Measurement of anomalously high hydration of (dA)n.(dT)n double helices in dilute solution

Different DNA sequences have different physical properties, which seem to be important for their biological function. In particular, (dA)n.(dT)n has many unusual features, which include resistance to conformational changes in a variable chemical environment, an unusual thermodynamics of interaction with ligands, and the inability to reassociate into nucleosomes. Short A.T base-pair runs also play a critical role in DNA bending. It is believed that hydration of DNA is an important factor in determining the physical chemical and biological properties of different regions of DNA. Until now, however, it has not been possible to study the details of the hydration of DNA in dilute solution with sufficient sensitivity and precision. Moreover, it was not known if different base sequences differ in the extent of their hydration. Indirect evidence that (dA)n.(dT)n can be hydrated to a greater extent than other DNA sequences may be inferred from a recent study of the binding of drugs to polynucleotides. Here we used a novel high-precision technique measuring ultrasonic velocity to obtain direct estimates of the extent of hydration of various oligo- and polynucleotides in dilute solution. We report that different DNA sequences differ in their hydration, and that (dA)n.(dT)n in particular has an anomalously high level of hydration.     

Poly(dA).poly(dT) is a B-type double helix with a distinctively narrow minor groove

The structure of poly(dA).poly(dT) currently arouses great interest, mainly because dAn.dTn stretches are associated with considerable DNA bending. Until recently the heteronomous DNA described by Arnott et al., with the poly(dA) and poly(dT) chains in A and B conformations respectively, was the only detailed model of this structure. Following our earlier studies of the interaction of DNA and monovalent ions, we examined the X-ray diffraction of the bivalent Ca2+ salt of poly(dA).poly(dT) (Ca-poly(dA).poly(dT)) and found no sign of a heteronomous structure: Ca-poly(dA).poly(dT) in fibres shows fully equivalent B-type conformations of the opposite sugar-phosphate chains. A revision of the structure of the sodium salt, Na-poly(dA).poly(dT), based on this result, yields only a slightly heteronomous structure with each chain in a B-type conformation, which is in much better agreement with the experimental data underlying the original heteronomous model. Both structures, Ca- and Na-poly(dA).poly(dT), have a minor groove narrower than that of the B form: this peculiarity seems to be very important for the interaction of poly(dA).poly(dT) and biologically significant molecules (including proteins and antibiotics). The specific base-pair positions in poly(dA).poly(dT) may account for the DNA bending adjacent to dAn.dTn tracts.     

The structure of an oligo(dA).oligo(dT) tract and its biological implications

Poly(dA).poly(dT) has unusual properties in that it cannot associate into nucleosomes and short, phased runs of it cause DNA bending. The crystal structure of a B-type DNA dodecamer containing a homopolymeric run of six A.T base pairs shows that this region possesses special structural features, including a system of bifurcated hydrogen bonds, which explains some of the properties of this simple homopolymer.     

Dependence of DNA helix flexibility on base composition

We have used triplet anisotropy decay techniques to study the flexibility of synthetic DNA fragments with different base pair compositions. We have found major differences in the torsional and bending stiffness of poly(dG) . poly(dC), poly(dA) . poly(dT) and poly(dA-dC) . poly(dT-dG). Poly(dG) . poly(dC) has a torsional modulus more than 40 times larger than poly(dA-dC) . poly(dT-dG), and approximately 20 times larger than poly(dA) . poly(dT). These differences imply that the torsional stiffness of DNA can vary greatly with base composition. The Young's modulus (bending stiffness) we have measured for poly(dG) . poly(dC) is at least twice that of poly(dA-dC) . poly(dT-dG) or random sequence DNA, and is at least threefold greater than that of poly(dA) . poly(dT). This implies that the bending stiffness of DNA is also strongly dependent on base composition. In light of this dramatic base composition dependence, we suggest here that such stiffness variation may lead to local variations in the stability of chromatin or other protein complexes that require bending or twisting of the DNA helix.     

