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Patricia Perez-Arnaiz, Daniel L Kaplan
Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by DDK in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and substantially decreased DNA replication. We also observed a substantially reduced RPA-ChIP signal at origins of replication, reduced levels of DDK-phosphorylated-Mcm2 and diminished GINS association with Mcm2-7 in vivo...
October 14, 2016: Journal of Molecular Biology
Benjamin Kick, Samantha Hensler, Florian Praetorius, Hendrik Dietz, Dirk Weuster-Botz
The bacteriophage M13 has found frequent applications in nanobiotechnology due to its chemically and genetically tunable protein surface and its ability to self-assemble into colloidal membranes. Additionally, its single-stranded (ss) genome is commonly used as scaffold for DNA origami. Despite the manifold uses of M13, upstream production methods for phage and scaffold ssDNA are underexamined with respect to future industrial usage. Here, the high-cell-density phage production with Escherichia coli as host organism was studied in respect of medium composition, infection time, multiplicity of infection and specific growth rate...
October 17, 2016: Biotechnology and Bioengineering
Qiang Su, Gilbert Nöll
The orientation of surface grafted dsDNA assemblies relative to the surface depends strongly on the nature of the employed thiol anchor. This was shown by ssDNA capture probe strands of 20 bases grafted to a gold surface by three dithiane rings or a single mercaptohexyl group. The capture probe strands were hybridized to one end of complementary ssDNA strands (target) comprising 40, 60, or 80 bases (T40, T60, and T80). At the other end of the targets a fluorophore-labeled reporter probe ssDNA strand of 20 bases was hybridized...
October 16, 2016: Chemistry: a European Journal
Sung-Jin Choi, Changill Ban
Structural elements are key elements for understanding single-stranded nucleic acid folding. Although various RNA structural elements have been documented, structural elements of single-stranded DNA (ssDNA) have rarely been reported. Herein, we determined a crystal structure of PvLDH in complex with a DNA aptamer called pL1. This aptamer folds into a hairpin-bulge contact by adopting three novel structural elements, viz, DNA T-loop-like motif, base-phosphate zipper, and DNA G·G metal ion zipper. Moreover, the pL1:PvLDH complex shows unique properties compared with other protein:nucleic acid complexes...
October 11, 2016: Scientific Reports
S Y New, S T Lee, X D Su
12 years after the introduction of DNA-templated silver nanoclusters (DNA-AgNCs), exciting progress has been made and yet we are still in the midst of trying to fully understand this nanomaterial. The prominent excellence of DNA-AgNCs is undoubtedly its modulatable emission property, of which how variation in DNA templates causes emission tuning remains elusive. Based on the up-to-date DNA-AgNCs, we aim to establish the correlation between the structure/sequence of DNA templates and emission behaviour of AgNCs...
October 7, 2016: Nanoscale
Hui Wang, Ke Ma, Bin Xu, Wenjing Tian
Aggregation-induced emission (AIE) probes have shown great potential applications in fluorescent sensing of biomolecules, and the integration of AIE probe and graphene oxide (GO) attracts intense interest in developing new tools for label-free and "turn-on" fluorescent biomolecular analysis. Herein, an ultrasensitive label-free and "turn-on" DNA sensing is realized by tuning the supramolecular interactions of AIE probe and GO with DNA. The investigation of supramolecular interactions of AIE probes and GO with DNA demonstrate that AIE probe with short alkyl chains substitute shows highest binding affinity with DNA strand, and GO with low oxidation degree possesses strong binding interactions to ssDNA and the highest fluorescence quenching efficiency...
September 26, 2016: Small
Thomas Wiegand, Riccardo Cadalbert, Carole Gardiennet, Joanna Timmins, Laurent Terradot, Anja Böckmann, Beat H Meier
DnaB helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the helicase. (13) C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding...
October 6, 2016: Angewandte Chemie
Martina B Schierling, Elena Doblhofer, Thomas Scheibel
Medical therapies are often accompanied by un-wanted side-effects or, even worse, targeted cells can develop drug resistance leading to an ineffective treatment. Therefore, drug delivery systems are under investigation to lower the risk thereof. Drug carriers should be biocompatible, biodegradable, nontoxic, non-immunogenic, and should show controllable drug loading and release properties. Previous studies qualified spider silk particles as drug delivery carriers, however, cellular uptake was only tested with unloaded spider silk particles...
