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Sample records for adenine-modified functionalized dna

  1. DNA: Structure and function

    DEFF Research Database (Denmark)

    Sinden, Richard R.; E. Pearson, Christopher; N. Potaman, Vladimir

    1998-01-01

    This chapter discusses the structure and function of DNA. DNA occupies a critical role in cells, because it is the source of all intrinsic genetic information. Chemically, DNA is a very stable molecule, a characteristic important for a macromolecule that may have to persist in an intact form...... for a long period of time before its information is accessed by the cell. Although DNA plays a critical role as an informational storage molecule, it is by no means as unexciting as a computer tape or disk drive. The structure of the DNA described by Watson and Crick in 1953 is a right handed helix of two...

  2. DNA structure and function.

    Science.gov (United States)

    Travers, Andrew; Muskhelishvili, Georgi

    2015-06-01

    The proposal of a double-helical structure for DNA over 60 years ago provided an eminently satisfying explanation for the heritability of genetic information. But why is DNA, and not RNA, now the dominant biological information store? We argue that, in addition to its coding function, the ability of DNA, unlike RNA, to adopt a B-DNA structure confers advantages both for information accessibility and for packaging. The information encoded by DNA is both digital - the precise base specifying, for example, amino acid sequences - and analogue. The latter determines the sequence-dependent physicochemical properties of DNA, for example, its stiffness and susceptibility to strand separation. Most importantly, DNA chirality enables the formation of supercoiling under torsional stress. We review recent evidence suggesting that DNA supercoiling, particularly that generated by DNA translocases, is a major driver of gene regulation and patterns of chromosomal gene organization, and in its guise as a promoter of DNA packaging enables DNA to act as an energy store to facilitate the passage of translocating enzymes such as RNA polymerase. © 2015 FEBS.

  3. Nanoarchitectonics with Porphyrin Functionalized DNA

    Science.gov (United States)

    2017-01-01

    Conspectus DNA is well-known as bearer of the genetic code. Since its structure elucidation nearly seven decades ago by Watson, Crick, Wilkins, and Franklin, much has been learned about its detailed structure, function, and genetic coding. The development of automated solid-phase synthesis, and with it the availability of synthetic DNA with any desired sequence in lengths of up to hundreds of bases in the best case, has contributed much to the advancement of the field of DNA research. In addition, classic organic synthesis has allowed introduction of a very large number of modifications in the DNA in a sequence specific manner, which have initially been targeted at altering the biological function of DNA. However, in recent years DNA has become a very attractive scaffold in supramolecular chemistry, where DNA is taken out of its biological role and serves as both stick and glue molecule to assemble novel functional structures with nanometer precision. The attachment of functionalities to DNA has led to the creation of supramolecular systems with applications in light harvesting, energy and electron transfer, sensing, and catalysis. Functional DNA is clearly having a significant impact in the field of bioinspired nanosystems. Of particular interest is the use of porphyrins in supramolecular chemistry and bionanotechnology, because they are excellent functional groups due to their electronic properties that can be tailored through chemical modifications of the aromatic core or through insertion of almost any metal of the periodic table into the central cavity. The porphyrins can be attached either to the nucleobase, to the phosphate group, or to the ribose moiety. Additionally, noncovalent templating through Watson–Crick base pairing forms an alternative and attractive approach. With this, the combination of two seemingly simple molecules gives rise to a highly complex system with unprecedented possibilities for modulation of function, and with it applications

  4. Functional footprinting of regulatory DNA.

    Science.gov (United States)

    Vierstra, Jeff; Reik, Andreas; Chang, Kai-Hsin; Stehling-Sun, Sandra; Zhou, Yuanyue; Hinkley, Sarah J; Paschon, David E; Zhang, Lei; Psatha, Nikoletta; Bendana, Yuri R; O'Neil, Colleen M; Song, Alexander H; Mich, Andrea K; Liu, Pei-Qi; Lee, Gary; Bauer, Daniel E; Holmes, Michael C; Orkin, Stuart H; Papayannopoulou, Thalia; Stamatoyannopoulos, George; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D; Stamatoyannopoulos, John A

    2015-10-01

    Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing-induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.

  5. Altruistic functions for selfish DNA

    National Research Council Canada - National Science Library

    Faulkner, Geoffrey J; Carninci, Piero

    2009-01-01

    .... In a major development, recent works have strongly contradicted this "selfish DNA" or "junk DNA" dogma by demonstrating that TEs use a host of novel promoters to generate RNA on a massive scale...

  6. Thermal stability of DNA functionalized gold nanoparticles.

    Science.gov (United States)

    Li, Feng; Zhang, Hongquan; Dever, Brittany; Li, Xing-Fang; Le, X Chris

    2013-11-20

    Therapeutic uses of DNA functionalized gold nanoparticles (DNA-AuNPs) have shown great potential and exciting opportunities for disease diagnostics and treatment. Maintaining stable conjugation between DNA oligonucleotides and gold nanoparticles under thermally stressed conditions is one of the critical aspects for any of the practical applications. We systematically studied the thermal stability of DNA-AuNPs as affected by organosulfur anchor groups and packing densities. Using a fluorescence assay to determine the kinetics of releasing DNA molecules from DNA-AuNPs, we observed an opposite trend between the temperature-induced and chemical-induced release of DNA from DNA-AuNPs when comparing the DNA-AuNPs that were constructed with different anchor groups. Specifically, the bidentate Au-S bond formed with cyclic disulfide was thermally less stable than those formed with thiol or acyclic disulfide. However, the same bidentate Au-S bond was chemically more stable under the treatment of competing thiols (mercaptohexanol or dithiothreitol). DNA packing density on AuNPs influenced the thermal stability of DNA-AuNPs at 37 °C, but this effect was minimum as temperature increased to 85 °C. With the improved understanding from these results, we were able to design a strategy to enhance the stability of DNA-AuNPs by conjugating double-stranded DNA to AuNPs through multiple thiol anchors.

  7. Light-induced functions in DNA.

    Science.gov (United States)

    Reisacher, Ulrike; Antusch, Linda; Hofsäß, Robert; Schwechheimer, Christian; Lehmann, Benjamin; Wagenknecht, Hans-Achim

    2017-10-01

    The chemical toolbox for synthetic modification by nucleotide building blocks and postsynthetic methods delivers light-induced functions to DNA in great variety and allows not only to initiate photoinduced processes but additionally the temporal and spatial control of these artificial functions. Herein, selected light-induced artificial functions in DNA are briefly summarized. This includes the postsynthetic 'photoclick' labeling strategy, benzophenone and acetophenone nucleosides as photosensitizers to induce [2+2] cycloadditions, molecular switches and energy transfer based fluorophore pairs, called "DNA traffic lights". Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Controlling Function and Structure with DNA

    DEFF Research Database (Denmark)

    Tørring, Thomas

    2011-01-01

    for constructing DNA-protein hybrids. The project is still ongoing, but our initial results and thoughts on the design are reported. The design was based on the concept of DNA directed chemistry, where stable covalent bonds are directed by weaker non-covalent bonds. Applying this concept, an aptamer for the human...... investigated on a two dimensional DNA origami platform. This was done by incorporating functional groups on the surface of the origami, and reacting these with biotin analogues carrying the complementary functional groups. Successful reactions could then be observed using atomic force microscopy after addition...... of the protein streptavidin. While the implementation of chemical functionalities on origami can be achieved during automated DNA synthesis, this is laborious and costly. In a separate research project we aimed at improving the accessibility by applying an enzymatic labelling method. We demonstrated that the DNA...

  9. Functionalization of DNA nanostructures with proteins.

    Science.gov (United States)

    Saccà, Barbara; Niemeyer, Christof M

    2011-12-01

    Proteins possess intrinsic functionalities, which have been optimized in billions of years of natural evolution. The conjugation of proteins with artificial nucleic acids allows one to further functionalize proteins with a synthetically accessible, physicochemically robust tag, which is addressable in a highly specific manner by Watson-Crick hybridization. The resulting DNA-protein conjugates can be advantageously used in a variety of applications, ranging from biomedical diagnostics to DNA-based nanofabrication. This critical review provides an overview on chemical approaches to the synthesis of DNA-protein conjugates and their applications in biomolecular nanosciences (96 references).

  10. Functional DNA nanostructures for theranostic applications.

    Science.gov (United States)

    Pei, Hao; Zuo, Xiaolei; Zhu, Dan; Huang, Qing; Fan, Chunhai

    2014-02-18

    There has been tremendous interest in constructing nanostructures by exploiting the unparalleled ability of DNA molecules in self-assembly. We have seen the appearance of many fantastic, "art-like" DNA nanostructures in one, two, or three dimensions during the last two decades. More recently, much attention has been directed to the use of these elegant nanoobjects for applications in a wide range of areas. Among them, diagnosis and therapy (i.e., theranostics) are of particular interest given the biological nature of DNA. One of the major barricades for the biosensor design lies in the restricted target accessibility at the solid-water interface. DNA nanotechnology provides a convenient approach to well control the biomolecule-confined surface to increase the ability of molecular recognition at the biosensing interface. For example, tetrahedral DNA nanostructures with thiol modifications can be self-assembled at the gold surface with high reproducibility. Since DNA tetrahedra are highly rigid and well-defined structures with atomic precision and versatile functionality, they provide scaffolds for anchoring of a variety of biomolecular probes (DNA, aptamers, peptides, and proteins) for biosensing. Significantly, this DNA nanostructure-based biosensing platform greatly increases target accessibility and improves the sensitivity for various types of molecular targets (DNA, RNA, proteins, and small molecules) by several orders of magnitude. In an alternative approach, DNA nanostructures provide a framework for the development of dynamic nanosensors that can function inside the cell. DNA tetrahedra are found to be facilely cell permeable and can sense and image specific molecules in cells. More importantly, these DNA nanostructures can be efficient drug delivery nanocarriers. Since they are DNA molecules by themselves, they have shown excellent cellular biocompatibility with minimal cytotoxicity. As an example, DNA tetrahedra tailored with CpG oligonucleotide drugs have

  11. Iterated function systems for DNA replication

    Science.gov (United States)

    Gaspard, Pierre

    2017-10-01

    The kinetic equations of DNA replication are shown to be exactly solved in terms of iterated function systems, running along the template sequence and giving the statistical properties of the copy sequences, as well as the kinetic and thermodynamic properties of the replication process. With this method, different effects due to sequence heterogeneity can be studied, in particular, a transition between linear and sublinear growths in time of the copies, and a transition between continuous and fractal distributions of the local velocities of the DNA polymerase along the template. The method is applied to the human mitochondrial DNA polymerase γ without and with exonuclease proofreading.

  12. Functional transferred DNA within extracellular vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Jin [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China); Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Jiangsu Province (China); Wu, Gengze [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China); Jose, Pedro A. [Division of Nephrology, Department of Medicine and Physiology, University of Maryland, School of Medicine, Baltimore, MD 21201 (United States); Zeng, Chunyu, E-mail: Chunyuzeng01@163.com [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China)

    2016-11-15

    Extracellular vesicles (EVs) are small membrane vesicles including exosomes and shedding vesicles that mediated a cell-to-cell communication. EVs are released from almost all cell types under both physiological and pathological conditions and incorporate nuclear and cytoplasmic molecules for intercellular delivery. Besides protein, mRNA, and microRNA of these molecules, as recent studies show, specific DNA are prominently packaged into EVs. It appears likely that some of exosomes or shedding vesicles, bearing nuclear molecules are released upon bubble-like blebs. Specific interaction of EVs with susceptible recipients performs the uptake of EVs into the target cells, discharging their cargo including nuclear and cytoplasmic macromolecules into the cytosol. These findings expand the nucleic acid content of EVs to include increased levels of specific DNA. Thus, EVs contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer and atherosclerosis. In this review, the focus is on the characteristics, biological functions, and roles in diseases of DNA within EVs. - Highlights: • This review is focused on the DNA within EVs including its characteristics, biological functions, and roles in diseases. • It is clear that DNA within EVs might have important physiological and pathological roles in various diseases. • Knowledge in this area may provides us alternative methods for disease diagnosis or therapy in the future.

  13. Mitochondrial DNA Alterations and Reduced Mitochondrial Function in Aging

    OpenAIRE

    Hebert, Sadie L.; Lanza, Ian R.; Nair, K. Sreekumaran

    2010-01-01

    Oxidative damage to mitochondrial DNA increases with aging. This damage has the potential to affect mitochondrial DNA replication and transcription which could alter the abundance or functionality of mitochondrial proteins. This review describes mitochondrial DNA alterations and changes in mitochondrial function that occur with aging. Age-related alterations in mitochondrial DNA as a possible contributor to the reduction in mitochondrial function are discussed.

  14. Function of BRCA1 at a DNA Replication Origin

    National Research Council Canada - National Science Library

    Lieberman, Paul

    2004-01-01

    ... and allow efficient repair of damaged DNA. In this proposal, we present preliminary data that BRCA1 functions in a DNA checkpoint response for the origin of Epstein-Barr Virus DNA replication (Ori P...

  15. DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair.

    OpenAIRE

    Mossi, R; Ferrari, E; Hübscher, U

    1998-01-01

    The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by th...

  16. Phylogenetic footprinting to find functional DNA elements.

    Science.gov (United States)

    Ganley, Austen R D; Kobayashi, Takehiko

    2007-01-01

    Phylogenetic footprinting is powerful technique for finding functional elements from sequence data. Functional elements are thought to have greater sequence constraint than nonfunctional elements, and, thus, undergo a slower rate of sequence change through time. Phylogenetic footprinting uses comparisons of homologous sequences from closely related organisms to identify "phylogenetic footprints," regions with slower rates of sequence change than background. This does not require prior characterization of the sequence in question, therefore, it can be used in a wide range of applications. In particular, it is useful for the identification of functional elements in noncoding DNA, which are traditionally difficult to detect. Here, we describe in detail how to perform a simple yet powerful phylogenetic footprinting analysis. As an example, we use ribosomal DNA repeat sequences from various Saccharomyces yeasts to find functional noncoding DNA elements in the intergenic spacer, and explain critical considerations in performing phylogenetic footprinting analyses, including the number of species and species range, and some of the available software. Our methods are broadly applicable and should appeal to molecular biologists with little experience in bioinformatics.

  17. Adsorption of DNA binding proteins to functionalized carbon nanotube surfaces with and without DNA wrapping.

    Science.gov (United States)

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-09-01

    We examined the adsorption of DNA binding proteins on functionalized, single-walled carbon nanotubes (SWNTs). When SWNTs were functionalized with polyethylene glycol (PEG-SWNT), moderate adsorption of protein molecules was observed. In contrast, nanotubes functionalized with CONH 2 groups (CONH 2 -SWNT) exhibited very strong interactions between the CONH 2 -SWNT and DNA binding proteins. Instead, when these SWNT surfaces were wrapped with DNA molecules (thymine 30-mers), protein binding was a little decreased. Our results revealed that DNA wrapped PEG-SWNT was one of the most promising candidates to realize DNA nanodevices involving protein reactions on DNA-SWNT surfaces. In addition, the DNA binding protein RecA was more adhesive than single-stranded DNA binding proteins to the functionalized SWNT surfaces.

  18. Regulation and function of DNA methylation in plants and animals

    Science.gov (United States)

    He, Xin-Jian; Chen, Taiping; Zhu, Jian-Kang

    2011-01-01

    DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. PMID:21321601

  19. Regulation and function of DNA methylation in plants and animals

    KAUST Repository

    He, Xinjian

    2011-02-15

    DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. © 2011 IBCB, SIBS, CAS All rights reserved.

  20. Dpb11 may function with RPA and DNA to initiate DNA replication.

    Science.gov (United States)

    Bruck, Irina; Dhingra, Nalini; Martinez, Matthew P; Kaplan, Daniel L

    2017-01-01

    Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation.

  1. Effect of varicocelectomy on sperm functional characteristics and DNA methylation.

    Science.gov (United States)

    Tavalaee, M; Bahreinian, M; Barekat, F; Abbasi, H; Nasr-Esfahani, M H

    2015-10-01

    In individuals with varicocele, DNA is damaged due to high level of oxidative stress, and varicocelectomy can overcome this effect. Damaged DNA is less liable to DNA methylation, and antioxidant therapy appears to have the potential to reduce sperm oxidative stress and DNA damage and thereby maintain DNA methylation, while effect of varicocelectomy on DNA methylation patterns has remained unclear. In the light of these considerations, we aimed to examine the effect of varicocelectomy on sperm DNA methylation and functional characteristics. Fifty-two men with left-sided varicocele (grade II &III) were included. Sperm parameters, DNA fragmentation, protamine deficiency, oxidative stress and global DNA methylation were evaluated before and 3 months after surgery. Our data show that sperm concentration, percentages of spermatozoon with abnormal morphology, DNA fragmentation, protamine deficiency and oxidative stress significantly improved after surgery. Percentage of sperm motility, global DNA methylation and intensity of DNA methylation also improved after surgery, although the differences were not significant when compared with before surgery. Categorisation of individuals to subgroups revealed that improvement of DNA methylation appears to take place in oligozoospermic individuals, which are more severely affected by state of varicocele. However, this is a preliminary study, and further studies are required to solidify this conclusion. © 2014 Blackwell Verlag GmbH.

  2. Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

    Energy Technology Data Exchange (ETDEWEB)

    Grierson, Patrick M. [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Acharya, Samir, E-mail: samir.acharya@osumc.edu [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

    2013-03-15

    Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

  3. The function of DNA methylation marks in social insects

    Directory of Open Access Journals (Sweden)

    Hongmei eLi-Byarlay

    2016-05-01

    Full Text Available The social arthropods are characterized by a caste-based division of labor that may be influenced by epigenetic effects. One of the most important and widely studied epigenetic mechanisms is DNA methylation. Advances in understanding of social insect genomes, including epigenetic marks, make it possible to assess the role of DNA methylation in social caste development and social behavior. In this mini review, I summarize and interpret recent findings regarding DNA methylation and discuss how DNA methylation might influence evolution of sociality. In particular, I focus on enzymes associated with DNA methylation, the functions of DNA methylation in caste determination, behavioral gene regulation, and the effects of DNA methylation on learning and memory. Finally, I highlight current challenges and predict future breakthroughs in the field of socioepigenomics.

  4. Topological aspects of DNA function and protein folding.

    Science.gov (United States)

    Stasiak, Andrzej; Bates, Andrew D; Buck, Dorothy E; Harris, Sarah A; Sumners, De Witt

    2013-04-01

    The Topological Aspects of DNA Function and Protein Folding international meeting provided an interdisciplinary forum for biological scientists, physicists and mathematicians to discuss recent developments in the application of topology to the study of DNA and protein structure. It had 111 invited participants, 48 talks and 21 posters. The present article discusses the importance of topology and introduces the articles from the meeting's speakers.

  5. A novel role of the Dna2 translocase function in DNA break resection.

    Science.gov (United States)

    Miller, Adam S; Daley, James M; Pham, Nhung Tuyet; Niu, Hengyao; Xue, Xiaoyu; Ira, Grzegorz; Sung, Patrick

    2017-03-01

    DNA double-strand break repair by homologous recombination entails nucleolytic resection of the 5' strand at break ends. Dna2, a flap endonuclease with 5'-3' helicase activity, is involved in the resection process. The Dna2 helicase activity has been implicated in Okazaki fragment processing during DNA replication but is thought to be dispensable for DNA end resection. Unexpectedly, we found a requirement for the helicase function of Dna2 in end resection in budding yeast cells lacking exonuclease 1. Biochemical analysis reveals that ATP hydrolysis-fueled translocation of Dna2 on ssDNA facilitates 5' flap cleavage near a single-strand-double strand junction while attenuating 3' flap incision. Accordingly, the ATP hydrolysis-defective dna2-K1080E mutant is less able to generate long products in a reconstituted resection system. Our study thus reveals a previously unrecognized role of the Dna2 translocase activity in DNA break end resection and in the imposition of the 5' strand specificity of end resection. © 2017 Miller et al.; Published by Cold Spring Harbor Laboratory Press.

  6. Resurrection of DNA Function In Vivo from an Extinct Genome

    OpenAIRE

    Pask, Andrew J; Behringer, Richard R.; Renfree, Marilyn B

    2008-01-01

    There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 ...

  7. Resurrection of DNA function in vivo from an extinct genome.

    Science.gov (United States)

    Pask, Andrew J; Behringer, Richard R; Renfree, Marilyn B

    2008-05-21

    There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

  8. Resurrection of DNA function in vivo from an extinct genome.

    Directory of Open Access Journals (Sweden)

    Andrew J Pask

    2008-05-01

    Full Text Available There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine, obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

  9. Structure and Function Study of Phi29 DNA packaging motor

    Science.gov (United States)

    Fang, Huaming

    molecules were required to bind to one short dsDNA molecule. The inhibitive curve of Walker B mutant gp16 analyzed by binomial distribution model showed that one inactive mutant gp16 in the gp16 ring could block the function of the motor and the stoichiometry of gp16 was six. These findings facilitate our understanding of the molecular mechanism of viral DNA packaging: a novel viral DNA packaging model "push through a one-way valve" was proposed. In this model, the connector functioned as a valve to allow DNA to enter but prevented it from sliding out during DNA packaging; the six subunits in the gp16 ring acted sequentially to push DNA into the connector channel. ATP binding of gp16 induced a conformation change with a high affinity for dsDNA. Then, the ATP was hydrolyzed which resulted in the movement of subdomains in this individual gp16 subunit and DNA was pushed forward, followed by the double helix of dsDNA being brought forward to the adjacent subunit in the gp16 ring. The elucidation of the viral DNA packaging mechanism holds great potential for developing artificial motors for delivering drugs and other molecular cargos.

