Z-DNA的形成及其与基因转录的关系

Z-DNA是一种非常独特的DNA二级结构.与B-DNA相比,Z-DNA最显著的结构特征是左手螺旋和磷酸-核糖骨架呈“zigzag”状. 虽然目前对Z-DNA功能的了解还不确切,但毫无疑问,Z-DNA与基因的转录和调控密切相关. 一方面,在体内Z-DNA在基因转录过程中产生;另一方面,分布于启动子等不同区域的Z-DNA又可以反过来调控基因的转录, 即Z-DNA能够增强一些基因转录,也能抑制某些基因的表达,但其调控机制还不清楚.这种调控似乎与Z-DNA在启动子中的位置、基因和细胞类型有关.研究Z-DNA的形成及其与基因转录的关系对理解基因转录调控理论具有十分重要的意义.     

Z-DNA功能研究的回顾和近况

自从Rich等在1979年首次发现左手螺旋的Z-DNA以后,人们对Z-DNA的研究不断深入,并推测它在基因转录、基因调控以及基因重组等方面有着重要的作用。最新研究表明Z-DNA与病毒的发病机制有关。这个研究结果可能蕴藏着关于这种另类DNA如何行使功能的答案,并可能由此成功研制能有效抗天花病毒的化合物。     

Z-DNA结合蛋白与Zα结构域

Z-DNA结合蛋白包括人、哺乳类和病毒中的ADAR1、DLM-1、E3L蛋白以及在鱼类中最新报道的PKR-like（PKZ）,其共同点是都含有Zα结构域。本文简述了这几种蛋白质的结构与功能,并着重分析Zα结构域与Z-DNA、Z-RNA、B-DNA等核酸分子的识别机制和亲和性。     

G-四链体:核酸的“新”结构

<正>核酸(包括DNA和RNA)是体内最重要一类生物大分子,DNA主要作为遗传信息载体,而RNA则作为信息中介分子、调节因子和酶等发挥多重生物学功能。核酸分子空间结构形式复杂、可变,可采用多种构象,在体内DNA主要为双螺旋结构,而RNA采取单链为主局部双螺旋结构,此外还存在一些特殊结构,如Z-DNA和G-四链体(G-quadruplex)等,这些结构对保证基因组完整性、基因表达和调控都具有重要作用。由于结构     

鲫鱼PKR-like Zα与d(GC)13质粒的结合及其适应性进化

Zα是一类能特异性识别与结合左旋DNA(Z-DNA)的蛋白质结构域,分布于不同物种的多种蛋白质中,其结构保守并分化自一个共同的祖先Z-结构域.适应性进化分析显示Zα没有经历正达尔文选择,表明与其结构对应Zα的功能相当保守.凝胶阻滞试验表明原核表达的鲫鱼PKR-like Zα多肽(CaPZα)与pMD18-T质粒结合,而却能与包含d(GC)13的重组质粒pMD18-T/(GC)13结合.同时,与ADARl Zα相比,CaPZα与d(GC)13质粒的结合能力较弱,即CaPZα1和CaPZα2子域单独不能结合d(GC)13质粒.这表明Zα功能虽然没有异化,但有很大的不同.实验结果还表明负超螺旋和d(GC)n可能都是正常生理条件下,DNA形成潜在Z-DNA必不可少的因素.定点突变试验证实38N与60W两个氨基酸位点对于CaPZα结合Z-DNA非常关键.     

The Zα domain of fish PKZ converts DNA hairpin with d（GC）n inserts to Z-conformation

