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Anna Szambowska, Ingrid Tessmer, Piotr Prus, Bernhard Schlott, Helmut Pospiech, Frank Grosse
Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA. Pull-down assays and surface plasmon resonance studies revealed that Cdc45-bound RPA complexed with ssDNA in the 8-10 nucleotide binding mode, but dissociated when RPA covered a 30-mer. Real-time analysis of RPA-ssDNA binding demonstrated that Cdc45 catalytically loaded RPA onto ssDNA...
January 18, 2017: Nucleic Acids Research
Priyamvada Jain, Babina Chakma, Sanjukta Patra, Pranab Goswami
A set of 90 mer long ssDNA candidates, with different degrees of cytosine (C-levels) (% and clusters) was analyzed for their function as suitable Ag-nanocluster (AgNC) nucleation scaffolds. The sequence (P4) with highest C-level (42.2%) emerged as the only candidate supporting the nucleation process as evident from its intense fluorescence peak at λ660 nm. Shorter DNA subsets derived from P4 with only stable hairpin structures could support the AgNC formation. The secondary hairpin structures were confirmed by PAGE, and CD studies...
March 1, 2017: Analytica Chimica Acta
Hui Yin Tan, Luke A Wilczek, Sasheen Pottinger, Maria Manosas, Cong Yu, Trong Nguyenduc, Piero R Bianco
The Escherichia coli single stranded DNA binding protein (SSB) is crucial for DNA replication, recombination and repair. Within each process, it has two seemingly disparate roles: it stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing and, forms complexes with a group of proteins known as the SSB-interactome. Key to both roles is the C-terminal, one-third of the protein, in particular the intrinsically disordered linker (IDL). Previously, we have shown using a series of linker deletion mutants that the IDL links both ssDNA and target protein binding by mediating interactions with the oligosaccharide/oligonucleotide binding fold in the target...
January 12, 2017: Protein Science: a Publication of the Protein Society
Phil De Luna, Sahar Mahshid, Jagotamoy Das, Binquan Luan, Edward H Sargent, Shana O Kelley, Ruhong Zhou
High-curvature electrodes facilitate rapid and sensitive detection of a broad class of molecular analytes. These sensors have reached detection limits not attained using bulk macroscale materials. It has been proposed that immobilized DNA probes are displayed at a high deflection angle on the sensor surface, allowing greater accessibility and more efficient hybridization. Here we report the first use of all-atom molecular dynamics simulations coupled with electrochemical experiments to explore the dynamics of ssDNA immobilized on high-curvature vs...
January 11, 2017: Nano Letters
Michael J Morten, Roland Gamsjaeger, Liza Cubeddu, Ruvini Kariawasam, Jose Peregrina, J Carlos Penedo, Malcolm F White
Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail...
January 10, 2017: Extremophiles: Life Under Extreme Conditions
Yulong Wang, Limin Wang, Juanjuan Xue, Jinbo Dong, Jia Cai, Xiude Hua, Minghua Wang, Cunzheng Zhang, Fengquan Liu
A signal-amplified lateral flow test strip (SA-LFTS) for the detection of Cu2+ in aqueous solution was constructed based on Cu+-catalyzed click chemistry and hybridization of single-stranded DNA (ssDNA). Alkyne and azide modified ssDNA acted as specific elements for Cu2+ recognition, and a chemical ligation product formed through Cu+-catalyzed alkyne-azide cycloaddition. Hybridization of ssDNA-labeled gold nanoparticles resulted in high sensitivity, and the output signal could be observed directly by the naked eye...
2017: PloS One
Shiling Gu, Qizhen Xue, Qin Liu, Mei Xiong, Wanneng Wang, Huidong Zhang
As one of the most common forms of oxidative DNA damage, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) generally leads to G:C to T:A mutagenesis. To study DNA replication encountering 8-oxoG by the sole DNA polymerase (Gp90) of Pseudomonasaeruginosa phage PaP1, we performed steady-state and pre-steady-state kinetic analyses of nucleotide incorporation opposite 8-oxoG by Gp90 D234A that lacks exonuclease activities on ssDNA and dsDNA substrates. Gp90 D234A could bypass 8-oxoG in an error-free manner, preferentially incorporate dCTP opposite 8-oxoG, and yield similar misincorporation frequency to unmodified G...
January 6, 2017: Genes
Cristina Gutiérrez Sánchez, Qiang Su, Sabine Wenderhold-Reeb, Gilbert Nöll
The multi-ligand binding flavoprotein dodecin is reconstituted on top of flavin-terminated oligonucleotide monolayers. A detailed quartz crystal microbalance with a dissipation monitoring (QCM-D) study showing how the length and flexibility of the oligonucleotide tethers influence the stability and the viscoelastic properties of the resulting DNA-protein layers is presented. Relatively dense protein layers can be obtained, if the length of the tethers is in the same range as the diameter of dodecin. When significantly longer tethers are used, less dense layers are formed...
June 24, 2016: RSC Advances
Jee Won Lee, Dohwan Lee, Yong Tae Kim, Eun Yeol Lee, Do Hyun Kim, Tae Seok Seo
This paper describes the development of a novel paper-based capillary electrophoresis (pCE) microdevice using mineral paper, which is durable, oil and tear resistant, and waterproof. The pCE device is inexpensive (~$1.6 per device for materials), simple to fabricate, lightweight, and disposable, so it is more adequate for point-of-care (POC) pathogen diagnostics than a conventional CE device made of glass, quartz, silicon or polymer. In addition, the entire fabrication process can be completed within 1h without using expensive clean room facilities and cumbersome photolithography procedures...