Molecular spectroscopy: complexity of excited-state dynamics in DNA

Absorption of ultraviolet light by DNA is known to lead to carcinogenic mutations, but the processes between photon absorption and the photochemical reactions are poorly understood. In their study of the excited-stated dynamics of model DNA helices using femtosecond transient absorption spectroscopy, Crespo-Hernández et al. observe that the picosecond component of the transient signals recorded for the adenine-thymine oligonucleotide (dA)18.(dT)18 is close to that for (dA)18, but quite different from that for (dAdT)9.(dAdT)9; from this observation, they conclude that excimer formation limits excitation energy to one strand at a time. Here we use time-resolved fluorescence spectroscopy to probe the excited-state dynamics, which reveals the complexity of these systems and indicates that the interpretation of Crespo-Hernández et al. is an oversimplification. We also comment on the pertinence of separating base stacking and base pairing in excited-state dynamics of double helices and question the authors' assignment of the long-lived signal component found for (dA)18.(dT)18 to adenine excimers.     

Estimation of wedge components in curved DNA

There are growing indications that the inherent curvature of DNA is important in protein-DNA recognition. The 10.5-base-pair (bp) periodicity of some dinucleotides first found in eukaryotic DNA sequences was interpreted as the expression of curvature of periodic segments of double-stranded DNA, the curvature resulting from co-orientation of periodically spaced 'wedges' between stacked base pairs. The wedge can be decomposed into roll and tilt components, opening towards a groove and a backbone respectively, both contributing to DNA curvature. The largest wedge was estimated to belong to the AA-TT dinucleotides. Recent work provided new experimental data on synthetic curved DNA. The authors tried to apply the wedge model to their results and met problems in doing so. We have found that taking into account both roll and tilt components of the AA-TT wedge, in the correct ratio, leads to remarkable consistency between the wedge model and the data.     

DNA bending at adenine . thymine tracts

Intrinsic bending of DNA molecules results from local structural polymorphism in regions of homopolymeric dA . dT which are at least 4 base pairs long; the A . T tracts must be repeated in phase with the helix screw. Bending, in the direction of base-pair tilt rather than roll, occurs at the junctions between the A . T tract and adjacent B-DNA, with a larger angle at the 3' than at the 5' end of the A tract.     

Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase

A functional immune system depends on the production of a wide range of immunoglobulin molecules. Immunoglobulin variable region (IgV) genes are diversified after gene rearrangement by hypermutation. In the DNA deamination model, we have proposed that deamination of dC residues to dU by activation-induced deaminase (AID) triggers this diversification. In hypermutating chicken DT40 B cells, most IgV mutations are dC --> dG/dA or dG --> dC/dT transversions, which are proposed to result from replication over sites of base loss produced by the excision activity of uracil-DNA glycosylase. Blocking the activity of uracil-DNA glycosylase should instead lead to replication over the dU lesion, resulting in dC --> dT (and dG --> dA) transitions. Here we show that expression in DT40 cells of a bacteriophage-encoded protein that inhibits uracil-DNA glycosylase shifts the pattern of IgV gene mutations from transversion dominance to transition dominance. This is good evidence that antibody diversification involves dC --> dU deamination within the immunoglobulin locus itself.     

三螺旋DNA分子久期方程的约化

利用矩阵分块技术和将二维矩阵化为一维矩阵的方法、明显地简化了poly(dT)·poly(dA)·poly(dT)的久期方程,大大节省了计算机内存空间和计算时间.这件方法可以用来计算具有螺旋对称性的所有巨分子的低频振动谱.     

Sequence-induced DNA curvature at the bacteriophage lambda origin of replication

DNA replication in bacteriophage lambda begins at a unique origin between residues 39,000 and 39,200 of the lambda genome. This segment of DNA serves a dual function since it also lies within the coding sequence of the lambda replication initiator protein O which binds origin DNA. The lambda origin sequence contains four 19-base-pair (bp) segments (iterons) which have dyad symmetry, followed by a 40-bp A + T-rich zone of highly asymmetrical base composition. It was noted earlier that lambda origin DNA exhibits an anomalous electrophoretic mobility on gels; that is, the length of DNA as determined by DNA sequencing is approximately 20% less than is predicted from electrophoretic mobility. Recent studies of kinetoplast minicircle DNA (K-DNA) from the protozoan Leishmania tarentolae have led to the proposal that sequence-induced DNA curvature could account for such electrophoretic anomalies by alteration of the shape of the DNA molecule. We now present evidence that the lambda origin contains a static curve.     

