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Mansoureh Haghighi, Hossein Khanahmad, Abbasali Palizban
The B-lymphocyte antigen (CD20) is a suitable target for single-stranded (ss) nucleic acid oligomer (aptamers). The aim of study was selection and characterization of a ssDNA aptamer against CD20 using Cell-Systematic Evolution of Ligands by Exponential Enrichment (Cell-SELEX). The cDNA clone of CD20 (pcDNA-CD20) was transfected to human embryonic kidney (HEK293T) cells. Ten rounds of Cell-SELEX was performed on recombinant HEK-CD20 cells. The final eluted ssDNA pool was amplified and ligated in T/A vector for cloning...
March 21, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Marta Markiewicz-Potoczny, Michael Lisby, David Lydall
Dna2 is a nuclease and helicase that functions redundantly with other proteins in Okazaki fragment processing, double strand break (DSB) resection and checkpoint kinase activation. Dna2 is an essential enzyme, required for yeast and mammalian cell viability. Here we report that numerous mutations affecting the DNA damage checkpoint suppress dna2Δ lethality in Saccharomyces cerevisiae dna2Δ cells are also suppressed by deletion of helicases, PIF1 and MPH1 , and by deletion of POL32 , a subunit of DNA polymerase δ...
March 20, 2018: Genetics
Lu Wang, Jian Kang, Liangzhong Lim, Yuanyuan Wei, Jianxing Song
TDP-43 inclusions are characterized by a large spectrum of neurodegenerative diseases such as ALS and Alzheimer's. Functionally, TDP-43 is engaged in forming dynamic granules via liquid-liquid phase separation (LLPS), which is now recognized to be a general principle for organizing a variety of cellular membrane-less organelles. TDP-43 is composed of the N-terminal domain (NTD) adopting an ubiquitin-like fold, two RRMs and C-terminal domain (CTD) with the low-complexity (LC) prion-like sequences. Previously, only the CTD was found to undergo LLPS to form dynamic liquid droplets with relatively small numbers and sizes...
March 16, 2018: Biochemical and Biophysical Research Communications
Isidoro Feliciello, Davor Zahradka, Ksenija Zahradka, Siniša Ivanković, Nikolina Puc, Damir Đermić
Double strand breaks (DSBs) in E. coli chromosome (such as those induced by gamma rays) are repaired by recombination repair, during which a certain amount of DNA gets degraded. We monitored DNA degradation in gamma-irradiated cells to assess processing of DSBs. DNA degradation in irradiated cells is regulated by RecA protein concentration and its affinity of ssDNA binding, as well as by exonucleases that trim 3'-terminated ss tails. Here we determined the effects of proteins that affect formation and stability of RecA nucleofilaments on DNA degradation and cell survival...
March 16, 2018: Biochimie
Christina Pfaff, Hans F Ehrnsberger, Maria Flores-Tornero, Brian B Soerensen, Thomas Schubert, Gernot Längst, Joachim Griesenbeck, Stefanie Sprunck, Marion Grasser, Klaus D Grasser
The regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step linking transcript synthesis and processing with translation. However, in plants, only few of the factors that act in the mRNA export pathway have been functionally characterised. Flowering plant genomes encode several members of the ALY protein family, which function as mRNA export factors in other organisms. Arabidopsis thaliana ALY1-4 are commonly detected in root and leaf cells, but are differentially expressed in reproductive tissue...
March 14, 2018: Plant Physiology
Walhan Alshaer, Nidaa A Ababneh, Mamon Hatmal, Heba Izmirli, Moujab Choukeife, Alaa Shraim, Nour Sharar, Aya Abu-Shiekah, Fadwa Odeh, Abeer Al Bawab, Abdalla Awidi, Said Ismail
[This corrects the article DOI: 10.1371/journal.pone.0189558.].
2018: PloS One
Xiubao Chang, Yuexian Hou
Genome editing is a powerful tool to modify a specific gene and to correct a disease-causing mutation. Recently developed new techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9), significantly facilitate the progression in this field. However, mutations associated with the double strand DNA breaks (DSBs) introduced by these systems hampered their direct usage in clinic. In order to prevent the mutations caused by DSBs, we have designed a novel mean to induce homology-directed recombination (HDR) without DSBs, i...
