Asymmetric DNA Research Articles

Asymmetric Cell Division of Fibroblasts is An Early Deterministic Step to Generate Elite Cells during Cell Reprogramming.

Cell reprogramming is considered a stochastic process, and it is not clear which cells are prone to be reprogrammed and whether a deterministic step exists. Here, asymmetric cell division (ACD) at the early stage of induced neuronal (iN) reprogramming is shown to play a deterministic role in generating elite cells for reprogramming. Within one day, fibroblasts underwent ACD, with one daughter cell being converted into an iN precursor and the other one remaining as a fibroblast. Inhibition of ACD significantly inhibited iN conversion. Moreover, the daughter cells showed asymmetric DNA segregation and histone marks during cytokinesis, and the cells inheriting newly replicated DNA strands during ACD became iN precursors. These results unravel a deterministic step at the early phase of cell reprogramming and demonstrate a novel role of ACD in cell phenotype change. This work also supports a novel hypothesis that daughter cells with newly replicated DNA strands are elite cells for reprogramming, which remains to be tested in various reprogramming processes.

Absolute and arbitrary orientation of single-molecule shapes

DNA origami is a modular platform for the combination of molecular and colloidal components to create optical, electronic, and biological devices. Integration of such nanoscale devices with microfabricated connectors and circuits is challenging: Large numbers of freely diffusing devices must be fixed at desired locations with desired alignment. We present a DNA origami molecule whose energy landscape on lithographic binding sites has a unique maximum. This property enabled device alignment within 3.2° on silica surfaces. Orientation was absolute (all degrees of freedom were specified) and arbitrary (the orientation of every molecule was independently specified). The use of orientation to optimize device performance was shown by aligning fluorescent emission dipoles within microfabricated optical cavities. Large-scale integration was demonstrated with an array of 3456 DNA origami with 12 distinct orientations that indicated the polarization of excitation light.

Orienting origami binding

DNA Origami Directing self-assembly for devices will require placing nanocomponents not only in the correct position on a surface but also in particular orientations. Gopinath et al. designed an asymmetric DNA origami, a “small moon” shape that binds to lithographically patterned sites on silica to within 3° of a target orientation angle. The authors were able to position and orient a molecular dipole within the resonant mode of an optical cavity. More than 3000 DNA origami were arranged in a single fabrication step in 12 different orientations to create a simple polarimeter. Science , this issue p. [eabd6179][1] [1]: /lookup/doi/10.1126/science.abd6179

Cancer Cell Fitness Is Dynamic.

Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynamic. Here we used the number of cells in colonies as an assessment of fitness and devised a novel method called Dynamic Fitness Analysis (DynaFit) to measure the dynamics in fitness over the course of colony formation. DynaFit is based on the variance in growth rate of a population of founder cells compared with the variance in growth rate of colonies with different sizes. DynaFit revealed that cell fitness in cancer cell lines, primary cancer cells, and fibroblasts under unhindered growth conditions is dynamic. Key cellular mechanisms such as ERK signaling and cell-cycle synchronization differed significantly among cells in colonies after 2 to 4 generations and became indistinguishable from randomly sampled cells regarding these features. In the presence of cytotoxic agents, colonies reduced their variance in growth rate when compared with their founder cell, indicating a dynamic nature in the capacity to survive and proliferate in the presence of a drug. This finding was supported by measurable differences in DNA damage and induction of senescence among cells of colonies. The presence of epigenetic modulators during the formation of colonies stabilized their fitness for at least four generations. Collectively, these results support the understanding that cancer cell fitness is dynamic and its modulation is a fundamental aspect to be considered in comprehending cancer cell biology and its response to therapeutic interventions. SIGNIFICANCE: Cancer cell fitness is dynamic over the course of the formation of colonies. This dynamic behavior is mediated by asymmetric mitosis, ERK activity, cell-cycle duration, and DNA repair capacity in the absence or presence of a drug.

The Emergence of the Bilateral Symmetry in Animals: A Review and a New Hypothesis

Most biological organisms exhibit different kinds of symmetry; an Animal (Metazoa), which is our Darwinist ancestor, has bilateral symmetry, and many plants exhibit rotational symmetry. It raises some questions: I. How can the evolution from an undifferentiated cell without bilateral symmetry to a complex biological organism with symmetry, which is based on asymmetric DNA and enzymes, lead to the bilateral symmetry? II. Is this evolution to an organism with bilateral symmetry obtained by other factors than DNA and enzymatic reactions? The existing literature about the evolution of the bilateral symmetry has been reviewed, and a new hypothesis has been formulated based on these reviews. The hypothesis is that the morphogenesis of biosystems is connected with the metabolism and that the oscillating kinetics in the Glycolysis have played a role in the polarity of the biological cells and in the establishment of the bilateral symmetry in Animals.

