本文關鍵詞：BLM解旋酶的酶學特性與DHBN結構研究　出處：《西北農林科技大學》2017年博士論文　論文類型：學位論文

  更多相關文章： BLM解旋酶 酶學特性 寡聚化 X-射線晶體衍射 DHBN(N端二聚化螺旋束)

【摘要】：以大腸桿菌recQ基因命名的RecQ家族解旋酶是對基因組穩(wěn)定性維持極為重要的分子馬達蛋白,其功能幾乎涉及到DNA復制、修復、重組、轉錄和端粒維持等代謝過程。在人類的五種RECQ解旋酶中,基因blm,wrn和recq4的突變分別會引起三種顯著不同的疾病病征:布魯姆綜合征(Bloom syndrome,BS)、沃納綜合征(Werner syndrome,WS)和先天性血管萎縮皮膚異色綜合征(Rothmund Thomson syndrome,RTS)。其中,BS病人細胞最典型的特點是呈現(xiàn)出比正常人約高10倍的姐妹染色單體交換率(Sister chromatid exchanges,SCEs),而且傾向于形成多種惡性腫瘤。由于所有已鑒定的RecQ家族解旋酶都具有結構和功能特點非常相似的C端解旋酶核心結構域,所以,N端的顯著差異可能是細胞中各解旋酶具有特異功能的重要因素。然而,截至目前,關于BLM N端結構研究的報導仍較欠缺,關于BLM全長蛋白結構的研究也因其穩(wěn)定性低、回收量少、大量純化耗資多等而受到限制。首先,鑒于以上不足,本研究在與已報導的BLM同源蛋白的序列和表達系統(tǒng)進行對比分析的基礎上,選取與人BLM解旋酶(homo sapiens BLM helicase,簡稱h BLM)親緣關系較近的模式生物雞BLM解旋酶(Gallus gallus BLM helicase,簡稱gBLM)為研究對象,優(yōu)化并確立了gBLM及其截短蛋白gBLM Core和gBLM CΔHRDC在大腸桿菌中的高水平表達和高產量純化方法體系,這可為gBLM酶學特性和生物物理實驗研究提供足量高質量的蛋白。通過對gBLM進行凝膠過濾層析、動態(tài)光散射(DLS)、熒光各向異性DNA結合實驗、快速停留DNA解旋實驗的酶學特性測定,研究得出以下結論:第一,gBLM是一種活力較強的非典型性DNA結構特異性解旋酶,不僅對具有3’尾鏈的DNA結構底物具有高親和性,而且可有效結合并解旋具有平末端結構的復制泡狀DNA底物,這暗示了gBLM潛在的加工DNA代謝中間物的生物學功能。第二,不同截短gBLM蛋白的活性對比分析顯示gBLM N端不僅與gBLM的多聚體形成相關,而且具有明顯的輔助結合復制叉和復制泡狀DNA底物的功能,對多種DNA結構底物的解旋也是必需的,這揭示了N端對BLM聚體和活性的調控作用。第三,DNA結合和解旋活性對比分析也顯示HRDC結構域呈現(xiàn)出明顯的輔助gBLM結合和解旋大多數(shù)復雜結構DNA底物的特性。因此,gBLM的大量高純度純化策略及相應的酶學特性分析結果可為hBLM的結構和分子機理研究提供一個新的參考模型。其次,基于以上gBLM的酶學特性分析,本研究進一步綜合利用生物信息學、生物化學和生物物理學方法對BLM的N端進行了深入研究,并取得了以下結果:第一,對數(shù)據(jù)庫中已測序的78種BLM同源蛋白的生物信息學分析發(fā)現(xiàn):BLM同源蛋白N端在物種間差異較大,序列保守性低,且柔性區(qū)域較多,推測BLM同源蛋白N端可能不具有保守的三維結構。然而,進一步的分析發(fā)現(xiàn)其中含2至3個二級結構元件的DHBN(Dimerization Helical Bundle in N-terminal domain,N端二聚化螺旋束)是存在于脊椎動物BLM同源蛋白N端中的唯一高度保守的結構域,這可能與其在進化中的重要生物功能相關。gDHBN結構穩(wěn)定性高的特點在不同截短gBLM N端蛋白的限制性蛋白水解實驗和LC-MS/MS鑒定中也得到了驗證。第二,通過對不同BLM N端截短蛋白的系統(tǒng)晶體初篩、優(yōu)化、X-射線晶體衍射與解析,研究分別獲得了hBLM、gBLM和p BLM(Pelecanus crispus BLM helicase)DHBN的高分辨率的晶體結構:hDHBN(2.0?)、gDHBN(2.7?)和pDHBN(1.4?)。進一步的相互作用力和生化特性對比分析顯示高度保守的DHBN結構域主要通過疏水作用力穩(wěn)定存在,并主要負責參與BLM解旋酶的二聚化。溶液中gDHBN的SAXS測定模型也較好地證實了DHBN的二聚體結構。第三,不同截短gBLM蛋白的凝膠過濾層析分析和DLS測定顯示高度保守的二聚化DHBN是BLM高聚體組裝的基本單位,在此基礎上,本研究提出了之前電鏡研究觀測的hBLM環(huán)狀結構模型。同時,不同截短gBLM蛋白的DNA解旋活性測定和DLS對比分析顯示伴隨著BLM從二聚體被組裝成六聚體,其DNA解旋速率與解旋幅度不斷降低,這暗示了DHBN結構域對DNA解旋過程的調控作用,也揭示了BLM聚體形態(tài)與DNA解旋活性之間的關系。有趣的是,穩(wěn)定的DHBN二聚體在BLM全長蛋白分子中可由ATP水解誘導解離,這與hBLM和WRN的ATP誘導解離性質相一致�？傊�,本研究不僅首次優(yōu)化確立了gBLM的大量高純度純化方法,分析了N端對其聚體和酶學活性的調節(jié)作用,而且也首次揭示了BLM DHBN的高度序列、結構保守性與多種潛在調控功能,這些研究不僅可深化對BLM同源蛋白酶學特性與結構相似性的理解,同時也可為深入認識細胞中hBLM的聚體組裝和結構機理提供重要參考依據(jù)。
[Abstract]:Named after the recQ gene of Escherichia coli RecQ helicase family is very important to maintain genomic stability of molecular motor proteins, its function involves nearly DNA replication, repair, recombination, transcription and telomere maintenance and other metabolic processes. In five kinds of human RECQ helicase, BLM gene, mutation of WRN and RECQ4 were caused by three different kinds of disease symptoms: Bloom syndrome (Bloom syndrome BS), Warner syndrome (Werner syndrome, WS) and congenital vascular atrophy poikiloderma syndrome (Rothmund Thomson syndrome, RTS). Among them, the most typical features of patients with BS cells is