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1.
Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification. 总被引:7,自引:5,他引:2
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Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases. 相似文献
2.
Single stranded DNA often forms stable secondary structures under physiological conditions. These DNA secondary structures play important physiological roles. However, the analysis of such secondary structure folded DNA is often complicated because of its high thermodynamic stability and slow hybridization kinetics. In this article, we demonstrate that Y-shaped junction probes could be used for rapid and highly efficient detection of secondary structure folded DNA. Our approach contained a molecular beacon (MB) probe and an assistant probe. In the absence of target, the MB probe failed to hybridize with the assistant probe. Whereas, the MB probe and the assistant probe could cooperatively unwind the secondary structure folded DNA target to form a ternary Y-shaped junction structure. In this condition, the MB probe was also opened, resulting in separating the fluorophores from the quenching moiety and emitting the fluorescence signal. This approach allowed for the highly sensitive detection of secondary structure folded DNA target, such as a tau specific DNA fragment related to Alzheimer's disease in this case. Additionally, this approach showed strong SNPs identifying capability. Furthermore, it was noteworthy that this newly proposed approach was capable of detecting secondary structure folded DNA target in cell lysate samples. 相似文献
3.
Determination of the elastic properties of short ssDNA molecules by mechanically folding and unfolding DNA hairpins
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The characterization of elastic properties of biopolymers is crucial to understand many molecular reactions determined by conformational bending fluctuations of the polymer. Direct measurement of such elastic properties using single‐molecule methods is usually hindered by the intrinsic tendency of such biopolymers to form high‐order molecular structures. For example, single‐stranded deoxyribonucleic acids (ssDNA) tend to form secondary structures such as local double helices that prevent the direct measurement of the ideal elastic response of the ssDNA. In this work, we show how to extract the ideal elastic response in the entropic regime of short ssDNA molecules by mechanically pulling two‐state DNA hairpins of different contour lengths. This is achieved by measuring the force dependence of the molecular extension and stiffness on mechanically folding and unfolding the DNA hairpin. Both quantities are fit to the worm‐like chain elastic model giving values for the persistence length and the interphosphate distance. This method can be used to unravel the elastic properties of short ssDNA and RNA sequences and, more generally, any biopolymer that can exhibit a cooperative two‐state transition between mechanically folded and unfolded states (such as proteins). © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1193–1199, 2014. 相似文献
4.
About 2% of mouse DNA reassociates at extremely low Cot values (10-7-10-6 mole-liter-1-sec). This DNA fraction was isolated with the aid of nuclease S1 and purified by chromatography on hydroxyapatite. The study of this fraction suggests that it has a structure of hairpin type. The DNA of the hairpins can hybridize with sequences forming the double-stranded regions in pre-mRNA. Probably at least some of the DNA of the hairpins, described as "reversed repeating sequences" of DNA, is transcribed with the formation of structures of hairpin type in pre-mRNA. 相似文献
5.
Mechanical unfolding of RNA structures, ranging from hairpins to ribozymes, using laser optical tweezer experiments have begun to reveal the features of the energy landscape that cannot be easily explored using conventional experiments. Upon application of constant force (f), RNA hairpins undergo cooperative transitions from folded to unfolded states whereas subdomains of ribozymes unravel one at a time. Here, we use a self-organized polymer model and Brownian dynamics simulations to probe mechanical unfolding at constant force and constant-loading rate of four RNA structures of varying complexity. For simple hairpins, such as P5GA, application of constant force or constant loading rate results in bistable cooperative transitions between folded and unfolded states without populating any intermediates. The transition state location (DeltaxFTS) changes dramatically as the loading rate is varied. At loading rates comparable to those used in laser optical tweezer experiments, the hairpin is plastic, with DeltaxFTS being midway between folded and unfolded states; whereas at high loading rates, DeltaxFTS moves close to the folded state, i.e., RNA is brittle. For the 29-nucleotide TAR RNA with the three-nucleotide bulge, unfolding occurs in a nearly two-state manner with an occasional pause in a high free energy metastable state. Forced unfolding of the 55 nucleotides of the Hepatitis IRES domain IIa, which has a distorted L-shaped structure, results in well-populated stable intermediates. The most stable force-stabilized intermediate represents straightening of the L-shaped structure. For these structures, the unfolding pathways can be predicted using the contact map of the native structures. Unfolding of a RNA motif with internal multiloop, namely, the 109-nucleotide prohead RNA that is part of the 29 DNA packaging motor, at constant value of rf occurs with three distinct rips that represent unraveling of the paired helices. The rips represent kinetic barriers to unfolding. Our work shows 1), the response of RNA to force is largely determined by the native structure; and 2), only by probing mechanical unfolding over a wide range of forces can the underlying energy landscape be fully explored. 相似文献
6.