  10. Visual optical biosensors based on DNA-functionalized polyacrylamide hydrogels.

    Science.gov (United States)

    Khimji, Imran; Kelly, Erin Y; Helwa, Youssef; Hoang, Michael; Liu, Juewen

    2013-12-15

    Biosensors are devices that can provide quantitative or semi-quantitative analytical information about target molecules, where molecular recognition is based on biomolecular interactions. In recent years, DNA has emerged as a useful molecule for biosensor development since DNA can not only recognize its complementary strand, but also metal ions, small molecules, proteins and cells utilizing DNA aptamer technology. Converting DNA binding events into useful biosensors often require sensor immobilization. Among the various materials for sensor immobilization, hydrogels are particularly attractive. Hydrogels are crosslinked hydrophilic polymer networks that undergo swelling in water. In a gel, DNA immobilization can take place in 3D, allowing for high DNA loading capacity. Hydrogels are transparent, offering low optical background. The gel volume is affected by many environmental parameters such as temperature, pH, ionic strength, and solvent composition. In this paper, we present a concise summary of recent developments in DNA-functionalized hydrogel biosensors for visual detection. Detailed methods for immobilizing DNA biosensors in monolithic polyacrylamide gels and gel microparticles are supplied. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Structural and Functional Coordination of DNA and Histone Methylation

    Science.gov (United States)

    Cheng, Xiaodong

    2014-01-01

    One of the most fundamental questions in the control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and recognized. This central process in controlling gene expression includes coordinated covalent modifications of DNA and its associated histones. This article focuses on structural aspects of enzymatic activities of histone (arginine and lysine) methylation and demethylation and functional links between the methylation status of the DNA and histones. An interconnected network of methyltransferases, demethylases, and accessory proteins is responsible for changing or maintaining the modification status of specific regions of chromatin. PMID:25085914

  12. Beyond the dna: a prototype for functional genomics

    Energy Technology Data Exchange (ETDEWEB)

    Albala, J

    2000-03-02

    A prototype oligonucleotide ''functional chip'' has been developed to screen novel DNA repair proteins for their ability to bind or alter different forms of DNA. This chip has been developed as a functional genomics screen for analysis of protein-DNA interactions for novel proteins identified from the Human Genome Project The process of novel gene identification that has ensued as a consequence of available sequence information is remarkable. The challenge how lies in determining the function of newly identified gene products in a time-and cost-effective high-throughput manner. The functional chip is generated by the robotic application of DNA spotted in a microarray format onto a glass slide. Individual proteins are then analyzed against the different form of DNA bound to the slide. Several prototype functional chips were designed to contain various DNA fragments tethered to a glass slide for analysis of protein-DNA binding or enzymatic activity of known proteins. The technology has been developed to screen novel, putative DNA repair proteins for their ability to bind various types of DNA alone and in concert with protein partners. An additional scheme has been devised to screen putative repair enzymes for their ability to process different types of DNA molecules. Current methods to analyze gene expression primarily utilize either of two technologies. The oligonucleotide chip, pioneered by Fodor and co-workers and Affymetrix, Inc., consists of greater than 64,000 oligonucleotides attached in situ to a glass support. The oligonucleotide chip has been used primarily to identify specific mutations in a given gene by hybridization against a fluorescently-labeled substrate. The second method is the microarray, whereby DNA targets are systematically arranged on a glass slide and then hybridized with fluorescently-labeled complex targets for gene expression analysis (Jordan, 1998). By this technique, a large amount of information can be obtained

  13. DNA Modifications: Function and Applications in Normal and Disease States

    Directory of Open Access Journals (Sweden)

    Vichithra R. B. Liyanage

    2014-10-01

    Full Text Available Epigenetics refers to a variety of processes that have heritable effects on gene expression programs without changes in DNA sequence. Key players in epigenetic control are chemical modifications to DNA, histone, and non-histone chromosomal proteins, which establish a complex regulatory network that controls genome function. Methylation of DNA at the fifth position of cytosine in CpG dinucleotides (5-methylcytosine, 5mC, which is carried out by DNA methyltransferases, is commonly associated with gene silencing. However, high resolution mapping of DNA methylation has revealed that 5mC is enriched in exonic nucleosomes and at intron-exon junctions, suggesting a role of DNA methylation in the relationship between elongation and RNA splicing. Recent studies have increased our knowledge of another modification of DNA, 5-hydroxymethylcytosine (5hmC, which is a product of the ten-eleven translocation (TET proteins converting 5mC to 5hmC. In this review, we will highlight current studies on the role of 5mC and 5hmC in regulating gene expression (using some aspects of brain development as examples. Further the roles of these modifications in detection of pathological states (type 2 diabetes, Rett syndrome, fetal alcohol spectrum disorders and teratogen exposure will be discussed.

  14. Functional DNA: Teaching Infinite Series through Genetic Analogy

    Science.gov (United States)

    Kowalski, R. Travis

    2011-01-01

    This article presents an extended analogy that connects infinite sequences and series to the science of genetics, by identifying power series as "DNA for a function." This analogy allows standard topics such as convergence tests or Taylor approximations to be recast in a "forensic" light as mathematical analogs of genetic concepts such as DNA…

  15. Establishment and functions of DNA methylation in the germline

    Science.gov (United States)

    Stewart, Kathleen R; Veselovska, Lenka; Kelsey, Gavin

    2016-01-01

    Epigenetic modifications established during gametogenesis regulate transcription and other nuclear processes in gametes, but also have influences in the zygote, embryo and postnatal life. This is best understood for DNA methylation which, established at discrete regions of the oocyte and sperm genomes, governs genomic imprinting. In this review, we describe how imprinting has informed our understanding of de novo DNA methylation mechanisms, highlight how recent genome-wide profiling studies have provided unprecedented insights into establishment of the sperm and oocyte methylomes and consider the fate and function of gametic methylation and other epigenetic modifications after fertilization. PMID:27659720

  16. Specificity and function of Archaeal DNA replication initiator proteins

    DEFF Research Database (Denmark)

    Samson, Rachel Y.; Xu, Yanqun; Gadelha, Catarina

    2013-01-01

    Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins...... to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels...... the protein's structure rather than that of the DNA template....

  17. Quelling targets the rDNA locus and functions in rDNA copy number control

    Directory of Open Access Journals (Sweden)

    Cecere Germano

    2009-02-01

    Full Text Available Abstract Background RNA silencing occurs in a broad range of organisms. Although its ancestral function is probably related to the genome defense mechanism against repetitive selfish elements, it has been found that RNA silencing regulates different cellular processes such as gene expression and chromosomal segregation. In Neurospora crassa, a RNA silencing mechanism, called quelling, acts to repress the expression of transgenes and transposons, but until now no other cellular functions have been shown to be regulated by this mechanism. Results Here, we detected by northern blotting endogenous short interfering RNA (siRNAs from the repetitive ribosomal DNA locus (rDNA that are loaded onto the argonaute protein QDE-2. Moreover, we found a bidirectional transcription that can generate double strand RNA (dsRNA molecules. Interestingly, quelling mutants have a reduced rDNA gene copy number. Conclusion Our finding could suggest a new biological function for RNA silencing in the maintenance of the integrity and stability of the Neurospora rDNA locus.

  18. NAD+ Modulates p53 DNA Binding Specificity and Function

    Science.gov (United States)

    McLure, Kevin G.; Takagi, Masatoshi; Kastan, Michael B.

    2004-01-01

    DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD+ binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B3) increases the rate of intracellular NAD+ synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD+ on p53, as a molecule structurally related to part of NAD+, TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B1), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53. PMID:15509798

  19. Optical Control of DNA Helicase Function through Genetic Code Expansion.

    Science.gov (United States)

    Luo, Ji; Kong, Muwen; Liu, Lili; Samanta, Subhas; Van Houten, Bennett; Deiters, Alexander

    2017-03-02

    Nucleotide excision repair (NER) is a general DNA repair mechanism that is capable of removing a wide variety of DNA lesions induced by physical or chemical insults. UvrD, a member of the helicase SF1 superfamily, plays an essential role in bacterial NER by unwinding the duplex DNA in the 3' to 5' direction to displace the lesion-containing strand. In order to achieve conditional control over NER, we generated a light-activated DNA helicase. This was achieved through a site-specific incorporation of a genetically encoded hydroxycoumarin lysine at a crucial position in the ATP-binding pocket of UvrD. The resulting caged enzyme was completely inactive in several functional assays. Moreover, enzymatic activity of the optically triggered UvrD was comparable to that of the wild-type protein, thus demonstrating excellent OFF to ON switching of the helicase. The developed approach provides optical control of NER, thereby laying a foundation for the regulation of ATP-dependent helicase functions in higher organisms. In addition, this methodology is applicable to the light-activation of a wide range of ATPases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Defining functional DNA elements in the human genome

    Science.gov (United States)

    Kellis, Manolis; Wold, Barbara; Snyder, Michael P.; Bernstein, Bradley E.; Kundaje, Anshul; Marinov, Georgi K.; Ward, Lucas D.; Birney, Ewan; Crawford, Gregory E.; Dekker, Job; Dunham, Ian; Elnitski, Laura L.; Farnham, Peggy J.; Feingold, Elise A.; Gerstein, Mark; Giddings, Morgan C.; Gilbert, David M.; Gingeras, Thomas R.; Green, Eric D.; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D.; Myers, Richard M.; Pazin, Michael J.; Ren, Bing; Stamatoyannopoulos, John A.; Weng, Zhiping; White, Kevin P.; Hardison, Ross C.

    2014-01-01

    With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease. PMID:24753594

  1. What cost mitochondria? The maintenance of functional mitochondrial DNA within and across generations

    NARCIS (Netherlands)

    Aanen, D.K.; Spelbrink, J.N.; Beekman, M.

    2014-01-01

    The peculiar biology of mitochondrial DNA (mtDNA) potentially has detrimental consequences for organismal health and lifespan. Typically, eukaryotic cells contain multiple mitochondria, each with multiple mtDNA genomes. The high copy number of mtDNA implies that selection on mtDNA functionality is

  2. Androgen receptor function links human sexual dimorphism to DNA methylation.

    Science.gov (United States)

    Ammerpohl, Ole; Bens, Susanne; Appari, Mahesh; Werner, Ralf; Korn, Bernhard; Drop, Stenvert L S; Verheijen, Frans; van der Zwan, Yvonne; Bunch, Trevor; Hughes, Ieuan; Cools, Martine; Riepe, Felix G; Hiort, Olaf; Siebert, Reiner; Holterhus, Paul-Martin

    2013-01-01

    Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS) due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

  3. Androgen receptor function links human sexual dimorphism to DNA methylation.

    Directory of Open Access Journals (Sweden)

    Ole Ammerpohl

    Full Text Available Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

  4. Functional demonstration of adaptive immunity in zebrafish using DNA vaccination

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lorenzen, Ellen; Einer-Jensen, Katja

    studies have documented existence of a classical innate immune response, there is mainly indirect evidence of functional adaptive immunity. To address this aspect, groups of zebrafish were vaccinated with DNA-vaccines against the rhabdoviruses VHSV, IHNV and SVCV. Seven weeks later, the fish were...... challenged with SVCV by immersion. Despite some variability between replicate aquaria, there was a protective effect of the homologous vaccine and no effect of the heterologous vaccines. The results therefore confirm the existence of not only a well developed but also a fully functional adaptive immune...

  5. Amine-functionalized magnetic mesoporous silica nanoparticles for DNA separation

    Energy Technology Data Exchange (ETDEWEB)

    Sheng, Wei; Wei, Wei; Li, Junjian; Qi, Xiaoliang; Zuo, Gancheng; Chen, Qi; Pan, Xihao; Dong, Wei, E-mail: weidong@njust.edu.cn

    2016-11-30

    Highlights: • Fe{sub 3}O{sub 4}@SiO{sub 2}@EDPS with uniform size and good dispersity is prepared. • We fabricated MMSN@EDPS with distinct core-shell–shell triple-layer composition. • DNA adsorption capacity of MMSN@EDPS is considerable. - Abstract: We report a modified approach for the functionalized magnetic mesoporous silica nanoparticles (MMSN) using polymer microspheres incorporated with magnetic nanoparticles in the presence of cetyltrimethylammonium bromide (CTAB) and the core-shell magnetic silica nanoparticles (MSN). These particles were functionalized with amino groups via the addition of aminosilane directly to the particle sol. We then evaluate their DNA separation abilities and find the capacity of DNA binding significantly increased (210.22 μg/mg) compared with normal magnetic silica spheres (138.44 μg/mg) by using an ultraviolet and visible spectrophotometer (UV). The morphologies, magnetic properties, particle size, pore size, core-shell structure and Zeta potential are characterized by Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometer (VSM), Transmission electron microscopy (TEM), Powder X-ray diffraction (XRD), and dynamic light scattering (DLS). This work demonstrates that our MMSN own an excellent potential application in bioseparation and drug delivery.

  6. Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Carolina Furtado

    Full Text Available The oxidative lesion 8-oxoguanine (8-oxoG is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1. This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1, the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1(-/- (CD138 to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H(2O(2. Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H(2O(2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.

  7. Specificity and Function of Archaeal DNA Replication Initiator Proteins

    Directory of Open Access Journals (Sweden)

    Rachel Y. Samson

    2013-02-01

    Full Text Available Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC that recruits the replicative helicase MCM(2-7 via Cdc6 and Cdt1. We find that the three origins in the single chromosome of the archaeon Sulfolobus islandicus are specified by distinct initiation factors. While two origins are dependent on archaeal homologs of eukaryal Orc1 and Cdc6, the third origin is instead reliant on an archaeal Cdt1 homolog. We exploit the nonessential nature of the orc1-1 gene to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the protein’s structure rather than that of the DNA template.

  8. WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

    Energy Technology Data Exchange (ETDEWEB)

    Yoshimura, Akari, E-mail: akari_yo@stu.musashino-u.ac.jp [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan); Kobayashi, Yume [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan); Tada, Shusuke [Department of Medical Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510 (Japan); Seki, Masayuki [Department of Biochemistry, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai-shi, Miyagi 981-8558 (Japan); Enomoto, Takemi [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan)

    2014-09-12

    Highlights: • The UV sensitivity of POLH{sup −/−} cells was suppressed by disruption of WRNIP1. • In WRNIP1{sup −/−/−}/POLH{sup −/−} cells, mutation frequencies and SCE after irradiation reduced. • WRNIP1 defect recovered rate of fork progression after irradiation in POLH{sup −/−} cells. • WRNIP1 functions upstream of Polη in the translesion DNA synthesis pathway. - Abstract: WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH{sup −/−}) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.

  9. The function of DNA binding protein nucleophosmin in AAV replication.

    Science.gov (United States)

    Satkunanathan, Stifani; Thorpe, Robin; Zhao, Yuan

    2017-10-01

    Adeno-associated viruses (AAV) contain minimal viral proteins necessary for their replication. During virus assembly, AAV acquire, inherently and submissively, various cellular proteins. Our previous studies identified the association of AAV vectors with the DNA binding protein nucleophosmin (NPM1). Nucleophosmin has been reported to enhance AAV infection by mobilizing AAV capsids into and out of the nucleolus, indicating the importance of NPM1 in the AAV life cycle; however the role of NPM1 in AAV production remains unknown. In this study, we systematically investigated NPM1 function on AAV production using NPM1 knockdown cells and revealing for the first time the presence of G-quadruplex DNA sequences (GQRS) in the AAV genome, the synergistic NPM1-GQRS function in AAV production and the significant enhancement of NPM1 gene knockdown on AAV vector production. Understanding the role of cellular proteins in the AAV life cycle will greatly facilitate high titre production of AAV vectors for clinical use. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  10. Polymerization amplified SPR-DNA assay on noncovalently functionalized graphene.

    Science.gov (United States)

    Yuan, Pei-Xin; Deng, Sheng-Yuan; Yao, Chuan-Guang; Wan, Ying; Cosnier, Serge; Shan, Dan

    2017-03-15

    A highly efficient surface plasmon resonance (SPR)-based DNA assay was developed, by employing noncovalently functionalized graphene nanosheets as a substrate, and enzymatic catalysis-induced polymerization as mass relay. The objective of this strategy was manifold: first of all, to sensitize the overall SPR output by in situ optimized electrogeneration of graphene thin-film, which was characterized by atomic force microscopic topography; secondly, to regulate the self-assembly and orientation of biotinylated capture probes on nickel-chelated nitrilotriacetic acid (NTA) scaffolds, that anchored onto graphene-supported pyrenyl derivatives; and lastly, to synergize the signal amplification via real-time conversion of the additive aniline into polyaniline precipitation by horseradish peroxidase-tagged reporters. With this setup, a precise and replicable DNA sensing platform for specific targets was achieved with a detection limit down to femtomolar, thus demonstrating a beneficial exploration and exploitation of two-dimensional nanomaterials as unique SPR infrastructure. The possibility of such ″bottom-up″ architecture mounted with ″top-down″ weight reactor would be most likely extensible and adaptable to protein determinations. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. DNA topoisomerase II modulates insulator function in Drosophila.

    Directory of Open Access Journals (Sweden)

    Edward Ramos

    2011-01-01

    Full Text Available Insulators are DNA sequences thought to be important for the establishment and maintenance of cell-type specific nuclear architecture. In Drosophila there are several classes of insulators that appear to have unique roles in gene expression. The mechanisms involved in determining and regulating the specific roles of these insulator classes are not understood. Here we report that DNA Topoisomerase II modulates the activity of the Su(Hw insulator. Downregulation of Topo II by RNAi or mutations in the Top2 gene result in disruption of Su(Hw insulator function. This effect is mediated by the Mod(mdg42.2 protein, which is a unique component of the Su(Hw insulator complex. Co-immunoprecipitation and yeast two-hybrid experiments show that Topo II and Mod(mdg42.2 proteins directly interact. In addition, mutations in Top2 cause a slight decrease of Mod(mdg42.2 transcript but have a dramatic effect on Mod(mdg42.2 protein levels. In the presence of proteasome inhibitors, normal levels of Mod(mdg42.2 protein and its binding to polytene chromosomes are restored. Thus, Topo II is required to prevent Mod(mdg42.2 degradation and, consequently, to stabilize Su(Hw insulator-mediated chromatin organization.

  12. DNA Topoisomerase II Modulates Insulator Function in Drosophila

    Science.gov (United States)

    Ramos, Edward; Torre, Eduardo A.; Bushey, Ashley M.; Gurudatta, B. V.; Corces, Victor G.

    2011-01-01

    Insulators are DNA sequences thought to be important for the establishment and maintenance of cell-type specific nuclear architecture. In Drosophila there are several classes of insulators that appear to have unique roles in gene expression. The mechanisms involved in determining and regulating the specific roles of these insulator classes are not understood. Here we report that DNA Topoisomerase II modulates the activity of the Su(Hw) insulator. Downregulation of Topo II by RNAi or mutations in the Top2 gene result in disruption of Su(Hw) insulator function. This effect is mediated by the Mod(mdg4)2.2 protein, which is a unique component of the Su(Hw) insulator complex. Co-immunoprecipitation and yeast two-hybrid experiments show that Topo II and Mod(mdg4)2.2 proteins directly interact. In addition, mutations in Top2 cause a slight decrease of Mod(mdg4)2.2 transcript but have a dramatic effect on Mod(mdg4)2.2 protein levels. In the presence of proteasome inhibitors, normal levels of Mod(mdg4)2.2 protein and its binding to polytene chromosomes are restored. Thus, Topo II is required to prevent Mod(mdg4)2.2 degradation and, consequently, to stabilize Su(Hw) insulator-mediated chromatin organization. PMID:21304601

  13. Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli.

    Science.gov (United States)

    El-Sagheer, Afaf H; Sanzone, A Pia; Gao, Rachel; Tavassoli, Ali; Brown, Tom

    2011-07-12

    A triazole mimic of a DNA phosphodiester linkage has been produced by templated chemical ligation of oligonucleotides functionalized with 5'-azide and 3'-alkyne. The individual azide and alkyne oligonucleotides were synthesized by standard phosphoramidite methods and assembled using a straightforward ligation procedure. This highly efficient chemical equivalent of enzymatic DNA ligation has been used to assemble a 300-mer from three 100-mer oligonucleotides, demonstrating the total chemical synthesis of very long oligonucleotides. The base sequences of the DNA strands containing this artificial linkage were copied during PCR with high fidelity and a gene containing the triazole linker was functional in Escherichia coli.

  14. A DNA 3′-phosphatase functions in active DNA demethylation in Arabidopsis

    OpenAIRE

    Martínez-Macías, María Isabel; Qian, Weiqiang; Miki, Daisuke; Pontes, Olga; Liu, Yunhua; Tang, Kai; Liu, Renyi; Morales-Ruiz, Teresa; Ariza, Rafael R.; Roldán-Arjona, Teresa; Zhu, Jian-Kang

    2012-01-01

    DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3′- and 5′-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3′-phosphate, allowing subsequent DNA pol...

  15. A Comparative Structure/Function Analysis of Two Type IV Pilin DNA Receptors Defines a Novel Mode of DNA Binding.

    Science.gov (United States)

    Berry, Jamie-Lee; Xu, Yingqi; Ward, Philip N; Lea, Susan M; Matthews, Stephen J; Pelicic, Vladimir

    2016-06-07

    DNA transformation is a widespread process allowing bacteria to capture free DNA by using filamentous nano-machines composed of type IV pilins. These proteins can act as DNA receptors as demonstrated by the finding that Neisseria meningitidis ComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when it contains the DNA-uptake sequence (DUS) motif abundant in this species genome, playing a role in its trademark ability to selectively take up its own DNA. Here, we report high-resolution structures for meningococcal ComP and Neisseria subflava ComPsub, which recognize different DUS motifs. We show that they are structurally identical type IV pilins that pack readily into filament models and display a unique DD region delimited by two disulfide bonds. Functional analysis of ComPsub defines a new mode of DNA binding involving the DD region, adapted for exported DNA receptors. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  16. Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions

    NARCIS (Netherlands)

    Martín-Pardillos, Ana; Tsaalbi-Shtylik, Anastasia; Chen, Si; Lazare, Seka; van Os, Ronald P; Dethmers-Ausema, Albertina; Fakouri, Nima Borhan; Bosshard, Matthias; Aprigliano, Rossana; van Loon, Barbara; Salvatori, Daniela C F; Hashimoto, Keiji; Dingemanse-van der Spek, Celia; Moriya, Masaaki; Rasmussen, Lene Juel; de Haan, Gerald; Raaijmakers, Marc H G P; de Wind, Niels

    2017-01-01

    Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem and

  17. Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA*

    Science.gov (United States)

    Zhou, Yayan; Hingorani, Manju M.

    2012-01-01

    Ring-shaped clamp proteins encircle DNA and affect the work of many proteins, notably processive replication by DNA polymerases. Crystal structures of clamps show several cationic residues inside the ring, and in a co-crystal of Escherichia coli β clamp-DNA, they directly contact the tilted duplex passing through (Georgescu, R. E., Kim, S. S., Yurieva, O., Kuriyan, J., Kong, X. P., and O'Donnell, M. (2008) Structure of a sliding clamp on DNA. Cell 132, 43–54). To investigate the role of these contacts in reactions involving circular clamps, we examined single arginine/lysine mutants of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) in replication factor C (RFC)-catalyzed loading of the clamp onto primer template DNA (ptDNA). Previous kinetic analysis has shown that ptDNA entry inside an ATP-activated RFC-PCNA complex accelerates clamp opening and ATP hydrolysis, which is followed by slow PCNA closure around DNA and product dissociation. Here we directly measured multiple steps in the reaction (PCNA opening, ptDNA binding, PCNA closure, phosphate release, and complex dissociation) to determine whether mutation of PCNA residues Arg-14, Lys-20, Arg-80, Lys-146, Arg-149, or Lys-217 to alanine affects the reaction mechanism. Contrary to earlier steady state analysis of these mutants (McNally, R., Bowman, G. D., Goedken, E. R., O'Donnell, M., and Kuriyan, J. (2010) Analysis of the role of PCNA-DNA contacts during clamp loading. BMC Struct. Biol. 10, 3), our pre-steady state data show that loss of single cationic residues can alter the rates of all DNA-linked steps in the reaction, as well as movement of PCNA on DNA. These results explain an earlier finding that individual arginines and lysines inside human PCNA are essential for polymerase δ processivity (Fukuda, K., Morioka, H., Imajou, S., Ikeda, S., Ohtsuka, E., and Tsurimoto, T. (1995) Structure-function relationship of the eukaryotic DNA replication factor, proliferating cell nuclear antigen

  18. The Function of DNA Methylation Marks in Social Insects

    OpenAIRE

    Li-Byarlay, Hongmei

    2016-01-01

    The social arthropods are characterized by a caste-based division of labor that may be influenced by epigenetic effects. One of the most important and widely studied epigenetic mechanisms is DNA methylation. Advances in understanding of social insect genomes, including epigenetic marks, make it possible to assess the role of DNA methylation in social caste development and social behavior. In this mini review, I summarize and interpret recent findings regarding DNA methylation and discuss how ...

  19. The human DNA ends proteome uncovers an unexpected entanglement of functional pathways.

    Science.gov (United States)

    Berthelot, Vivien; Mouta-Cardoso, Gildas; Hégarat, Nadia; Guillonneau, François; François, Jean-Christophe; Giovannangeli, Carine; Praseuth, Danièle; Rusconi, Filippo

    2016-06-02

    DNA ends get exposed in cells upon either normal or dysfunctional cellular processes or molecular events. Telomeres need to be protected by the shelterin complex to avoid junctions occurring between chromosomes while failing topoisomerases or clustered DNA damage processing may produce double-strand breaks, thus requiring swift repair to avoid cell death. The rigorous study of the great many proteins involved in the maintenance of DNA integrity is a challenging task because of the innumerous unspecific electrostatic and/or hydrophobic DNA-protein interactions that arise due to the chemical nature of DNA. We devised a technique that discriminates the proteins recruited specifically at DNA ends from those that bind to DNA because of a generic affinity for the double helix. Our study shows that the DNA ends proteome comprises proteins of an unexpectedly wide functional spectrum, ranging from DNA repair to ribosome biogenesis and cytoskeleton, including novel proteins of undocumented function. A global mapping of the identified proteome on published DNA repair protein networks demonstrated the excellent specificity and functional coverage of our purification technique. Finally, the native nucleoproteic complexes that assembled specifically onto DNA ends were shown to be endowed with a highly efficient DNA repair activity. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. What cost mitochondria? The maintenance of functional mitochondrial DNA within and across generations.