2018: International Journal of Biochemistry and Molecular Biology
Alex Bronstein, Shay Bramson, Keren Shemesh, Batia Liefshitz, Martin Kupiec
Proper DNA damage repair is one of the most vital and fundamental functions of every cell. Several different repair mechanisms exist to deal with various types of DNA damage, in various stages of the cell cycle and under different conditions. Homologous recombination is one of the most important repair mechanisms in all organisms. Srs2, a regulator of homologous recombination, is a DNA helicase involved in DNA repair, cell cycle progression and genome integrity. Srs2 can remove Rad51 from ssDNA, and is thought to inhibit unscheduled recombination...
March 12, 2018: G3: Genes—Genomes—Genetics
Alicia Del Prado, Elsa Franco-Echevarría, Beatriz González, Luis Blanco, Margarita Salas, Miguel de Vega
Most replicative DNA polymerases (DNAPs) are endowed with a 3'-5' exonuclease activity to proofread the polymerization errors, governed by four universally conserved aspartate residues belonging to the Exo I, Exo II, and Exo III motifs. These residues coordinate the two metal ions responsible for the hydrolysis of the last phosphodiester bond of the primer strand. Structural alignment of the conserved exonuclease domain of DNAPs from families A, B, and C has allowed us to identify an additional and invariant aspartate, located between motifs Exo II and Exo III...
March 12, 2018: Proceedings of the National Academy of Sciences of the United States of America
Xiao-Mei Shi, Li-Ping Mei, Qian Wang, Wei-Wei Zhao, Jing-Juan Xu, Hong-Yuan Chen
We report herein the energy transfer (ET) between semiconducting polymer dots (Pdots) and gold nanoparticles (Au NPs) in a photoelectrochemical (PEC) system and its feasibility for cathodic bioanalysis application. Specifically, COOH-capped Pdots were first fabricated and then assembled onto the indium-tin oxide (ITO) surface, followed by the modification of single-strand (ss) DNA probe (pDNA). After the DNA hybridization with the Au NP-tethered complementary ssDNA (Au NP-tDNA), the Au NPs were brought into the close proximity of Pdots...
March 13, 2018: Analytical Chemistry
Jon Gil-Ranedo, Eva Hernando, Noelia Valle, Laura Riolobos, Beatriz Maroto, José M Almendral
The T1 parvovirus Minute Virus of Mice (MVM) was used to study the roles that phosphorylation and N-terminal domains (Nt) configuration of capsid subunits may play in icosahedral nuclear viruses assembly. In synchronous MVM infection, capsid subunits newly assembled as two types of cytoplasmic trimeric intermediates (3VP2, and 1VP1:2VP2) harbored a VP1 phosphorylation level fivefold higher than that of VP2, and hidden Nt. Upon nuclear translocation at S phase, VP1-Nt became exposed in the heterotrimer and subsequent subviral assembly intermediates...
March 7, 2018: Virology
Jinan Tuma Sabah, Razauden Mohamed Zulkifli, Shafinaz Shahir, Farediah Ahmed, Mohammed Rafiq Abdul Kadir, Zarita Zakaria
Distinctive bioactivities possessed by luteolin (3', 4', 5, 7-tetrahydroxy-flavone) are advantageous for sundry practical applications. This paper reports the in vitro selection and characterization of single stranded-DNA (ssDNA) aptamers, specific for luteolin (LUT). 76-mer library containing 1015 randomized ssDNA were screened via systematic evolution of ligands by exponential enrichment (SELEX). The recovered ssDNA pool from the 8th round was amplified with unlabeled primers and cloned into PSTBlue-1 vector prior to sequencing...
March 6, 2018: Analytical Biochemistry
Eleni P Mimitou, Scott Keeney
During meiosis, the specialized cell division giving rise to gametes, numerous DNA double-strand breaks (DSBs) are introduced at multiple places throughout the genome by the topoisomerase-like protein Spo11. Homologous recombination, a highly conserved DSB repair pathway, is employed for their repair and ensures the formation of chiasmata and the proper segregation of homologous chromosomes. In the initial steps of recombination, end resection takes place, wherein Spo11 is endonucleolytically released and the 5'-terminal strands of each DSB are exonucleolytically processed, exposing the ssDNA necessary to identify a homologous repair template...