The Effect of a Rearward Conservative Force and a Dragged Load on a Synthetic Nano-Motor Inspired by Kinesin

The object of our research is to design and measure the performance of synthetic protein constructs that mimic biological nano-motors. In a previous article we presented a persistent symmetric nano-walker design named Synthetic Kinesin Inspired Protein (SKIP). Here, we extend this design to an asymmetric, directional and processive nano-motor construct called SKIP-4R and describe and contrast the effect of a rearward conservative force and a dragged load on the motor properties of the system. Unlike kinesin, SKIP-4R's motor movement is controlled by clocked ligand pulses that activate the binding and unbinding of four different repressor proteins (RA, RB, RC, RD) to an asymmetric DNA track. To simplify future synthesis and assembly, we use a redesigned structure of SKIP (Roberta Davies, private communication) in terms of a composite of two short and two longer coiled coils (rods), each linking a different repressor RA, RB, RC or RD flexibly to a central hub. The motion of SKIP-4R was simulated using Langevin dynamics in the overdamped limit both for a rearward conservative force and a dragged load and we found that the motor both stalled and then reversed in the former case, whereas it simply stalled for large drag coefficients in the latter case. We also show that SKIP-4R can be reduced to a simpler motor (SKIP-3R) when one short rod is removed. The effects of both a rearward conservative force and a dragged load on the motion of SKIP-3R and SKIP-4R are described in detail.

DNA Template Strand Segregation in Whole Zebrafish

Asymmetric inheritance of sister chromatids has long been predicted to be linked to discordant fates of daughter cells and even hypothesized to minimize accumulation of mutations in stem cells. Here, we use (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU), bromodeoxyuridine (BrdU), and light-sheet microscopy to track embryonic DNA in whole zebrafish. Larval development resulted in rapid depletion of older, template DNA strands from stem cell niches in retina, brain and intestine. Prolonged label retention occurred in quiescent progenitors that resumed replication in later development. Quantifying >100 daughter cell pairs throughout the body showed no evidence of asymmetric template strand segregation, making it improbable that asymmetric DNA segregation prevents mutational burden.

The interplay between asymmetric and symmetric DNA loop extrusion.

Chromosome compaction is essential for reliable transmission of genetic information. Experiments suggest that ∼1000-fold compaction is driven by condensin complexes that extrude chromatin loops, by progressively collecting chromatin fiber from one or both sides of the complex to form a growing loop. Theory indicates that symmetric two-sided loop extrusion can achieve such compaction, but recent single-molecule studies (Golfier et al., 2020) observed diverse dynamics of condensins that perform one-sided, symmetric two-sided, and asymmetric two-sided extrusion. We use simulations and theory to determine how these molecular properties lead to chromosome compaction. High compaction can be achieved if even a small fraction of condensins have two essential properties: a long residence time and the ability to perform two-sided (not necessarily symmetric) extrusion. In mixtures of condensins I and II, coupling two-sided extrusion and stable chromatin binding by condensin II promotes compaction. These results provide missing connections between single-molecule observations and chromosome-scale organization.

Replication Stress Induces ATR/CHK1-Dependent Nonrandom Segregation of Damaged Chromosomes

Nonrandom DNA segregation (NDS) is a mitotic event in which sister chromatids carrying the oldest DNA strands are inherited exclusively by one of the two daughter cells. Although this phenomenon has been observed across various organisms, the mechanism and physiological relevance of this event remain poorly defined. Here, we demonstrate that DNA replication stress can trigger NDS in human cells. This biased inheritance of old template DNA is associated with the asymmetric DNA damage response (DDR), which derives at least in part from telomeric DNA. Mechanistically, we reveal that the ATR/CHK1 signaling pathway plays an essential role in mediating NDS. We show that this biased segregation process leads to cell-cycle arrest and cell death in damaged daughter cells inheriting newly replicated DNA. These data therefore identify a key role for NDS in the maintenance of genomic integrity within cancer cell populations undergoing replication stress due to oncogene activation.

Detection of DNA Double-Strand Breaks and Chromosome Translocations Using Ligation-Mediated PCR and Inverse PCR.

Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allow detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location.