    Science.gov (United States)

    Aanen, Duur K; Spelbrink, Johannes N; Beekman, Madeleine

    2014-07-05

    The peculiar biology of mitochondrial DNA (mtDNA) potentially has detrimental consequences for organismal health and lifespan. Typically, eukaryotic cells contain multiple mitochondria, each with multiple mtDNA genomes. The high copy number of mtDNA implies that selection on mtDNA functionality is relaxed. Furthermore, because mtDNA replication is not strictly regulated, within-cell selection may favour mtDNA variants with a replication advantage, but a deleterious effect on cell fitness. The opportunities for selfish mtDNA mutations to spread are restricted by various organism-level adaptations, such as uniparental transmission, germline mtDNA bottlenecks, germline selection and, during somatic growth, regular alternation between fusion and fission of mitochondria. These mechanisms are all hypothesized to maintain functional mtDNA. However, the strength of selection for maintenance of functional mtDNA progressively declines with age, resulting in age-related diseases. Furthermore, organismal adaptations that most probably evolved to restrict the opportunities for selfish mtDNA create secondary problems. Owing to predominantly maternal mtDNA transmission, recombination among mtDNA from different individuals is highly restricted or absent, reducing the scope for repair. Moreover, maternal inheritance precludes selection against mtDNA variants with male-specific effects. We finish by discussing the consequences of life-history differences among taxa with respect to mtDNA evolution and make a case for the use of microorganisms to experimentally manipulate levels of selection. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  1. The skeletal function of non-genic nuclear DNA: new evidence from ancient cell chimaeras.

    Science.gov (United States)

    Cavalier-Smith, T; Beaton, M J

    1999-01-01

    DNA can be divided functionally into three categories: (1) genes--which code for proteins or specify non-messenger RNAs; (2) semons--short specific sequences involved in the replication, segregation, recombination or specific attachments of chromosomes, or chromosome regions (e.g. loops or domains) or selfish genetic elements; (3) secondary DNA--which does not function by means of specific sequences. Probably more than 90% of DNA in the biosphere is secondary DNA present in the nuclei of plants and phytoplankton. The amount of genic DNA is related to the complexity of the organism, whereas the amount of secondary DNA increases proportionally with cell volume, and not with complexity. This correlation is most simply explained by the skeletal DNA hypothesis, according to which nuclear DNA functions as the basic framework for the assembly of the nucleus and the total genomic DNA content functions (together with relatively invariant folding rules) in determining nuclear volumes. Balanced growth during the cell cycle requires the cytonuclear ratio to be basically constant, irrespective of cell volume; thus nuclear volumes, and therefore the overall genome size, have to be evolutionarily adjusted to changing cell volumes for optimal function. Bacteria, mitochondria, chloroplasts and viruses have no nuclear envelope; and the skeletal DNA hypothesis simply explains why secondary DNA is essentially absent from them but present in large cell nuclei. Hitherto it has been difficult to refute the alternative hypothesis that nuclear secondary DNA (whether 'junk' or selfish DNA) accumulates merely by mutation pressure, and that selection for economy is not strong enough to eliminate it, whereas accumulation in mitochondria and plastids is prevented by intracellular replicative competition between their multiple genomes. New data that discriminate clearly between these explanations for secondary DNA come from cryptomonads and chlorarachneans, two groups of algae that originated

  2. Injection molded nanofluidic chips: Fabrication method and functional tests using single-molecule DNA experiments

    DEFF Research Database (Denmark)

    Utko, Pawel; Persson, Karl Fredrik; Kristensen, Anders

    2011-01-01

    We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels.......We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels....

  3. Whole genome duplications and a 'function' for junk DNA? Facts and hypotheses.

    Directory of Open Access Journals (Sweden)

    Reiner A Veitia

    Full Text Available BACKGROUND: The lack of correlation between genome size and organismal complexity is understood in terms of the massive presence of repetitive and non-coding DNA. This non-coding subgenome has long been called "junk" DNA. However, it might have important functions. Generation of junk DNA depends on proliferation of selfish DNA elements and on local or global DNA duplication followed by genic non-functionalization. METHODOLOGY/PRINCIPAL FINDINGS: Evidence from genomic analyses and experimental data indicates that Whole Genome Duplications (WGD are often followed by a return to the diploid state, through DNA deletions and intra/interchromosomal rearrangements. We use simple theoretical models and simulations to explore how a WGD accompanied by sequence deletions might affect the dosage balance often required among several gene products involved in regulatory processes. We find that potential genomic deletions leading to changes in nuclear and cell volume might potentially perturb gene dosage balance. CONCLUSIONS/SIGNIFICANCE: The potentially negative impact of DNA deletions can be buffered if deleted genic DNA is, at least temporarily, replaced by repetitive DNA so that the nuclear/cell volume remains compatible with normal living. Thus, we speculate that retention of non-functionalized non-coding DNA, and replacement of deleted DNA through proliferation of selfish elements, might help avoid dosage imbalances in cycles of polyploidization and diploidization, which are particularly frequent in plants.

  4. Conformation of nanoconfined DNA as a function of ATP, AMP, CTP, Mg2+, and dye binding

    Science.gov (United States)

    Roushan, Maedeh; Riehn, Robert

    2014-03-01

    DNA molecules stretch in nanochannels with a channel cross-section of 100x100 nm2, thereby allowing analysis by observation of a fluorescent dye. The length and configuration of DNA can be directly observed, and the effect of different DNA-binding proteins on DNA configuration can be studied. Recently, we reported on the ability of T4 ligase to transiently manipulate DNA as a function of ATP and magnesium exposure. In this process we have extensively probed the interactions of dyes and enzyme co-factors with DNA under nanoconfinement. We find negligible effects if DNA is visualized using groove-binding dyes such as DAPI. However, if an intercalating dye (YOYO-1) is used, we find a significant shortening of the DNA in the presence of ATP that we attribute to an interaction of dye and ATP (as well as AMP and CTP). We did not record a noticeable effect due to Mg2+.

  5. Left-handed Z-DNA: structure and function

    Science.gov (United States)

    Herbert, A.; Rich, A.

    1999-01-01

    Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

  6. Evaluation of Fluorescent Analogs of Deoxycytidine for Monitoring DNA Transitions from Duplex to Functional Structures

    Directory of Open Access Journals (Sweden)

    Yogini P. Bhavsar

    2011-01-01

    Full Text Available Topological variants of single-strand DNA (ssDNA structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC and 1,3-diaza-2-oxophenoxazine (tC∘, can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC∘ is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

  7. The DnaA Cycle in Escherichia coli: Activation, Function and Inactivation of the Initiator Protein

    Directory of Open Access Journals (Sweden)

    Tsutomu Katayama

    2017-12-01

    Full Text Available This review summarizes the mechanisms of the initiator protein DnaA in replication initiation and its regulation in Escherichia coli. The chromosomal origin (oriC DNA is unwound by the replication initiation complex to allow loading of DnaB helicases and replisome formation. The initiation complex consists of the DnaA protein, DnaA-initiator-associating protein DiaA, integration host factor (IHF, and oriC, which contains a duplex-unwinding element (DUE and a DnaA-oligomerization region (DOR containing DnaA-binding sites (DnaA boxes and a single IHF-binding site that induces sharp DNA bending. DiaA binds to DnaA and stimulates DnaA assembly at the DOR. DnaA binds tightly to ATP and ADP. ATP-DnaA constructs functionally different sub-complexes at DOR, and the DUE-proximal DnaA sub-complex contains IHF and promotes DUE unwinding. The first part of this review presents the structures and mechanisms of oriC-DnaA complexes involved in the regulation of replication initiation. During the cell cycle, the level of ATP-DnaA level, the active form for initiation, is strictly regulated by multiple systems, resulting in timely replication initiation. After initiation, regulatory inactivation of DnaA (RIDA intervenes to reduce ATP-DnaA level by hydrolyzing the DnaA-bound ATP to ADP to yield ADP-DnaA, the inactive form. RIDA involves the binding of the DNA polymerase clamp on newly synthesized DNA to the DnaA-inactivator Hda protein. In datA-dependent DnaA-ATP hydrolysis (DDAH, binding of IHF at the chromosomal locus datA, which contains a cluster of DnaA boxes, results in further hydrolysis of DnaA-bound ATP. SeqA protein inhibits untimely initiation at oriC by binding to newly synthesized oriC DNA and represses dnaA transcription in a cell cycle dependent manner. To reinitiate DNA replication, ADP-DnaA forms oligomers at DnaA-reactivating sequences (DARS1 and DARS2, resulting in the dissociation of ADP and the release of nucleotide-free apo-DnaA, which then

  8. Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

    Science.gov (United States)

    Wu, Yuliang; Sommers, Joshua A.; Suhasini, Avvaru N.; Leonard, Thomas; Deakyne, Julianna S.; Mazin, Alexander V.; Shin-ya, Kazuo; Kitao, Hiroyuki

    2010-01-01

    Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair. PMID:20639400

  9. RPA binds histone H3-H4 and functions in DNA replication-coupled nucleosome assembly.

    Science.gov (United States)

    Liu, Shaofeng; Xu, Zhiyun; Leng, He; Zheng, Pu; Yang, Jiayi; Chen, Kaifu; Feng, Jianxun; Li, Qing

    2017-01-27

    DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication. Copyright © 2017, American Association for the Advancement of Science.

  10. RNA-directed DNA methylation: Mechanisms and functions

    KAUST Repository

    Mahfouz, Magdy M.

    2010-07-01

    Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response to disease states, growth, developmental and stress signals. RdDM machinery is composed of proteins that produce and modify 24-nt- long siRNAs, recruit the RdDM complex to genomic targets, methylate DNA and remodel chromatin. The final DNA methylation pattern is determined by either DNA methyltransferase alone or by the combined action of DNA methyltransferases and demethylases. The dynamic interaction between RdDM and demethylases may render the plant epigenome plastic to growth, developmental, and environmental cues. The epigenome plasticity may allow the plant genome to assume many epigenomes and to have the right epigenome at the right time in response to intracellular or extracellular stimuli. This review discusses recent advances in RdDM research and considers future perspectives.

  11. Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks

    Directory of Open Access Journals (Sweden)

    Pierre Caron

    2015-11-01

    Full Text Available DNA double-strand breaks (DSBs elicit the so-called DNA damage response (DDR, largely relying on ataxia telangiectasia mutated (ATM and DNA-dependent protein kinase (DNA-PKcs, two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.

  12. Structural and Functional Regulation of DNA: Geometry, Topology and Methylation

    Science.gov (United States)

    Auclair, C.

    The work of Rosalind Franklin, then Watson and Crick [1], established the architecture of deoxyribose nucleic acid (DNA), carrier of all genetic information. The idea that DNA was structurally organised in the form of a double helix comprising two antiparallel and complementary polymer chains was one of the great scientific discoveries of the twentieth century. It revealed not only the way in which genetic information is stored, but also the mechanism by which the genetic code is read, and the way this code can be faultlessly copied from one cell to another during cell division.

  13. DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

    Science.gov (United States)

    Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

    2017-09-01

    Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Synthesis of nucleobase-functionalized carbon nanotubes and their hybridization with single-stranded DNA.

    Science.gov (United States)

    Hwu, Jih Ru; Kapoor, Mohit; Li, Rou-Ying; Lin, Yung-Chieh; Horng, Jia-Cherng; Tsay, Shwu-Chen

    2014-12-01

    For the first time ssDNA (25-aptamer of mixed dA, dT, dG, and dC) was wrapped around functionalized single-walled carbon nanotubes (SWCNTs), whose external surfaces were attached to multiple triazole-(ethylene glycol)-dA ligands. This method of hybridization involved the formation of hydrogen bonds between dT of ssDNA and dA of functionalized SWCNTs. It deviates from the reported π-π stacking between the nucleobases of DNA and the external sidewalls of nanotubes. The structural properties of the functionalized SWCNTs and its ssDNA complex were characterized by spectroscopic (including CD and Raman), thermogravimetric, and microscopic (TEM) methods. The results thus obtained establish a new platform of DNA delivery by use of nanotubes as a new vehicle with great potential in biomedical applications and drug development. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. [Pathways for maintenance of mitochondrial DNA integrity and mitochondrial functions in cells exposed to ionizing radiation].

    Science.gov (United States)

    Gaziev, A I

    2013-01-01

    The analytical review deals with the results of studies devoted to mitochondrial DNA (mtDNA) disorders, the development of oxidative stress and possible pathways for the maintenance of mitochondrial functions in cells exposed to ionizing radiation (IR). Mitochondrial functions, which are closely related to the integrity of mtDNA, play a key role in many cellular processes. A wide range of degenerative diseases, carcinogenesis, and aging is associated with disturbances in mtDNA. MtDNA and the mitochondrion as a whole are increasingly considered as sensitive targets for cancer radio-chemotherapy. Knowledge of post-radiation processes in the mitochondria also facilitates creation of possible additional ways to reduce the radiation reaction of the organism. Injuries and mutations in mtDNA occur with a greater frequency than in the nuclear DNA (nDNA) in cells exposed to IR and other genotoxicants. On the other hand, functionally active copies of mtDNA can persist and survive in the cells exposed to clinically relevant doses of radiation. This safety is ensured by numerous copies of mtDNA in the cell, and due to their shielding from the effects of reactive oxygen (and nitrogen) species (ROS) by nucleoid proteins and by the operation of base excision repair in mitochondria. However, the generation of ROS increases in the mitochondria of cells exposed to IR. The increased generation of ROS in mitochondria can sometimes persist up to several days after the exposure of cells. The prolonged increased generation of ROS may be due to the involvement in the electron transport chain of the complexes of aberrant proteins expressed by the genes of mutated mtDNA copies. This may lead to the additional DNA damage, mitochondrial dysfunction, and instability of the nuclear genome. However, the development of oxidative stress can be restrained by antioxidant systems in the mitochondria. The key role here is played by activation of Mn-SOD2 and the protein p53. In addition, activation of

  16. Dissolving microneedles for DNA vaccination: Improving functionality via polymer characterization and RALA complexation.

    Science.gov (United States)

    Cole, Grace; McCaffrey, Joanne; Ali, Ahlam A; McBride, John W; McCrudden, Cian M; Vincente-Perez, Eva M; Donnelly, Ryan F; McCarthy, Helen O

    2017-01-02

    DNA vaccination holds the potential to treat or prevent nearly any immunogenic disease, including cancer. To date, these vaccines have demonstrated limited immunogenicity in vivo due to the absence of a suitable delivery system which can protect DNA from degradation and improve transfection efficiencies in vivo. Recently, microneedles have been described as a novel physical delivery technology to enhance DNA vaccine immunogenicity. Of these devices, dissolvable microneedles promise a safe, pain-free delivery system which may simultaneously improve DNA stability within a solid matrix and increase DNA delivery compared to solid arrays. However, to date little work has directly compared the suitability of different dissolvable matrices for formulation of DNA-loaded microneedles. Therefore, the current study examined the ability of 4 polymers to formulate mechanically robust, functional DNA loaded dissolvable microneedles. Additionally, complexation of DNA to a cationic delivery peptide, RALA, prior to incorporation into the dissolvable matrix was explored as a means to improve transfection efficacies following release from the polymer matrix. Our data demonstrates that DNA is degraded following incorporation into PVP, but not PVA matrices. The complexation of DNA to RALA prior to incorporation into polymers resulted in higher recovery from dissolvable matrices, and increased transfection efficiencies in vitro. Additionally, RALA/DNA nanoparticles released from dissolvable PVA matrices demonstrated up to 10-fold higher transfection efficiencies than the corresponding complexes released from PVP matrices, indicating that PVA is a superior polymer for this microneedle application.

  17. PIG3 Functions in DNA Damage Response through Regulating DNA-PKcs Homeostasis

    OpenAIRE

    Li, Bing; Shang, Zeng-Fu; Yin, Jiao-Jiao; Xu, Qin-Zhi; Liu, Xiao-Dan; Wang, Yu; Zhang, Shi-Meng; Guan, Hua; Zhou, Ping-Kun

    2013-01-01

    The p53-inducible gene 3 (PIG3) recently has been reported to be a new player in DNA damage signaling and response, but the crucial mechanism remains unclear. In the present study, the potential mechanism of PIG3 participation in the DNA damage response induced by ionizing radiation (IR) was investigated in multiple cell lines with depleted expression of PIG3 transiently or stably by the small interference RNA and lentivirus-mediated shRNA expression strategies. PIG3 knockdown led to an abnor...

  18. Establishment and functions of DNA methylation in the germline

    DEFF Research Database (Denmark)

    Stewart-Morgan, Kathleen; Veselovska, Lenka; Kelsey, Gavin

    2016-01-01

    Epigenetic modifications established during gametogenesis regulate transcription and other nuclear processes in gametes, but also have influences in the zygote, embryo and postnatal life. This is best understood for DNA methylation which, established at discrete regions of the oocyte and sperm ge...

  19. Functional DNA-containing nanomaterials: cellular applications in biosensing, imaging, and targeted therapy.

    Science.gov (United States)

    Liang, Hao; Zhang, Xiao-Bing; Lv, Yifan; Gong, Liang; Wang, Ruowen; Zhu, Xiaoyan; Yang, Ronghua; Tan, Weihong

    2014-06-17

    CONSPECTUS: DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell's nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer-micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand-receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA-nanomaterial complexes can enter cells more easily than free single

  20. An AP endonuclease functions in active DNA demethylation and gene imprinting in Arabidopsis [corrected].

    Directory of Open Access Journals (Sweden)

    Yan Li

    2015-01-01

    Full Text Available Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l+/-zdp-/- mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.

  1. Predicting functionality of protein-DNA interactions by integrating diverse evidence

    DEFF Research Database (Denmark)

    Ucar, Duygu; Beyer, A.; Parthasarathy, S.

    2009-01-01

    with motif binding sites, nucleosome occupancy and mRNA expression datasets within a probabilistic framework. This framework was specifically tailored for the identification of functional and nonfunctional DNA binding events. Using this, we estimate that only 50% of condition-specific protein-DNA binding...... in budding yeast is functional. We further investigated the molecular factors determining the functionality of protein-DNA interactions under diverse growth conditions. Our analysis suggests that the functionality of binding is highly condition-specific and highly dependent on the presence of specific......Chromatin immunoprecipitation (ChIP-chip) experiments enable capturing physical interactions between regulatory proteins and DNA in vivo. However, measurement of chromatin binding alone is not sufficient to detect regulatory interactions. A detected binding event may not be biologically relevant...

  2. The Functional Role of TopBP1 in DNA Maintenance at Mitosis

    DEFF Research Database (Denmark)

    Pedersen, Rune Troelsgaard

    . This active processing was found to be an underlying mechanism of CFS expression. A final advance was the description of how DNA damage, arising as a consequence of replication stress in S-phase, was shielded in 53BP1 nuclear bodies (NBs), preventing untimely DNA repair during the subsequent G1-phase. We...... of active DNA synthesis at the G2/M transition where TopBP1 promotes DNA synthesis. (ii) TopBP1 colocalizes with the scaffold protein and structure-selective nuclease subunit SLX4, and is required for SLX4 recruitment to chromatin in mitosis. Depletion of TopBP1 at mitosis greatly induces formation...... of unreplicated DNA and DNA-repair intermediates. These loci can manifest themselves as breaks and gaps on metaphase chromosomes, which often coincide with specific chromosome loci termed common fragile sites (CFSs). Additionally, underreplicated loci function as physical links between sister chromatids, which...

  3. Hydrodynamic size of DNA/cationic gemini surfactant complex as a function of surfactant structure.

    Science.gov (United States)

    Devínsky, Ferdinand; Pisárcik, Martin; Lacko, Ivan

    2009-06-01

    The present study deals with the determination of hydrodynamic size of DNA/cationic gemini surfactant complex in sodium bromide solution using the dynamic light scattering method. Cationic gemini surfactants with polymethylene spacer of variable length were used for the interaction with DNA. The scattering experiments were performed at constant DNA and sodium bromide concentrations and variable surfactant concentration in the premicellar and micellar regions as a function of surfactant spacer length. It was found that the DNA conformation strongly depends on the polymethylene spacer length as well as on the surfactant concentration relative to the surfactant critical micelle concentration. Gemini surfactant molecules with 4 methylene groups in the spacer were found to be the least efficient DNA compacting agent in the region above the surfactant cmc. Gemini molecules with the shortest spacer length (2 methylene groups) and the longest spacer length (8 methylene groups) investigated showed the most efficient DNA compaction ability.

  4. DNA-Damage-Induced Type I Interferon Promotes Senescence and Inhibits Stem Cell Function

    Directory of Open Access Journals (Sweden)

    Qiujing Yu

    2015-05-01

    Full Text Available Expression of type I interferons (IFNs can be induced by DNA-damaging agents, but the mechanisms and significance of this regulation are not completely understood. We found that the transcription factor IRF3, activated in an ATM-IKKα/β-dependent manner, stimulates cell-autonomous IFN-β expression in response to double-stranded DNA breaks. Cells and tissues with accumulating DNA damage produce endogenous IFN-β and stimulate IFN signaling in vitro and in vivo. In turn, IFN acts to amplify DNA-damage responses, activate the p53 pathway, promote senescence, and inhibit stem cell function in response to telomere shortening. Inactivation of the IFN pathway abrogates the development of diverse progeric phenotypes and extends the lifespan of Terc knockout mice. These data identify DNA-damage-response-induced IFN signaling as a critical mechanism that links accumulating DNA damage with senescence and premature aging.

  5. Imparting the unique properties of DNA into complex material architectures and functions

    Science.gov (United States)

    Xu, Phyllis F.; Noh, Hyunwoo; Lee, Ju Hun; Domaille, Dylan W.; Nakatsuka, Matthew A.; Goodwin, Andrew P.; Cha, Jennifer N.

    2014-01-01

    While the remarkable chemical and biological properties of DNA have been known for decades, these properties have only been imparted into materials with unprecedented function much more recently. The inimitable ability of DNA to form programmable, complex assemblies through stable, specific, and reversible molecular recognition has allowed the creation of new materials through DNA’s ability to control a material’s architecture and properties. In this review we discuss recent progress in how DNA has brought unmatched function to materials, focusing specifically on new advances in delivery agents, devices, and sensors. PMID:25525408

  6. Functionally homologous DNA replication genes in fission and budding yeast

    OpenAIRE

    Sánchez, Mar; Calzada, Arturo; Bueno, Avelino

    1999-01-01

    The cdc18+ gene of the fission yeast Schizosaccharomyces pombe is involved in the initiation of DNA replication as well as in coupling the S phase to mitosis. In this work, we show that the Saccharomyces cerevisiae CDC6 gene complements cdc18-K46 ts and cdc18 deletion mutant S. pombe strains. The budding yeast gene suppresses both the initiation and the checkpoint defects associated with the lack of cdc18+. The Cdc6 protein interacts in vivo with Cdc2 kinase complexes. Interestingly, Cdc6 is ...

