论文总字数：27758字

摘 要

Dcn1蛋白是真核细胞中蛋白质类泛素化（neddylation）修饰途径的E3连接酶，真核细胞cullin蛋白在neddylation系统中作为脚手架蛋白，复制叉相关的Cul4蛋白极有可能是与Dcn1蛋白相互作用的候选cullin蛋白，通过调节染色体黏连的建立与维系来影响有丝分裂M期染色体分离的忠实性。近年酵母研究发现Dcn1缺失细胞株（dcn1△）在M期染色体分离出现差错，而且此异常与染色体分离必需蛋白如黏连蛋白cohesin等密切相关，因此解析生理条件下Dcn1与胞内其它蛋白的相互作用具有重要的生物学意义和医学前景。基于此，本项目通过分子生物学方法分离出目标蛋白，为揭示Dcn1经由Cul4蛋白的类泛素化修饰而调控染色体分离的机制研究奠定基础。

本项目在同源重组，利用醋酸锂法制备酵母感受态细胞（野生型及dcn1缺失型），并将带有同源臂的含Myc-tag的DNA片段转化并整合至酵母的基因组上，使酵母细胞的Cul4蛋白的C-末端标记上Myc-tag，然后提取对数期中期的酵母细胞裂解物，通过SDS-PAGE分离其Cul4-Myc融合蛋白，以便为后期研究酵母Dcn1蛋白对Cul4的类泛素化状态的影响奠定技术基础。

关键词：类泛素修饰，Dcn1，Cul4

Abstract

Dcn1 protein is an E3 ligase of the protein neddylation modification pathway in eukaryotic cells. The cullin protein acts as a scaffolding protein in the neddylation system. The replication fork -related Cul4 protein is most likely a candidate cullin protein that interacts with Dcn1 protein, and affects the fidelity of mitotic chromosome separation by regulating the establishment and maintenance of chromosome cohesion. Studies in fission yeast have found that Dcn1-deficient cell line (dcn1△) is dysfunctional in mitosis (M phase) chromosome segregation, and this abnormality is closely related to chromosome-segregation key proteins such as cohesin. Therefore, the protein-protein interaction between Dcn1 and its interacting partners under physiological conditions has important biological and medical prospects. In light of this, this project isolates the target protein by molecular biological methods, to lay the foundation for revealing that Dcn1 may regulates chromosome segregation via neddylation of Cul4 protein.

Based on the theory of homologous recombination, this project, via acetate lithium procotol, prepared yeast competent cells (wild type and dcn1 deletion type fission yeast), and transformed into yeast cells Myc-tag-containing DNA fragments with homologous arms, which were integrated into the spcific sites of the yeast genomic DNA. In this way, the C-terminus of the Cul4 protein was tagged with the Myc-epitope. The expressed proteins in the lysates of yeast cells at mid-log phase, were separated by SDS-PAGE. This study would lay the foundation for further elucidating the effect of yeast Dcn1 protein on the neddylation state of Cul4.

KEY WORDS: neddylation, Dcn1, Cul4

目 录

摘要 I

Abstract II

第一章 类泛素化修饰简介 2

1.1 类泛素化修饰的基本过程 2

1.2 类泛素化修饰的相关酶类 2

1.2.1 NEDD8活化酶(E1) 3

1.2.2 NEDD8结合酶(E2) 3

1.2.3 NEDD8连接酶(E3) 3

1.2.4 Deneddylation及前体加工酶 3

1.3 类泛素化修饰的底物 4

1.3.1 Cullin家族 4

1.3.2 其他底物 4

1.4 类泛素化修饰的生物学功能 4

1.4.1 调节蛋白质间的相互作用 5

1.4.2 调节转录因子活性 5

第二章 材料与方法 6

2.1 酵母细胞的转化 6

2.1.1 实验材料与器械 6

2.1.2 用于同源重组的PCR产物的获取 10

2.1.3 酵母细胞的转化 11

2.1.4 菌落PCR验证 12

2.2 表达蛋白的SDS-PAGE 14

2.2.1 实验材料与器械 14

2.2.2 蛋白的提取 16

2.2.3 SDS-PAGE 17

第三章 结果与讨论 19

3.1 酵母的转化 19

3.1.1 用于同源重组的PCR产物的电泳图谱 19

3.1.2 菌落PCR的电泳图谱 20

3.2 表达蛋白的SDS-PAGE 21

3.3 小结 22

参考文献 23

致谢 25

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