repair mechanism and physiological functions of A-EJ are still investigating. Poly-ADP-ribose polymerase (PARP ) family is an known factor that function in the repair of single strand DNA breaks. Some research showed that PARP can take part in the end joining of DSBs in the Ku70 or DNA-PKcs deficient cells. And someone thought that PARP complete with Ku to bind to DSBs and then lead the DSBs repair. But the concrete function and mechanism of PARP is still obscure. This project took advantage of CRISPR-Cas9 technology to knock out PARP1 and PARP2 gene respectively in NHEJ-lacking CH12 cell line (lig4-null CH12). And we got the LIG4^-/-PARP1^-/- CH12 cell line and LIG4^-/-PARP2^-/- CH12 cell line. In the stimulation of CD40, TGF-β and IL-4, cells were promoted to process CSR (Class Switch Recombination). After detection of IgA positive cells, we found that the CSR level didn’t appear an obvious change after the deletion of PARP1 and PARP2 in the LIG4^-/- cells. And it’s confusing that there is a slight increase of CSR level when PARP1 was knocked out in LIG4^-/- CH12 cells. These results probably results from the function redundancy and overlap between PARP1 and PARP2. When either one is lacking, the other one would back up the repair activity of DSBs, which keep the CSR level in a relatively stable state. Altogether, these results showed that PARP1 and PARP2 double knockout cell lines is essential for the elucidation of A-EJ mechanism.

Key words：alternative end joining; PARP; class switch recombination;

目 录

摘要 I

Abstract II

1. 绪论 2

2. 材料与方法 4

2.1 质粒 4

2.2 sgRNA的设计和克隆 5

2.3 细胞培养 7

2.4 质粒电穿孔转染 8

2.5 质粒的扩增、抽提 8

2.6 基因组DNA的提取 11

2.7 目的片段的PCR 13

2.8 PCR产物的割胶回收纯化 13

2.9 Surveyor法检测sgRNA活性 14

2.10 核酸电泳分析 15

2.11 IgA染色 16

3. 结果与讨论 16

3.1 sgRNA的设计与克隆 16

3.2 质粒电穿孔转染CH12细胞 17

3.3 Surveyor法检测sgRNA的活性 19

3.4 阳性克隆的筛选 20

3.5 PARP1^-/-、PARP2^-/-、POLQ^-/-细胞的筛选 29

3.6 CSR检测 32

4. 结论 37

致谢 38

参考文献 39

1. 绪论

我们生活在一个充满DNA损伤因子的环境之中，每时每刻我们的遗传物质都受到各种有害物质带来的DNA损伤。这种损伤主要是在外源环境诸如各类辐射（紫外，电离辐射等）或细胞内代谢产物如活性氧等物质的作用下产生^[1]。如果不能及时正确地修复，DNA的损伤会导致细胞周期的停滞或细胞的死亡。而不恰当的修复往往也会造成染色体的异常，进而影响DNA的复制和基因的表达。在众多的DNA损伤中，DNA双链断裂（DNA Strand Breaks，DSBs）的损害尤为严重。因其直接破坏了染色体的物理连续性，如不能及时修复，可能会因染色体的丢失而造成细胞衰老、凋亡、癌变等。

真核细胞中能够识别并修复DSBs的途径主要有俩种，同源重组（homologous recombination，HR）和经典的非同源末端连接（classical non-homologous end joining，C-NHEJ）^[2]。HR修复DSBs需要通过同源序列作为模板，这种模板通常由姐妹染色单体和同源染色体来提供，因此HR的修复被局限在DNA复制后的S期和G2期^[3]。NHEJ作为一种不依赖于同源序列的修复方式，可以整个细胞周期中发挥作用，是一种基于末端结构的修复方式，与HR相比NHEJ由于在修复过程中引入了小的删除和插入可能会造成一定的突变，但是在很大程度上防止了DSBs周围大片段DNA的丢失，保证了基因组的稳定性^[4]。

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