CUL4B在调控细胞增殖中的作用及其机制研究

发布时间：2018-01-19 00:29

本文关键词： CUL4B DNA复制 CDC6 细胞周期 Cyclin E　出处：《山东大学》2011年硕士论文　论文类型：学位论文

【摘要】：DNA复制是细胞正常分裂、增殖的核心内容。在进化过程中,细胞建立并发展了一个十分精细而复杂的细胞周期调控系统：通过G1/S、G2/M转换调控DNA复制。DNA复制包括起始、延伸和终止三个过程,多种细胞周期调控蛋白和促进细胞增殖的生长因子参与了此进程的调控。这些调控蛋白相互作用形成一个特异的细胞周期依赖的DNA复制调控系统,保证DNA复制的有序进行。DNA复制的起始是整个DNA复制过程的关键步骤,这一过程中前复制起始复合物(pre-replication complex, Pre-RC)发挥着核心的调控作用。DNA复制通常都是从复制原点(origin of replieatiom)开始进行。在G1期,细胞在复制原点处形成一个蛋白复合体,称为前复制起始复合物(Pre-RC)。组成Pre-RC的蛋白包括复制起始位点识别复合体(origin recognition complex, ORC)、细胞分裂周期蛋白6 (cell division cycle-6, cdc6)、cdc10依赖性转录因子1(cdc10 dependent transcript 1, cdt1)和微染色体维持蛋白MCM (minichromosome maintence proteins)。Pre-RC受到包括细胞周期调控蛋白复合物Cyclin/CDK在内的多种调控系统的控制,确保在一个细胞周期中DNA只能复制一次。 CUL4B属于Cullin基因家族,该家族成员是泛素E3连接酶CULLIN-RING (CULLIN-RING ubiquitin Ligases, CRLs)的重要组成部分,主要发挥结构支架的功能。CRLs是目前已知的最大一类泛素连接酶,它参与调节包括细胞周期、转录、信号传导、生长发育以及DNA修复等几乎所有的生理活动。CUL4B基因突变能够导致一种X连锁智力低下综合征,CUL4B丧失功能突变的女性携带者体内只有活性X染色体携带正常基因的细胞能有效增殖而得以保留。我们的前期研究发现抑制CUL4B表达可导致细胞增殖障碍,细胞周期蛋白Cyclin E累积,细胞出现S期阻滞,提示CUL4B在细胞增殖和DNA复制过程中发挥重要作用。为进一步研究CUL4B参与调控细胞周期功能的分子学机制,我们首先利用Real-time PCR和、Vestern blot分析方法检测了不同细胞周期CUL4B的表达情况。发现在细胞周期的各个时期,CUL4B在mRNA及蛋白表达水平没有明显差别,提示CUL4B表达量不随细胞周期的变化而明显改变。此外,对内源和外源CUL4B的检测结果均表明CUL4B在不同的细胞周期都主要分于在细胞核。利用Western blot对分离得到的细胞各组分检测,我们发现CUL4B是一种染色质结合蛋白,同时CUL4B与DNA合成位点共定位。利用DNA fiber技术研究了CUL4B对DNA复制的起始,延伸和终止的影响,发现抑制CUL4B表达导致DNA复制起始障碍,提示CUL4B在参与调控DNA复制起始过程中发挥重要作用。因此,我们又对CUL4B参与调控DNA复制的分子机制进行了研究。结果发现CULAB的表达下调导致CDC6,MCM2与染色质的结合障碍并促进CDC6的核内降解。此外,CUL4B对CDC6的调控不依赖于细胞周期以及CDC6的磷酸化。有趣的是,我们发现尽管CUL4A与CUL4B高度同源,但CUL4B与CUL4A在调控DNA复制中的功能可能有所不同,而且CUL4B对CDC6的调控并不依赖于CUL4B的羧基末端而依赖于其特异性的氨基末端。综上所述,本研究揭示了CULAB在调控细胞周期中发挥重要功能,并对CULAB调控DNA复制起始的分子机制进行了初步研究,发现CUL4B通过维持前复制起始复合物完整性而参与调控DNA复制起始。
[Abstract]:DNA replication is the core content of normal cell division, proliferation of cells. In the process of evolution, establishment and development of a cell cycle is very fine and complicated control system: through G1/S, G2/M conversion regulation of DNA replication.DNA replication including initiation, elongation and termination of three kinds of process, regulation of cell cycle regulatory proteins and promote cell the proliferation of growth factors involved in this process. The regulation of DNA replication system in cell cycle of these proteins interact with each other to form a specific manner, to ensure the orderly conduct of the DNA replication initiation of.DNA replication is a key step in the whole process of DNA replication, in the process of pre initiation complex (pre-replication complex Pre-RC) play the core of the regulation of.DNA replication is usually from the origin of replication (origin of replieatiom) began. In the period of G1 cells in the replication origin form a protein complex The body, before the initiation complex is called (Pre-RC). The Pre-RC protein includes identification of replication initiation site complex (origin recognition complex, ORC), cell cycle protein 6 (cell division cycle-6, Cdc6), CDC10 dependent transcription factor 1 (CDC10 dependent 1 transcript, Cdt1) MCM and minichromosome maintenance protein (minichromosome maintence proteins).Pre-RC is controlled by a variety of regulatory systems including the regulation of cell cycle protein complexes, Cyclin/CDK, to ensure that in a cell cycle in DNA can be copied only once.