PKZ, protein kinase containing Z-DNA domains, is a novel member of the vertebrate eIF2α kinase family. Containing a catalytic domain in C-terminus and two Z-DNA binding domains （Zαl and Zα2） in N-terminus, PKZ can be acti- vated through the binding of Zα to Z-DNA. However, the regulatory function of PKZ Zα remains to be established. Here, to understand the impact of PKZ Zα on DNA con- formational transition, wild-type ZαdZα2 and 11 mutant proteins were expressed and purified. At the same time, several different lengths of DNA hairpins-d（GC）nT4（GC）, （n = 2-6） and an RNA hairpin-r（GC）6T4（GC）6 were synthesized. The effects of ZαdZα2 and mutant proteins on the conformation of these synthetic DNA or RNA hairpins were investigated by using circular dichroism spectrum and gel mobility shift assays. The results showed that DNA hair- pins retained a conventional B-DNA conformation in the absence of ZαdZα2, while some of the DNA hairpins （n 〉 3） were converted to Z-conformation under Zαd Zα2 induction. The tendency was proportionally associated with the increas- ing amount of GC repeat. In comparison with ZodZα2, ZαdZαd rather than Zα2ZαL2 displayed a higher ability in converting d（GC）6T4（GC）6 from B- to Z-DNA. These results demonstrated that Zcd sub-domain played a more essential role in the process of B-Z conformational transition than Zα2 sub-domain did. Mutant proteins （K34A, N38A, R39A, Y42A, P57A, P58A, and W60A） could not convert d（GC）6T4（GC）6 into Z-DNA, whereas S35A or K56A retained some partial activities. Interestingly, ZαlZα2 was also able to induce r（GC）6T4（GC）6 RNA from A-conform- ation to Z-conformation under appropriate conditions.     

Z-DNA结合蛋白与Zα结构域

Z-DNA结合蛋白包括人、哺乳类和病毒中的ADAR1,DLM-1.E3L蛋白以及在鱼类中最新报道的PKR-like(PKZ),其共同点是都含有za结构域.本文简述了这几种蛋白质的结构与功能,并着重分析Za结构域与Z-DNA,Z-RNA、B-DNA 等核酸分子的识别机制和亲和性.     

Z-DNA的发现者一里奇

1953年,沃森和克里克双螺旋模型的提出标志着现代分子生物学诞生,从而使生命科学研究进入了一个崭新的时代。在随后的一段时间内,生命科学领域取得了一系列辉煌的成就,诞生了大量科学大师和伟大科学家。许多工作都获得了诺贝尔奖,对今天的分子生物学研究产生了巨大的影响。但是,一些未获诺贝尔奖的研究成果同样推动了分子生物学的发展。比如在RNA表达和调控的研究领域,20世纪50～70年代的RNA结构解析与Z-DNA的发现就是重要的基础。做出这些重要发现的就是美国著名生物物理学家亚历山大·里奇（AlexanderRich）（图1）。     

双股寡聚d(G-C)6 DNA Z-构象的形成及其构象转换的动态研究

研究揭示,生物信息的形成,传递与DNA构象的多样性,特别是其中的左手螺旋Z-构象DNA（Z-DNA）相关.在机体DNA链中,普遍存在的特异序列结构d（C-G）n和d（G-C）n片段易形成Z-构象.但对d（G-C）n序列结构的寡聚体Oligo-d（G-C）n,（n小于8）能转换形成Z-DNA片段少见报道.为促进对Z-DNA尤其是其中的短片段Z-DNA与生物功能的相关性研究,我们对合成并纯化后的寡聚体Oligo-d（G-C）n,n分别为4,6,8,10, 及Oligo-d（C-G）6和多聚体poly-d（G-C）500-900进行Z-构象的形成和其构象转换的比较研究.研究结果发现：①d（GpCpGpCpGpCpGpCpGpCpGpC）是d（G-C）n序列结构中能转换形成Z-构象的最短片段（n＝6）.其转换成Z-构象能力有链长依赖性（poly d（G-C）500-900易于Oligo-d（G-C）6）;②Oligo-d（G-C）6的Z-构象形成能力因溶液中的介质性质不同而异.Co（NH3）3＋〉Mg2＋〉Na＋;C1O-4〉Cl-,因此要求盐溶液的浓度差异很大.③PH7.2,室温条件下,在MgCl2, NaClO4, NaCl溶液浓度分别由0 mol/L增至6.0 mol/L,Oligo-d（G-C）6的B、Z构象转换都出现：B-构象相对稳定期,B-、Z-构象转换跃迁期和Z-构象相对稳定期.每个阶段要求跨越的盐浓度变迁范围也因所用介质而异.当溶液中Oligo-d（G-C）6 B-构象、Z-构象各占50%（θ1/2）时,其盐浓度分别为1.72 mol/L（MgCl2）,2.88 mol/L（NaClO4）,3.85 mol/L（NaCl）.④Oligo-d（G-C）6的B-,Z-构象转换程度受盐浓度影响：当Oligo-d（G-C）6处于最适条件和不同盐溶液其浓度为θ（12）浓度时,温度由8 ℃→22 ℃,在MgCl2,NaClO4溶液中的Oligo-d（G-C）6形成Z-构象能力增加,当由22 ℃→60 ℃,MgCl2溶液中的Z-构象Oligo-d（G-C）6加速增加,而在NaClO4溶液中则是急速向B-型Oligo-d（G-C）6方向转换;温度变化对处于NaCl溶液中的Oligo-d（G-C）6B-、Z-构象相对平衡影响较小.⑤甲基化胞嘧啶即Oligo-d（G-mC）6或d（mC-G）6均增大Z-构象形成能力.⑥在4 mol/L MgCl2溶液中的Oligo-d（G-C）6或Oligo-d（C-G）6或poly d（G-C）500-900的UVab谱、UVcd谱均显示出非B-型或Z-型DNA的新谱型.并且有链长依赖性和因溶液浓度改变出现构象可逆性转变.提示在Oligo-d（G-C）6的构象转换过程中可能存在新构象＂X＂型,即BZX构象转换模式.     