showing a normal person is 10 times higher than the sister chromatid single exchange rate (Sister chromatid exchanges, SCEs), and tend to form a variety of malignant tumors. Because of all the identified RecQ family helicases have structural and functional characteristics are very similar to the C end of the helicase core The knot domain, therefore, significant differences at N may be an important factor in the helicase cells have a specific function. However, up to now, on the BLM N terminal structure research report is still lacking, a full-length BLM protein structure research because of its low stability, recovery is less, and the purification of a large number of high cost limited. First of all, in view of the above problems, in this study, with reported BLM homologous protein sequences and the basis of the comparative analysis of the expression system, selection and human BLM unwindase (Homo sapiens BLM helicase h, referred to as BLM) is more closely related to the biological model of chicken BLM (Gallus Gallus BLM helicase helicase, gBLM) as the research object, optimize and establish the high level expression of gBLM and gBLM Core and gBLM C truncated protein HRDC in Escherichia coli and high yield purification method for gBLM system, the enzymatic characteristics and biological physics experiment To provide sufficient high quality protein. By gel filtration chromatography on gBLM (DLS), dynamic light scattering, fluorescence anisotropy DNA binding assay and determination of enzymatic properties of fast time DNA unwinding experiments, the research draws the following conclusions: first, gBLM is a strong activity of the atypical DNA structure specific helicase, not only DNA has the structure of substrate on the 3 'tail chain has a high affinity, and can be effectively combined and untwisting has a flat structure at the end of the replication bubble DNA substrate, suggesting the potential biological functions of gBLM processing DNA metabolic intermediates. Second, analysis and comparison of different activity of truncated gBLM protein showed that the gBLM N not only with the end gBLM multimer formation, but also has obvious auxiliary combination of replication forks and replication bubble DNA substrates, the substrate DNA structure of a variety of unwinding is also necessary, which reveals the N end of BLM dimer and live Regulation of sex. Third, DNA binding and solution analysis also showed that HRDC activity of spin domain showed obvious characteristics of auxiliary gBLM binding and unwinding the most complex structure of DNA substrate. Therefore, gBLM high purity purification strategy and corresponding enzymatic characteristics analysis can provide a new reference model for structure hBLM and molecular mechanism study. Secondly, based on the above analysis of enzymatic properties of gBLM, this study further comprehensive utilization of bioinformatics, biochemistry and biophysics are studied on the BLM end of the N, and achieved the following results: first, 78 kinds of