An electron microscopic study of mouse foldback DNA. 总被引:14,自引:0,他引:14
Foldback DNA is defined by its rapid, concentration-independent renaturation, consistent with intramolecular base pairing of inverted repeat sequences. Foldback DNA, isolated from renatured mouse main band DNA by hydroxyapatite chromatography, is spread for electron microscopy by the formamide isodenaturing technique. A large fraction of the molecules can be recognized as intramolecular "hairpins"--structures in which complementary sequences on a single DNA strand form base-paired "stem" regions analogous to tRNA stems. The stem regions of the hairpins have a wide distribution of lengths, averaging about 1000 base pairs. About 60% of the stem regions terminate in single-stranded loops, ranging from 400 to many thousands of nucleotides in length, while 40% of the hairpins do not have discernible loops. There are about 40,000 hairpin-forming sequences in the main band portion of the mouse haploid genome. They appear to be either clustered in groups or confined to about one third of the DNA, rather than uniformly or randomly distributed. Another large fraction of the molecules seen in foldback DNA consists of linear structures, some of which are probably also hairpins. The electron microscopic results, along with simple theoretical considerations, make possible a better interpretation of our previous studies of the yield and S1 nuclease resistance of mouse foldback DNA. 相似文献
7.
8.
Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures. 相似文献
9.
Repetitive DNA sequences are abundant in eukaryotic genomes, and many of these sequences have the potential to adopt non-B DNA conformations. Genes harboring non-B DNA structure-forming sequences increase the risk of genetic instability and thus are associated with human diseases. In this review, we discuss putative mechanisms responsible for genetic instability events occurring at these non-B DNA structures, with a focus on hairpins, left-handed Z-DNA, and intramolecular triplexes or H-DNA. Slippage and misalignment are the most common events leading to DNA structure-induced mutagenesis. However, a number of other mechanisms of genetic instability have been proposed based on the finding that these structures not only induce expansions and deletions, but can also induce DNA strand breaks and rearrangements. The available data implicate a variety of proteins, such as mismatch repair proteins, nucleotide excision repair proteins, topoisomerases, and structure specific-nucleases in the processing of these mutagenic DNA structures. The potential mechanisms of genetic instability induced by these structures and their contribution to human diseases are discussed. 相似文献
10.
DNA topoisomerase II is an enzyme that specializes in DNA disentanglement. It catalyzes the interconversion of DNA between different topological states. This event requires the passage of one duplex through another one via a transient double-strand break. Topoisomerase II is able to process any type of DNA, including structures such as DNA juxtapositions (crossovers), DNA hairpins or cruciforms, which are recognized with high specificity. In this review, we focused our attention on topoisomerase II recognizing DNA substrates that possess particular geometries. A strong cleavage site, as we identified in pBR322 DNA in the presence of ellipticine (site 22), appears to be characterized by a cruciform structure formed from two stable hairpins. The same sequence could also constitute a four-way junction structure stabilized by interactions involving ATC sequences. The latter have been shown to be able to promote Holliday junctions. We reviewed the recent literature that deals with the preferential recognition of crossovers by various topoisomerases. The single molecule relaxation experiments have demonstrated the differential abilities of the topoisomerases to recognize crossovers. It appears that enzymes, which distinguish the chirality of the crossovers, possess specialized domains dedicated to this function. We also stress that the formation of crossovers is dependent on the presence of adequate stabilizing sequences. Investigation of the impact of such structures on enzyme activity is important in order to both improve our knowledge of the mechanism of action of the topoisomerase II and to develop new inhibitors of this enzyme. 相似文献
11.
