甘油对单链构象多态性分析的影响

单链构象多态性(single strand conformation poly-morphism,SSCP)分析是以PCR技术为基础检测DNA多态性的一种方法。其基本原理是:单链DNA在进行不含变性剂的中性聚丙烯酰胺凝胶电泳(PAGE)时,迁移率除与DNA单链的长短有关外,更主要取决于DNA单链所形成的具有一定空间结构的构象。这种构象由DNA单链碱基顺序决定,其稳定性靠分子内部局部顺序的相互作用来维持。相同长度的单链因其顺序的不同,甚至单个碱基的不同,所形成的构象不同,导致迁移率的变化而出现泳动变位(mobili-ty shift)。该方法自建立起来不断地发展、完善,应用范围也日益广泛。特别是在人类癌基因和抑癌基因突变的检测、基因诊断、连锁分析、基因作图等领域成效突出。     

DNA单链二级结构预测与单链构象多态性分析的一致性研究

单链构象多态性(SSCP)分析是一种简便,快速检测DNA突变的方法,它在基因突变检测、遗传分析、进化研究等领域有着广泛的应用价值.但是这种方法的突变检出率随DNA序列不同而变化,一般只能达到70%～80%.这主要是有的碱基突变对单链DNA的构象影响较小,不能通过SSCP检测出来.将计算机对DNA二级结构的预测结果和实验结果作了对比,发现二者有很高的一致性.这一结果表明计算机的DNA单链二级结构预测分析可用于PCR-SSCP分析的辅助设计,提高SSCP的突变检出率.     

黏附细胞中生物大分子结构变化的显微Raman光谱

鉴于细胞黏附作用的重要生物学意义, 选择了T淋巴细胞(Jurkat)为实验对象, 用先进的显微Raman光谱技术, 同时获得用其他方法难以得到的活细胞内数种生物大分子的构象信息. 通过比较单个和黏附细胞的显微Raman光谱的差异发现, 当细胞黏附时, 细胞内生物分子(DNA, 蛋白质, 碳水化合物, 脂类)的构象有不同的变化: (ⅰ) 双链DNA的骨架保持有序的B型或修改的B型构象, 然而它的脱氧核糖和碱基(A, G, C, T)的一些基团被修饰. (ⅱ) 蛋白质构象的主、侧链变化明显不同, 属于a螺旋和b折叠的一些谱线强度下降而b回折的一些谱线强度却明显增加; 酪氨酸和色氨酸从“埋藏状态”变成“暴露状态”; 它的巯基基团的谱线强度也有增加; 二硫键的构象从两种变成3种, 表明细胞黏附引起该蛋白质主链的氢键体系和侧链环境的变化. (ⅲ) 碳水化合物的一些基团同时被修饰. (ⅳ) 膜脂双层的构象变化很明显, 随着黏附细胞数目的增加, 链间侧向相互作用序参数逐渐减少, 提示细胞黏附导致细胞膜的流动性和离子通透性增加.     

DNA和RNA的种类名称及缩写

随着遗传物质脱氧核糖核酸(DNA)和核糖核酸(RNA)的深入研究,具有新的结构及功能特点的DNA、RNA不断揭示,因此其名称,尤其是缩写名称愈用愈多,愈用愈复杂。下面将近年来书刊中出现的一些DNA、RNA种类及名称简述如下: (一)DNA(Deoxyribonucleic acid) A—DNA A型脱氧核糖核酸。指相对湿度低于70％时的DNA分子构象。 B—DNA B型脱氧核糖核酸。指相对湿度在90％左右时,DNA在含水解质中的类晶型分子构象。     

质粒pUC18 DNA分子构象的多样性与线圈型超螺旋DNA

从大肠杆菌HB101细胞制备的质粒pUC18 DNA可被双向琼脂糖凝胶电泳分离成6组电泳行为不同的结构成分.超螺旋pUC18 DNA的两种结构形式:互缠型超螺旋与线圈型超螺旋构象同时存在于这种DNA样品中.在离子强度等环境条件改变时这两种DNA构象可以发生互变.DNA拓扑异构酶能改变线圈型DNA在样品中的含量.线圈型pUC18 DNA的电子显微镜表观为面包圈形,直径约45nm.实验事实表明线圈型超螺旋DNA是不依赖组蛋白而独立存在的一种结构实体.     

