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。     

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.     

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.     

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）。     

核酸结合蛋白的一维运动及其生物学功能

许多动力学研究已经观察到核酸结合蛋白在大分子核酸上的一维运动。尽管这些运动通常并不依赖特定的核酸序列,但它们在各种生命活动中具有重要作用。例如,转录因子通过一维运动靶向启动子或增强子区域并调控基因表达、DNA修复蛋白通过一维运动检测DNA损伤和传递修复信号、染色体结构维持蛋白通过一维运动调控染色体的结构等。当前,揭示核酸结合蛋白一维运动的生理功能和意义仍然是核酸生物学研究的一大挑战。该文将介绍核酸结合蛋白一维运动的模式,探讨单分子技术在实时观测核酸结合蛋白一维运动中的应用及其优势,并总结最近发现的核酸结合蛋白一维运动的生物学功能。     

组蛋白修饰及其生物学效应

组蛋白是染色质的主要成分之一,其氨基端的氨基酸残基可以被共价修饰,进而改变染色质构型,导致转录激活或基因沉默。组蛋白修饰除了简单地调控基因表达,更在于它可以招募蛋白复合体,影响下游蛋白,从而参与细胞分裂、细胞凋亡和记忆形成,甚至影响免疫系统和炎症反应等。不仅如此,最近的研究表明,组蛋白修饰与CTD密码、生物节律、DNA修复之间也存在一定的联系。这些发现证明了组蛋白修饰的重要性。在组蛋白的密码形成与密码破译、修饰级联与招募蛋白质过程中,蛋白复合体的特殊结构域起到的中介作用都是无法替代的。因此,这些特殊结构域将是了解"组蛋白密码"的关键。目前质谱分析等技术的广泛应用,正使得许多新的结构域不断被发现。文章旨在对组蛋白密码的基本内容作一述评,同时对可能的研究热点进行展望。     

T-STAR的结构及生物学功能

T-STAR基因定位于染色体8q24.2,其表达产物为分子量约55kDa的Sam68样蛋白,是STAR(signaltransductionandactivatorofRNA)家族新成员,具有RNA结合蛋白特征性的结合位点和酪氨酸磷酸化功能域。可能通过酪氨酸激酶信号转导系统和pre-mRNA的选择性剪接、加工等途径,参与了精子的发生、细胞的增殖调控、转化细胞的永生化过程并可能与某些疾病有关。     

甘蓝中BcpLH同源基因的克隆及其表达分析

根据大白菜ＢｃｐＬＨ基因设计引物,用ＰＣＲ方法从甘蓝结球期的茎尖组织中克隆到了大白菜ＢｃｐＬＨ基因的同源基因ＢｏＬＨ１。序列分析表明,ＢｏＬＨ１基因含有３个内含子,ｃＤＮＡ编码２４５个氨基酸,编码的蛋白中存在两个双链ＲＮＡ结合蛋白结构域;Ｓｏｕｔｈｅｒｎ杂交结果显示在甘蓝基因组有１个以上的ＢｏＬＨ１基因拷贝。ＰＣＲ表达分析表明,甘蓝ＢｏＬＨ１同源基因主要在结球期的茏尖组织、球叶和包叶中表达。推测Ｂ     

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     

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.     

PCBP1在基因表达过程中的功能和作用机理

Poly（C）-binding protein 1 （PCBP 1）是一种RNA结合蛋白,其蛋白质相对分子质量约为38000。PCBP1含三个KH（hnRNP K homology）结构域,这些结构域对其结合RNA有重要作用。PCBP1的功能主要是参与基因转录及转录后调节,如前体mRNA的剪切、mRNA稳定性、mRNA翻译过程的沉默或增强等。本文主要对PCBP1参与的信号通路以及与人类疾病的关系进行综述,试图阐明PCBP1的生物学功能和作用机理。     

NMR study of hydrogen exchange during the B-Z transition of a DNA duplex induced by the Zα domains of yatapoxvirus E3L

The Yaba-like disease viruses (YLDV) are members of the Yatapoxvirus family and have double-stranded DNA genomes. The E3L protein, which is essential for pathogenesis in the vaccinia virus, consists of two domains: an N-terminal Z-DNA binding domain and a C-terminal RNA binding domain. The crystal structure of the E3L orthologue of YLDV (yabZα_E3L) bound to Z-DNA revealed that the overall structure of yabZα_E3L and its interaction with Z-DNA are very similar to those of hZα_ADAR1. Here we have performed NMR hydrogen exchange experiments on the complexes between yabZα_E3L and d(CGCGCG)₂ with a variety of protein-to-DNA molar ratios. This study revealed that yabZα_E3L could efficiently change the B-form helix of the d(CGCGCG)₂ to left-handed Z-DNA via the active-mono B-Z transition pathway like hZα_ADAR1.     

Sequence discrimination of the Zα domain of human ADAR1 during B-Z transition of DNA duplexes

The Zα domain of human ADAR1 (Zα_ADAR1) preferentially binds Z-DNA rather than B-DNA with high binding affinity. Zα_ADAR1 binds to the Z-conformation of both non-CG-repeat DNA duplexes and a d(CGCGCG)₂ duplex similarly. We performed NMR experiments on complexes between the Zα_ADAR1 and non-CG-repeat DNA duplexes, d(CACGTG)₂ or d(CGTACG)₂, with a variety of protein-DNA molar ratios. Comparison of these results with those from the analysis of d(CGCGCG)₂ in the previous study suggests that Zα_ADAR1 exhibits the sequence preference of d(CGCGCG)₂ ? d(CACGTG)₂ > d(CGTACG)₂ through multiple sequence discrimination steps during the B-Z transition.     

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.     

A BC200-derived element and Z-DNA as structural markers in annexin I genes: Relevance to Alu evolution and annexin tetrad formation

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.