Nanopore translocation experiments are increasingly applied to probe the secondary structures of RNA and DNA molecules. Here, we report two vital steps toward establishing nanopore translocation as a tool for the systematic and quantitative analysis of polynucleotide folding: 1), Using α-hemolysin pores and a diverse set of different DNA hairpins, we demonstrate that backward nanopore force spectroscopy is particularly well suited for quantitative analysis. In contrast to forward translocation from the vestibule side of the pore, backward translocation times do not appear to be significantly affected by pore-DNA interactions. 2), We develop and verify experimentally a versatile mesoscopic theoretical framework for the quantitative analysis of translocation experiments with structured polynucleotides. The underlying model is based on sequence-dependent free energy landscapes constructed using the known thermodynamic parameters for polynucleotide basepairing. This approach limits the adjustable parameters to a small set of sequence-independent parameters. After parameter calibration, the theoretical model predicts the translocation dynamics of new sequences. These predictions can be leveraged to generate a baseline expectation even for more complicated structures where the assumptions underlying the one-dimensional free energy landscape may no longer be satisfied. Taken together, backward translocation through α-hemolysin pores combined with mesoscopic theoretical modeling is a promising approach for label-free single-molecule analysis of DNA and RNA folding. 相似文献
12.
About 70% of the RNA tetra-loop sequences identified in ribosomal RNAs from different organisms fall into either (UNCG) or (GNRA) families (where N = A, C, G, or U; and R = A or G). RNA hairpins with these loop sequences form unusually stable tetra-loop structures. We have studied the RNA hairpin GGAC(UUCG)GUCC and several sequence variants to determine the effect of changing the loop sequence and the loop-closing base pair on the thermodynamic stability of (UNCG) tetra-loops. The hairpin GGAG(CUUG)CUCC with the conserved loop G(CUUG)C was also unusually stable. We have determined melting temperatures (Tm), and obtained thermodynamic parameters for DNA hairpins with sequences analogous to stable RNA hairpins with (UNCG), C(GNRA)G, C(GAUA)G, and G(CUUG)C loops. DNA hairpins with (TTCG), (dUdUCG), and related sequences in the loop, unlike their RNA counterparts, did not form unusually stable hairpins. However, DNA hairpins with the consensus loop sequence C(GNRA)G were very stable compared to hairpins with C(TTTT)G or C(AAAA)G loops. The C(GATA)G and G(CTTG)C loops were also extra stable. The relative stabilities of the unusually stable DNA hairpins are similar to those observed for their RNA analogs. 相似文献
13.
Using the NDB database, we calculated geometrical parameters that were needed to reproduce crystal structures of short DNA fragments in a phosphorus atom representation. The geometrical parameters were included in a software generating tertiary structures of, for example, the Escherichia coli and human chromosome 21 molecules of DNA whose complete nucleotide sequences are deposited in the EMBL and related databases. Both molecules were found to be heavily folded and composed of domains. A more elaborate version of the present approach will make analysis and comparison possible of tertiary structures of genomic DNA molecules of various chromosomes to identify the chromosome evolutionary and functional relationships. 相似文献
14.
A novel class of nucleolar RNAs from Tetrahymena. 总被引:1,自引:0,他引:1
We describe a family of at least four nucleolar RNAs (snoRNAs) from the ciliate, Tetrahymena. The snoRNAs are 120-140 nucleotides long, moderately AU-rich and contain no modified nucleotides. Their 5' ends are blocked by a cap of unknown nature. The snoRNAs can be folded into similar secondary structures consisting of two hairpins separated by a single-stranded AU-rich spacer. The sequences and secondary structures show no extensive sequence or secondary structure resemblance to any other small RNAs in the public databases. 相似文献
15.
16.
The inverted repeated sequences (foldback DNA) of yeast nuclear DNA have been examined by electron microscopy and hydroxyapatite chromatography. Of the inverted repeat structures seen in the electron microscope, 34% were hairpins and 66% had a single stranded loop at the end of a duplex stem. The number average length of the repeat was 0.3 kb and the single stranded loop was 1.6 kb. It is estimated that there are approximately 250 inverted repeats per haploid genome. A statistical analysis of the frequency of molecules containing multiple inverted repeats showed that these sequences are non-randomly distributed. The distribution of inverted repeats was also examined by measuring the fraction of total DNA in the foldback fraction that bound to hydroxyapatite as a function of single strand fragment size. This analysis also indicated that the inverted repeats are clustered. Renaturation kinetic analysis of isolated foldback and inverted repeat stem sequence DNA showed that these sequences are enriched for repetitive DNA. 相似文献
17.
