Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks

RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3' tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.     

Structural basis for gate-DNA recognition and bending by type IIA topoisomerases

Type II topoisomerases disentangle DNA to facilitate chromosome segregation, and represent a major class of therapeutic targets. Although these enzymes have been studied extensively, a molecular understanding of DNA binding has been lacking. Here we present the structure of a complex between the DNA-binding and cleavage core of Saccharomyces cerevisiae Topo II (also known as Top2) and a gate-DNA segment. The structure reveals that the enzyme enforces a 150 degrees DNA bend through a mechanism similar to that of remodelling proteins such as integration host factor. Large protein conformational changes accompany DNA deformation, creating a bipartite catalytic site that positions the DNA backbone near a reactive tyrosine and a coordinated magnesium ion. This configuration closely resembles the catalytic site of type IA topoisomerases, reinforcing an evolutionary link between these structurally and functionally distinct enzymes. Binding of DNA facilitates opening of an enzyme dimerization interface, providing visual evidence for a key step in DNA transport.     

Single-molecule analysis of DNA uncoiling by a type II topoisomerase

Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment. This enables them to untangle DNA and relax the interwound supercoils (plectonemes) that arise in twisted DNA. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled. By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the damping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.     

Chromosome length influences replication-induced topological stress

During chromosome duplication the parental DNA molecule becomes overwound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression, this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake. Despite these insights it remains largely unknown how replication-induced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of Saccharomyces cerevisiae chromosomes. This highlights the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter, chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin-related Smc5/6 complex. The frequency of chromosomal association sites of the Smc5/6 complex increases in response to chromosome lengthening, chromosome circularization, or inactivation of topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings that form behind the replication machinery.     

Newly replicated DNA is associated with DNA topoisomerase II in cultured rat prostatic adenocarcinoma cells

DNA topoisomerases have been proposed to function in a variety of genetic processes in both prokaryotes and eukaryotes. Here, we have assessed the role of DNA topoisomerase II in mammalian DNA replication by determining the proximity of newly synthesized DNA to covalent enzyme-DNA complexes generated by treating cultured rat prostatic adenocarcinoma cells with teniposide. Teniposide (VM-26), an epipodophyllotoxin, is known to interact with mammalian DNA topoisomerase II so as to trap the enzyme in a covalent complex with DNA. We have found that the teniposide-induced trapping of such complexes requires MgCl2, is stimulated by ATP and is inhibited by novobiocin. The formation of covalent complexes seems to be reversible on removal of teniposide. Furthermore, analysis of the covalent complexes formed between 3H-thymidine pulse-labelled DNA and topoisomerase II following teniposide treatment reveals a direct association of the enzyme with nascent DNA fragments. Our results suggest that DNA topoisomerase II may interact with newly replicated daughter DNA molecules near DNA replication forks in mammalian cells.     

Translocation step size and mechanism of the RecBC DNA helicase

DNA helicases are ubiquitous enzymes that unwind double-stranded DNA. They are a diverse group of proteins that move in a linear fashion along a one-dimensional polymer lattice--DNA--by using a mechanism that couples nucleoside triphosphate hydrolysis to both translocation and double-stranded DNA unwinding to produce separate strands of DNA. The RecBC enzyme is a processive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses slightly more than one ATP molecule per base pair unwound. Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme translocates along only one strand of duplex DNA in the 3'-->5' direction. The translocating enzyme will traverse, or 'step' across, single-stranded DNA gaps in defined steps that are 23 (+/-2) nucleotides in length. This step is much larger than the amount of double-stranded DNA that can be unwound using the free energy derived from hydrolysis of one molecule of ATP, implying that translocation and DNA unwinding are separate events. We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, inchworm-like mechanism that may have parallels for translocation by other linear motor proteins.     

Complex shapes self-assembled from single-stranded DNA tiles

Programmed self-assembly of strands of nucleic acid has proved highly effective for creating a wide range of structures with desired shapes. A particularly successful implementation is DNA origami, in which a long scaffold strand is folded by hundreds of short auxiliary strands into a complex shape. Modular strategies are in principle simpler and more versatile and have been used to assemble DNA or RNA tiles into periodic and algorithmic two-dimensional lattices, extended ribbons and tubes, three-dimensional crystals, polyhedra and simple finite two-dimensional shapes. But creating finite yet complex shapes from a large number of uniquely addressable tiles remains challenging. Here we solve this problem with the simplest tile form, a 'single-stranded tile' (SST) that consists of a 42-base strand of DNA composed entirely of concatenated sticky ends and that binds to four local neighbours during self-assembly. Although ribbons and tubes with controlled circumferences have been created using the SST approach, we extend it to assemble complex two-dimensional shapes and tubes from hundreds (in some cases more than one thousand) distinct tiles. Our main design feature is a self-assembled rectangle that serves as a molecular canvas, with each of its constituent SST strands--folded into a 3 nm-by-7 nm tile and attached to four neighbouring tiles--acting as a pixel. A desired shape, drawn on the canvas, is then produced by one-pot annealing of all those strands that correspond to pixels covered by the target shape; the remaining strands are excluded. We implement the strategy with a master strand collection that corresponds to a 310-pixel canvas, and then use appropriate strand subsets to construct 107 distinct and complex two-dimensional shapes, thereby establishing SST assembly as a simple, modular and robust framework for constructing nanostructures with prescribed shapes from short synthetic DNA strands.     

