BLM解旋酶的酶学特性与DHBN结构研究

发布时间：2018-01-08 00:14

本文关键词：BLM解旋酶的酶学特性与DHBN结构研究　出处：《西北农林科技大学》2017年博士论文　论文类型：学位论文

【摘要】：以大肠杆菌recQ基因命名的RecQ家族解旋酶是对基因组稳定性维持极为重要的分子马达蛋白,其功能几乎涉及到DNA复制、修复、重组、转录和端粒维持等代谢过程。在人类的五种RECQ解旋酶中,基因blm,wrn和recq4的突变分别会引起三种显著不同的疾病病征:布鲁姆综合征(Bloom syndrome,BS)、沃纳综合征(Werner syndrome,WS)和先天性血管萎缩皮肤异色综合征(Rothmund Thomson syndrome,RTS)。其中,BS病人细胞最典型的特点是呈现出比正常人约高10倍的姐妹染色单体交换率(Sister chromatid exchanges,SCEs),而且倾向于形成多种恶性肿瘤。由于所有已鉴定的RecQ家族解旋酶都具有结构和功能特点非常相似的C端解旋酶核心结构域,所以,N端的显著差异可能是细胞中各解旋酶具有特异功能的重要因素。然而,截至目前,关于BLM N端结构研究的报导仍较欠缺,关于BLM全长蛋白结构的研究也因其稳定性低、回收量少、大量纯化耗资多等而受到限制。首先,鉴于以上不足,本研究在与已报导的BLM同源蛋白的序列和表达系统进行对比分析的基础上,选取与人BLM解旋酶(homo sapiens BLM helicase,简称h BLM)亲缘关系较近的模式生物鸡BLM解旋酶(Gallus gallus BLM helicase,简称gBLM)为研究对象,优化并确立了gBLM及其截短蛋白gBLM Core和gBLM CΔHRDC在大肠杆菌中的高水平表达和高产量纯化方法体系,这可为gBLM酶学特性和生物物理实验研究提供足量高质量的蛋白。通过对gBLM进行凝胶过滤层析、动态光散射(DLS)、荧光各向异性DNA结合实验、快速停留DNA解旋实验的酶学特性测定,研究得出以下结论:第一,gBLM是一种活力较强的非典型性DNA结构特异性解旋酶,不仅对具有3’尾链的DNA结构底物具有高亲和性,而且可有效结合并解旋具有平末端结构的复制泡状DNA底物,这暗示了gBLM潜在的加工DNA代谢中间物的生物学功能。第二,不同截短gBLM蛋白的活性对比分析显示gBLM N端不仅与gBLM的多聚体形成相关,而且具有明显的辅助结合复制叉和复制泡状DNA底物的功能,对多种DNA结构底物的解旋也是必需的,这揭示了N端对BLM聚体和活性的调控作用。第三,DNA结合和解旋活性对比分析也显示HRDC结构域呈现出明显的辅助gBLM结合和解旋大多数复杂结构DNA底物的特性。因此,gBLM的大量高纯度纯化策略及相应的酶学特性分析结果可为hBLM的结构和分子机理研究提供一个新的参考模型。其次,基于以上gBLM的酶学特性分析,本研究进一步综合利用生物信息学、生物化学和生物物理学方法对BLM的N端进行了深入研究,并取得了以下结果:第一,对数据库中已测序的78种BLM同源蛋白的生物信息学分析发现:BLM同源蛋白N端在物种间差异较大,序列保守性低,且柔性区域较多,推测BLM同源蛋白N端可能不具有保守的三维结构。然而,进一步的分析发现其中含2至3个二级结构元件的DHBN(Dimerization Helical Bundle in N-terminal domain,N端二聚化螺旋束)是存在于脊椎动物BLM同源蛋白N端中的唯一高度保守的结构域,这可能与其在进化中的重要生物功能相关。gDHBN结构稳定性高的特点在不同截短gBLM N端蛋白的限制性蛋白水解实验和LC-MS/MS鉴定中也得到了验证。第二,通过对不同BLM N端截短蛋白的系统晶体初筛、优化、X-射线晶体衍射与解析,研究分别获得了hBLM、gBLM和p BLM(Pelecanus crispus BLM helicase)DHBN的高分辨率的晶体结构:hDHBN(2.0?)、gDHBN(2.7?)和pDHBN(1.4?)。进一步的相互作用力和生化特性对比分析显示高度保守的DHBN结构域主要通过疏水作用力稳定存在,并主要负责参与BLM解旋酶的二聚化。溶液中gDHBN的SAXS测定模型也较好地证实了DHBN的二聚体结构。第三,不同截短gBLM蛋白的凝胶过滤层析分析和DLS测定显示高度保守的二聚化DHBN是BLM高聚体组装的基本单位,在此基础上,本研究提出了之前电镜研究观测的hBLM环状结构模型。同时,不同截短gBLM蛋白的DNA解旋活性测定和DLS对比分析显示伴随着BLM从二聚体被组装成六聚体,其DNA解旋速率与解旋幅度不断降低,这暗示了DHBN结构域对DNA解旋过程的调控作用,也揭示了BLM聚体形态与DNA解旋活性之间的关系。有趣的是,稳定的DHBN二聚体在BLM全长蛋白分子中可由ATP水解诱导解离,这与hBLM和WRN的ATP诱导解离性质相一致。总之,本研究不仅首次优化确立了gBLM的大量高纯度纯化方法,分析了N端对其聚体和酶学活性的调节作用,而且也首次揭示了BLM DHBN的高度序列、结构保守性与多种潜在调控功能,这些研究不仅可深化对BLM同源蛋白酶学特性与结构相似性的理解,同时也可为深入认识细胞中hBLM的聚体组装和结构机理提供重要参考依据。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q75

【相似文献】