单链构象多态性分析与应用

单链构象多态性(single-strand conformational poly-morphism,SSCP)是一种简单、方便,且十分有效的分析核酸(DNA或RNA)变异的方法和技术,是Orita等在1989年首先建立的,其基本原理是单链核酸于非变性聚丙烯酰胺凝胶电泳的迁移率与核酸的一级结构和构象密切相关,核酸序列的变异,甚至单个碱基的改变,都可能改变核酸片段的构象,从而改变核酸的迁移速率,因此可以将“变异DNA”与正常的DNA区分开来。尽管目前还难以根据SSCP电泳迁移率的     

R-藻红蛋白介导的光敏反应对DNA分子的生物学效应

藻红蛋白(phycoerythrin, PE)是海藻中的重要捕光色素蛋白,具有强荧光性,易溶于水.在藻体内能将捕获的光能传递给光合反应中心; 在体外则能将光能传递给周围环境中的氧分子,产生如单线态氧等活性氧组分,可用来介导光动力效应治疗癌症.将纯化的藻红蛋白加入到瘤细胞培养基中,数小时后,采用488 nm波长的氩离子激光辐照,MTT法检测细胞存活数,计算细胞存活率. ³H-TdR掺入实验观察细胞DNA的合成.结果表明,藻红蛋白介导的光动力反应能够有效地抑制肿瘤细胞DNA合成并杀伤癌细胞.随着藻红蛋白浓度增加,DNA合成下降,瘤细胞存活率降低.将藻红蛋白加入到pUC18质粒溶液中,随之进行激光辐照,琼脂糖电泳结果可见pUC18构象由超螺旋（supercoiled）向带切口的环形构象(relax)转换.结果提示：通过改变或影响DNA构象,抑制细胞DNA合成可能是藻红蛋白介导肿瘤光动力治疗的途径之一.     

肠杆菌4.5SRNA基因单链构象多态性的比较研究

本文对大肠杆菌(Escherichia coli)不同菌株和肠杆菌科(Enterobacteriaceae)不同属其他三种菌株,即普通变形菌(Proteus vulgaris)、粘质沙雷氏菌(Serratia marcescens)和产气肠杆菌(Enterobacter aerogenes),分别进行4.5S RNA基因聚合酶链式反应(PCR),然后对扩增产物作依赖于序列的单链构象多态性(SSCP)分析.实验结果表明,上述细菌4.5S RNA基因的大小和正链构象均无可觉察的差异,仅产气肠杆菌的负链构象有明显不同.由此可见4.5S RNA基因在进化上相当保守,产气肠杆菌4.5S RNA基因的序列虽有改变,仍能维持其有义链的基本构象.     

脱血红素细胞色素c与膜结合及插膜时的构象研究

应用特殊的单分子层样品制备技术,分别制备了与中性、酸性磷脂膜结合的和完全插膜的鸡心脱血红素细胞色素c样品,并运用圆二色谱(CD)、表面衰减全反射Fourier变换红外光谱(ATR-FTIR)对膜上蛋白的构象进行了鉴定.研究结果表明,蛋白在与膜结合及插入阶段的构象是不同的,膜界面性质的不同也会对蛋白的构象产生不同的诱导,在酸性磷脂DSPG膜表面,该蛋白是以α螺旋和β折叠混合的构象形式结合;而在中性磷脂DSPC膜表面是以β折叠为主的构象形式结合.插入 DOPG单分子层内时则是 α螺旋为主的构象形式.     

部分柔性的核酸双螺旋构象的模型化研究

基于核酸结构单元的基本思想,在分析给出任意碱基对片段的自由度集合和两碱基对片段的简化自由度集合的基础上,建立了DNA/m5cDNA/RNA双螺旋结构的理论模型和两碱基对部分柔性的构象计算方法.利用本方法获得的标准B-DNA双螺旋构象参数与实验基本一致.根据DNA嵌插受体的统计性实验约束,优化产生了适用于阿霉素类抗癌药的三碱基对DNA片段的理论嵌插受体模型.     