  7. Epigenetic features in the oyster Crassostrea gigas suggestive of functionally relevant promoter DNA methylation in invertebrates

    Science.gov (United States)

    Rivière, Guillaume

    2014-01-01

    DNA methylation is evolutionarily conserved. Vertebrates exhibit high, widespread DNA methylation whereas invertebrate genomes are less methylated, predominantly within gene bodies. DNA methylation in invertebrates is associated with transcription level, alternative splicing, and genome evolution, but functional outcomes of DNA methylation remain poorly described in lophotrochozoans. Recent genome-wide approaches improve understanding in distant taxa such as molluscs, where the phylogenetic position, and life traits of Crassostrea gigas make this bivalve an ideal model to study the physiological and evolutionary implications of DNA methylation. We review the literature about DNA methylation in invertebrates and focus on DNA methylation features in the oyster. Indeed, though our MeDIP-seq results confirm predominant intragenic methylation, the profiles depend on the oyster's developmental and reproductive stage. We discuss the perspective that oyster DNA methylation could be biased toward the 5′-end of some genes, depending on physiological status, suggesting important functional outcomes of putative promoter methylation from cell differentiation during early development to sustained adaptation of the species to the environment. PMID:24778620

  8. Epigenetic features in the oyster Crassostrea gigas suggestive of functionally relevant promoter DNA methylation in invertebrates.

    Directory of Open Access Journals (Sweden)

    Guillaume eRiviere

    2014-04-01

    Full Text Available DNA methylation is evolutionarily conserved. Vertebrates exhibit high, widespread DNA methylation whereas invertebrate genomes are less methylated, predominantly within gene bodies. DNA methylation in invertebrates is associated with transcription level, alternative splicing and genome evolution, but functional outcomes of DNA methylation remain poorly described in lophotrochozoans. Recent genome-wide approaches improve understanding in distant taxa such as molluscs, where the phylogenetic position and life traits of Crassostrea gigas make this bivalve an ideal model to study the physiological and evolutionary implications of DNA methylation. We review the literature about DNA methylation in invertebrates and focus on DNA methylation features in the oyster. Indeed, though our MeDIP-seq results confirm predominant intragenic methylation, the profiles depend on the oyster’s developmental and reproductive stage. We discuss the perspective that oyster DNA methylation could be biased toward the 5’-end of some genes, depending on physiological status, suggesting important functional outcomes of putative promoter methylation from cell differentiation during early development to sustained adaptation of the species to the environment.

  9. Epigenetic features in the oyster Crassostrea gigas suggestive of functionally relevant promoter DNA methylation in invertebrates.

    Science.gov (United States)

    Rivière, Guillaume

    2014-01-01

    DNA methylation is evolutionarily conserved. Vertebrates exhibit high, widespread DNA methylation whereas invertebrate genomes are less methylated, predominantly within gene bodies. DNA methylation in invertebrates is associated with transcription level, alternative splicing, and genome evolution, but functional outcomes of DNA methylation remain poorly described in lophotrochozoans. Recent genome-wide approaches improve understanding in distant taxa such as molluscs, where the phylogenetic position, and life traits of Crassostrea gigas make this bivalve an ideal model to study the physiological and evolutionary implications of DNA methylation. We review the literature about DNA methylation in invertebrates and focus on DNA methylation features in the oyster. Indeed, though our MeDIP-seq results confirm predominant intragenic methylation, the profiles depend on the oyster's developmental and reproductive stage. We discuss the perspective that oyster DNA methylation could be biased toward the 5'-end of some genes, depending on physiological status, suggesting important functional outcomes of putative promoter methylation from cell differentiation during early development to sustained adaptation of the species to the environment.

  10. Co-transcriptional production of RNA–DNA hybrids for simultaneous release of multiple split functionalities

    Science.gov (United States)

    Afonin, Kirill A.; Desai, Ravi; Viard, Mathias; Kireeva, Maria L.; Bindewald, Eckart; Case, Christopher L.; Maciag, Anna E.; Kasprzak, Wojciech K.; Kim, Taejin; Sappe, Alison; Stepler, Marissa; KewalRamani, Vineet N.; Kashlev, Mikhail; Blumenthal, Robert; Shapiro, Bruce A.

    2014-01-01

    Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA–DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA–RNA, RNA–DNA and DNA–DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates. PMID:24194608

  11. A Green's Function Approach to Simulate DNA Damage by the Indirect Effect

    Science.gov (United States)

    Plante, Ianik; Cicinotta, Francis A.

    2013-01-01

    The DNA damage is of fundamental importance in the understanding of the effects of ionizing radiation. DNA is damaged by the direct effect of radiation (e.g. direct ionization) and by indirect effect (e.g. damage by.OH radicals created by the radiolysis of water). Despite years of research, many questions on the DNA damage by ionizing radiation remains. In the recent years, the Green's functions of the diffusion equation (GFDE) have been used extensively in biochemistry [1], notably to simulate biochemical networks in time and space [2]. In our future work on DNA damage, we wish to use an approach based on the GFDE to refine existing models on the indirect effect of ionizing radiation on DNA. To do so, we will use the code RITRACKS [3] developed at the NASA Johnson Space Center to simulate the radiation track structure and calculate the position of radiolytic species after irradiation. We have also recently developed an efficient Monte-Carlo sampling algorithm for the GFDE of reversible reactions with an intermediate state [4], which can be modified and adapted to simulate DNA damage by free radicals. To do so, we will use the known reaction rate constants between radicals (OH, eaq, H,...) and the DNA bases, sugars and phosphates and use the sampling algorithms to simulate the diffusion of free radicals and chemical reactions with DNA. These techniques should help the understanding of the contribution of the indirect effect in the formation of DNA damage and double-strand breaks.

  12. Age-related mitochondrial DNA depletion and the impact on pancreatic Beta cell function.

    Science.gov (United States)

    Nile, Donna L; Brown, Audrey E; Kumaheri, Meutia A; Blair, Helen R; Heggie, Alison; Miwa, Satomi; Cree, Lynsey M; Payne, Brendan; Chinnery, Patrick F; Brown, Louise; Gunn, David A; Walker, Mark

    2014-01-01

    Type 2 diabetes is characterised by an age-related decline in insulin secretion. We previously identified a 50% age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. The purpose of this study was to mimic this degree of mtDNA depletion in MIN6 cells to determine whether there is a direct impact on insulin secretion. Transcriptional silencing of mitochondrial transcription factor A, TFAM, decreased mtDNA levels by 40% in MIN6 cells. This level of mtDNA depletion significantly decreased mtDNA gene transcription and translation, resulting in reduced mitochondrial respiratory capacity and ATP production. Glucose-stimulated insulin secretion was impaired following partial mtDNA depletion, but was normalised following treatment with glibenclamide. This confirms that the deficit in the insulin secretory pathway precedes K+ channel closure, indicating that the impact of mtDNA depletion is at the level of mitochondrial respiration. In conclusion, partial mtDNA depletion to a degree comparable to that seen in aged human islets impaired mitochondrial function and directly decreased insulin secretion. Using our model of partial mtDNA depletion following targeted gene silencing of TFAM, we have managed to mimic the degree of mtDNA depletion observed in aged human islets, and have shown how this correlates with impaired insulin secretion. We therefore predict that the age-related mtDNA depletion in human islets is not simply a biomarker of the aging process, but will contribute to the age-related risk of type 2 diabetes.

  13. Computational and experimental studies of reassociating RNA/DNA hybrids containing split functionalities.

    Science.gov (United States)

    Afonin, Kirill A; Bindewald, Eckart; Kireeva, Maria; Shapiro, Bruce A

    2015-01-01

    Recently, we developed a novel technique based on RNA/DNA hybrid reassociation that allows conditional activation of different split functionalities inside diseased cells and in vivo. We further expanded this idea to permit simultaneous activation of multiple different functions in a fully controllable fashion. In this chapter, we discuss some novel computational approaches and experimental techniques aimed at the characterization, design, and production of reassociating RNA/DNA hybrids containing split functionalities. We also briefly describe several experimental techniques that can be used to test these hybrids in vitro and in vivo. 2015 Published by Elsevier Inc.

  14. Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents.

    Directory of Open Access Journals (Sweden)

    2005-08-01

    Full Text Available The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.

  15. Genome-wide requirements for resistance to functionally distinct DNA-damaging agents.

    Directory of Open Access Journals (Sweden)

    William Lee

    2005-08-01

    Full Text Available The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of approximately 4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.

  16. Complex ABCC8 DNA variations in congenital hyperinsulinism: lessons from functional studies

    DEFF Research Database (Denmark)

    Muzyamba, Morris; Farzaneh, Tabasum; Behe, Phillip

    2007-01-01

    singly or in combination led to intracellular retention of the channel complex and loss of function. By contrast, V1572I is trafficked appropriately and is functional, consistent with a mechanism of reduction to hemizygosity of paternal ABCC8 in focal disease. V1572I is likely to be a benign DNA variant...

  17. Reassembly of functionally intact environmental DNA-derived biosynthetic gene clusters.

    Science.gov (United States)

    Kallifidas, Dimitris; Brady, Sean F

    2012-01-01

    Only a small fraction of the bacterial diversity present in natural microbial communities is regularly cultured in the laboratory. Those bacteria that remain recalcitrant to culturing cannot be examined for the production of bioactive secondary metabolites using standard pure-culture approaches. The screening of genomic DNA libraries containing DNA isolated directly from environmental samples (environmental DNA (eDNA)) provides an alternative approach for studying the biosynthetic capacities of these organisms. One drawback of this approach has been that most eDNA isolation procedures do not permit the cloning of DNA fragments of sufficient length to capture large natural product biosynthetic gene clusters in their entirety. Although the construction of eDNA libraries with inserts big enough to capture biosynthetic gene clusters larger than ∼40kb remains challenging, it is possible to access large gene clusters by reassembling them from sets of smaller overlapping fragments using transformation-associated recombination in Saccharomyces cerevisiae. Here, we outline a method for the reassembly of large biosynthetic gene clusters from captured sets of overlapping soil eDNA cosmid clones. Natural product biosynthetic gene clusters reassembled using this approach can then be used directly for functional heterologous expression studies. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein.

    Science.gov (United States)

    Perugino, Giuseppe; Miggiano, Riccardo; Serpe, Mario; Vettone, Antonella; Valenti, Anna; Lahiri, Samarpita; Rossi, Franca; Rossi, Mosè; Rizzi, Menico; Ciaramella, Maria

    2015-10-15

    Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. DNA-based visual majority logic gate with one-vote veto function.

    Science.gov (United States)

    Fan, Daoqing; Wang, Kun; Zhu, Jinbo; Xia, Yong; Han, Yanchao; Liu, Yaqing; Wang, Erkang

    2015-03-01

    A molecular logic gate is a basic element and plays a key role in molecular computing. Herein, we have developed a label-free and enzyme-free three-input visual majority logic gate which is realized for the first time according to DNA hybridization only, without DNA replacement and enzyme catalysis. Furthermore, a one-vote veto function was integrated into the DNA-based majority logic gate, in which one input has priority over other inputs. The developed system can also implement multiple basic and cascade logic gates.

  20. Functional annotation of 19,841 Populus nigra full-length enriched cDNA clones

    Science.gov (United States)

    Nanjo, Tokihiko; Sakurai, Tetsuya; Totoki, Yasushi; Toyoda, Atsushi; Nishiguchi, Mitsuru; Kado, Tomoyuki; Igasaki, Tomohiro; Futamura, Norihiro; Seki, Motoaki; Sakaki, Yoshiyuki; Shinozaki, Kazuo; Shinohara, Kenji

    2007-01-01

    Background Populus is one of favorable model plants because of its small genome. Structural genomics of Populus has reached a breakpoint as nucleotides of the entire genome have been determined. Reaching the post genome era, functional genomics of Populus is getting more important for well-comprehended plant science. Development of bioresorce serving functional genomics is making rapid progress. Huge efforts have achieved deposits of expressed sequence tags (ESTs) in various plant species consequently accelerating functional analysis of genes. ESTs from full-length cDNA clones are especially powerful for accurate molecular annotation. We promoted collection and annotation of the ESTs from Populus full-length enriched cDNA clones as part of functional genomics of tree species. Results We have been collecting the full-length enriched cDNA of the female poplar (Populus nigra var. italica) for years. By sequencing P. nigra full-length (PnFL) cDNA libraries, we generated about 116,000 5'-end or 3'-end ESTs corresponding to 19,841 nonredundant PnFL clones. Population of PnFL cDNA clones represents 44% of the predicted genes in the Populus genome. Conclusion Our resource of P. nigra full-length enriched clones is expected to provide valuable tools to gain further insight into genome annotation and functional genomics in Populus. PMID:18053163

  1. Functional annotation of 19,841 Populus nigra full-length enriched cDNA clones

    Directory of Open Access Journals (Sweden)

    Seki Motoaki

    2007-12-01

    Full Text Available Abstract Background Populus is one of favorable model plants because of its small genome. Structural genomics of Populus has reached a breakpoint as nucleotides of the entire genome have been determined. Reaching the post genome era, functional genomics of Populus is getting more important for well-comprehended plant science. Development of bioresorce serving functional genomics is making rapid progress. Huge efforts have achieved deposits of expressed sequence tags (ESTs in various plant species consequently accelerating functional analysis of genes. ESTs from full-length cDNA clones are especially powerful for accurate molecular annotation. We promoted collection and annotation of the ESTs from Populus full-length enriched cDNA clones as part of functional genomics of tree species. Results We have been collecting the full-length enriched cDNA of the female poplar (Populus nigra var. italica for years. By sequencing P. nigra full-length (PnFL cDNA libraries, we generated about 116,000 5'-end or 3'-end ESTs corresponding to 19,841 nonredundant PnFL clones. Population of PnFL cDNA clones represents 44% of the predicted genes in the Populus genome. Conclusion Our resource of P. nigra full-length enriched clones is expected to provide valuable tools to gain further insight into genome annotation and functional genomics in Populus.

  2. DNA barcoding of life: a classification of uses according to function and scale after ten years of development

    OpenAIRE

    Nancai Pei; Bufeng Chen

    2013-01-01

    DNA barcoding technology provides molecular information, standard dataset platforms, and universal technical regulations for modern biological research. We briefly review the history of DNA barcoding between 2003 and 2012, and classify DNA barcoding into three types of biological function: basic function (e.g., storing data, and identifying species), extending function (e.g., building phylogenies, serving specific subjects, and compiling biological atlas) and potential function (e.g., reveali...

  3. Tcf4 Regulates Synaptic Plasticity, DNA Methylation, and Memory Function

    Directory of Open Access Journals (Sweden)

    Andrew J. Kennedy

    2016-09-01

    Full Text Available Human haploinsufficiency of the transcription factor Tcf4 leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS, which is associated with severe language impairment and development delay. Here, we demonstrate that Tcf4 haploinsufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4(+/− mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, we found that small-molecule HDAC inhibitors normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4-deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified “memory-associated” genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, we observed that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4(+/− mice.

  4. Defining the functional footprint for recognition and repair of deaminated DNA.

    Science.gov (United States)

    Baldwin, Michael R; O'Brien, Patrick J

    2012-12-01

    Spontaneous deamination of DNA is mutagenic, if it is not repaired by the base excision repair (BER) pathway. Crystallographic data suggest that each BER enzyme has a compact DNA binding site. However, these structures lack information about poorly ordered termini, and the energetic contributions of specific protein-DNA contacts cannot be inferred. Furthermore, these structures do not reveal how DNA repair intermediates are passed between enzyme active sites. We used a functional footprinting approach to define the binding sites of the first two enzymes of the human BER pathway for the repair of deaminated purines, alkyladenine DNA glycosylase (AAG) and AP endonuclease (APE1). Although the functional footprint for full-length AAG is explained by crystal structures of truncated AAG, the footprint for full-length APE1 indicates a much larger binding site than is observed in crystal structures. AAG turnover is stimulated in the presence of APE1, indicating rapid exchange of AAG and APE1 at the abasic site produced by the AAG reaction. The coordinated reaction does not require an extended footprint, suggesting that each enzyme engages the site independently. Functional footprinting provides unique information relative to traditional footprinting approaches and is generally applicable to any DNA modifying enzyme or system of enzymes.

  5. Multiplexed aptasensors and amplified DNA sensors using functionalized graphene oxide: application for logic gate operations.

    Science.gov (United States)

    Liu, Xiaoqing; Aizen, Ruth; Freeman, Ronit; Yehezkeli, Omer; Willner, Itamar

    2012-04-24

    Graphene oxide (GO) is implemented as a functional matrix for developing fluorescent sensors for the amplified multiplexed detection of DNA, aptamer-substrate complexes, and for the integration of predesigned DNA constructs that activate logic gate operations. Fluorophore-labeled DNA strands acting as probes for two different DNA targets are adsorbed onto GO, leading to the quenching of the luminescence of the fluorophores. Desorption of the probes from the GO, through hybridization with the target DNAs, leads to the fluorescence of the respective label. By coupling exonuclease III, Exo III, to the system, the recycling of the target DNAs is demonstrated, and this leads to the amplified detection of the DNA targets (detection limit 5 × 10(-12) M). Similarly, adsorption of fluorophore-functionalized aptamers against thrombin or ATP onto the GO leads to the desorption of the aptamer-substrate complexes from GO and to the triggering of the luminescence corresponding to the respective fluorophore, thus, allowing the multiplexed analysis of the aptamer-substrate complexes. By designing functional fluorophore-labeled DNA constructs and their interaction with GO, in the presence (or absence) of nucleic acids, or two different substrates for aptamers, as inputs, the activation of the "OR" and "AND" logic gates is demonstrated.

  6. DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition.

    Science.gov (United States)

    Liu, Xiao; Dang, Yunkun; Matsu-Ura, Toru; He, Yubo; He, Qun; Hong, Christian I; Liu, Yi

    2017-07-20

    Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify DNA replication as a critical process in the circadian mechanism. Published by Elsevier Inc.

  7. DNA methylation and cognitive functioning in healthy older adults

    NARCIS (Netherlands)

    Schiepers, O.J.G.; Boxtel, van M.P.J.; Groot, R.H.M.; Jolles, J.; Kok, F.J.; Verhoef, P.; Durga, J.

    2012-01-01

    Long-term supplementation with folic acid may improve cognitive performance in older individuals. The relationship between folate status and cognitive performance might be mediated by changes in methylation capacity, as methylation reactions are important for normal functioning of the brain.

  8. Functional characterization of a conserved archaeal viral operon revealing single-stranded DNA binding, annealing and nuclease activities

    DEFF Research Database (Denmark)

    Guo, Yang; Kragelund, Birthe Brandt; White, Malcolm F.

    2015-01-01

    encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U......-shaped binding path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5'→3' ssDNA exonuclease activity, in addition...... for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair....

  9. Oxidative cleavage of DNA mediated by hybrid metalloporphyrin-ellipticine molecules and functionalized metalloporphyrin precursors.

    Science.gov (United States)

    Ding, L; Etemad-Moghadam, G; Meunier, B

    1990-08-28

    The nuclease activity of functionalized metalloporphyrins 1-8 and hybrid metalloporphyrin-ellipticine molecules 10-16 in the presence of potassium monopersulfate (KHSO5) or magnesium monoperoxyphthalate (MMPP), water-soluble oxygen atom donors at physiological pH, toward double-stranded phi X174 DNA is reported. The DNA cleavage efficiency as a function of the nature of functionalized metalloporphyrins, the length of the linkage between the two parts of the hybrid molecule, viz., metalloporphyrin and 9-methoxyellipticine, the nature of the central metal atom (Mn, Fe, or Zn) the ionic strength, and the nature of the oxygen donor has been studied. Single-strand breaks (SSBs) are observed on double-stranded DNA with a short incubation time of 2 min in the presence of manganese derivatives of both metalloporphyrins and hybrid molecules. Owing to their cytotoxic and nuclease activity, these new water-soluble hybrid molecules may be considered as efficient bleomycin models based on cationic metalloporphyrins.

  10. Accumulation of Mitochondrial DNA Mutations Disrupts Cardiac Progenitor Cell Function and Reduces Survival.

    Science.gov (United States)

    Orogo, Amabel M; Gonzalez, Eileen R; Kubli, Dieter A; Baptista, Igor L; Ong, Sang-Bing; Prolla, Tomas A; Sussman, Mark A; Murphy, Anne N; Gustafsson, Åsa B

    2015-09-04

    Transfer of cardiac progenitor cells (CPCs) improves cardiac function in heart failure patients. However, CPC function is reduced with age, limiting their regenerative potential. Aging is associated with numerous changes in cells including accumulation of mitochondrial DNA (mtDNA) mutations, but it is unknown how this impacts CPC function. Here, we demonstrate that acquisition of mtDNA mutations disrupts mitochondrial function, enhances mitophagy, and reduces the replicative and regenerative capacities of the CPCs. We show that activation of differentiation in CPCs is associated with expansion of the mitochondrial network and increased mitochondrial oxidative phosphorylation. Interestingly, mutant CPCs are deficient in mitochondrial respiration and rely on glycolysis for energy. In response to differentiation, these cells fail to activate mitochondrial respiration. This inability to meet the increased energy demand leads to activation of cell death. These findings demonstrate the consequences of accumulating mtDNA mutations and the importance of mtDNA integrity in CPC homeostasis and regenerative potential. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Function of the Plant DNA Polymerase Epsilon in Replicative Stress Sensing, a Genetic Analysis.

    Science.gov (United States)

    Pedroza-García, José-Antonio; Mazubert, Christelle; Del Olmo, Ivan; Bourge, Mickael; Domenichini, Séverine; Bounon, Rémi; Tariq, Zakia; Delannoy, Etienne; Piñeiro, Manuel; Jarillo, José A; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2017-03-01

    Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis (Arabidopsis thaliana). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote. © 2017 American Society of Plant Biologists. All Rights Reserved.

  12. [Design of artificial DNA binding proteins toward control and elucidation of cellular functions].

    Science.gov (United States)

    Imanishi, Miki

    2012-01-01

    An artificial transcription factor that can regulate the expression of specific genes at a desired time is very useful for research in chemical biology, cell biology, and future gene therapy. A C2H2 zinc finger motif, one of zinc-containing proteins, is known as the most ubiquitous DNA binding motif. The motif is attractive for designing artificial transcription factors with desired DNA binding specificities because of its characteristic DNA binding properties: (1) recognition of 3 bp per motif, (2) tandemly connected modular structure, and (3) binding to non-palindrome sequences as a monomer. Taking advantage of these properties, artificial DNA binding proteins with new DNA binding characteristics have been designed. By changing the linker region between two 3-zinc finger domains, artificial 6-zinc finger proteins were developed and shown to skip DNA sequences. Zinc-responsive transcription factors were created by altering one of the zinc ligands. An artificial zinc finger transcription factor targeting a core clock gene induced phase shifts of the cellular "circadian rhythm". Herein, I will summarize creation and function of the above-mentioned artificial zinc finger-type DNA binding proteins and transcription factors.