Reproductive characteristics of two Triturus species (Amphibia: Caudata)

During three consecutive years, we compared the reproductive characteristics and oviposition dynamics of two crested newt species, Triturus ivanbureschi and T. macedonicus. These two well-defined species are of special interest because of complex interactions at their contact zone, which include hybridization, species replacement and asymmetric mitochondrial DNA introgression. In common garden experiments, females were introduced to conspecific males, to males of other species and to hybrid males. We monitored the total number and size of the deposited eggs, as well as the dynamics and duration of oviposition during three consecutive years. The number of deposited eggs of Triturus species was much higher than previously reported. Triturus macedonicus lay more eggs that were of larger size in comparison to T. ivanbureschi. Also, the onset of T. macedonicus oviposition was considerably delayed compared to T. ivanbureschi. These are fundamental data, important for understanding complex species interactions in their contact zones.

Transcription-induced formation of extrachromosomal DNA during yeast ageing.

Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper-resistance gene CUP1. Transcription of the tandemly repeated CUP1 gene causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2, Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant, we find that CUP1 eccDNA does not replicate efficiently, and high-copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.

Transposon molecular domestication and the evolution of the RAG recombinase.

Domestication of a transposon to give rise to the RAG1/RAG2 recombinase and V(D)J recombination was a pivotal event in the evolution of the jawed vertebrate adaptive immune system. The evolutionary adaptations that transformed the ancestral RAG transposase into a RAG recombinase with appropriately regulated DNA cleavage and transposition activities are not understood. Here, beginning with cryo-electron microscopy structures of RAG’s evolutionary relative, the ProtoRAG transposase from amphioxus, we identify amino acid residues and domains whose acquisition or loss underpins RAG’s propensity for coupled cleavage, preference for asymmetric DNA substrates, and inability to perform transposition in cells. In particular, we identify two jawed-vertebrate-specific adaptations—arginine 848 in RAG1 and an acidic region in RAG2—that together suppress RAG-mediated transposition more than 1000-fold. Our findings reveal a two-tiered mechanism for suppression of RAG-mediated transposition, illuminate the forces at work during the evolution of V(D)J recombination, and provide insight into the principles governing transposon molecular domestication.

Azouracil and Its Cu(II)-Catalyzed Cyclization to an Anticancer Active Triazole Derivative: Symmetrical and Asymmetrical Reductive Cleavage, DNA Interaction, and Molecular Docking Studies.

The 6-amino-1,3-dimethyl uracil-based azo derivative (p-carboxy phenylazouracil, L11) undergoes Cu(II)-catalyzed cyclization to a triazole derivative, namely, 1,3-dimethyl-8-(p-carboxy phenyl) azapurine (L11P). Interestingly, the azo functionality of L11 undergoes both symmetrical and asymmetrical reductive cleavage at two different reaction conditions. The chloride salts of Mn(II), Ni(II), and Pd(II) catalyze reductive cleavage of an azo moiety in an asymmetric manner, producing a new uracil hydrazine derivative (A3). On the other hand, hydrazine catalyzes symmetrical reductive cleavage of the azo moiety of L11, resulting in 5,6-diamino-1,3-dimethyl uracil (A2) along with the starting p-aminobenzoic acid (A1). Time-dependent density functional theoretical (TD-DFT) studies provide optimized geometries of L11, L11P, and A3 along with their orbital energies. The L11 and L11P bind firmly to genomic DNA of E. coli with a site size n ∼ 9 and n ∼ 8. The L11P shows anticancer activity on selected murine lymphoma cancer cell lines (DL, YAC1, and 2PK3). In addition, its antiproliferative activity is measured with several cancer cell lines and found hemocompatible toward blood cells. Corresponding molecular docking studies of L11P with caspase-3 (cysteine-aspartic proteases) unlock their mode of interaction.

Histopathological Image Retrieval Based on Asymmetric Residual Hash and DNA Coding