September 20, 2016: Biomaterials Science
Sivaraman Subramaniam, Axel Erler, Jun Fu, Andrea Kranz, Jing Tang, Mohanraj Gopalswamy, Saminathan Ramakrishnan, Adrian Keller, Guido Grundmeier, Daniel Müller, Michael Sattler, A Francis Stewart
Single strand annealing proteins (SSAPs) like Redβ initiate homologous recombination by annealing complementary DNA strands. We show that C-terminally truncated Redβ, whilst still able to promote annealing and nucleoprotein filament formation, is unable to mediate homologous recombination. Mutations of the C-terminal domain were evaluated using both single- and double stranded (ss and ds) substrates in recombination assays. Mutations of critical amino acids affected either dsDNA recombination or both ssDNA and dsDNA recombination indicating two separable functions, one of which is critical for dsDNA recombination and the second for recombination per se...
October 6, 2016: Scientific Reports
Leah Elizabeth Page, Xi Zhang, Christina Marie Tyrakowski, Chiun-Teh Ho, Preston Todd Snee
A quantum dot-based ratiometrically responsive fluorescent sensor for unlabeled single-stranded DNA (ssDNA) is reported. Several technical issues concerning the development of high yield ssDNA-QD conjugation chemistry were addressed. The DNA sensor was synthesized by conjugating methacrylic phosphoramidite-functional oligonucleotides to water-soluble cadmium zinc sulfide core/zinc sulfide shell quantum dots (CdZnS/ZnS QDs). Duplex DNA was formed when the QD-bound ssDNA was incubated with its complement. Next, titration with PicoGreen resulted in FRET energy transfer from the dot to the dsDNA intercalating dye...
September 26, 2016: Analyst
Mona Alibolandi, Marzieh Mohammadi, Seyed Mohammad Taghdisi, Mohammad Ramezani, Khalil Abnous
In the current study, dextran (DEX) was covalently conjugated to the surface of nano-GO sheets, making stable biocompatible dextran coated GO (GO-DEX). The prepared GO-DEX was nontoxic to 4T1 mammary carcinoma cell line at concentrations up to 300μg/mL. AS1411 aptamer, a ssDNA aptamer which can improve the intracellular uptake by nucleolin recognition, also has been introduced to hydroxyl groups of DEX in GO-DEX to produce GO-DEX-Apt. Moreover, curcumin (CUR), a natural polyphenol, found in the rhizomes of Curcuma longa (turmeric) which shows antineoplastic effects, was loaded onto the GO-DEX and GO-DEX-Apt via π-π stacking interactions with a high loading capacity (∼29wt%)...
January 2, 2017: Carbohydrate Polymers
Subir Sarker, María C Terrón, Yogesh Khandokar, David Aragão, Joshua M Hardy, Mazdak Radjainia, Manuel Jiménez-Zaragoza, Pedro J de Pablo, Fasséli Coulibaly, Daniel Luque, Shane R Raidal, Jade K Forwood
The assembly and regulation of viral capsid proteins into highly ordered macromolecular complexes is essential for viral replication. Here, we utilize crystal structures of the capsid protein from the smallest and simplest known viruses capable of autonomously replicating in animal cells, circoviruses, to establish structural and mechanistic insights into capsid morphogenesis and regulation. The beak and feather disease virus, like many circoviruses, encode only two genes: a capsid protein and a replication initiation protein...
October 4, 2016: Nature Communications
Andrew A Kelso, Steven D Goodson, Leah E Watts, LeAnna L Ledford, Sarah M Waldvogel, J Nathaniel Diehl, Shivani B Shah, Amanda F Say, Julie D White, Michael G Sehorn
Homologous recombination (HR) is a template-driven repair pathway that mends DNA double-stranded breaks (DSBs), and thus helps to maintain genome stability. The RAD51 recombinase facilitates DNA joint formation during HR, but to accomplish this task, RAD51 must be loaded onto the single-stranded DNA. DSS1, a candidate gene for split hand/split foot syndrome, provides the ability to recognize RPA-coated ssDNA to the tumor suppressor BRCA2, which is complexed with RAD51. Together BRCA2-DSS1 displace RPA and load RAD51 onto the ssDNA...
September 30, 2016: Nucleic Acids Research
Wonbae Lee, John P Gillies, Davis Jose, Brett A Israels, Peter H von Hippel, Andrew H Marcus
Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex...