Expansion of trinucleotide repeats (TNR) has been implicated in the emergence of neurodegenerative diseases. Formation of non-B conformations such as hairpins by these repeat sequences during DNA replication and/or repair has been proposed as a contributing factor to expansion. In this work we employed a combination of fluorescence, chemical probing, optical melting, and gel shift assays to characterize the structure of a series of (CTG)(n) sequences and the kinetic parameters describing their interaction with a complementary sequence. Our structure-based experiments using chemical probing reveal that sequences containing an even or odd number of CTG repeats adopt stem-loop hairpins that differ from one another by the absence or presence of a stem overhang. Furthermore, we find that this structural difference dictates the rate at which the TNR hairpins convert to duplex with a complementary CAG sequence. Indeed, the rate constant describing conversion to (CAG)(10)/(CTG)(n) duplex is slower for sequences containing an even number of CTG repeats than for sequences containing an odd number of repeats. Thus, when both the CAG and CTG hairpins have an even number of the repeats, they display a longer lifetime relative to when the CTG hairpin has an odd number of repeats. The difference in lifetimes observed for these TNR hairpins has implications toward their persistence during DNA replication or repair events and could influence their predisposition toward expansion. Taken together, these results contribute to our understanding of trinucleotide repeats and the factors that regulate persistence of hairpins in these repetitive sequences and conversion to canonical duplex. 相似文献
18.
V S Mikha?lov S N Voronova S B Akopov P K Kuldyev A M Atrazhev 《Molekuliarnaia biologiia》1991,25(1):240-249
Depending on the ionic environment the replicative complex of silkworm Bombyx mori, containing DNA polymerase alpha and primase, catalyzes on single-stranded DNA of phage M13 a NTP-dependent synthesis or elongation of preformed primers. In the presence of NTPs and dNTPs at conditions optimal for the NTP-dependent synthesis the replicative complex synthesizes on M13 DNA oligoribonucleotides of 9-11 residues, which serve as primers for polymerization of DNA. The length of RNA-primers synthesized by primase of the complex depends on concentration of dNTP but does not depend on activity of DNA polymerase alpha. During elongation of exogenic primers annealed to M13 DNA the complex is processive synthesizing DNA fragments of dozens residues without dissociation from the template. Double-stranded structures in DNA such as "hairpins" appear to be barriers for driving of the complex along the template and cause pauses in elongation. DNA-binding proteins the SSB of Escherichia coli or the p32 of phage T4 destabilize double-stranded regions in DNA and eliminate elongation pauses corresponding to these regions. The replicative complex is able to fill in single-stranded gaps in DNA completely and to perform slowly the synthesis with displacement of one of parent strands in duplexes via repeated cycles of binding to the primer-template, limited elongation and dissociation. 相似文献
19.
DNA library design for molecular computation. 总被引:1,自引:0,他引:1
A novel approach to designing a DNA library for molecular computation is presented. The method is employed for encoding binary information in DNA molecules. It aims to achieve a practical discrimination between perfectly matched DNA oligomers and those with mismatches in a large pool of different molecules. The approach takes into account the ability of DNA strands to hybridize in complex structures like hairpins, internal loops, or bulge loops and computes the stability of the hybrids formed based on thermodynamic data. A dynamic programming algorithm is applied to calculate the partition function for the ensemble of structures, which play a role in the hybridization reaction. The applicability of the method is demonstrated by the design of a twelve-bit DNA library. The library is constructed and experimentally tested using molecular biology tools. The results show a high level of specific hybridization achieved for all library words under identical conditions. The method is also applicable for the design of primers for PCR, DNA sequences for isothermal amplification reactions, and capture probes in DNA-chip arrays. The library could be applied for integrated DNA computing of twelve-bit instances of NP-complete combinatorial problems by multi-step DNA selection in microflow reactors. 相似文献
20.
CTG triplet repeat sequences have been found to form slipped-strand structures leading to self-expansion during DNA replication. The lengthening of these repeats causes the onset of neurodegenerative diseases, such as myotonic dystrophy. In this study, electrophoretic and NMR spectroscopic studies have been carried out to investigate the length and the structural roles of CTG repeats in affecting the hairpin formation propensity. Direct NMR evidence has been successfully obtained the first time to support the presence of three types of hairpin structures in sequences containing 1–10 CTG repeats. The first type contains no intra-loop hydrogen bond and occurs when the number of repeats is less than four. The second type has a 4 nt TGCT-loop and occurs in sequences with even number of repeats. The third type contains a 3 nt CTG-loop and occurs in sequences with odd number of repeats. Although stabilizing interactions have been identified between CTG repeats in both the second and third types of hairpins, the structural differences observed account for the higher hairpin formation propensity in sequences containing even number of CTG repeats. The results of this study confirm the hairpin loop structures and explain how slippage occurs during DNA replication. 相似文献