  13. Functional evaluation of malaria Pfs25 DNA vaccine by in vivo electroporation in olive baboons.

    Science.gov (United States)

    Kumar, Rajesh; Nyakundi, Ruth; Kariuki, Thomas; Ozwara, Hastings; Nyamongo, Onkoba; Mlambo, Godfree; Ellefsen, Barry; Hannaman, Drew; Kumar, Nirbhay

    2013-06-28

    Plasmodium falciparum Pfs25 antigen, expressed on the surface of zygotes and ookinetes, is one of the leading targets for the development of a malaria transmission-blocking vaccine (TBV). Our laboratory has been evaluating DNA plasmid based Pfs25 vaccine in mice and non-human primates. Previously, we established that in vivo electroporation (EP) delivery is an effective method to improve the immunogenicity of DNA vaccine encoding Pfs25 in mice. In order to optimize the in vivo EP procedure and test for its efficacy in more clinically relevant larger animal models, we employed in vivo EP to evaluate the immune response and protective efficacy of Pfs25 encoding DNA vaccine in nonhuman primates (olive baboons, Papio anubis). The results showed that at a dose of 2.5mg DNA vaccine, antibody responses were significantly enhanced with EP as compared to without EP resulting in effective transmission blocking efficiency. Similar immunogenicity enhancing effect of EP was also observed with lower doses (0.5mg and 1mg) of DNA plasmids. Further, final boosting with a single dose of recombinant Pfs25 protein resulted in dramatically enhanced antibody titers and significantly increased functional transmission blocking efficiency. Our study suggests priming with DNA vaccine via EP along with protein boost regimen as an effective method to elicit potent immunogenicity of malaria DNA vaccines in nonhuman primates and provides the basis for further evaluation in human volunteers. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Photoligation of self-assembled DNA constructs containing anthracene-functionalized 2'-amino-LNA monomers

    DEFF Research Database (Denmark)

    Pasternak, Karol; Pasternak, Anna; Gupta, Pankaj

    2011-01-01

    Efficient synthesis of a novel anthracene-functionalized 2'-amino-LNA phosphoramidite derivative is described together with its incorporation into oligodeoxynucleotides. Two DNA strands with the novel 2'-N-anthracenylmethyl-2'-amino-LNA monomers can be effectively cross-linked by photoligation...... at 366nm in various types of DNA constructs. Successful application of three differently functionalized 2'-amino-LNA monomers in self-assembled higher ordered structures for simultaneous cross-linking and monitoring of assembly formation is furthermore demonstrated....

  15. Structure, function and evolution of the animal mitochondrial replicative DNA helicase.

    Science.gov (United States)

    Kaguni, Laurie S; Oliveira, Marcos T

    2016-01-01

    The mitochondrial replicative DNA helicase is essential for animal mitochondrial DNA (mtDNA) maintenance. Deleterious mutations in the gene that encodes it cause mitochondrial dysfunction manifested in developmental delays, defects and arrest, limited life span, and a number of human pathogenic phenotypes that are recapitulated in animals across taxa. In fact, the replicative mtDNA helicase was discovered with the identification of human disease mutations in its nuclear gene, and based upon its deduced amino acid sequence homology with bacteriophage T7 gene 4 protein (T7 gp4), a bi-functional primase-helicase. Since that time, numerous investigations of its structure, mechanism, and physiological relevance have been reported, and human disease alleles have been modeled in the human, mouse, and Drosophila systems. Here, we review this literature and draw evolutionary comparisons that serve to shed light on its divergent features.

  16. Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions

    DEFF Research Database (Denmark)

    Martín-Pardillos, Ana; Tsaalbi-Shtylik, Anastasia; Chen, Si

    2017-01-01

    Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem......-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly......), the postreplicative bypass of damaged nucleotides. Rev1 hematopoietic stem and progenitor cells displayed compromised proliferation, and replication stress that could be rescued with an antioxidant. The additional disruption of Xpc, essential for global-genome nucleotide excision repair (ggNER) of helix...

  17. DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

    Directory of Open Access Journals (Sweden)

    Katherine S Lawrence

    2015-04-01

    Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

  18. Uncoupling of satellite DNA and centromeric function in the genus Equus.

    Directory of Open Access Journals (Sweden)

    Francesca M Piras

    2010-02-01

    Full Text Available In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1 several centromeres, including the previously described evolutionary new centromeres (ENCs, seem to be devoid of satellite DNA, and 2 satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.

  19. Uncoupling of Satellite DNA and Centromeric Function in the Genus Equus

    Science.gov (United States)

    Magnani, Elisa; Bertoni, Livia; Attolini, Carmen; Khoriauli, Lela; Raimondi, Elena; Giulotto, Elena

    2010-01-01

    In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs. PMID:20169180

  20. Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.

    Directory of Open Access Journals (Sweden)

    Kaitlin M Stimpson

    Full Text Available The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA. The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2 causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.

  1. Inspiration from chemical photography: accelerated photoconversion of AgCl to functional silver nanoparticles mediated by DNA.

    Science.gov (United States)

    Wang, Guoqing; Nishio, Takashi; Sato, Masato; Ishikawa, Ayako; Nambara, Katsuyuki; Nagakawa, Keita; Matsuo, Yasutaka; Niikura, Kenichi; Ijiro, Kuniharu

    2011-09-07

    We demonstrate a facile approach for converting AgCl to functional silver nanoparticles (AgNPs) via photoreduction in the presence of DNA. The resulting AgNPs are biofunctionalized, and exhibit photostable luminescence and DNA-specific Raman signatures, showing high potential for use in DNA-directed recognition and advanced bioimaging. This journal is © The Royal Society of Chemistry 2011

  2. Multi-scale coding of genomic information: From DNA sequence to genome structure and function

    Energy Technology Data Exchange (ETDEWEB)

    Arneodo, Alain, E-mail: alain.arneodo@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Vaillant, Cedric, E-mail: cedric.vaillant@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Audit, Benjamin, E-mail: benjamin.audit@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Argoul, Francoise, E-mail: francoise.argoul@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); D' Aubenton-Carafa, Yves, E-mail: daubenton@cgm.cnrs-gif.f [Centre de Genetique Moleculaire, CNRS, Allee de la Terrasse, 91198 Gif-sur-Yvette (France); Thermes, Claude, E-mail: claude.thermes@cgm.cnrs-gif.f [Centre de Genetique Moleculaire, CNRS, Allee de la Terrasse, 91198 Gif-sur-Yvette (France)

    2011-02-15

    Understanding how chromatin is spatially and dynamically organized in the nucleus of eukaryotic cells and how this affects genome functions is one of the main challenges of cell biology. Since the different orders of packaging in the hierarchical organization of DNA condition the accessibility of DNA sequence elements to trans-acting factors that control the transcription and replication processes, there is actually a wealth of structural and dynamical information to learn in the primary DNA sequence. In this review, we show that when using concepts, methodologies, numerical and experimental techniques coming from statistical mechanics and nonlinear physics combined with wavelet-based multi-scale signal processing, we are able to decipher the multi-scale sequence encoding of chromatin condensation-decondensation mechanisms that play a fundamental role in regulating many molecular processes involved in nuclear functions.

  3. DNA Sequence Constraints Define Functionally Active Steroid Nuclear Receptor Binding Sites in Chromatin.

    Science.gov (United States)

    Coons, Laurel A; Hewitt, Sylvia C; Burkholder, Adam B; McDonnell, Donald P; Korach, Kenneth S

    2017-10-01

    Gene regulatory programs are encoded in the sequence of the DNA. Since the completion of the Human Genome Project, millions of gene regulatory elements have been identified in the human genome. Understanding how each of those sites functionally contributes to gene regulation, however, remains a challenge for nearly every field of biology. Transcription factors influence cell function by interpreting information contained within cis-regulatory elements in chromatin. Whereas chromatin immunoprecipitation-sequencing has been used to identify and map transcription factor-DNA interactions, it has been difficult to assign functionality to the binding sites identified. Thus, in this study, we probed the transcriptional activity, DNA-binding competence, and functional activity of select nuclear receptor mutants in cellular and animal model systems and used this information to define the sequence constraints of functional steroid nuclear receptor cis-regulatory elements. Analysis of the architecture within sNR chromatin interacting sites revealed that only a small fraction of all sNR chromatin-interacting events is associated with transcriptional output and that this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides. These findings define the transcriptional grammar necessary to predict functionality from regulatory sequences, with a multitude of future implications. Copyright © 2017 Endocrine Society.

  4. Functions of FUS/TLS From DNA Repair to Stress Response: Implications for ALS

    Directory of Open Access Journals (Sweden)

    Reddy Ranjith Kumar Sama

    2014-08-01

    Full Text Available Fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS is a multifunctional DNA-/RNA-binding protein that is involved in a variety of cellular functions including transcription, protein translation, RNA splicing, and transport. FUS was initially identified as a fusion oncoprotein, and thus, the early literature focused on the role of FUS in cancer. With the recent discoveries revealing the role of FUS in neurodegenerative diseases, namely amyotrophic lateral sclerosis and frontotemporal lobar degeneration, there has been a renewed interest in elucidating the normal functions of FUS. It is not clear which, if any, endogenous functions of FUS are involved in disease pathogenesis. Here, we review what is currently known regarding the normal functions of FUS with an emphasis on DNA damage repair, RNA processing, and cellular stress response. Further, we discuss how ALS-causing mutations can potentially alter the role of FUS in these pathways, thereby contributing to disease pathogenesis.

  5. Lung function discordance in monozygotic twins and associated differences in blood DNA methylation

    DEFF Research Database (Denmark)

    Bolund, Anneli C S; Starnawska, Anna; Miller, Martin R

    2017-01-01

    design, we correlated intra-pair differences in cross-sectional and longitudinal lung function with intra-pair blood DNA methylation differences at follow-up by linear regression analyses adjusted for sex, age, BMI, smoking, and blood cell composition measured for each individual with the use of flow......Background: Lung function is an important predictor of morbidity and mortality, with accelerated lung function decline reported to have immense consequences for the world's healthcare systems. The lung function decline across individual's lifetime is a consequence of age-related changes in lung...... anatomical structure and combination of various environmental factors; however, the exact molecular mechanisms contributing to this decline are not fully understood. DNA methylation is an epigenetic modification that changes across individual's lifetime, as well as allows for interplay between environmental...

  6. Colloidal Dancers: Designing networks of DNA-functionalized colloids for non-random walks

    Science.gov (United States)

    Gehrels, Emily W.; Rogers, W. Benjamin; Zeravcic, Zorana; Manoharan, Vinothan N.

    2014-03-01

    We present experimental developments of a system of DNA-functionalized colloidal particles with the goal of creating directed motion (`dancing') along patterned substrates in response to temperature cycling. We take advantage of toehold exchange in the design of the DNA sequences that mediate the colloidal interactions to produce broadened, flat, or even re-entrant binding and unbinding transitions between the particles and substrate. Using this new freedom of design, we devise systems where, by thermal ratcheting, we can externally control the direction of motion and sequence of steps of the colloidal dancer. In comparison to DNA-based walkers, which move autonomously and whose motion is controlled by the substrate, our colloidal dancers respond to external driving, and their motion can be controlled in situ. Our use of DNA-functionalized colloidal particles instead of pure DNA systems also enables walking on the mesoscale in contrast to the molecular length scales previously demonstrated, allowing for the future prospect of directed transport over larger distances.

  7. Mitochondrial DNA and Functional Investigations into the Radiosensitivity of Four Mouse Strains

    Directory of Open Access Journals (Sweden)

    Steven B. Zhang

    2014-01-01

    Full Text Available We investigated whether genetic radiosensitivity-related changes in mtDNA/nDNA ratios are significant to mitochondrial function and if a material effect on mtDNA content and function exists. BALB/c (radiosensitive, C57BL/6 (radioresistant, and F1 hybrid mouse strains were exposed to total body irradiation. Hepatic genomic DNA was extracted, and mitochondria were isolated. Mitochondrial oxygen consumption, ROS, and calcium-induced mitochondrial swelling were measured. Radiation influenced strain-specific survival in vivo. F1 hybrid survival was influenced by maternal input. Changes in mitochondrial content corresponded to survival in vivo among the 4 strains. Calcium-induced mitochondrial swelling was strain dependent. Isolated mitochondria from BALB/c mice were significantly more sensitive to calcium overload than mitochondria from C57BL/6 mice. Maternal input partially influenced the recovery effect of radiation on calcium-induced mitochondrial swelling in F1 hybrids; the hybrid with a radiosensitive maternal lineage exhibited a lower rate of recovery. Hybrids had a survival rate that was biased toward maternal input. mtDNA content and mitochondrial permeability transition pores (MPTP measured in these strains before irradiation reflected a dominant input from the parent. After irradiation, the MPTP opened sooner in radiosensitive and hybrid strains, likely triggering intrinsic apoptotic pathways. These findings have important implications for translation into predictors of radiation sensitivity/resistance.

  8. Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

    Science.gov (United States)

    García-Rodríguez, Néstor; Wong, Ronald P.; Ulrich, Helle D.

    2016-01-01

    Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and flexible responses, for which posttranslational protein modifications that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance specifically as they relate to DNA replication. We will consider cases where the modifiers act during regular, i.e., unperturbed stages of replication, such as initiation, fork progression, and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse. PMID:27242895

  9. The impact of 6-thioguanine incorporation into DNA on the function of DNA methyltransferase Dnmt3a.

    Science.gov (United States)

    Kirsanova, Olga V; Sergeev, Alexander V; Yasko, Ivan S; Gromova, Elizaveta S

    2017-06-03

    The incorporation of chemotherapeutic agent 6-thioguanine ((S)G) into DNA is a prerequisite for its cytotoxic action. This modification of DNA impedes the activity of enzymes involved in DNA repair and replication. Here, using hemimethylated DNA substrates we demonstrated that DNA methylation by Dnmt3a-CD is reduced if DNA is damaged by the incorporation of (S)G into one or two CpG sites separated by nine base pairs. An increase in the number of (S)G substitutions did not enhance the effect. Dnmt3a-CD binding to either of (S)G-containing DNA substrates was not distorted. Our results suggest that (S)G incorporation into DNA may influence epigenetic regulation via DNA methylation.

  10. Structure-function analysis of human enzymes initiating nucleobase repair in DNA and RNA

    OpenAIRE

    Sundheim, Ottar

    2008-01-01

    In humans, there are four known glycosylases that initiate repair of uracils in DNA. These are UNG, TDG, SMUG1, and MBD4. It was proposed that the replication independent SMUG1 was the main enzyme initiating removal of deaminated cytosine, whereas UNG2 was responsible for replication associated repair of mis-incorporated dUTP (Nilsen et al., 2001). We aimed at elucidating the specific function of the two main human uracil-DNA glycosylases in vitro and in vivo to further clarify their distinct...

  11. Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

    Science.gov (United States)

    Horvath, Beatrix M; Kourova, Hana; Nagy, Szilvia; Nemeth, Edit; Magyar, Zoltan; Papdi, Csaba; Ahmad, Zaki; Sanchez-Perez, Gabino F; Perilli, Serena; Blilou, Ikram; Pettkó-Szandtner, Aladár; Darula, Zsuzsanna; Meszaros, Tamas; Binarova, Pavla; Bogre, Laszlo; Scheres, Ben

    2017-05-02

    The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  12. The impact of arginine-modified chitosan-DNA nanoparticles on the function of macrophages

    Science.gov (United States)

    Liu, Lanxia; Bai, Yuanyuan; Song, Chunni; Zhu, Dunwan; Song, Liping; Zhang, Hailing; Dong, Xia; Leng, Xigang

    2010-06-01

    It has been demonstrated that incorporation of arginine moieties into chitosan significantly elevates the transgenic efficacy of the chitosan. However, little is known about the impact of arginine-modified chitosan on the function of macrophages, which play a vitally important role in the inflammatory response of the body to foreign substances, especially particulate substances. This study was designed to investigate the impact of arginine-modified chitosan/DNA nanoparticles on the function of the murine macrophage through observation of phagocytic activity and production of pro-inflammatory cytokines (IL-1β, IL-6, IL-10, IL-12, and TNF-α). Results showed that both chitosan/DNA nanoparticles and arginine-modified chitosan/DNA nanoparticles, containing 20 μg/mL DNA, were internalized by almost all the macrophages in contact. This led to no significant changes, compared to the non-exposure group, in production of cytokines and phagocytic activity of the macrophages 24 h post co-incubation, whereas exposure to LPS induced obviously elevated cytokine production and phagocytic activity, suggesting that incorporation of arginine moieties into chitosan does not have a negative impact on the function of the macrophages.

  13. The impact of arginine-modified chitosan-DNA nanoparticles on the function of macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Liu Lanxia; Bai Yuanyuan; Song Chunni; Zhu Dunwan; Song Liping; Zhang Hailing; Dong Xia; Leng Xigang, E-mail: lengxg@bme.org.c [Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Laboratory of Bioengineering (China)

    2010-06-15

    It has been demonstrated that incorporation of arginine moieties into chitosan significantly elevates the transgenic efficacy of the chitosan. However, little is known about the impact of arginine-modified chitosan on the function of macrophages, which play a vitally important role in the inflammatory response of the body to foreign substances, especially particulate substances. This study was designed to investigate the impact of arginine-modified chitosan/DNA nanoparticles on the function of the murine macrophage through observation of phagocytic activity and production of pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-10, IL-12, and TNF-{alpha}). Results showed that both chitosan/DNA nanoparticles and arginine-modified chitosan/DNA nanoparticles, containing 20 {mu}g/mL DNA, were internalized by almost all the macrophages in contact. This led to no significant changes, compared to the non-exposure group, in production of cytokines and phagocytic activity of the macrophages 24 h post co-incubation, whereas exposure to LPS induced obviously elevated cytokine production and phagocytic activity, suggesting that incorporation of arginine moieties into chitosan does not have a negative impact on the function of the macrophages.

  14. Synthesis of a drug delivery vehicle for cancer treatment utilizing DNA-functionalized gold nanoparticles

    Science.gov (United States)

    Brann, Tyler

    The treatment of cancer with chemotherapeutic agents has made great strides in the last few decades but still introduces major systemic side effects. The potent drugs needed to kill cancer cells often cause irreparable damage to otherwise healthy organs leading to further morbidity and mortality. A therapy with intrinsic selective properties and/or an inducible activation has the potential to change the way cancer can be treated. Gold nanoparticles (GNPs) are biocompatible and chemically versatile tools that can be readily functionalized to serve as molecular vehicles. The ability of these particles to strongly absorb light with wavelengths in the therapeutic window combined with the heating effect of surface plasmon resonance makes them uniquely suited for noninvasive heating in biologic applications. Specially designed DNA aptamers have shown their ability to serve as drug carriers through intercalation as well as directly acting as therapeutic agents. By combining these separate molecules a multifaceted drug delivery vehicle can be created with great potential as a selective and controllable treatment for cancer. Oligonucleotide-coated GNPs have been created using spherical GNPs but little work has been reported using gold nanoplates in this way. Using the Diasynth method gold nanoplates were produced to absorb strongly in the therapeutic near infrared (nIR) window. These particles were functionalized with two DNA oligonucleotides: one serving as an intercalation site for doxorubicin, and another, AS1411, serving directly as an anticancer targeting/therapeutic agent. These functional particles were fully synthesized and processed along with confirmation of DNA functionalization and doxorubicin intercalation. Doxorubicin is released via denaturation of the DNA structure into which doxorubicin is intercalated upon the heating of the gold nanoplate well above the DNA melting temperature. This temperature increase, due to light stimulation of surface plasmon

  15. A multi-functional guanine derivative for studying the DNA G-quadruplex structure.

    Science.gov (United States)

    Ishizuka, Takumi; Zhao, Pei-Yan; Bao, Hong-Liang; Xu, Yan

    2017-10-23

    In the present study, we developed a multi-functional guanine derivative, 8FG, as a G-quadruplex stabilizer, a fluorescent probe for the detection of G-quadruplex formation, and a 19F sensor for the observation of the G-quadruplex. We demonstrate that the functional nucleoside bearing a 3,5-bis(trifluoromethyl)benzene group at the 8-position of guanine stabilizes the DNA G-quadruplex structure and fluoresces following the G-quadruplex formation. Furthermore, we show that the functional sensor can be used to directly observe DNA G-quadruplexes by 19F-NMR in living cells. To our knowledge, this is the first study showing that the nucleoside derivative simultaneously allows for three kinds of functions at a single G-quadruplex DNA. Our results suggest that the multi-functional nucleoside derivative can be broadly used for studying the G-quadruplex structure and serves as a powerful tool for examining the molecular basis of G-quadruplex formation in vitro and in living cells.

  16. Detection of change in elastic properties of a stretched DNA by using correlation functions of fluctuations

    Science.gov (United States)

    Murayama, Yoshihiro; Yogiashi, Yusuke; Iwamoto, Takuya; Mitsuhashi, Yoshitake; Homma, Hirofumi

    2013-02-01

    We measured fluctuations of single DNA molecules stretched by dual trap optical tweezers, and obtained its elastic properties, the force and its derivative, by calculating auto- and cross-correlation functions of the fluctuations. We investigated suitable range of the spring constant of optical tweezers for the detection of the change in the elastic properties. We found that the prediction of worm-like chain model for the elastic properties agrees with the experimentally obtained values when the spring constant of optical tweezers is 2-5 pN/μm in our setup. Moreover, we introduced a fluorescent dye, YOYO-1, into the flow chamber, and the change of the elastic properties of the single DNA molecule was observed. The results show that we can detect the changes of not only the force but also its derivative for the DNA stretched at a fixed extension.

  17. Charge-transfer excited states in aqueous DNA: Insights from many-body Green's function theory.

    Science.gov (United States)

    Yin, Huabing; Ma, Yuchen; Mu, Jinglin; Liu, Chengbu; Rohlfing, Michael

    2014-06-06

    Charge-transfer (CT) excited states play an important role in the excited-state dynamics of DNA in aqueous solution. However, there is still much controversy on their energies. By ab initio many-body Green's function theory, together with classical molecular dynamics simulations, we confirm the existence of CT states at the lower energy side of the optical absorption maximum in aqueous DNA as observed in experiments. We find that the hydration shell can exert strong effects (∼1  eV) on both the electronic structure and CT states of DNA molecules through dipole electric fields. In this case, the solvent cannot be simply regarded as a macroscopic screening medium as usual. The influence of base stacking and base pairing on the CT states is also discussed.

  18. Distinct functions of human RecQ helicases during DNA replication.

    Science.gov (United States)

    Urban, Vaclav; Dobrovolna, Jana; Janscak, Pavel

    2017-06-01

    DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. RecQ helicases promote recovery of replication forks being stalled due to different replication roadblocks of either exogenous or endogenous source. They prevent generation of aberrant replication fork structures and replication fork collapse, and are involved in proper checkpoint signaling. The essential role of human RecQ helicases in the genome maintenance during DNA replication is underlined by association of defects in their function with cancer predisposition. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. A DNA aptamer recognising a malaria protein biomarker can function as part of a DNA origami assembly

    OpenAIRE

    Maia Godonoga; Ting-Yu Lin; Azusa Oshima; Koji Sumitomo; Marco S. L. Tang; Yee-Wai Cheung; Andrew B. Kinghorn; Roderick M. Dirkzwager; Cunshan Zhou; Akinori Kuzuya; Julian A. Tanner; Jonathan G. Heddle

    2016-01-01

    DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target...

  20. DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization

    Directory of Open Access Journals (Sweden)

    Philipp Heissig

    2016-01-01

    Full Text Available siRNA and microRNA are promising therapeutic agents, which are engaged in a natural mechanism called RNA interference that modulates gene expression posttranscriptionally. For intracellular delivery of such nucleic acid triggers, we use sequence-defined cationic polymers manufactured through solid phase chemistry. They consist of an oligoethanamino amide core for siRNA complexation and optional domains for nanoparticle shielding and cell targeting. Due to the small size of siRNA, electrostatic complexes with polycations are less stable, and consequently intracellular delivery is less efficient. Here we use DNA oligomers as adaptors to increase size and charge of cargo siRNA, resulting in increased polyplex stability, which in turn boosts transfection efficiency. Extending a single siRNA with a 181-nucleotide DNA adaptor is sufficient to provide maximum gene silencing aided by cationic polymers. Interestingly, this simple strategy was far more effective than merging defined numbers (4–10 of siRNA units into one DNA scaffolded construct. For DNA attachment, the 3′ end of the siRNA passenger strand was beneficial over the 5′ end. The impact of the attachment site however was resolved by introducing bioreducible disulfides at the connection point. We also show that DNA adaptors provide the opportunity to readily link additional functional domains to siRNA. Exemplified by the covalent conjugation of the endosomolytic influenza peptide INF-7 to siRNA via a DNA backbone strand and complexing this construct with a targeting polymer, we could form a highly functional polyethylene glycol–shielded polyplex to downregulate a luciferase gene in folate receptor–positive cells.

