本文關(guān)鍵詞： CUL4B DNA復(fù)制 CDC6 細(xì)胞周期 Cyclin E　出處：《山東大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：DNA復(fù)制是細(xì)胞正常分裂、增殖的核心內(nèi)容。在進化過程中,細(xì)胞建立并發(fā)展了一個十分精細(xì)而復(fù)雜的細(xì)胞周期調(diào)控系統(tǒng)：通過G1/S、G2/M轉(zhuǎn)換調(diào)控DNA復(fù)制。DNA復(fù)制包括起始、延伸和終止三個過程,多種細(xì)胞周期調(diào)控蛋白和促進細(xì)胞增殖的生長因子參與了此進程的調(diào)控。這些調(diào)控蛋白相互作用形成一個特異的細(xì)胞周期依賴的DNA復(fù)制調(diào)控系統(tǒng),保證DNA復(fù)制的有序進行。DNA復(fù)制的起始是整個DNA復(fù)制過程的關(guān)鍵步驟,這一過程中前復(fù)制起始復(fù)合物(pre-replication complex, Pre-RC)發(fā)揮著核心的調(diào)控作用。DNA復(fù)制通常都是從復(fù)制原點(origin of replieatiom)開始進行。在G1期,細(xì)胞在復(fù)制原點處形成一個蛋白復(fù)合體,稱為前復(fù)制起始復(fù)合物(Pre-RC)。組成Pre-RC的蛋白包括復(fù)制起始位點識別復(fù)合體(origin recognition complex, ORC)、細(xì)胞分裂周期蛋白6 (cell division cycle-6, cdc6)、cdc10依賴性轉(zhuǎn)錄因子1(cdc10 dependent transcript 1, cdt1)和微染色體維持蛋白MCM (minichromosome maintence proteins)。Pre-RC受到包括細(xì)胞周期調(diào)控蛋白復(fù)合物Cyclin/CDK在內(nèi)的多種調(diào)控系統(tǒng)的控制,確保在一個細(xì)胞周期中DNA只能復(fù)制一次。 CUL4B屬于Cullin基因家族,該家族成員是泛素E3連接酶CULLIN-RING (CULLIN-RING ubiquitin Ligases, CRLs)的重要組成部分,主要發(fā)揮結(jié)構(gòu)支架的功能。CRLs是目前已知的最大一類泛素連接酶,它參與調(diào)節(jié)包括細(xì)胞周期、轉(zhuǎn)錄、信號傳導(dǎo)、生長發(fā)育以及DNA修復(fù)等幾乎所有的生理活動。CUL4B基因突變能夠?qū)е乱环NX連鎖智力低下綜合征,CUL4B喪失功能突變的女性攜帶者體內(nèi)只有活性X染色體攜帶正常基因的細(xì)胞能有效增殖而得以保留。我們的前期研究發(fā)現(xiàn)抑制CUL4B表達可導(dǎo)致細(xì)胞增殖障礙,細(xì)胞周期蛋白Cyclin E累積,細(xì)胞出現(xiàn)S期阻滯,提示CUL4B在細(xì)胞增殖和DNA復(fù)制過程中發(fā)揮重要作用。 為進一步研究CUL4B參與調(diào)控細(xì)胞周期功能的分子學(xué)機制,我們首先利用Real-time PCR和、Vestern blot分析方法檢測了不同細(xì)胞周期CUL4B的表達情況。發(fā)現(xiàn)在細(xì)胞周期的各個時期,CUL4B在mRNA及蛋白表達水平?jīng)]有明顯差別,提示CUL4B表達量不隨細(xì)胞周期的變化而明顯改變。此外,對內(nèi)源和外源CUL4B的檢測結(jié)果均表明CUL4B在不同的細(xì)胞周期都主要分于在細(xì)胞核。利用Western blot對分離得到的細(xì)胞各組分檢測,我們發(fā)現(xiàn)CUL4B是一種染色質(zhì)結(jié)合蛋白,同時CUL4B與DNA合成位點共定位。利用DNA fiber技術(shù)研究了CUL4B對DNA復(fù)制的起始,延伸和終止的影響,發(fā)現(xiàn)抑制CUL4B表達導(dǎo)致DNA復(fù)制起始障礙,提示CUL4B在參與調(diào)控DNA復(fù)制起始過程中發(fā)揮重要作用。因此,我們又對CUL4B參與調(diào)控DNA復(fù)制的分子機制進行了研究。結(jié)果發(fā)現(xiàn)CULAB的表達下調(diào)導(dǎo)致CDC6,MCM2與染色質(zhì)的結(jié)合障礙并促進CDC6的核內(nèi)降解。此外,CUL4B對CDC6的調(diào)控不依賴于細(xì)胞周期以及CDC6的磷酸化。有趣的是,我們發(fā)現(xiàn)盡管CUL4A與CUL4B高度同源,但CUL4B與CUL4A在調(diào)控DNA復(fù)制中的功能可能有所不同,而且CUL4B對CDC6的調(diào)控并不依賴于CUL4B的羧基末端而依賴于其特異性的氨基末端。 綜上所述,本研究揭示了CULAB在調(diào)控細(xì)胞周期中發(fā)揮重要功能,并對CULAB調(diào)控DNA復(fù)制起始的分子機制進行了初步研究,發(fā)現(xiàn)CUL4B通過維持前復(fù)制起始復(fù)合物完整性而參與調(diào)控DNA復(fù)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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

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