基于多输出端差动差分电流传送器的模拟电感

为在电路中消除无源电感器，以利于实现电路全集成，提出了一种基于多输出端差动差分电流传送器(MDDCC)的模拟电感电路，并用其和差动差分电流传送器(DDCC)实现的模拟电感构成了连续时间高通滤波器。用PSPICE仿真分析比较了两种模拟电感构成的高通滤波器的特性，仿真结果表明，提出的电路方案在组成高通滤波器时幅频特性优于DDCC。     

DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase

Helicases are molecular motors that use the energy of nucleoside 5'-triphosphate (NTP) hydrolysis to translocate along a nucleic acid strand and catalyse reactions such as DNA unwinding. The ring-shaped helicase of bacteriophage T7 translocates along single-stranded (ss)DNA at a speed of 130 bases per second; however, T7 helicase slows down nearly tenfold when unwinding the strands of duplex DNA. Here, we report that T7 DNA polymerase, which is unable to catalyse strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase up to similar speeds compared to its translocation along ssDNA. The helicase rate of stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between T7 DNA polymerase and the carboxy-terminal residues of T7 helicase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The strand displacement DNA synthesis by the DNA polymerase depends on the unwinding activity of the helicase, which provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.     

Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).     

Height measurement of DNA molecules with lift mode AFM

Atomic force microscopy (AFM) can be used to im-age and study the local properties of the sample surfacethrough tip/sample interactions. There are three operationmodes: contact mode, non-contact mode and tappingmode. Mostly, the measured heights of dsDNA were only0.7—0.8 nm[1—8] in air by tapping mode atomic force mi-croscopy (TMAFM), which were much less than the gen-erally accepted diameter of 2.0—2.2 nm[9]. In TMAFM, stable imaging is available when the tip is closer to sample surf…     

颈动脉药物滴注治疗急性脑梗塞疗效对比

对急性脑梗塞(acute cerebral infarction,ACI)60例,随机分为颈动脉加压滴注瘫康灵组和常规治疗对照组,治疗后效果有显著差异(P<0.01)。颈动脉药物滴注组效果明显。     

海藻酸钠溶液溶胶——凝胶相转变的温度效应与流变学研究

本文考察了０．０３ｇ／ｍｌ的海藻酸的溶液中加入ＣＯ２＋后体系的粘度与离子浓度和温度的关系,结果表明,体系在升温过程中存在着分子簇构型的转变,即由分子内交联形成的分子簇型向分子间交联形式的转变过程,这种转变不具有温度的可逆性．不同交联离子浓度下体系的剪切行为表明,在体系的表现粘度（ηα与剪切速率（γ）的幂次公式ηα～γｎ中,幂指数ｎ在－０．３１～－０．５８之间,并从分子链交叠和分子交联的角度对这一结果进行了定性的剖析．     

基于DNA链置换反应的编码器逻辑运算模型研究

作为自组装DNA计算领域中一门新技术,DNA链置换反应在分子计算领域得到了广泛的应用.基于自组装DNA计算原理,设计了对应不同逻辑门的DNA分子电路.基于DNA链置换反应机理构建了编码器逻辑电路的分子计算模型.当输入DNA分子信号链时,将不同分子浓度比的DNA分子逻辑门电路混合,借助分子间的特异性杂交反应及分子间链置换反应,最终可输出信号链分子.Visual DSD仿真结果表明了本文设计的编码器逻辑计算模型的可行性与准确性.为拓展分子逻辑电路的应用做出有益的探索.     

Electrical conduction through DNA molecules

The question of whether DNA is able to transport electrons has attracted much interest, particularly as this ability may play a role as a repair mechanism after radiation damage to the DNA helix. Experiments addressing DNA conductivity have involved a large number of DNA strands doped with intercalated donor and acceptor molecules, and the conductivity has been assessed from electron transfer rates as a function of the distance between the donor and acceptor sites. But the experimental results remain contradictory, as do theoretical predictions. Here we report direct measurements of electrical current as a function of the potential applied across a few DNA molecules associated into single ropes at least 600 nm long, which indicate efficient conduction through the ropes. We find that the resistivity values derived from these measurements are comparable to those of conducting polymers, and indicate that DNA transports electrical current as efficiently as a good semiconductor. This property, and the fact that DNA molecules of specific composition ranging in length from just a few nucleotides to chains several tens of micrometres long can be routinely prepared, makes DNA ideally suited for the construction of mesoscopic electronic devices.