  1. B-chromosome ribosomal DNA is functional in the grasshopper Eyprepocnemis plorans.

    Science.gov (United States)

    Ruiz-Estévez, Mercedes; López-León, Ma Dolores; Cabrero, Josefa; Camacho, Juan Pedro M

    2012-01-01

    B-chromosomes are frequently argued to be genetically inert elements, but activity for some particular genes has been reported, especially for ribosomal RNA (rRNA) genes whose expression can easily be detected at the cytological level by the visualization of their phenotypic expression, i.e., the nucleolus. The B(24) chromosome in the grasshopper Eyprepocnemis plorans frequently shows a nucleolus attached to it during meiotic prophase I. Here we show the presence of rRNA transcripts that unequivocally came from the B(24) chromosome. To detect these transcripts, we designed primers specifically anchoring at the ITS-2 region, so that the reverse primer was complementary to the B chromosome DNA sequence including a differential adenine insertion being absent in the ITS2 of A chromosomes. PCR analysis carried out on genomic DNA showed amplification in B-carrying males but not in B-lacking ones. PCR analyses performed on complementary DNA showed amplification in about half of B-carrying males. Joint cytological and molecular analysis performed on 34 B-carrying males showed a close correspondence between the presence of B-specific transcripts and of nucleoli attached to the B chromosome. In addition, the molecular analysis revealed activity of the B chromosome rDNA in 10 out of the 13 B-carrying females analysed. Our results suggest that the nucleoli attached to B chromosomes are actively formed by expression of the rDNA carried by them, and not by recruitment of nucleolar materials formed in A chromosome nucleolar organizing regions. Therefore, B-chromosome rDNA in E. plorans is functional since it is actively transcribed to form the nucleolus attached to the B chromosome. This demonstrates that some heterochromatic B chromosomes can harbour functional genes.

  2. B-chromosome ribosomal DNA is functional in the grasshopper Eyprepocnemis plorans.

    Directory of Open Access Journals (Sweden)

    Mercedes Ruiz-Estévez

    Full Text Available B-chromosomes are frequently argued to be genetically inert elements, but activity for some particular genes has been reported, especially for ribosomal RNA (rRNA genes whose expression can easily be detected at the cytological level by the visualization of their phenotypic expression, i.e., the nucleolus. The B(24 chromosome in the grasshopper Eyprepocnemis plorans frequently shows a nucleolus attached to it during meiotic prophase I. Here we show the presence of rRNA transcripts that unequivocally came from the B(24 chromosome. To detect these transcripts, we designed primers specifically anchoring at the ITS-2 region, so that the reverse primer was complementary to the B chromosome DNA sequence including a differential adenine insertion being absent in the ITS2 of A chromosomes. PCR analysis carried out on genomic DNA showed amplification in B-carrying males but not in B-lacking ones. PCR analyses performed on complementary DNA showed amplification in about half of B-carrying males. Joint cytological and molecular analysis performed on 34 B-carrying males showed a close correspondence between the presence of B-specific transcripts and of nucleoli attached to the B chromosome. In addition, the molecular analysis revealed activity of the B chromosome rDNA in 10 out of the 13 B-carrying females analysed. Our results suggest that the nucleoli attached to B chromosomes are actively formed by expression of the rDNA carried by them, and not by recruitment of nucleolar materials formed in A chromosome nucleolar organizing regions. Therefore, B-chromosome rDNA in E. plorans is functional since it is actively transcribed to form the nucleolus attached to the B chromosome. This demonstrates that some heterochromatic B chromosomes can harbour functional genes.

  3. mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling

    Directory of Open Access Journals (Sweden)

    Riikka H. Hämäläinen

    2015-06-01

    Full Text Available mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function.

  4. Histone-DNA contacts in structure/function relationships of nucleosomes as revealed by crosslinking

    Energy Technology Data Exchange (ETDEWEB)

    Usachenko, S.I. [Univ. of California, Davis, CA (United States); Bradbury, E.M. [Los Alamos National Lab., NM (United States). Life Science Div.]|[Univ. of California, Davis, CA (United States)

    1998-12-31

    The magnitude of the problem of understanding the structure/function relationships of eukaryotic chromosomes can be appreciated from the fact that the human diploid genome contains more than 2 meters of DNA packaged into 46 chromosomes, each at metaphase being several microns in length. Each chromatid of a chromosome contains a single DNA molecule several centimeters in length. In addition to the DNA, chromosomes contain an equal weight of histones and an equal weight of non-histone chromosomal proteins. These histones are the major chromosomal structural proteins. The non-histone chromosomal proteins are involved in the DNA processes of transcription and replication, in chromosome organization and in nuclear architecture. Polytene chromosomes with their bands and interbands and puffs of active genetic loci provide visual evidence for long range order as do the bands and interbands of mammalian metaphase chromosomes. The gentle removal of histones and all but the most tightly bound 2--3% of non-histone proteins from metaphase chromosomes revealed by electron microscopy a residual protein scaffold constraining a halo of DNA loops extending out from the scaffold.

  5. DNA duplex-supported artificial esterase mimicking by cooperative grafting functional groups.

    Science.gov (United States)

    Xu, Liang; Ji, Chuanshi; Bai, Yu; He, Junlin; Liu, Keliang

    2013-05-10

    The molecular structures of enzyme mimics may be modified to optimize their catalytic properties. In this study, to generate artificial enzyme mimics, Watson-Crick base paired DNA duplexes were designed as scaffolds which were assembled by nucleotides modified with specific functional groups. This process allowed various functional groups to be precisely assembled at different sites on the duplexes. By using this strategy, the 5-[2-(1H-imidazolyl-4)-(E)-ethylene]-2'-deoxythymidine (1) analog with the 5-substituted imidazolyl group was incorporated into single strands of DNA. Upon DNA duplex formation, several combinations of the imidazolyl group were formed. Using p-nitrophenyl acetate as the substrate of the catalytic reaction, we evaluated the hydrolysis capabilities of the imidazolyl assemblies. The catalytic ability was closely related to the distribution of imidazolyl groups in the DNA duplex. The most effective catalytic center was that of the duplex O5-O6 construct with three imidazolyl groups. This construct displayed bell-shaped pH-dependent and Mg(2+)-independent kinetic curves, which are typical characteristics of imidazolyl-mediated catalytic reactions. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Coumestan inhibits radical-induced oxidation of DNA: is hydroxyl a necessary functional group?

    Science.gov (United States)

    Xi, Gao-Lei; Liu, Zai-Qun

    2014-06-18

    Coumestan is a natural tetracycle with a C═C bond shared by a coumarin moiety and a benzofuran moiety. In addition to the function of the hydroxyl group on the antioxidant activity of coumestan, it is worth exploring the influence of the oxygen-abundant scaffold on the antioxidant activity as well. In this work, seven coumestans containing electron-withdrawing and electron-donating groups were synthesized to evaluate the abilities to trap 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS(•+)), 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH), and galvinoxyl radical, respectively, and to inhibit the oxidations of DNA mediated by (•)OH, Cu(2+)/glutathione (GSH), and 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), respectively. It was found that all of the coumestans used herein can quench the aforementioned radicals and can inhibit (•)OH-, Cu(2+)/GSH-, and AAPH-induced oxidations of DNA. In particular, substituent-free coumestan exhibits higher ability to quench DPPH and to inhibit AAPH-induced oxidation of DNA than Trolox. In addition, nonsubstituted coumestan shows a similar ability to inhibit (•)OH- and Cu(2+)/GSH-induced oxidations of DNA relative to that of Trolox. The antioxidant effectiveness of the coumestan can be attributed to the lactone in the coumarin moiety and, therefore, a hydroxyl group may not be a necessary functional group for coumestan to be an antioxidant.

  7. A Microneedle Functionalized with Polyethyleneimine and Nanotubes for Highly Sensitive, Label-Free Quantification of DNA

    Science.gov (United States)

    Saadat-Moghaddam, Darius; Kim, Jong-Hoon

    2017-01-01

    The accurate measure of DNA concentration is necessary for many DNA-based biological applications. However, the current methods are limited in terms of sensitivity, reproducibility, human error, and contamination. Here, we present a microneedle functionalized with polyethyleneimine (PEI) and single-walled carbon nanotubes (SWCNTs) for the highly sensitive quantification of DNA. The microneedle was fabricated using ultraviolet (UV) lithography and anisotropic etching, and then functionalized with PEI and SWCNTs through a dip coating process. The electrical characteristics of the microneedle change with the accumulation of DNA on the surface. Current-voltage measurements in deionized water were conducted to study these changes in the electrical properties of the sensor. The sensitivity test found the signal to be discernable from the noise level down to 100 attomolar (aM), demonstrating higher sensitivity than currently available UV fluorescence and UV absorbance based methods. A microneedle without any surface modification only had a 100 femtomolar (fM) sensitivity. All measurement results were consistent with fluorescence microscopy. PMID:28812987

  8. Naturally occurring mitochondrial DNA haplotypes exhibit metabolic differences: insight into functional properties of mitochondria.

    Science.gov (United States)

    Pichaud, Nicolas; Ballard, J William O; Tanguay, Robert M; Blier, Pierre U

    2012-10-01

    Linking the mitochondrial genotype and the organismal phenotype is of paramount importance in evolution of mitochondria. In this study, we determined the differences in catalytic properties of mitochondria dictated by divergences in the siII and siIII haplogroups of Drosophila simulans using introgressions of siII mtDNA type into the siIII nuclear background. We used a novel in situ method (permeabilized fibers) that allowed us to accurately measure the consumption of oxygen by mitochondria in constructed siII-introgressed flies and in siIII-control flies. Our results showed that the catalytic capacity of the electron transport system is not impaired by introgressions, suggesting that the functional properties of mitochondria are tightly related to the mtDNA haplogroup and not to the nuclear DNA or to the mito-nuclear interactions. This is the first study, to our knowledge, that demonstrates a naturally occurring haplogroup can confer specific functional differences in aspects of mitochondrial metabolism. This study illustrates the importance of mtDNA changes on organelle evolution and highlights the potential bioenergetic and metabolic impacts that divergent mitochondrial haplogroups may have upon a wide variety of species including humans. © 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.

  9. A DNA aptamer recognising a malaria protein biomarker can function as part of a DNA origami assembly.

    Science.gov (United States)

    Godonoga, Maia; Lin, Ting-Yu; Oshima, Azusa; Sumitomo, Koji; Tang, Marco S L; Cheung, Yee-Wai; Kinghorn, Andrew B; Dirkzwager, Roderick M; Zhou, Cunshan; Kuzuya, Akinori; Tanner, Julian A; Heddle, Jonathan G

    2016-02-19

    DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology.

  10. Neutralizing the function of a β-globin-associated cis-regulatory DNA element using an artificial zinc finger DNA-binding domain.

    Science.gov (United States)

    Barrow, Joeva J; Masannat, Jude; Bungert, Jörg

    2012-10-30

    Gene expression is primarily regulated by cis-regulatory DNA elements and trans-interacting proteins. Transcription factors bind in a DNA sequence-specific manner and recruit activities that modulate the association and activity of transcription complexes at specific genes. Often, transcription factors belong to families of related proteins that interact with similar DNA sequences. Furthermore, genes are regulated by multiple, sometimes redundant, cis-regulatory elements. Thus, the analysis of the role of a specific DNA regulatory sequence and the interacting proteins in the context of intact cells is challenging. In this study, we designed and functionally characterized an artificial DNA-binding domain that neutralizes the function of a cis-regulatory DNA element associated with adult β-globin gene expression. The zinc finger DNA-binding domain (ZF-DBD), comprising six ZFs, interacted specifically with a CACCC site located 90 bp upstream of the transcription start site (-90 β-ZF-DBD), which is normally occupied by KLF1, a major regulator of adult β-globin gene expression. Stable expression of the -90 β-ZF-DBD in mouse erythroleukemia cells reduced the binding of KLF1 with the β-globin gene, but not with locus control region element HS2, and led to reduced transcription. Transient transgenic embryos expressing the -90 β-ZF-DBD developed normally but revealed reduced expression of the adult β-globin gene. These results demonstrate that artificial DNA-binding proteins lacking effector domains are useful tools for studying and modulating the function of cis-regulatory DNA elements.

  11. Optimal functional levels of activation-induced deaminase specifically require the Hsp40 DnaJa1

    Science.gov (United States)

    Orthwein, Alexandre; Zahn, Astrid; Methot, Stephen P; Godin, David; Conticello, Silvestro G; Terada, Kazutoyo; Di Noia, Javier M

    2012-01-01

    The enzyme activation-induced deaminase (AID) deaminates deoxycytidine at the immunoglobulin genes, thereby initiating antibody affinity maturation and isotype class switching during immune responses. In contrast, off-target DNA damage caused by AID is oncogenic. Central to balancing immunity and cancer is AID regulation, including the mechanisms determining AID protein levels. We describe a specific functional interaction between AID and the Hsp40 DnaJa1, which provides insight into the function of both proteins. Although both major cytoplasmic type I Hsp40s, DnaJa1 and DnaJa2, are induced upon B-cell activation and interact with AID in vitro, only DnaJa1 overexpression increases AID levels and biological activity in cell lines. Conversely, DnaJa1, but not DnaJa2, depletion reduces AID levels, stability and isotype switching. In vivo, DnaJa1-deficient mice display compromised response to immunization, AID protein and isotype switching levels being reduced by half. Moreover, DnaJa1 farnesylation is required to maintain, and farnesyltransferase inhibition reduces, AID protein levels in B cells. Thus, DnaJa1 is a limiting factor that plays a non-redundant role in the functional stabilization of AID. PMID:22085931

  12. Maleimide-Functionalized PEI600 Grafted Polyurethane: Synthesis, Nano-Complex Formation with DNA and Thiol-Conjugation of the Complexes for Dual DNA Transfection

    Directory of Open Access Journals (Sweden)

    Wei-Chih Hung

    2015-10-01

    Full Text Available A polyurethane (PU grafted with small molecular weight polyethylenimine (PEI600 was synthesized. This PU-PEI600 can assemble DNA via electrostatic interactions into nano-sized polymer/DNA complexes. The complexes exhibited great transfection efficiency in delivering DNA along with a reduced cell toxicity comparing to commercial PEI25k (Mw ~25,000. In order to establish a system for concurrently delivering two different DNA or RNA molecules for cell reprogramming (e.g., induced pluripotent stem cells or the necessity of multi-expression (e.g., double knock down, the PU-PEI600 was further functionalized with maleimide molecules. The novel PU-PEI600-maleimide would still effectively interact with assigned DNA and different functions of PU-PEI600-maleimide/DNA complexes were self-conjugated in presence of a dithiol molecule (1,6-hexanedithiol. In this study, two reporter genes (pEGFP-C2 and pLanRFP-N were used and evidence of green/red fluorescence co-expression in cells was observed. This article brings a new concept and a practical method for a plurality of different DNA molecules that are more efficient to be concurrently delivered and co-expressed. This method is very helpful in studying cellular multi-regulation or in the treatment of disease with multiple gene defects in vivo.

  13. Modeling structure-function relationships in synthetic DNA sequences using attribute grammars.

    Directory of Open Access Journals (Sweden)

    Yizhi Cai

    2009-10-01

    Full Text Available Recognizing that certain biological functions can be associated with specific DNA sequences has led various fields of biology to adopt the notion of the genetic part. This concept provides a finer level of granularity than the traditional notion of the gene. However, a method of formally relating how a set of parts relates to a function has not yet emerged. Synthetic biology both demands such a formalism and provides an ideal setting for testing hypotheses about relationships between DNA sequences and phenotypes beyond the gene-centric methods used in genetics. Attribute grammars are used in computer science to translate the text of a program source code into the computational operations it represents. By associating attributes with parts, modifying the value of these attributes using rules that describe the structure of DNA sequences, and using a multi-pass compilation process, it is possible to translate DNA sequences into molecular interaction network models. These capabilities are illustrated by simple example grammars expressing how gene expression rates are dependent upon single or multiple parts. The translation process is validated by systematically generating, translating, and simulating the phenotype of all the sequences in the design space generated by a small library of genetic parts. Attribute grammars represent a flexible framework connecting parts with models of biological function. They will be instrumental for building mathematical models of libraries of genetic constructs synthesized to characterize the function of genetic parts. This formalism is also expected to provide a solid foundation for the development of computer assisted design applications for synthetic biology.

  14. Rapid DNA Synthesis During EarlyDrosophilaEmbryogenesis Is Sensitive to Maternal Humpty Dumpty Protein Function.

    Science.gov (United States)

    Lesly, Shera; Bandura, Jennifer L; Calvi, Brian R

    2017-11-01

    Problems with DNA replication cause cancer and developmental malformations. It is not fully understood how DNA replication is coordinated with development and perturbed in disease. We had previously identified the Drosophila gene humpty dumpty ( hd ), and showed that null alleles cause incomplete DNA replication, tissue undergrowth, and lethality. Animals homozygous for the missense allele, hd 272-9 , were viable, but adult females had impaired amplification of eggshell protein genes in the ovary, resulting in the maternal effects of thin eggshells and embryonic lethality. Here, we show that expression of an hd transgene in somatic cells of the ovary rescues amplification and eggshell synthesis but not embryo viability. The germline of these mothers remain mutant for the hd 272-9 allele, resulting in reduced maternal Hd protein and embryonic arrest during mitosis of the first few S/M nuclear cleavage cycles with chromosome instability and chromosome bridges. Epistasis analysis of hd with the rereplication mutation plutonium indicates that the chromosome bridges of hd embryos are the result of a failed attempt to segregate incompletely replicated sister chromatids. This study reveals that maternally encoded Humpty dumpty protein is essential for DNA replication and genome integrity during the little-understood embryonic S/M cycles. Moreover, the two hd 272-9 maternal-effect phenotypes suggest that ovarian gene amplification and embryonic cleavage are two time periods in development that are particularly sensitive to mild deficits in DNA replication function. This last observation has broader relevance for interpreting why mild mutations in the human ortholog of humpty dumpty and other DNA replication genes cause tissue-specific malformations of microcephalic dwarfisms. Copyright © 2017 by the Genetics Society of America.

  15. Essential Function of Dicer in Resolving DNA Damage in the Rapidly Dividing Cells of the Developing and Malignant Cerebellum

    Directory of Open Access Journals (Sweden)

    Vijay Swahari

    2016-01-01

    Full Text Available Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells.

  16. Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance.

    Science.gov (United States)

    Hishiki, Asami; Hara, Kodai; Ikegaya, Yuzu; Yokoyama, Hideshi; Shimizu, Toshiyuki; Sato, Mamoru; Hashimoto, Hiroshi

    2015-05-22

    HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3'-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3'-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Proper functioning of the GINS complex is important for the fidelity of DNA replication in yeast.

    Science.gov (United States)

    Grabowska, Ewa; Wronska, Urszula; Denkiewicz, Milena; Jaszczur, Malgorzata; Respondek, Aleksandra; Alabrudzinska, Malgorzata; Suski, Catherine; Makiela-Dzbenska, Karolina; Jonczyk, Piotr; Fijalkowska, Iwona J

    2014-05-01

    The role of replicative DNA polymerases in ensuring genome stability is intensively studied, but the role of other components of the replisome is still not fully understood. One of such component is the GINS complex (comprising the Psf1, Psf2, Psf3 and Sld5 subunits), which participates in both initiation and elongation of DNA replication. Until now, the understanding of the physiological role of GINS mostly originated from biochemical studies. In this article, we present genetic evidence for an essential role of GINS in the maintenance of replication fidelity in Saccharomyces cerevisiae. In our studies we employed the psf1-1 allele (Takayama et al., 2003) and a novel psf1-100 allele isolated in our laboratory. Analysis of the levels and specificity of mutations in the psf1 strains indicates that the destabilization of the GINS complex or its impaired interaction with DNA polymerase epsilon increases the level of spontaneous mutagenesis and the participation of the error-prone DNA polymerase zeta. Additionally, a synergistic mutator effect was found for the defects in Psf1p and in the proofreading activity of Pol epsilon, suggesting that proper functioning of GINS is crucial for facilitating error-free processing of terminal mismatches created by Pol epsilon. © 2014 John Wiley & Sons Ltd.

  18. Molecular Dynamics Studies of Self-Assembling Biomolecules and DNA-functionalized Gold Nanoparticles

    Science.gov (United States)

    Cho, Vince Y.

    This thesis is organized as following. In Chapter 2, we use fully atomistic MD simulations to study the conformation of DNA molecules that link gold nanoparticles to form nanoparticle superlattice crystals. In Chapter 3, we study the self-assembly of peptide amphiphiles (PAs) into a cylindrical micelle fiber by using CGMD simulations. Compared to fully atomistic MD simulations, CGMD simulations prove to be computationally cost-efficient and reasonably accurate for exploring self-assembly, and are used in all subsequent chapters. In Chapter 4, we apply CGMD methods to study the self-assembly of small molecule-DNA hybrid (SMDH) building blocks into well-defined cage-like dimers, and reveal the role of kinetics and thermodynamics in this process. In Chapter 5, we extend the CGMD model for this system and find that the assembly of SMDHs can be fine-tuned by changing parameters. In Chapter 6, we explore superlattice crystal structures of DNA-functionalized gold nanoparticles (DNA-AuNP) with the CGMD model and compare the hybridization.

  19. Dual functions of ASCIZ in the DNA base damage response and pulmonary organogenesis.

    Directory of Open Access Journals (Sweden)

    Sabine Jurado

    2010-10-01

    Full Text Available Zn²(+-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn²(+-finger protein (ASCIZ; also known as ATMIN and ZNF822 was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis.

  20. Dual functions of ASCIZ in the DNA base damage response and pulmonary organogenesis.

    Science.gov (United States)

    Jurado, Sabine; Smyth, Ian; van Denderen, Bryce; Tenis, Nora; Hammet, Andrew; Hewitt, Kimberly; Ng, Jane-Lee; McNees, Carolyn J; Kozlov, Sergei V; Oka, Hayato; Kobayashi, Masahiko; Conlan, Lindus A; Cole, Timothy J; Yamamoto, Ken-Ichi; Taniguchi, Yoshihito; Takeda, Shunichi; Lavin, Martin F; Heierhorst, Jörg

    2010-10-21

    Zn²(+)-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn²(+)-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis.

  1. Agrobacterium rhizogenes pRi8196 T-DNA: mapping and DNA sequence of functions involved in mannopine synthesis and hairy root differentiation.

    Science.gov (United States)

    Hansen, G; Larribe, M; Vaubert, D; Tempé, J; Biermann, B J; Montoya, A L; Chilton, M D; Brevet, J

    1991-01-01

    This paper presents the map and DNA sequence analysis of pRi8196 transferred DNA (T-DNA) genes encoding root-inducing and mannopine synthesis functions. A canonical 24-base-pair border repeat as well as two "pseudoborders" are present at the functional right T-DNA border. To the left of this border are homologs of the mas1' and mas2' genes of TR pRiA4. Next to these are five open reading frames (ORFs) homologous to ORFs 10-14 of TL of pRiA4. ORFs 10-12 (rolA, rolB, and rolC) are less related to their pRiA4 homologs than are the other large ORFs analyzed here. In contrast to T-DNA genes of pRiA4, pRi8196 T-DNA ORFs 11 and 12 (rolB and rolC) are sufficient to induce hairy roots on carrot disks. Images PMID:1909028

  2. Production of ROS and its effects on mitochondrial and nuclear DNA, human spermatozoa, and sperm function

    Directory of Open Access Journals (Sweden)

    Hardi Darmawan

    2007-06-01

    Full Text Available Over the past few decades many researchers studying the causes of male infertility have recently focused on the role played by reactive oxygen species (ROS – highly reactive oxidizing agents belonging to the class of free radicals. If ROS levels rise, oxidative stress (OS occurs, which results in oxygen and oxygen derived oxidants, and in turn increases the rates of cellular damage. In human, ROS are produced by a variety of semen components, and antioxidants in the seminal fluid keep their level balance. Small amounts of ROS help spermatozoa acquire their necessary fertilizing capabilities. Many researches showed that ROS attack DNA integrity in the sperm nucleus by causing base modification, DNA strand breaks, and chromatin cross linking. The DNA damage induced excessive levels of ROS and might accelerate the process of germ cell apoptosis leading to a decline in sperm counts associated with male infertility. This paper will review the molecular (cellular origins of ROS in human semen, how ROS damage sperm nuclear DNA, and how such DNA damage contributes to male infertility. Increased ROS production by spermatozoa is associated with a decreased mitochondrial membrane potential (MMP, which is an important indicator of functional integrity of the spermatozoa. Germ cell apoptosis is essential for normal spermatogenesis and its dysregulation may lead to male infertility. Thus, understanding the causes and mechanisms of germ cell apoptosis is of major importance in preventing male reproductive problems. Levels of apoptosis in mature spermatozoa that were significantly correlated with levels of seminal ROS determined by chemiluminescence assay indicate the linkage between ROS and male fertility problems. (Med J Indones 2007; 16:127-33 Keywords: Apoptosis, infertility, free radicals

  3. DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Huang, Dongqing; Piening, Brian D; Kennedy, Jacob J; Lin, Chenwei; Jones-Weinert, Corey W; Yan, Ping; Paulovich, Amanda G

    2016-05-01

    In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance. Copyright © 2016 by the Genetics Society of America.