Site-specific excision repair of 1-nitrosopyrene-induced DNA adducts at the nucleotide level in the HPRT gene of human fibroblasts: effect of adduct conformation on the pattern of site-specific repair.

Studies showing that different types of DNA adducts are repaired in human cells at different rates suggest that DNA adduct conformation is the major determinant of the rate of nucleotide excision repair. However, recent studies of repair of cyclobutane pyrimidine dimers or benzo[a]pyrene diol epoxide (BPDE)-induced adducts at the nucleotide level in DNA of normal human fibroblasts indicate that the rate of repair of the same adduct at different nucleotide positions can vary up to 10-fold, suggesting an important role for local DNA conformation. To see if site-specific DNA repair is a common phenomenon for bulky DNA adducts, we determined the rate of repair of 1-nitrosopyrene (1-NOP)-induced adducts in exon 3 of the hypoxanthine phosphoribosyltransferase gene at the nucleotide level using ligation-mediated PCR. To distinguish between the contributions of adduct conformation and local DNA conformation to the rate of repair, we compared the results obtained with 1-NOP with those we obtained previously using BPDE. The principal DNA adduct formed by either agent involves guanine. We found that rates of repair of 1-NOP-induced adducts also varied significantly at the nucleotide level, but the pattern of site-specific repair differed from that of BPDE-induced adducts at the same guanine positions in the same region of DNA. The average rate of excision repair of 1-NOP adducts in exon 3 was two to three times faster than that of BPDE adducts, but at particular nucleotides the rate was slower or faster than that of BPDE adducts or, in some cases, equal to that of BPDE adducts. These results indicate that the contribution of the local DNA conformation to the rate of repair at a particular nucleotide position depends upon the specific DNA adduct involved. However, the data also indicate that the conformation of the DNA adduct is not the only factor contributing to the rate of repair at different nucleotide positions. Instead, the rate of repair at a particular nucleotide position depends on the interaction between the specific adduct conformation and the local DNA conformation at that nucleotide.     

Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

G-rich telomeric DNA sequences can form G-quadruplex structures. The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and a shortened derivative (UP1) are active in telomere length regulation, and it has been reported that UP1 can unwind G-quadruplex structures. Here, we investigate the interaction of hnRNP A1 with G-quadruplex DNA structures containing the human telomere repeat (TTAGGG) by gel retardation assays, ensemble fluorescence energy transfer (FRET) spectroscopy, and single molecule FRET microscopy. Our biochemical experiments show that hnRNP A1 binds well to the G-quadruplex telomeric DNA. Ensemble and single molecule FRET measurements provide further insight into molecular conformation: the telomeric DNA overhang is found to be in a folded state in the absence of hnRNP A1 and to remain predominantly in a compact state when complexed with hnRNP A1. This finding is in contrast to the previously reported crystal structures of UP1-telomere DNA complexes where the DNA oligo within the protein-DNA complex is in a fully open conformation.     

Replication protein A phosphorylation and the cellular response to DNA damage

Defects in cellular DNA metabolism have a direct role in many human disease processes. Impaired responses to DNA damage and basal DNA repair have been implicated as causal factors in diseases with DNA instability like cancer, Fragile X and Huntington's. Replication protein A (RPA) is essential for multiple processes in DNA metabolism including DNA replication, recombination and DNA repair pathways (including nucleotide excision, base excision and double-strand break repair). RPA is a single-stranded DNA-binding protein composed of subunits of 70-, 32- and 14-kDa. RPA binds ssDNA with high affinity and interacts specifically with multiple proteins. Cellular DNA damage causes the N-terminus of the 32-kDa subunit of human RPA to become hyper-phosphorylated. Current data indicates that hyper-phosphorylation causes a change in RPA conformation that down-regulates activity in DNA replication but does not affect DNA repair processes. This suggests that the role of RPA phosphorylation in the cellular response to DNA damage is to help regulate DNA metabolism and promote DNA repair.     