鲫鱼PKZ Zα结构域与d(GC)、d(TA)重组质粒的结合

Zα是能够特异性识别并结合左旋DNA(Z-DNA)的蛋白结构域,首先在人ADAR1中鉴定,随后又在ZBP-1(DLM-1)和E3L等蛋白质中发现了该结构域．鲫鱼PKZ是首次报道的具有Zα结构域的鱼类eIF2α激酶．为深入了解鲫鱼PKZ Zα的功能,原核表达并亲和层析纯化了3种多肽,即野生型PZα(Zα1Zα2)、替换型P(Zα1)2(Zα1Zα1)和点突变型（PZαK34A、PZαS35A、PZαR39A、PZαP57A）．同时,构建了含有d(GC)6、d(GC)13、d(TA)13特殊插入序列的3种重组质粒,用于体外模拟Z-DNA．凝胶阻滞实验分析了3种多肽分别与重组质粒的亲和性结果表明,PZα和P(Zα1)2能够与d(GC)重组质粒结合,并且随着多肽含量的增加,阻滞效应越明显．与野生型PZα相比,替换型P(Zα1)2结合d(GC)重组质粒的能力更强PZα和P(Zα1)2还能微弱地与d(TA)13重组质粒结合．点突变型多肽都不能与重组质粒结合,暗示鲫鱼PKZ Zα结构域中这4个氨基酸残基在结合核酸分子的过程中非常关键．该文结果有利于进一步揭示鱼类PKZ Zα结构域与Z-DNA结合的分子机理．     

5-Methylcytosine containing CG decamer as Z-DNA embedded sequence for a potential Z-DNA binding protein probe

Abstract

Attempting to elucidate biological significance of the left-handed Z-DNA is a research challenge due to Z-DNA potential role in many diseases. Discovery of Z-DNA binding proteins has ignited the interest in search for Z-DNA functions. Biosensor with Z-DNA forming probe can be useful to study the interaction between Z-DNA conformation and Z-DNA binding proteins. In this study, 5-methylcytosine (^mC) containing CG decamers were characterized for their suitability to form Z-DNA and to be used in Z-DNA forming probe. The 5′-thiol oligonucleotide embedded with 5′-^mCG^mCG^mCG^mCG^m CG-3′ was designed and developed as a potential Z-DNA forming probe for Z-DNA binding protein screening.     

Amine free crystal structure: the crystal structure of d(CGCGCG)2 and methylamine complex crystal

We succeeded in the crystallization of d(CGCGCG)2 and methylamine Complex. The crystal was clear and of sufficient size to collect the X-ray crystallographic data up to 1.0 A resolution using synchrotron radiation. As a result of X-ray crystallographic analysis of 2Fo-Fc map was much clear and easily traced. It is the first time monoamine co-crystallizes with d(CGCGCG)2. However, methylamine was not found from the complex crystal of d(CGCGCG)2 and methylamine. Five Mg ions were found around d(CGCGCG)2 molecules. These Mg ions neutralized the anion of 10 values of the phosphate group of DNA with five Mg2+. DNA stabilized only by a metallic ion and there is no example of analyzing the X-ray crystal structure like this. Mg ion stabilizes the conformation of Z-DNA. To use monoamine for crystallization of DNA, we found that we can get only d(CGCGCG)2 and Mg cation crystal. Only Mg cation can stabilize the conformation of Z-DNA. The method of using the monoamine for the crystallization of DNA can be applied to the crystallization of DNA of long chain of length in the future like this.     