BLM homologous protein sequence database has biological information science the analysis found that BLM homologous protein N terminal differences between species, low sequence conservation, and more flexible regions, suggesting that BLM homologous protein N terminal may not have conserved structure. However, a Further analysis found that including 2 to 3 grade two structural elements of DHBN (Dimerization Helical Bundle in N-terminal domain, N terminal dimerization helix bundle) is found in vertebrate BLM homologue N end only in the highly conserved domains, which may be related to the evolution of the important characteristics related to the stability of.GDHBN structure of biological function high have been verified in different truncated gBLM N terminal protein limited proteolysis experiments and identification of LC-MS/MS. Second, the system of different BLM crystal N truncated protein screening, optimization, and analysis of X-ray crystal diffraction studies were obtained from X-, hBLM, gBLM and P (BLM Pelecanus crispus BLM helicase) the high-resolution crystal structure of DHBN: hDHBN (2?), gDHBN (2.7?) and pDHBN (1.4?). The analysis of interactions and biochemical properties compared to show highly conserved DHBN domain mainly by hydrophobic Stable force exists, and is mainly responsible for participating in the BLM helicase. The dimerization of gDHBN in solution was determined by SAXS model but also confirmed the DHBN two dimer structure. Third, gel filtration chromatography and DLS analysis of different truncated gBLM protein showed highly conserved DHBN dimerization is a basic unit of BLM polymer assembly, on the basis of, this study proposes hBLM ring structure model before electron microscopy observation. At the same time, different truncated gBLM protein DNA helicase activity assay and DLS analysis showed that compared with BLM from two dimers were assembled into six dimers, the DNA helicase unwinding rate and amplitude decreases, suggesting that the regulation of DHBN domain of DNA unwinding process, BLM dimer form and DNA relationship between the activity of spinning solution revealed. Interestingly, stable DHBN two dimer by ATP hydrolysis induced dissociation in BLM full-length protein molecules, The hBLM and WRN ATP induced dissociation properties consistent. In conclusion, this study not only for the first time to establish the optimization gBLM a large number of high purity purification method, analysis of the regulatory role of N terminal dimer and its enzymatic activity, but also for the first time reveals high sequence BLM DHBN structure, conservative and several potential regulatory functions these studies can not only deepen the homologous BLM protease characteristics and structure similarity of the understanding, but also provide important reference for understanding the mechanism of dimer assembly and structure of hBLM in cells.

【學位授予單位】：西北農林科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q75

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