  4. Functionalized Nanostructures: Redox-Active Porphyrin Anchors for Supramolecular DNA Assemblies

    KAUST Repository

    Börjesson, Karl

    2010-09-28

    We have synthesized and studied a supramolecular system comprising a 39-mer DNA with porphyrin-modified thymidine nucleosides anchored to the surface of large unilamellar vesicles (liposomes). Liposome porphyrin binding characteristics, such as orientation, strength, homogeneity, and binding site size, was determined, suggesting that the porphyrin is well suited as a photophysical and redox-active lipid anchor, in comparison to the inert cholesterol anchor commonly used today. Furthermore, the binding characteristics and hybridization capabilities were studied as a function of anchor size and number of anchoring points, properties that are of importance for our future plans to use the addressability of these redox-active nodes in larger DNA-based nanoconstructs. Electron transfer from photoexcited porphyrin to a lipophilic benzoquinone residing in the lipid membrane was characterized by steady-state and time-resolved fluorescence and verified by femtosecond transient absorption. © 2010 American Chemical Society.

  5. In vitro transcription and translation inhibition via DNA functionalized gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Conde, J; Baptista, P V [Centro de Investigacao em Genetica Molecular Humana (CIGMH), Departamento de Ciencias da Vida, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); De la Fuente, J M, E-mail: pmvb@fct.unl.pt [Instituto de Nanociencia de Aragon, Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza (Spain)

    2010-12-17

    The use of gold nanoparticles (AuNPs) has been gaining momentum as vectors for gene silencing strategies, combining the AuNPs' ease of functionalization with DNA and/or siRNA, high loading capacity and fast uptake by target cells. Here, we used AuNP functionalized with thiolated oligonucleotides to specifically inhibit transcription in vitro, demonstrating the synergetic effect between AuNPs and a specific antisense sequence that blocks the T7 promoter region. Also, AuNPs efficiently protect the antisense oligonucleotide against nuclease degradation, which can thus retain its inhibitory potential. In addition, we demonstrate that AuNPs functionalized with a thiolated oligonucleotide complementary to the ribosome binding site and the start codon, effectively shut down in vitro translation. Together, these two approaches can provide for a simple yet robust experimental set up to test for efficient gene silencing of AuNP-DNA conjugates. What is more, these results show that appropriate functionalization of AuNPs can be used as a dual targeting approach to an enhanced control of gene expression-inhibition of both transcription and translation.

  6. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

    2009-05-11

    The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

  7. A chemiluminescence biosensor based on the adsorption recognition function between Fe3O4@SiO2@GO polymers and DNA for ultrasensitive detection of DNA

    Science.gov (United States)

    Sun, Yuanling; Li, Jianbo; Wang, Yanhui; Ding, Chaofan; Lin, Yanna; Sun, Weiyan; Luo, Chuannan

    2017-05-01

    In this work, a chemiluminescence (CL) biosensor was prepared for ultrasensitive determination of deoxyribonucleic acid (DNA) based on the adsorption recognition function between core-shell Fe3O4@SiO2 - graphene oxide (Fe3O4@SiO2@GO) polymers and DNA. The Fe3O4@SiO2@GO polymers were composed by GO and magnetite nanoparticles. And the core-shell polymers were confirmed by Scanning Electron Microscope (SEM), X-Ray Powder Diffraction (XRD) and Fourier Transform Infrared (FTIR). Then Fe3O4@SiO2@GO was modified by DNA. Based on the principle of complementary base, Fe3O4@SiO2@GO-DNA was introduced to the CL system and the selectivity, sensitivity of DNA detection was significantly improved. The adsorption properties of Fe3O4@SiO2@GO to DNA were researched through the adsorption equilibrium, adsorption kinetic and thermodynamics. Under optimized CL conditions, DNA could be assayed with the linear concentration range of 5.0 × 10- 12-2.5 × 10- 11 mol/L. The detection limit was 1.7 × 10- 12 mol/L (3δ) and the relative standard deviation (RSD) was 3.1%. The biosensor was finally used for the determination of DNA in laboratory samples and recoveries ranged from 99% to 103%. The satisfactory results revealed the potential application of Fe3O4@SiO2@GO-DNA-CL biosensor in the diagnosis and the treatment of human genetic diseases.

  8. Doping Level of Boron-Doped Diamond Electrodes Controls the Grafting Density of Functional Groups for DNA Assays.

    Science.gov (United States)

    Švorc, Ĺubomír; Jambrec, Daliborka; Vojs, Marian; Barwe, Stefan; Clausmeyer, Jan; Michniak, Pavol; Marton, Marián; Schuhmann, Wolfgang

    2015-09-02

    The impact of different doping levels of boron-doped diamond on the surface functionalization was investigated by means of electrochemical reduction of aryldiazonium salts. The grafting efficiency of 4-nitrophenyl groups increased with the boron levels (B/C ratio from 0 to 20,000 ppm). Controlled grafting of nitrophenyldiazonium was used to adjust the amount of immobilized single-stranded DNA strands at the surface and further on the hybridization yield in dependence on the boron doping level. The grafted nitro functions were electrochemically reduced to the amine moieties. Subsequent functionalization with a succinic acid introduced carboxyl groups for subsequent binding of an amino-terminated DNA probe. DNA hybridization significantly depends on the probe density which is in turn dependent on the boron doping level. The proposed approach opens new insights for the design and control of doped diamond surface functionalization for the construction of DNA hybridization assays.

  9. DNA-PKcs-interacting protein KIP binding to TRF2 is required for the maintenance of functional telomeres.

    Science.gov (United States)

    Khadka, Prabhat; Lee, Ji Hoon; Baek, Seung Han; Oh, Sue Young; Chung, In Kwon

    2014-10-01

    Human telomeres associate with shelterin, a six-protein complex that protects chromosome ends from being recognized as sites of DNA damage. The shelterin subunit TRF2 (telomeric repeat-binding factor 2) protects telomeres by facilitating their organization into the protective capping structure. We have reported previously that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit)-interacting protein KIP associates with telomerase through an interaction with hTERT (human telomerase reverse transcriptase). In the present study, we identify KIP as a novel interacting partner of TRF2. KIP is able to interact with both TRF2 and DNA-PKcs at telomeres. Because KIP is required for the association between TRF2 and DNA-PKcs, the interplay of these three proteins may provide a mechanism for the recruitment of DNA-PKcs to telomeres. We also show that KIP binding to TRF2 enhances the telomere-binding activity of TRF2, suggesting that KIP acts as a positive regulator of TRF2 function. Furthermore, depletion of KIP induces DNA-damage response foci at telomeres, thereby leading to induction of growth arrest, cellular senescence and altered cell cycle distribution. Collectively, our findings suggest that KIP, in addition to its association with catalytically active telomerase, plays important roles in the maintenance of functional telomeres and the regulation of telomere-associated DNA-damage response. Thus KIP represents a new pathway for modulating telomerase and telomere function in cancer.

  10. Mutant analysis of Cdt1's function in suppressing nascent strand elongation during DNA replication in Xenopus egg extracts.

    Science.gov (United States)

    Nakazaki, Yuta; Tsuyama, Takashi; Azuma, Yutaro; Takahashi, Mikiko; Tada, Shusuke

    2017-09-02

    The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255-620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255-289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Genetic and functional diversity of ubiquitous DNA viruses in selected Chinese agricultural soils

    Science.gov (United States)

    Han, Li-Li; Yu, Dan-Ting; Zhang, Li-Mei; Shen, Ju-Pei; He, Ji-Zheng

    2017-03-01

    Viral community structures in complex agricultural soils are largely unknown. Electron microscopy and viromic analyses were conducted on six typical Chinese agricultural soil samples. Tailed bacteriophages, spherical and filamentous viral particles were identified by the morphological analysis. Based on the metagenomic analysis, single-stranded DNA viruses represented the largest viral component in most of the soil habitats, while the double-stranded DNA viruses belonging to the Caudovirales order were predominanted in Jiangxi-maize soils. The majority of functional genes belonged to the subsystem “phages, prophages, transposable elements, and plasmids”. Non-metric multidimensional analysis of viral community showed that the environment medium type was the most important driving factor for the viral community structure. For the major viral groups detected in all samples (Microviridae and Caudovirales), the two groups gathered viruses from different sites and similar genetic composition, indicating that viral diversity was high on a local point but relatively limited on a global scale. This is a novel report of viral diversity in Chinese agricultural soils, and the abundance, taxonomic, and functional diversity of viruses that were observed in different types of soils will aid future soil virome studies and enhance our understanding of the ecological functions of soil viruses.

  12. SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair

    DEFF Research Database (Denmark)

    Kanu, N.; Grönroos, E.; Martinez, P.

    2015-01-01

    proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal......-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication......, suppression of replication stress and the coordination of DNA repair....

  13. An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.

    Science.gov (United States)

    MacConnell, Andrew B; Price, Alexander K; Paegel, Brian M

    2017-03-13

    DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.

  14. Functional importance of the DNA binding activity of Candida albicans Czf1p.

    Directory of Open Access Journals (Sweden)

    Ivana Petrovska

    Full Text Available The human opportunistic pathogen Candida albicans undergoes a reversible morphological transition between the yeast and hyphal states in response to a variety of signals. One such environmental trigger is growth within a semisolid matrix such as agar medium. This growth condition is of interest because it may mimic the growth of C. albicans in contact with host tissue during infection. During growth within a semisolid matrix, hyphal growth is positively regulated by the transcriptional regulator Czf1p and negatively by a second key transcriptional regulator, Efg1p. Genetic studies indicate that Czf1p, a member of the zinc-cluster family of transcriptional regulators, exerts its function by opposing the inhibitory influence of Efg1p on matrix-induced filamentous growth. We examined the importance of the two known activities of Czf1p, DNA-binding and interaction with Efg1p. We found that the two activities were separable by mutation allowing us to demonstrate that the DNA-binding activity of Czf1p was essential for its role as a positive regulator of morphogenesis. Surprisingly, however, interactions with Efg1p appeared to be largely dispensable. Our studies provide the first evidence of a key role for the DNA-binding activity of Czf1p in the morphological yeast-to-hyphal transition triggered by matrix-embedded growth.

  15. Preparation of functional spherical polysilsesquioxane/gold nanoparticle composites and their applications in DNA assay

    Science.gov (United States)

    Jung, Jung A.; Kim, Young Baek; Kim, Young A.; Ryu, Seung Bum; Kim, Veronica

    2011-06-01

    Functional spherical solid and hollow particles of polysilsesquioxanes (PSQs) containing amine, thiol, and vinyl groups were prepared by polymerizing organotrialkoxysilanes (OTASs) containing corresponding chemical groups. Fluorescent PSQ particles were prepared by physically entrapping Rhodamine 6G, Coumarin 7, and Fluoresceine sodium salts. The intensity of fluorescent light increased initially with increasing amount of entrapped fluorophores and then leveled off or decreased slightly after reaching a maximum value. PSQ particles containing gold nanoparticles (GNPs), both inside and on the surface, were prepared by the in situ reduction of gold ions by the PSQ particles. When the reduction reaction was carried out for extended periods of time, the GNP that had formed inside the poly(3-mercaptopropyl)silsesquioxane (PMPSQ) and polyvinylsilsesequioxane (PVSQ) particles underwent interesting morphological changes. PSQ particles containing amine and thiol groups fixed the GNPs on the surface, which could be utilized further in binding amine-modified oligo-DNA strands. The aggregation of PSQ/GNP particles combined with complementary oligo-DNA strands was examined to demonstrate that these particles could be applied to DNA assays and isolation. The particles were characterized by scanning electron microscopy, transmission electron microscopy, solid state nuclear magnetic resonance spectroscopy, ultraviolet/visible spectroscopy, and fluorescence microscopy.

  16. De-novo protein function prediction using DNA binding and RNA binding proteins as a test case.

    Science.gov (United States)

    Peled, Sapir; Leiderman, Olga; Charar, Rotem; Efroni, Gilat; Shav-Tal, Yaron; Ofran, Yanay

    2016-11-21

    Of the currently identified protein sequences, 99.6% have never been observed in the laboratory as proteins and their molecular function has not been established experimentally. Predicting the function of such proteins relies mostly on annotated homologs. However, this has resulted in some erroneous annotations, and many proteins have no annotated homologs. Here we propose a de-novo function prediction approach based on identifying biophysical features that underlie function. Using our approach, we discover DNA and RNA binding proteins that cannot be identified based on homology and validate these predictions experimentally. For example, FGF14, which belongs to a family of secreted growth factors was predicted to bind DNA. We verify this experimentally and also show that FGF14 is localized to the nucleus. Mutating the predicted binding site on FGF14 abrogated DNA binding. These results demonstrate the feasibility of automated de-novo function prediction based on identifying function-related biophysical features.

  17. Metabolism and function of hepatitis B virus cccDNA: Implications for the development of cccDNA-targeting antiviral therapeutics.

    Science.gov (United States)

    Guo, Ju-Tao; Guo, Haitao

    2015-10-01

    Persistent hepatitis B virus (HBV) infection relies on the stable maintenance and proper functioning of a nuclear episomal form of the viral genome called covalently closed circular (ccc) DNA. One of the major reasons for the failure of currently available antiviral therapeutics to achieve a cure of chronic HBV infection is their inability to eradicate or inactivate cccDNA. In this review article, we summarize our current understanding of cccDNA metabolism in hepatocytes and the modulation of cccDNA by host pathophysiological and immunological cues. Perspectives on the future investigation of cccDNA biology, as well as strategies and progress in therapeutic elimination and/or transcriptional silencing of cccDNA through rational design and phenotypic screenings, are also discussed. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B." Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Variable DNA Methylation Is Associated with Chronic Obstructive Pulmonary Disease and Lung Function

    Science.gov (United States)

    Qiu, Weiliang; Baccarelli, Andrea; Carey, Vincent J.; Boutaoui, Nadia; Bacherman, Helene; Klanderman, Barbara; Rennard, Stephen; Agusti, Alvar; Anderson, Wayne; Lomas, David A.

    2012-01-01

    Rationale: Chronic obstructive pulmonary disease (COPD) is associated with local (lung) and systemic (blood) inflammation and manifestations. DNA methylation is an important regulator of gene transcription, and global and specific gene methylation marks may vary with cigarette smoke exposure. Objectives: To perform a comprehensive assessment of methylation marks in DNA from subjects well phenotyped for nonneoplastic lung disease. Methods: We conducted array-based methylation screens, using a test-replication approach, in two family-based cohorts (n = 1,085 and 369 subjects). Measurements and Main Results: We observed 349 CpG sites significantly associated with the presence and severity of COPD in both cohorts. Seventy percent of the associated CpG sites were outside of CpG islands, with the majority of CpG sites relatively hypomethylated. Gene ontology analysis based on these 349 CpGs (330 genes) suggested the involvement of a number of genes responsible for immune and inflammatory system pathways, responses to stress and external stimuli, as well as wound healing and coagulation cascades. Interestingly, our observations include significant, replicable associations between SERPINA1 hypomethylation and COPD and lower average lung function phenotypes (combined P values: COPD, 1.5 × 10−23; FEV1/FVC, 1.5 × 10−35; FEV1, 2.2 × 10−40). Conclusions: Genetic and epigenetic pathways may both contribute to COPD. Many of the top associations between COPD and DNA methylation occur in biologically plausible pathways. This large-scale analysis suggests that DNA methylation may be a biomarker of COPD and may highlight new pathways of COPD pathogenesis. PMID:22161163

  19. Natural DNA transformation is functional in Lactococcus lactis ssp. cremoris KW2.

    Science.gov (United States)

    David, Blandine; Radziejwoski, Amandine; Toussaint, Frédéric; Fontaine, Laetitia; Henry de Frahan, Marie; Patout, Cédric; van Dillen, Sabine; Boyaval, Patrick; Horvath, Philippe; Fremaux, Christophe; Hols, Pascal

    2017-06-16

    Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis ssp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a low amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCELactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis ssp. cremoris KW2 by the overproduction of the master competence

  20. DnaA and the timing of chromosome replication in Es-cherichia coli as a function of growth rate

    Directory of Open Access Journals (Sweden)

    Grant Matthew AA

    2011-12-01

    Full Text Available Abstract Background In Escherichia coli, overlapping rounds of DNA replication allow the bacteria to double in faster times than the time required to copy the genome. The precise timing of initiation of DNA replication is determined by a regulatory circuit that depends on the binding of a critical number of ATP-bound DnaA proteins at the origin of replication, resulting in the melting of the DNA and the assembly of the replication complex. The synthesis of DnaA in the cell is controlled by a growth-rate dependent, negatively autoregulated gene found near the origin of replication. Both the regulatory and initiation activity of DnaA depend on its nucleotide bound state and its availability. Results In order to investigate the contributions of the different regulatory processes to the timing of initiation of DNA replication at varying growth rates, we formulate a minimal quantitative model of the initiator circuit that includes the key ingredients known to regulate the activity of the DnaA protein. This model describes the average-cell oscillations in DnaA-ATP/DNA during the cell cycle, for varying growth rates. We evaluate the conditions under which this ratio attains the same threshold value at the time of initiation, independently of the growth rate. Conclusions We find that a quantitative description of replication initiation by DnaA must rely on the dependency of the basic parameters on growth rate, in order to account for the timing of initiation of DNA replication at different cell doubling times. We isolate two main possible scenarios for this, depending on the roles of DnaA autoregulation and DnaA ATP-hydrolysis regulatory process. One possibility is that the basal rate of regulatory inactivation by ATP hydrolysis must vary with growth rate. Alternatively, some parameters defining promoter activity need to be a function of the growth rate. In either case, the basal rate of gene expression needs to increase with the growth rate, in

  1. Enzymatic Synthesis, Amplification, and Application of DNA with a Functionalized Backbone.

    Science.gov (United States)

    Chen, Tingjian; Romesberg, Floyd E

    2017-11-06

    The ability to amplify DNA along with its unprecedented sequence control has led to its use for different applications, but all are limited by the properties available to natural nucleotides. We previously reported the evolution of polymerase SFM4-3, which better tolerates 2'-modified substrates. To explore the utility of SFM4-3, we now report the characterization of its recognition of substrates with 2'-azido, 2'-chloro, 2'-amino, or arabinose sugars. We find that SFM4-3 can efficiently synthesize polymers composed of these nucleotides, and most interestingly, that SFM4-3 can also PCR amplify these modified oligonucleotides. When combined with post-amplification modification, the latter allows for the exponential amplification of polymers that may be functionalized with desired moieties arrayed in a controlled fashion, the utility of which we demonstrate with extensive small molecule functionalization and the production and initial characterization of a novel DNA hydrogel. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets

    Science.gov (United States)

    Xiang, Yu; Lu, Yi

    2011-09-01

    Portable, low-cost and quantitative detection of a broad range of targets at home and in the field has the potential to revolutionize medical diagnostics and environmental monitoring. Despite many years of research, very few such devices are commercially available. Taking advantage of the wide availability and low cost of the pocket-sized personal glucose meter—used worldwide by diabetes sufferers—we demonstrate a method to use such meters to quantify non-glucose targets, ranging from a recreational drug (cocaine, 3.4 µM detection limit) to an important biological cofactor (adenosine, 18 µM detection limit), to a disease marker (interferon-gamma of tuberculosis, 2.6 nM detection limit) and a toxic metal ion (uranium, 9.1 nM detection limit). The method is based on the target-induced release of invertase from a functional-DNA-invertase conjugate. The released invertase converts sucrose into glucose, which is detectable using the meter. The approach should be easily applicable to the detection of many other targets through the use of suitable functional-DNA partners (aptamers, DNAzymes or aptazymes).

  3. Epigenome-wide association studies identify DNA methylation associated with kidney function.

    Science.gov (United States)

    Chu, Audrey Y; Tin, Adrienne; Schlosser, Pascal; Ko, Yi-An; Qiu, Chengxiang; Yao, Chen; Joehanes, Roby; Grams, Morgan E; Liang, Liming; Gluck, Caroline A; Liu, Chunyu; Coresh, Josef; Hwang, Shih-Jen; Levy, Daniel; Boerwinkle, Eric; Pankow, James S; Yang, Qiong; Fornage, Myriam; Fox, Caroline S; Susztak, Katalin; Köttgen, Anna

    2017-11-03

    Chronic kidney disease (CKD) is defined by reduced estimated glomerular filtration rate (eGFR). Previous genetic studies have implicated regulatory mechanisms contributing to CKD. Here we present epigenome-wide association studies of eGFR and CKD using whole-blood DNA methylation of 2264 ARIC Study and 2595 Framingham Heart Study participants to identify epigenetic signatures of kidney function. Of 19 CpG sites significantly associated (P < 1e-07) with eGFR/CKD and replicated, five also associate with renal fibrosis in biopsies from CKD patients and show concordant DNA methylation changes in kidney cortex. Lead CpGs at PTPN6/PHB2, ANKRD11, and TNRC18 map to active enhancers in kidney cortex. At PTPN6/PHB2 cg19942083, methylation in kidney cortex associates with lower renal PTPN6 expression, higher eGFR, and less renal fibrosis. The regions containing the 243 eGFR-associated (P < 1e-05) CpGs are significantly enriched for transcription factor binding sites of EBF1, EP300, and CEBPB (P < 5e-6). Our findings highlight kidney function associated epigenetic variation.