September 30, 2016: Nucleic Acids Research
Anukana Bhattacharjee, Jason Stewart, Mary Chaiken, Carolyn M Price
Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function...
September 2016: PLoS Genetics
Andrew A Kelso, Steven D Goodson, Suchitra Chavan, Amanda F Say, Audrey Turchick, Deepti Sharma, LeAnna L Ledford, Erin Ratterman, Kristin Leskoske, Ada V King, Christopher C Attaway, Yura Bandera, Stephen H Foulger, Alexander V Mazin, Lesly A Temesvari, Michael G Sehorn
The protozoan parasite responsible for human amoebiasis is Entamoeba histolytica. An important facet of the life cycle of E. histolytica involves the conversion of the mature trophozoite to a cyst. This transition is thought to involve homologous recombination (HR), which is dependent upon the Rad51 recombinase. Here, a biochemical characterization of highly purified ehRad51 protein is presented. The ehRad51 protein preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA...
September 24, 2016: Molecular and Biochemical Parasitology
Sheng Lin, Lihua Lu, Tian-Shu Kang, Jean-Louis Mergny, Chung-Hang Leung, Dik-Lung Ma
Sialic acid (Sia) binding immunoglobulin (Ig)-like lectin-5 (Siglec-5) is a type-I transmembrane protein, and it has been demonstrated as a biomarker of granulocytic maturation and acute myeloid leukemia phenotype. Herein we aimed to construct a method that could sensitively detect Siglec-5 by taking advantage of the high affinity and selectivity of the K19 aptamer for its cognate target, and the selective interaction of luminescent iridium(III) transition metal complexes with G-quadruplex DNA. The iridium(III) complex 1 [Ir(tpyd)2(2,9-dmphen)]PF6 (where tpyd =2-(m-tolyl)pyridine; 2,9-dmphen =2,9-dimethyl-1,10-phenanthroline) was synthesized, and it displayed high luminescence for G-quadruplex DNA compared to dsDNA and ssDNA...
October 6, 2016: Analytical Chemistry
Shyamal Subramanyam, Mohammed Ismail, Ipshita Bhattacharya, Maria Spies
The DNA strand exchange protein RAD51 facilitates the central step in homologous recombination, a process fundamentally important for accurate repair of damaged chromosomes, restart of collapsed replication forks, and telomere maintenance. The active form of RAD51 is a nucleoprotein filament that assembles on single-stranded DNA (ssDNA) at the sites of DNA damage. The c-Abl tyrosine kinase and its oncogenic counterpart BCR-ABL fusion kinase phosphorylate human RAD51 on tyrosine residues 54 and 315. We combined biochemical reconstitutions of the DNA strand exchange reactions with total internal reflection fluorescence microscopy to determine how the two phosphorylation events affect the biochemical activities of human RAD51 and properties of the RAD51 nucleoprotein filament...
October 11, 2016: Proceedings of the National Academy of Sciences of the United States of America
Maher Al Rwahnih, Olufemi J Alabi, Nathaniel M Westrick, Deborah Golino, Adib Rowhani
A novel virus was detected in grapevines by Illumina sequencing during the screening of two table grape (Vitis vinifera) accessions, cvs. Black Beet and Nagano Purple, from South Korea. The monopartite circular ssDNA genome sequence was subsequently confirmed by rolling cycle amplification, cloning and Sanger sequencing. Complete viral genomic sequences from both accessions ranging in length from 2,903 to 2,907 nucleotides contained the conserved nonanucleotide sequence TAATATT↓AC and other motifs typical of the family Geminiviridae...
September 27, 2016: Phytopathology
Hongliang Tan, Gonge Tang, Zhixiong Wang, Qian Li, Jie Gao, Shimeng Wu
Metal-organic frameworks (MOFs) have emerged as very fascinating functional materials due to their tunable nature and diverse applications. In this work, we prepared a magnetic porous carbon (MPC) nanocomposite by employing iron-containing MOFs (MIL-88A) as precursors through a one-pot thermolysis method. It was found that the MPC can absorb selectively single-stranded DNA (ssDNA) probe to form MPC/ssDNA complex and subsequently quench the labelled fluorescent dye of the ssDNA probe, which is resulted from the synergetic effect of magnetic nanoparticles and carbon matrix...
October 12, 2016: Analytica Chimica Acta
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