December 28, 2016: Biosensors & Bioelectronics
Remco P Crefcoeur, Omar Zgheib, Thanos D Halazonetis
DNA replication stress has the potential to compromise genomic stability and, therefore, cells have developed elaborate mechanisms to detect and resolve problems that may arise during DNA replication. The presence of single-stranded DNA (ssDNA) is often associated with DNA replication stress and serves as a signal for both checkpoint and repair responses. In this study, we exploited a CRISPR-Cas9 system to induce regions of ssDNA in the genome. Specifically, single-guide RNAs bearing sequence complementarity to human telomeric repeats, were used to target nuclease-deficient Cas9 (dCas9) to telomeres...
2017: PloS One
Christopher W Lennon, Matthew Stanger, Marlene Belfort
Inteins (or protein introns) autocatalytically excise themselves through protein splicing. We challenge the long-considered notion that inteins are merely molecular parasites and posit that some inteins evolved to regulate host protein function. Here we show substrate-induced and DNA damage-induced splicing, in which an archaeal recombinase RadA intein splices dramatically faster and more accurately when provided with ssDNA. This unprecedented example of intein splicing stimulation by the substrate of the invaded host protein provides compelling support in favor of inteins acting as pause buttons to arrest protein function until needed; then, an immediate activity switch is triggered, representing a new form of post-translational control...
December 28, 2016: Genes & Development
Yangang Pan, Zhiqiang Sun, Atanu Maiti, Tapan Kanai, Hiroshi Matsuo, Ming Li, Reuben S Harris, Luda S Shlyakhtenko, Yuri L Lyubchenko
The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of the HIV-1 virus in the absence of viral infectivity factor (Vif). The molecular mechanism of A3G antiviral activity is primarily attributed to deamination of single-stranded DNA (ssDNA); however the non-deamination mechanism also contributes to HIV-1 restriction. The interaction of A3G with ssDNA and RNA is reguired for its antiviral activity. Here we used atomic force microscopy (AFM) to directly visualize A3G-RNA and A3G-ssDNA complexes and compare to each other...
December 28, 2016: Biochemistry
Bret D Wallace, Zachary Berman, Geoffrey A Mueller, Yunfeng Lin, Timothy Chang, Sara N Andres, Jessica L Wojtaszek, Eugene F DeRose, C Denise Appel, Robert E London, Shan Yan, R Scott Williams
The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3'-5' nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9...
December 27, 2016: Proceedings of the National Academy of Sciences of the United States of America
Devashish Das, Maryam Faridounnia, Lidija Kovacic, Robert Kaptein, Rolf Boelens, Gert E Folkers
The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of the DNA damage. Functional studies have provided insight into the binding of ERCC1-XPF to various DNA substrates. However, since no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the Helix-hairpin-Helix domains of XPF and ERCC-XPF and show that the binding to ss/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF...
December 27, 2016: Journal of Biological Chemistry
Patrick Ruff, Roberto A Donnianni, Eleanor Glancy, Julyun Oh, Lorraine S Symington
DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR)...
December 20, 2016: Cell Reports
Zhisong Lu, Ying Wang, Dan Xu, Lei Pang
A DNA origami-based platform for detecting aflatoxin B1 has been constructed with the assistance of aptamer probes and its complementary ssDNA-modified gold nanoparticles. This work may open new horizons for the application of DNA origami in the detection of a variety of small molecules.
December 23, 2016: Chemical Communications: Chem Comm
Simon Roux, Natalie E Solonenko, Vinh T Dang, Bonnie T Poulos, Sarah M Schwenck, Dawn B Goldsmith, Maureen L Coleman, Mya Breitbart, Matthew B Sullivan
BACKGROUND: Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation)...
2016: PeerJ
Kun Li, Chen-Lin Xiu, Li-Ming Gao, Ming Shi, Yue Zhai
OBJECTIVE: To obtain DNA aptamers with a highly specific affinity to HIV gp41 antigen using SELEX screening for detection of HIV. METHODS: The specific DNA aptamers of HIV gp41 antigen were screened from the double-stranded DNA derived from the single-stranded DNA (ssDNA) library with agarose beads as the supportive medium and HIV gp41 antigen as the target molecule using SELEX technique and real-time quantitative PCR. RESULTS: The secondary ssDNA library obtained after 6 rounds of screening was amplified by PCR to obtain dsDNA...
December 20, 2016: Nan Fang Yi Ke da Xue Xue Bao, Journal of Southern Medical University
Gaofei Lu, Gregory R Bluemling, Paul Collop, Michael Hager, Damien Kuiper, Bharat P Gurale, George R Painter, Abel De La Rosa, Alexander A Kolykhalov
Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barre Syndrome cases. Currently, no vaccine or therapeutic options exist for the prevention or treatment of ZIKV infections. In this report, we have expressed, purified and characterized full-length NS5 and the polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we have developed an in vitro non-radioactive primer extension assay employing a fluorescently labeled primer-template pair...
December 19, 2016: Antimicrobial Agents and Chemotherapy
Chun Guo, Ryan Hili
We describe the development and analysis of the T4 DNA ligase-catalyzed DNA templated polymerization of pentanucleotides modified with peptide fragments toward the generation of ssDNA-scaffolded peptides. A high-throughput duplex DNA sequencing method was developed to facilitate the determination of fidelity for various codon sets and library sizes used during the polymerization process. With this process, we identified several codon sets that enable the efficient and sequence-specific incorporation of peptide fragments along a ssDNA template at fidelities up to 99% and with low sequence bias...
December 28, 2016: Bioconjugate Chemistry
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