8-Oxo-7,8-dihydrodeoxyadenosine: the first example of a native DNA lesion that stabilizes human telomeric G-quadruplex DNA

Native DNA lesions in general destabilize DNA secondary structures such as duplex and G-quadruplex because they disrupt optimized interactions in DNA defined by nature. In this paper, we report the first example of a native DNA lesion (8-oxo-7,8-dihydrodeoxyadenosine, OxodA) that stabilizes human telomeric G-quadruplex DNA. CD thermal denaturation studies explicitly displayed increased melting temperatures of telomeric G-quadruplex DNAs that contain OxodA(s) in different DNA loops, suggesting enhanced thermal stability. Conformation studies of G-quadruplex DNAs containing OxodA(s) in the loops using CD and native PAGE revealed that they adopt a similar antiparallel conformation in Na(+) but have much more versatile conformations in K(+). According to computational calculations, the observed stabilization may result from the tight binding of K(+) into the pocket formed by the O8 of OxodA and its loop. The study reported here may provide better understanding of the effect of DNA lesions on G-quadruplex stability and conformation.     

Structural polymorphism of intramolecular quadruplex of human telomeric DNA: effect of cations, quadruplex-binding drugs and flanking sequences

G-quadruplex structures formed in the telomeric DNA are thought to play a role in the telomere function. Drugs that stabilize the G-quadruplexes were shown to have anticancer effects. The structures formed by the basic telomeric quadruplex-forming unit G(3)(TTAG(3))(3) were the subject of multiple studies. Here, we employ (125)I-radioprobing, a method based on analysis of the distribution of DNA breaks after decay of (125)I incorporated into one of the nucleotides, to determine the fold of the telomeric DNA in the presence of TMPyP4 and telomestatin, G-quadruplex-binding ligands and putative anticancer drugs. We show that d[G(3)(TTAG(3))(3)(125)I-CT] adopts basket conformation in the presence of NaCl and that addition of either of the drugs does not change this conformation of the quadruplex. In KCl, the d[G(3)(TTAG(3))(3)(125)I-CT] is most likely present as a mixture of two or more conformations, but addition of the drugs stabilize the basket conformation. We also show that d[G(3)(TTAG(3))(3)(125)I-CT] with a 5'-flanking sequence folds into (3+1) type 2 conformation in KCl, while in NaCl it adopts a novel (3+1) basket conformation with a diagonal central loop. The results demonstrate the structural flexibility of the human telomeric DNA; and show how cations, quadruplex-binding drugs and flanking sequences can affect the conformation of the telomeric quadruplex.     

Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.     

Structure of the DNA Duplex d(ATTAAT)2 with Hoogsteen Hydrogen Bonds

The traditional Watson-Crick base pairs in DNA may occasionally adopt a Hoogsteen conformation, with a different organization of hydrogen bonds. Previous crystal structures have shown that the Hoogsteen conformation is favored in alternating AT sequences of DNA. Here we present new data for a different sequence, d(ATTAAT)₂, which is also found in the Hoogsteen conformation. Thus we demonstrate that other all-AT sequences of DNA with a different sequence may be found in the Hoogsteen conformation. We conclude that any all-AT sequence might acquire this conformation under appropriate conditions. We also compare the detailed features of DNA in either the Hoogsteen or Watson-Crick conformations.     

Locking the ATP-operated clamp of DNA gyrase: probing the mechanism of strand passage

DNA gyrase catalyses DNA supercoiling by passing one segment of DNA (the T segment) through another (the G segment) in a reaction coupled to the binding and hydrolysis of ATP. The N-terminal domains of the gyrase B dimer constitute an ATP-operated clamp that is proposed to capture the T segment during the DNA supercoiling reaction. We have locked this clamp in the closed conformation using the non-hydrolysable ATP analogue ADPNP (5'-adenylyl beta,gamma-imidodiphosphate). The clamp-locked enzyme is able to bind and cleave DNA, albeit at a reduced level. Although the locked enzyme is not capable of carrying out DNA supercoiling, it can catalyse limited DNA relaxation, consistent with the ability to complete one strand passage event per enzyme molecule via entry of the T segment through the exit gate of the enzyme. The DNA-protein complex of the clamp-locked enzyme has a conformation that differs from the normal positively wrapped conformation of the gyrase-DNA complex. These experiments confirm the role of the ATP-operated clamp in the strand-passage reactions of gyrase and suggest a model for the interaction of DNA with gyrase in which a conformation with the T segment in equilibrium across the DNA gate can be achieved via T-segment entry through the ATP-operated clamp or through the exit gate.