2018: Methods in Enzymology
Davide Moiani, Daryl A Ronato, Chris A Brosey, Andrew S Arvai, Aleem Syed, Jean-Yves Masson, Elena Petricci, John A Tainer
For inhibitor design, as in most research, the best system is question dependent. We suggest structurally defined allostery to design specific inhibitors that target regions beyond active sites. We choose systems allowing efficient quality structures with conformational changes as optimal for structure-based design to optimize inhibitors. We maintain that evolutionarily related targets logically provide molecular avatars, where this Sanskrit term for descent includes ideas of functional relationships and of being a physical embodiment of the target's essential features without requiring high sequence identity...
2018: Methods in Enzymology
Stan J Maassen, Mark Vincent de Ruiter, Saskia Lindhoud, Jeroen J L M Cornelissen
Understanding the assembly pathway of viruses can contribute to creating monodisperse virus-based materials. In this study the Cowpea Chlorotic Mottle Virus is used to determine the interactions between the capsid proteins of viruses and their cargo. The assembly of the capsid proteins in the presence of different lengths of short single-stranded DNA is studied at neutral pH, where the protein-protein interactions are weak. Chromatography, electrophoresis, microscopy, and light scattering shows that the assembly efficiency and speed of the particles increases with increasing length of oligonucleotides...
March 8, 2018: Chemistry: a European Journal
Yanmei Si, Yaocai Bai, Xiaojie Qin, Jun Li, Wenwan Zhong, Zhijun Xiao, Jishan Li, Yadong Yin
A novel ratiometric surface-enhanced Raman scattering (SERS) nanosensor has been developed to probe the activity of endonuclease under in vitro and in living cells conditions. The optimized alloyed Au/Ag nanoparticles (Au/AgNPs) were synthesized as the SERS substrate, which combined the superior properties of both the pure Au and Ag nanoparticles: they exhibit excellent plasmonic property with high chemical stability and low cytotoxicity. They were then employed for quantitative detection of endonuclease through functionalization with single-stranded DNA (ssDNA) carrying 3-(4-(phenylethynyl)benzylthio) propanoic acid (PEB) as endonuclease-responsive SERS signaling molecule, and 4-thiol phenylacetylene (TPA) as the internal standard SERS signaling molecule...
March 5, 2018: Analytical Chemistry
Maja Sidstedt, Johannes Hedman, Erica L Romsos, Leticia Waitara, Lars Wadsö, Carolyn R Steffen, Peter M Vallone, Peter Rådström
Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity...
March 5, 2018: Analytical and Bioanalytical Chemistry
Eui-Hwan Choi, Seobin Yoon, Keun P Kim
Homologous recombination (HR), which ensures accurate DNA replication and strand-break repair, is necessary to preserve embryonic stem cell (ESC) self-renewal. However, little is known about how HR factors modulate ESC differentiation and replication stress-associated DNA breaks caused by unique cell-cycle progression. Here, we report that ESCs utilize Rad51-dependent HR to enhance viability and induce rapid proliferation through a replication-coupled pathway. In addition, ESC differentiation was shown to be enhanced by ectopic expression of a subset of recombinases...
February 8, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Atefeh Sarafan Sadeghi, Mohammad Mohsenzadeh, Khalil Abnous, Seyed Mohammad Taghdisi, Mohammad Ramezani
Specific ssDNA aptamers for the antibiotic florfenicol (FF) were developed from an enriched nucleotide library using magnetic beads-based SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique with high-binding affinity. After 12 rounds of selection, thirty-six sequences were obtained that were then divided into five major families, according to the primary sequence similarity. Binding affinity analyses of three fluorescently tagged aptamers belonging to different families demonstrated that the dissociation constants (Kd ) were in the low nanomolar range (Kd = 52...
May 15, 2018: Talanta
Tomasz Stenzel, Daria Dziewulska, Brejnev M Muhire, Penelope Hartnady, Simona Kraberger, Darren P Martin, Arvind Varsani
Circoviruses are circular single-stranded DNA (ssDNA) viruses that infect a variety of animals, both domestic and wild. Circovirus infection in birds is associated with immunosuppression and this in turn predisposes the infected animals to secondary infections that can lead to mortality. Farmed geese ( Anser anser ) in many parts of the world are infected with circoviruses. The majority of the current genomic information for goose circoviruses (GoCVs) ( n = 40) are from birds sampled in China and Taiwan, and only two genome sequences are available from Europe (Germany and Poland)...
March 2, 2018: Viruses
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