Structural details of an adenine tract that does not cause DNA to bend

Runs of adenines (adenine tracts) have been implicated as the main determinant of sequence-directed DNA bending. The most widely used experimental test for bending relies on the observation that bent DNA migrates more slowly than straight DNA on a polyacrylamide electrophoresis gel. It was shown recently that the polymer (GTTTTAAAAC)n runs with normal mobility on a gel, whereas (GAAAATTTTC)n runs more slowly and thus appears to be strongly bent. The observation that these similar sequences, which differ only in the order of the adenine and thymine tracts, adopt such different shapes offers a stringent test of theories to explain DNA bending. Although the wedge model for DNA bending has recently been elaborated to explain the gel mobilities of these molecules, we wished to determine experimentally the structural basis for the difference in bending. We report here measurements of the frequency of cleavage by the hydroxyl radical at each nucleotide of cloned versions of the two polymers (see Fig. 1). We show that the TTTTAAAA sequence does not display the cleavage pattern that is associated with bent DNA, whereas the AAAATTTT sequence does. The observed sequence dependence of the cleavage pattern of an adenine tract is at odds with current models for DNA bending, which assume that adenine tracts always adopt the same conformation.     

核酸的分子静电势──Ⅳ 自补十二聚合物序列d（Cp Gp Cp Gp Ap AP Tp Tp Cp Gp CP G）的分子静电势

本文介绍了Ｂ－ＤＮＡ自补十二聚合物序列ｄ（ＣｐＧｐＣｐＯｐＡｐＡｐＴｐＴｐＣｐＯｐＣｐＧ）的分子静电势的计算结果．讨论了这种结构对性能的影响．这些结果与其晶体的性质有关．尤其注意到寡核苷酸（ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ）的螺旋中心的静电势比其末端要深．这是如此短的螺旋的一般特性，而与碱基序列无关，但与ＤＮＡ寡聚体的反应性有重要关系．     

青岛文昌鱼神经胚中期cDNA文库的构建

提取青岛文昌鱼18小时神经胚中期mRNA,以5＇脱磷的NotI-oligo(dT)18为引物,反转录合成cDNA.双链cDNA的5＇端钝端连接上带EcoRI突出末端的衔接头,再经NotI酶切,在cDNA的3＇端形成NotI突出末端.以SizeSep离心层析柱除去400bp以下的小分子,与带有NotI和EcoRI突出末端并经过5＇端脱磷的λExCellNotI/EcoRI/CIP载体DNA进行连接,经体外包装和感染NM522宿主菌,得到了3.6×10     

Structure refined to 2A of a nicked DNA octanucleotide complex with DNase I

The cutting rates of bovine pancreatic deoxyribonuclease I (DNase I) vary along a given DNA sequence, indicating that the enzyme recognizes sequence-dependent structural variations of the DNA double-helix. In an attempt to define the helical parameters determining this sequence-dependence, we have co-crystallized a complex of DNase I with a self-complementary octanucleotide and refined the crystal structure at 2 A resolution. This structure confirms the basic features of an early model, namely that an exposed loop of DNase I binds in the minor groove of B-type DNA and that interactions do occur with the backbone of both strands. Nicked octamer duplexes that have lost a dinucleotide from the 3'-end of one strand are hydrogen-bonded across a two-fold axis in the crystal to form a quasi-continuous double helix of 14 base pairs. The DNA 14-mer has a B-type conformation and shows substantial distortion of both local and overall helix parameters, induced mainly by the tight interaction of Y73 and R38 in the unusually wide minor groove. Directly coupled to the widening of the groove by approximately 3A is a 21.5 degree bend of the DNA away from the bound enzyme towards the major groove, suggesting that both DNA stiffness and groove width are important in determining the sequence-dependence of the enzyme cutting rate. A second cut of the DNA which is induced by diffusion of Mn2+ into the co-crystals suggests that there are two active sites in DNase I separated by more than 15A.     