Z-DNA crystallography

We review the effect of sequence on the structure of left-handed Z-DNA in single crystals. The various substituent groups that define a nucleotide base as guanine, cytosine, thymine, or adenine affect both the DNA conformation and the organization of solvent around the duplex. These are discussed in terms of their effect on the ability of sequences to adopt the unusual Z-DNA structure. In addition, the experimental and theoretical methods used to treat DNA hydration are discussed as they relate to the stability of Z-DNA. Finally, we argue that Z-DNA, as defined by the crystal conformation, is sufficient in itself to account for the physical properties of left-handed conformations observed in polymers and in genomic sequences. © 1997 John Wiley & Sons, Inc. Biopoly 44: 65–90, 1997     

Importance of DNA conformation in the reaction with cis-dichlorodiammine platinum (II)

Cis-dichlorodiammine platinum (II) has been reacted with synthetic polynucleotides either in B or in Z conformation. The binding of cis-dichlorodiammine platinum (II) stabilizes the Z conformation when reacted with poly (dG-m⁵dC) ·poly (dG-m⁵dC) in the Z conformation as shown by circular dichroism and by the antibodies to Z-DNA. On the other hand, the binding of cis-dichlorodiammine platinum (II) stabilizes a new conformation when reacted with poly(dG-dC)·poly(dG-dC) or poly (dG-m⁵dC)·poly(dG-m5dC) in the B conformation. The antibodies to Z-DNA bind to these platinated polynucleotides. In rabbits, the injection of platinated poly (dG-dC) poly (dG-dC) induces the synthesis of antibodies which recognize Z-DNA. In low salt conditions, the circular dichroism spectra of these platinated polynucleotides differ from those of B-DNA or Z-DNA. The characteristic³¹P nuclear magnetic resonance spectrum of Z-DNA is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

Reduced 4,5',8-trimethylpsoralen cross-linking of left-handed Z-DNA stabilized by DNA supercoiling

Z-DNA-forming sequences, (GT)21, (GT)12ATGT, and (CG)6TA(CG)6, were cloned into plasmids. These sequences formed left-handed Z-DNA conformations under torsional tension from negative supercoiling of DNA. 4,5',8-Trimethylpsoralen, on absorption of 360-nm light, forms monoadducts and interstrand cross-links in DNA that exists in the B-helical conformation. Trimethylpsoralen cross-links were introduced into the potential Z-DNA-forming sequences in relaxed DNA when these sequences existed as B-form DNA. In supercoiled DNA when these sequences existed in the Z conformation, the rate of cross-linking was greatly reduced, and trimethylpsoralen did not form monoadducts appreciably to Z-DNA. As an internal control in these experiments, the rates of cross-linking of the Z-DNA-forming sequences were measured relative to that of an adjacent, cloned sequence that could not adopt a Z conformation. The initial relative rates of cross-linking to Z-DNA-forming sequences were dependent on the superhelical density of the DNA, and the rates were ultimately reduced by factors of 10-15 for Z-DNA in highly supercoiled plasmids. This differential rate of cross-linking provides a novel assay for Z-DNA. Initial application of this assay in vivo suggests that a substantial fraction of (CG)6TA(CG)6, which existed as Z-DNA in plasmid molecules purified from cells, existed in the B conformation in vivo.     

Chemical detection of Z-DNA within the maize Adh1 promoter

Z-DNA is a left-handed helix which can form within tracts of alternating purines and pyrimidines. Tracts of potential Z-DNA identified by sequence inspection are often noted within regulatory portions of genes, but evidence that these tracts of sequence actually exist as Z-DNA is very limited, and not available for any plant gene. In this study, the chemical probes osmium tetroxide, diethylpyrocarbonate and hydroxylamine were used to show that a tract of alternating purines and pyrimidines in the Adh1 promoter (from -311 to -325) actually assumes a Z-DNA conformation under superhelical stress in vitro.