Histopathological image retrieval is a key technology for computer-aided diagnosis. However, patients are reluctant to reveal their privacy in histopathological image retrieval. In order to further improve the effectiveness and safety of histopathological image retrieval, this paper proposes a new histopathological retrieval scheme based on asymmetric residual hash (ARH) and DNA coding techniques. In this paper, we first present a novel ARH for histopathological image retrieval to improve the effectiveness of histopathological search scheme, and then we use the 5-D hyperchaotic system to protect patient privacy. Specifically, the contribution consists of four aspects: 1) A histopathology ciphertext domain search scheme was proposed to improve the performance of computer-aided diagnosis. 2) An asymmetric approach was implemented to process histopathological query points and database points, and a novel asymmetric residual hash algorithm was first proposed to improve the accuracy and speed of histopathological image retrieval. 3) The 5-D hyperchaotic system and DNA coding technique are applied to histopathological image retrieval to protect patient privacy. 4) The loss function is constructed and optimized to learn network parameters and hash codes. The simulation experiment was performed on three datasets (Kimia Path24, Kimia Path960, and Malaria), and the results proved the effectiveness of the ARH algorithm. In addition, our proposed search method can resist common types of attacks during histopathological data transmission. Specifically, the MAP of the ARH is 0.9678 on the KIMIA Path24 with the value of hyperparameter is 125 and the length of hash code is 32. The MAP of the ARH is 0.966 on the KIMIA Path960 with the value of hyperparameter is 225 and the length of hash code is 32. The MAP of the ARH is 0.9482 on the Malaria with the value of hyperparameter is 10 and the length of hash code is 24.

Design and Analysis of DNA Encryption and Decryption Technique based on Asymmetric Cryptography System

Security of sensitive information at the time of transmission over public channels is one of the critical issues in digital society. The DNA-based cryptography technique is a new paradigm in the cryptography field that is used to protect data during transmission. In this paper we introduce the asymmetric DNA cryptography technique for encrypting and decrypting plain-texts. This technique is based on the concept of data dependency, dynamic encoding and asymmetric cryptosystem (i.e. RSA algorithm). The asymmetric cryptosystem is used solely to initiate the encryption and decryption processes that are completely conducted using DNA computing. The basic idea is to create a dynamic DNA table based on the plaintext, using multi-level security, data dependency and generating 14 dynamic round keys. The proposed technique is implemented using the JAVA platform and its efficiency is examined in terms of avalanche property. The evaluation process proves that the proposed technique outperforms the RSA algorithm in terms of avalanche property.

Integrative single-cell omics analyses reveal epigenetic heterogeneity in mouse embryonic stem cells.

Embryonic stem cells (ESCs) consist of a population of self-renewing cells displaying extensive phenotypic and functional heterogeneity. Research towards the understanding of the epigenetic mechanisms underlying the heterogeneity among ESCs is still in its initial stage. Key issues, such as how to identify cell-subset specifically methylated loci and how to interpret the biological meanings of methylation variations remain largely unexplored. To fill in the research gap, we implemented a computational pipeline to analyze single-cell methylome and to perform an integrative analysis with single-cell transcriptome data. According to the origins of variation in DNA methylation, we determined the genomic loci associated with allelic-specific methylation or asymmetric DNA methylation, and explored a beta mixture model to infer the genomic loci exhibiting cell-subset specific methylation (CSM). We observed that the putative CSM loci in ESCs are significantly enriched in CpG island (CGI) shelves and regions with histone marks for promoter and enhancer, and the genes hosting putative CSM loci show wide-ranging expression among ESCs. More interestingly, the putative CSM loci may be clustered into co-methylated modules enriching the binding motifs of distinct sets of transcription factors. Taken together, our study provided a novel tool to explore single-cell methylome and transcriptome to reveal the underlying transcriptional regulatory networks associated with epigenetic heterogeneity of ESCs.

Simplified detection of the asymmetric polymerase chain reaction-amplified DNA and its application in the target identification

Screening paper currency for Mycobacterium tuberculosis using loop-mediated isothermal amplificationManoj Nimesh, Rigzin Kang, Deepali Joon

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress

The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs invivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations invitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands.

Asymmetric DNA methylation between sister chromatids of metaphase chromosomes in mouse embryos upon bisphenol A action

Earlier we showed that asymmetric methylation of sister chromatids (AMSC) was a specific characteristic of differentiation potency, and supposed that AMSC could be a useful marker of environmental impact connected with differentiation and/or dedifferentiation. Here we investigated the level of AMSC in chromosomes and the nuclei methylation in mouse preimplantation and postimplantation embryos, in comparison with the undifferentiated cells of mouse embryonal carcinoma cell line F9, and human differentiated HEK293 cells upon BPA influence. We found that exposure of mouse preimplantation embryos to BPA caused a significant decrease in the level of AMSC in chromosomes and the nuclei methylation. The BPA exposure of potentially differentiating F9 cells had no any influence on DNA methylation in nuclei but significantly decreased the number of AMSC. The level of DNA methylation and AMSC in HEK293 cells were not also changed. These data indicate that BPA exerts significant influence on differentiating and potentially differentiable cells. The most sensitive BPA targets are preimplantation embryos and stem cells.