入口楔形对中厚板侧弯影响规律

为了研究轧件入口楔形对中厚板侧弯的影响规律,提高侧弯控制精度与效率,针对国内某中板厂精轧机组四辊轧机的生产实际情况,采用基于双悬臂梁的影响函数法开发了不对称条件下辊系辊弹性变形计算模块,阐述了该模块的离散化方法、关键数学模型及向量与矩阵的确定以及轧辊弹性变形的求解方法.基于现场实际数据进行入口楔形不对称条件下的模拟计算,得出了入口轧件楔形对出口轧件弯曲半径的影响规律,指出轧辊刚性倾斜是控制侧弯的有效手段,并建立了来料存在楔形时的目标楔形量计算模型及侧弯控制模型.     

Single-site enzymatic cleavage of yeast genomic DNA mediated by triple helix formation.

Physical mapping of chromosomes would be facilitated by methods of breaking large DNA into manageable fragments, or cutting uniquely at genetic markers of interest. Key issues in the design of sequence-specific DNA cleaving reagents are the specificity of binding, the generalizability of the recognition motif, and the cleavage yield. Oligonucleotide-directed triple helix formation is a generalizable motif for specific binding to sequences longer than 12 base pairs within DNA of high complexity. Studies with plasmid DNA show that triple helix formation can limit the operational specificity of restriction enzymes to endonuclease recognition sequences that overlap oligonucleotide-binding sites. Triple helix formation, followed by methylase protection, triple helix-disruption, and restriction endonuclease digestion produces near quantitative cleavage at the single overlapping triple helix-endonuclease site. As a demonstration that this technique may be applicable to the orchestrated cleavage of large genomic DNA, we report the near quantitative single-site enzymatic cleavage of the Saccharomyces cerevisiae genome mediated by triple helix formation. The 340-kilobase yeast chromosome III was cut uniquely at an overlapping homopurine-EcoRI target site 27 base pairs long to produce two expected cleavage products of 110 and 230 kilobases. No cleavage of any other chromosome was detected. The potential generalizability of this technique, which is capable of near quantitative cleavage at a single site in at least 14 megabase pairs of DNA, could enable selected regions of chromosomal DNA to be isolated without extensive screening of genomic libraries.     

与大豆叶片衰老相关的cDNA的克隆

植物叶片衰老是一个受遗传控制的细胞降解过程,最终导致叶片的死亡。一些研究结果已表明,蛋白酶基因的表达与叶片衰老过程相关,其中一些基因的表达具有衰老特异性。以半胱氨酸蛋白基因特异性引物和寡聚ｄＴ为引物,用ＲＴ－ＰＣＲ方法分别扩增来源于绿叶和诱导衰老叶片ｍＲＮＡ的互补ＤＮＡ（ｃＤＮＡ）,然后进行琼脂糖凝胶电泳,差异显示出一条衰老叶片样品所特有的ｃＤＮＡ带,将此ｃＤＮＡ进行了克隆和序列分析。分析结果证明     

Tying a molecular knot with optical tweezers.

Filamentous structures are abundant in cells. Relatively rigid filaments, such as microtubules and actin, serve as intracellular scaffolds that support movement and force, and their mechanical properties are crucial to their function in the cell. Some aspects of the behaviour of DNA, meanwhile, depend critically on its flexibility-for example, DNA-binding proteins can induce sharp bends in the helix. The mechanical characterization of such filaments has generally been conducted without controlling the filament shape, by the observation of thermal motions or of the response to external forces or flows. Controlled buckling of a microtubule has been reported, but the analysis of the buckled shape was complicated. Here we report the continuous control of the radius of curvature of a molecular strand by tying a knot in it, using optical tweezers to manipulate the strand's ends. We find that actin filaments break at the knot when the knot diameter falls below 0.4 microm. The pulling force at breakage is around 1 pN, two orders of magnitude smaller than the tensile stress of a straight filament. The flexural rigidity of the filament remained unchanged down to this diameter. We have also knotted a single DNA molecule, opening up the possibility of studying curvature-dependent interactions with associated proteins. We find that the knotted DNA is stronger than actin.