CUL4B belongs to Cullin gene family, the family members are ubiquitin E3 ligase (CULLIN-RING CULLIN-RING ubiquitin Ligases, CRLs) is an important part of the main function of.CRLs structure is currently the largest known class of ubiquitin ligase, which is involved in the regulation of cell cycle, signal transduction, transcription, development and DNA repair almost all the physiological activities of.CUL4B gene mutation can cause a syndrome X linked mental retardation, CUL4B loss of function mutation in female carriers only active X chromosomes carry normal cell proliferation and gene can be effectively preserved. Our preliminary study showed that inhibition of CUL4B expression can lead to cell proliferation, cell cycle protein Cyclin E accumulation, S cell arrest, suggesting that CUL4B play an important role in cell proliferation and DNA replication process.
For the further study of molecular mechanism of CUL4B involved in cell cycle regulation function, we first use the Real-time PCR and Vestern blot analysis method to detect the expression of CUL4B in different cell cycle. Found at different stages of the cell cycle, CUL4B in mRNA and protein expression level was not significantly different, suggesting that CUL4B expression did not change with the cell cycle and obvious change. In addition, the detection of endogenous and exogenous CUL4B showed CUL4B in different cell cycle are mainly in the nucleus. The use of Western blot on the isolated cells were detected, we found that CUL4B is a chromatin binding protein, while CUL4B and DNA loci were synthesis of CUL4B on location. The initiation of DNA replication by DNA fiber technology, extension and termination, that inhibits CUL4B induced DNA replication initiation disorder expression, suggesting that CUL4B involved in the regulation of DNA Play an important role in the process of replication initiation. Therefore, we have to CUL4B the molecular mechanism involved in the regulation of DNA replication were studied. The results showed that CULAB expression led to CDC6, MCM2 and nuclear chromatin degradation with obstacles and promote CDC6. In addition, CUL4B does not depend on the regulation of CDC6 cell cycle and CDC6 phosphorylation. Interestingly, we found that although CUL4A and CUL4B are highly homologous, but CUL4B and CUL4A in the regulation of DNA replication in the function may be different amino terminal carboxyl terminal CUL4B and the regulation of CDC6 is not dependent on the CUL4B depends on its specificity.
To sum up, this study reveals that CULAB plays an important role in regulating cell cycle, and the molecular mechanism of CULAB regulating DNA replication initiation is preliminarily studied. It is found that CUL4B participates in the regulation of DNA replication initiation by maintaining the integrity of replication initiation complex.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

【参考文献】