  4. Methamidophos alters sperm function and DNA at different stages of spermatogenesis in mice

    Energy Technology Data Exchange (ETDEWEB)

    Urióstegui-Acosta, Mayrut; Hernández-Ochoa, Isabel [Departamento de Toxicología, CINVESTAV-IPN, D.F. (Mexico); Sánchez-Gutiérrez, Manuel [Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Hidalgo (Mexico); Piña-Guzmán, Belem [Instituto Politécnico Nacional-UPIBI, D.F. (Mexico); Rafael-Vázquez, Leticia; Solís-Heredia, M.J.; Martínez-Aguilar, Gerardo [Departamento de Toxicología, CINVESTAV-IPN, D.F. (Mexico); Quintanilla-Vega, Betzabet, E-mail: mquintan@cinvestav.mx [Departamento de Toxicología, CINVESTAV-IPN, D.F. (Mexico)

    2014-09-15

    Methamidophos (MET) is a highly toxic organophosphate (OP) pesticide that is widely used in developing countries. MET has male reproductive effects, including decreased fertility. We evaluated MET effects on sperm quality, fertilization and DNA integrity, exploring the sensitivity of different stages of spermatogenesis. Adult male mice received MET (3.75 or 5 mg/kg-bw/ip/day/4 days) and were euthanized 1, 28 or 45 days post-treatment (dpt) to evaluate MET's effects on epididymal maturation, meiosis or mitosis, respectively. Spermatozoa were obtained from the cauda epididymis–vas deferens and were evaluated for sperm quality, acrosome reaction (AR; Coomassie staining), mitochondrial membrane potential (by JC-1), DNA damage (comet assay), oxidative damage (malondialdehyde (MDA) production), in vitro fertilization and protein phosphorylation (immunodetection), and erythrocyte acetylcholinesterase (AChE) activity. At 1-dpt, MET inhibited AChE (43–57%) and increased abnormal cells (6%). While at 28- and 45-dpt, sperm motility and viability were significantly reduced with an increasing MET dose, and abnormal morphology increased at 5 mg/kg/day/4 days. MDA and mitochondrial activity were not affected at any dose or time. DNA damage (OTM and %DNA) was observed at 5 mg/kg/day/4 days in a time-dependent manner, whereas both parameters were altered in cells from mice exposed to 3.75 mg/kg/day/4 days only at 28-dpt. Depending on the time of collection, initial-, spontaneous- and induced-AR were altered at 5 mg/kg/day/4 days, and the fertilization capacity also decreased. Sperm phosphorylation (at serine and tyrosine residues) was observed at all time points. Data suggest that meiosis and mitosis are the more sensitive stages of spermatogenesis for MET reproductive toxicity compared to epididymal maturation. - Highlights: • Methamidophos alters sperm cell function at different stages of spermatogenesis. • Testicular stages of spermatogenesis are more sensitive to

  5. Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains.

    Directory of Open Access Journals (Sweden)

    Nicolas Siaud

    2011-12-01

    Full Text Available The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR. Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.

  6. Global DNA hypomethylation has no impact on lung function or serum inflammatory and fibrosis cytokines in asbestos-exposed population.

    Science.gov (United States)

    Yu, Min; Lou, Jianlin; Xia, Hailing; Zhang, Min; Zhang, Yixiao; Chen, Junqiang; Zhang, Xing; Ying, Shibo; Zhu, Lijin; Liu, Lihong; Jia, Guang

    2017-04-01

    To examine the effect of asbestos exposure on global DNA methylation and determine whether lung function and inflammatory and fibrosis biomarkers are correlated with the methylation state. A total of 26 healthy subjects without asbestos exposure (Group 1), 47 healthy subjects with exposure (Group 2), and 52 subjects with benign asbestos-related disorders (ARDs) (Group 3) participated in this cross-sectional study. Blood global 5-methylcytosine (5mC) and serum TNF-α, collagen IV, CCL5 and CC16 concentrations were analyzed using enzyme-linked immunosorbent assay-like assays. Spirometric maneuvers were performed to assess lung function. Decreased 5mC levels were observed in Groups 2 and 3 compared to Group 1, irrespective of lung function (p asbestos exposure. Asbestos exposure causes global DNA hypomethylation. DNA hypomethylation has no influence on serum biomarkers and lung function in asbestos-exposed population with or without pleural and pulmonary parenchymal abnormalities.

  7. DNA methylation profile associated with rapid decline in kidney function: findings from the CRIC Study

    Science.gov (United States)

    Wing, Maria R.; Devaney, Joseph M.; Joffe, Marshall M.; Xie, Dawei; Feldman, Harold I.; Dominic, Elizabeth A.; Guzman, Nicolas J.; Ramezani, Ali; Susztak, Katalin; Herman, James G.; Cope, Leslie; Harmon, Brennan; Kwabi-Addo, Bernard; Gordish-Dressman, Heather; Go, Alan S.; He, Jiang; Lash, James P.; Kusek, John W.; Raj, Dominic S.

    2014-01-01

    Background Epigenetic mechanisms may be important in the progression of chronic kidney disease (CKD). Methods We studied the genome-wide DNA methylation pattern associated with rapid loss of kidney function using the Infinium HumanMethylation 450 K BeadChip in 40 Chronic Renal Insufficiency (CRIC) study participants (n = 3939) with the highest and lowest rates of decline in estimated glomerular filtration rate. Results The mean eGFR slope was 2.2 (1.4) and −5.1 (1.2) mL/min/1.73 m2 in the stable kidney function group and the rapid progression group, respectively. CpG islands in NPHP4, IQSEC1 and TCF3 were hypermethylated to a larger extent in subjects with stable kidney function (P-values of 7.8E−05 to 9.5E−05). These genes are involved in pathways known to promote the epithelial to mesenchymal transition and renal fibrosis. Other CKD-related genes that were differentially methylated are NOS3, NFKBIL2, CLU, NFKBIB, TGFB3 and TGFBI, which are involved in oxidative stress and inflammatory pathways (P-values of 4.5E−03 to 0.046). Pathway analysis using Ingenuity Pathway Analysis showed that gene networks related to cell signaling, carbohydrate metabolism and human behavior are epigenetically regulated in CKD. Conclusions Epigenetic modifications may be important in determining the rate of loss of kidney function in patients with established CKD. PMID:24516231

  8. Integral parametrization of the Kinetics of Crosslink production in plasmid DNA as a function of 8-methoxypsoralen concentration

    Energy Technology Data Exchange (ETDEWEB)

    Vidania, R. de; Paramio, J. M.; Bauluz, C.

    1986-07-01

    In this paper we present results of crosslink production in pBR322 DNA along a wide range of 8-methoxypsoralen (8-MOP) concentration. Experimental data were obtained as DNA renaturation percentages, from the shift in hyperchromicity after a temperature-dependent denaturation-renaturation process. the experimental results showed a three-stage profile when represented as a function of the natural logarithms of 8-MOP concentration. an integral parametrization which allows a simultaneous fit of the three observed stages is presented here. the theoretical values of crosslink production determined from the fit are useful to asses the genotoxicity of psoralen-induced crosslinks in plasmid DNA. (Author) 24 refs.

  9. An RNA Recognition Motif-Containing Protein Functions in Meiotic Silencing by Unpaired DNA

    Directory of Open Access Journals (Sweden)

    Dilini A. Samarajeewa

    2017-08-01

    Full Text Available Meiotic silencing by unpaired DNA (MSUD is a biological process that searches pairs of homologous chromosomes (homologs for segments of DNA that are unpaired. Genes found within unpaired segments are silenced for the duration of meiosis. In this report, we describe the identification and characterization of Neurospora crassa sad-7, a gene that encodes a protein with an RNA recognition motif (RRM. Orthologs of sad-7 are found in a wide range of ascomycete fungi. In N. crassa, sad-7 is required for a fully efficient MSUD response to unpaired genes. Additionally, at least one parent must have a functional sad-7 allele for a cross to produce ascospores. Although sad-7-null crosses are barren, sad-7Δ strains grow at a wild-type (wt rate and appear normal under vegetative growth conditions. With respect to expression, sad-7 is transcribed at baseline levels in early vegetative cultures, at slightly higher levels in mating-competent cultures, and is at its highest level during mating. These findings suggest that SAD-7 is specific to mating-competent and sexual cultures. Although the role of SAD-7 in MSUD remains elusive, green fluorescent protein (GFP-based tagging studies place SAD-7 within nuclei, perinuclear regions, and cytoplasmic foci of meiotic cells. This localization pattern is unique among known MSUD proteins and raises the possibility that SAD-7 coordinates nuclear, perinuclear, and cytoplasmic aspects of MSUD.

  10. Effective delivery of anti-miRNA DNA oligonucleotides by functionalized gold nanoparticles.

    Science.gov (United States)

    Kim, Jae-Hong; Yeom, Ji-Hyun; Ko, Jeong-Jae; Han, Min Su; Lee, Kangseok; Na, Soon-Young; Bae, Jeehyeon

    2011-09-20

    MicroRNAs (miRNAs) are gaining recognition as essential regulators involved in many biological processes, and they are emerging as therapeutic targets for treating disease. Here, we introduce a method for effective delivery of anti-miRNA oligonucleotides (AMOs) using functionalized gold nanoparticles (AuNPs). To demonstrate the ability of AMOs to silence miRNA, we selected miR-29b, which is known to downregulate myeloid cell leukemia-1 (MCL-1), a factor responsible for promoting cell survival. We first generated AuNPs coated with cargo DNA, which was then coupled to complementary DNA linked to an antisense miR-29b sequence. When the AuNPs were delivered into HeLa cells, MCL-1 protein and mRNA levels were increased significantly. Furthermore, apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was inhibited, proving that AMOs targeting miR-29b were effectively delivered by our innovative AuNP. In addition, we provided evidence that AuNP could deliver other AMOs against miR-21 into two independent cell lines, KGN and 293T, suggesting that the AuNP conjugates can be versatile for any AMO and cell type. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Sequence conservation in avian CR1: an interspersed repetitive DNA family evolving under functional constraints.

    Science.gov (United States)

    Chen, Z Q; Ritzel, R G; Lin, C C; Hodgetts, R B

    1991-07-01

    CR1 is a short interspersed repetitive DNA element originally identified in the domestic chicken (Gallus gallus). However, unlike virtually all other such sequences described to date, CR1 is not confined to one or a few closely related species. It is probably a ubiquitous component of the avian genome, having been detected in representatives of nine orders encompassing a wide spectrum of the class Aves. This identification was made possible by using the polymerase chain reaction (PCR), which revealed interspecific similarities not detected by conventional Southern analysis. DNA sequence comparisons between a CR1 element isolated from a sarus crane (Grus antigone) and those isolated from an emu (Dromaius novaehollandiae) showed that two short highly conserved regions are present. These are included within two regions previously characterized in the CR1 units of domestic fowl. One of these behaves as a transcriptional silencer and the other is a binding site for a nuclear protein. Our observations suggest that CR1 has evolved under functional constraints and that interspersed repetitive sequences as a class may constitute a more significant component of the eukaryotic genome than is generally acknowledged.

  12. Induction of DNA synthesis and apoptosis are separable functions of E2F-1

    DEFF Research Database (Denmark)

    Phillips, A C; Bates, S; Ryan, K M

    1997-01-01

    The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic...... response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant...... apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E...

  13. DNA binding domains in diverse nuclear receptors function as nuclear export signals.

    Science.gov (United States)

    Black, B E; Holaska, J M; Rastinejad, F; Paschal, B M

    2001-11-13

    The nuclear receptor superfamily of transcription factors directs gene expression through DNA sequence-specific interactions with target genes. Nuclear import of these receptors involves recognition of a nuclear localization signal (NLS) by importins, which mediate translocation into the nucleus. Nuclear receptors lack a leucine-rich nuclear export signal (NES), and export is insensitive to leptomycin B, indicating that nuclear export is not mediated by Crm1. We set out to define the NES in the glucocorticoid receptor (GR) and to characterize the export pathway. We found that the 69 amino acid DNA binding domain (DBD) of GR, which is unrelated to any known NES, is necessary and sufficient for export. Mutational analysis revealed that a 15 amino acid sequence between the two zinc binding loops in the GR-DBD confers nuclear export to a GFP reporter protein, and alanine-scanning mutagenesis was used to identify the residues within this sequence that are critical for export. The DBD is highly related (41%-88% identity) in steroid, nonsteroid, and orphan nuclear receptors, and we found that the DBDs from ten different nuclear receptors all function as export signals. DBD-dependent nuclear export is saturable, and prolonged nuclear localization of the GR increases its transcriptional activity. Multiple members of the nuclear receptor superfamily use a common pathway to exit the nucleus. We propose that NLS-mediated import and DBD-mediated export define a shuttling cycle that integrates the compartmentalization and activity of nuclear receptors.

  14. The F-plasmid TraI protein contains three functional domains required for conjugative DNA strand transfer.

    Science.gov (United States)

    Matson, Steven W; Ragonese, Heather

    2005-01-01

    The F-plasmid-encoded TraI protein, also known as DNA helicase I, is a bifunctional protein required for conjugative DNA transfer. The enzyme catalyzes two distinct but functionally related reactions required for the DNA processing events associated with conjugation: the site- and strand-specific transesterification (relaxase) reaction that provides the nick required to initiate strand transfer and a processive 5'-to-3' helicase reaction that provides the motive force for strand transfer. Previous studies have identified the relaxase domain, which encompasses the first approximately 310 amino acids of the protein. The helicase-associated motifs lie between amino acids 990 and 1450. The function of the region between amino acids 310 and 990 and the region from amino acid 1450 to the C-terminal end is unknown. A protein lacking the C-terminal 252 amino acids (TraIDelta252) was constructed and shown to have essentially wild-type levels of transesterase and helicase activity. In addition, the protein was capable of a functional interaction with other components of the minimal relaxosome. However, TraIDelta252 was not able to support conjugative DNA transfer in genetic complementation experiments. We conclude that TraIDelta252 lacks an essential C-terminal domain that is required for DNA transfer. We speculate this domain may be involved in essential protein-protein interactions with other components of the DNA transfer machinery.

  15. DNA microarrays: from structural genomics to functional genomics. The applications of gene chips in dermatology and dermatopathology.

    Science.gov (United States)

    Sellheyer, Klaus; Belbin, Thomas J

    2004-11-01

    The human genome project was successful in sequencing the entire human genome and ended earlier than expected. The vast genetic information now available will have far-reaching consequences for medicine in the twenty-first century. The knowledge gained from the mapping and sequencing of human genes on a genome-wide scale--commonly referred to as structural genomics--is prerequisite for studies that focus on the functional aspects of genes. A recently invented technique, known as gene chip, or DNA microarray, technology, allows the study of the function of thousands of genes at once, thereby opening the door to the new field of functional genomics. At its core, the DNA microarray utilizes a unique feature of DNA known as complementary hybridization. As such, it is not different from Southern (DNA) blot or northern (RNA) blot hybridizations, or the polymerase chain reaction, with the exception that it allows expression profiling of the entire human genome in a single hybridization experiment. The article highlights the principles, technology, and applications of DNA microarrays as they pertain to the field of dermatology and dermatopathology. The most important applications are the gene expression profiling of skin cancer, especially of melanoma. Other potential applications include gene expression profiling of inflammatory skin diseases, the mutational analysis of genodermatoses, and polymorphism screening, as well as drug development and chemosensitivity prediction. cDNA microarrays will shape the diagnostic approach of the dermatology and the dermatopathology of the future and may lead to new therapeutic options.

  16. Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Carmen Fernandez-Fernandez

    Full Text Available DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA. We found that the expression of the DnaA(R357A mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

  17. New Insights into 5hmC DNA Modification: Generation, Distribution and Function

    Directory of Open Access Journals (Sweden)

    Dong-Qiao Shi

    2017-07-01

    Full Text Available Dynamic DNA modifications, such as methylation/demethylation on cytosine, are major epigenetic mechanisms to modulate gene expression in both eukaryotes and prokaryotes. In addition to the common methylation on the 5th position of the pyrimidine ring of cytosine (5mC, other types of modifications at the same position, such as 5-hydroxymethyl (5hmC, 5-formyl (5fC, and 5-carboxyl (5caC, are also important. Recently, 5hmC, a product of 5mC demethylation by the Ten-Eleven Translocation family proteins, was shown to regulate many cellular and developmental processes, including the pluripotency of embryonic stem cells, neuron development, and tumorigenesis in mammals. Here, we review recent advances on the generation, distribution, and function of 5hmC modification in mammals and discuss its potential roles in plants.

  18. The multiple nuclear functions of BRCA1: transcription, ubiquitination and DNA repair.

    Science.gov (United States)

    Starita, Lea M; Parvin, Jeffrey D

    2003-06-01

    Interest in BRCA1 stems from its role as a tumour suppressor in breast and ovarian cancer. Intensive research in BRCA1 has revealed little about its specific role in cancer; rather, this protein has been implicated in a multitude of important cellular processes. The diverse biochemical activities of BRCA1 combine to protect the genome from damage. New data reveal that BRCA1 transcriptionally regulates some DNA-repair genes, and, in addition, new roles for BRCA1 have been identified in heterochromatin formation on the X chromosome, double-strand-break repair, and ubiquitination. These diverse activities of BRCA1 may be linked in a single pathway, or BRCA1 might function in multiple nuclear processes.

  19. Kinetic control of the coverage of oil droplets by DNA-functionalized colloids.

    Science.gov (United States)

    Joshi, Darshana; Bargteil, Dylan; Caciagli, Alessio; Burelbach, Jerome; Xing, Zhongyang; Nunes, André S; Pinto, Diogo E P; Araújo, Nuno A M; Brujic, Jasna; Eiser, Erika

    2016-08-01

    We report a study of reversible adsorption of DNA-coated colloids on complementary functionalized oil droplets. We show that it is possible to control the surface coverage of oil droplets using colloidal particles by exploiting the fact that, during slow adsorption, compositional arrest takes place well before structural arrest occurs. As a consequence, we can prepare colloid-coated oil droplets with a "frozen" degree of loading but with fully ergodic colloidal dynamics on the droplets. We illustrate the equilibrium nature of the adsorbed colloidal phase by exploring the quasi-two-dimensional phase behavior of the adsorbed colloids under the influence of depletion interactions and present simulations of a simple model that illustrates the nature of the compositional arrest and the structural ergodicity.

  20. Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA.

    Directory of Open Access Journals (Sweden)

    Philippe A Parone

    Full Text Available Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS. At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.

  1. Synthesis, characterization, DNA interaction and potential applications of gold nanoparticles functionalized with Acridine Orange fluorophores.

    Science.gov (United States)

    Biver, Tarita; Eltugral, Nurettin; Pucci, Andrea; Ruggeri, Giacomo; Schena, Alberto; Secco, Fernando; Venturini, Marcella

    2011-04-28

    Two new water-soluble gold nanoparticles (AO-TEG-Au and AO-PEG-Au NPs) are prepared and characterized. They are stabilized by thioalkylated oligoethylene glycols and functionalized with fluorescent Acridine Orange (AO) derivatives. Despite the different core sizes (11.8 and 3.9 nm respectively) and shell composition, they are both well dispersed and are stable in water, even if some self-aggregation is observed in the case of AO-TEG-Au NPs. However, AO-PEG-Au NPs show much lower emission efficiency with respect to AO-TEG-Au NPs. Spectrophotometric and spectrofluorometric experiments indicate that both types of nanoparticle are able to bind to calf thymus DNA, either by external binding or partial intercalation. Preliminary FACS flow cytometry tests seem to indicate that the AO-TEG-Au nanoparticle is able to cross the cell membrane where it is absorbed by Chinese hamster ovary (CHO) cells at the picomolar concentration level.

  2. C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

    Directory of Open Access Journals (Sweden)

    Angela Brieger

    Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

  3. The fission yeast inhibitor of growth (ING) protein Png1p functions in response to DNA damage.

    Science.gov (United States)

    Chen, Jian-Qiang; Li, Yang; Pan, Xian; Lei, Bing-Kun; Chang, Cheng; Liu, Zheng-Xun; Lu, Hong

    2010-05-21

    In budding yeast and human cells, ING (inhibitor of growth) tumor suppressor proteins play important roles in response to DNA damage by modulating chromatin structure through collaborating with histone acetyltransferase or histone deacetylase complexes. However, the biological functions of ING family proteins in fission yeast are poorly defined. Here, we report that Png1p, a fission yeast ING homolog protein, is required for cell growth under normal and DNA-damaged conditions. Png1p was further confirmed to regulate histone H4 acetylation through collaboration with the MYST family histone acetyltransferase 1 (Mst1). Additionally, both fission yeast PNG1 and MST1 can functionally complement their budding yeast correspondence homologs YNG2 and ESA1, respectively. These results suggest that ING proteins in fission yeast might also conserve function, similar to ING proteins in budding yeast and human cells. We also showed that decreased acetylation in Deltapng1 cells resulted in genome-wide down-regulation of 756 open reading frames, including the central DNA repair gene RAD22. Overexpression of RAD22 partially rescued the png1 mutant phenotype under both normal and DNA-damaged conditions. Furthermore, decreased expression of RAD22 in Deltapng1 cells was confirmed to be caused by decreased H4 acetylation at its promoter. Altogether, these results indicate that Png1p is required for histone H4 acetylation and functions upstream of RAD22 in the DNA damage response pathway.

  4. Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF₆]: Enhanced Efficiency in Bacterial Gene Transformation.

    Science.gov (United States)

    Soni, Sarvesh K; Sarkar, Sampa; Mirzadeh, Nedaossadat; Selvakannan, P R; Bhargava, Suresh K

    2015-04-28

    The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 μL of IL ([Bmim][PF6]) and 1 μg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

  5. A DNA nanocapsule with aptamer-controlled open-closure function for targeted delivery

    DEFF Research Database (Denmark)

    Bentin, Thomas

    2012-01-01

    A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces.......A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces....

  6. Dual functions of α-ketoglutarate dehydrogenase E2 in the Krebs cycle and mitochondrial DNA inheritance in Trypanosoma brucei.

    Science.gov (United States)

    Sykes, Steven E; Hajduk, Stephen L

    2013-01-01

    The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.

  7. Investigation of plasma-functionalized multiwalled carbon nanotube film and its application of DNA sensor for Legionella pneumophila detection.

    Science.gov (United States)

    Park, Eun Jin; Lee, Jun-Yong; Kim, Jun Hyup; Lee, Cheol Jin; Kim, H Stanley; Min, Nam Ki

    2010-08-15

    A novel multiwall carbon nanotube (MWCNT) electrode functionalized with oxygen plasma treatment was prepared and characterized, and its DNA sensing ability for Legionella pneumophila (L. pneumophila) detection was examined using electrochemical measurement. A well-patterned MWCNT working electrode (WE) on a Pt track was fabricated using photolithography, transfer methods and an etching technique. The MWCNT WE was functionalized by oxygen plasma treatment prior to applying for DNA sensor. The surface morphology of the plasma-functionalized MWCNT (pf-MWCNT) WEs were observed by scanning electron microscope (SEM) and the change of chemical composition was characterized by X-ray photoelectron spectroscopy (XPS), and electrochemical measurements were performed using CV with ferricyanide/ferrocyanide redox couple. Effective areas of working electrodes were calculated to be 0.00453 cm(2) for pristine MWCNT electrode and 0.00747-0.00874 cm(2) for pf-MWCNT electrodes with different plasma treatment times. Differential pulse voltammetry (DPV) was carried out in methylene blue solution for DNA sensing. The pf-MWCNT based DNA sensor was successfully operated in a target concentration range of 10 pM to 100 nM and had a lower detection limit than a pristine MWCNT based DNA sensor. Copyright 2010 Elsevier B.V. All rights reserved.

  8. Electrochemical Characterization of O2 Plasma Functionalized Multi-Walled Carbon Nanotube Electrode for Legionella pneumophila DNA Sensor

    Science.gov (United States)

    Park, Eun Jin; Lee, Jun-Yong; Hyup Kim, Jun; Kug Kim, Sun; Lee, Cheol Jin; Min, Nam Ki

    2010-08-01

    An electrochemical DNA sensor for Legionella pneumophila detection was constructed using O2 plasma functionalized multi-walled carbon nanotube (MWCNT) film as a working electrode (WE). The cyclic voltammetry (CV) results revealed that the electrocatalytic activity of plasma functionalized MWCNT (pf-MWCNT) significantly changed depending on O2 plasma treatment time due to some oxygen containing functional groups on the pf-MWCNT surface. Scanning electron microscope (SEM) images and X-ray photoelectron spectroscopy (XPS) spectra were also presented the changes of their surface morphologies and oxygen composition before and after plasma treatment. From a comparison study, it was found that the pf-MWCNT WEs had higher electrocatalytic activity and more capability of probe DNA immobilization: therefore, electrochemical signal changes by probe DNA immobilization and hybridization on pf-MWCNT WEs were larger than on Au WEs. The pf-MWCNT based DNA sensor was able to detect a concentration range of 10 pM-100 nM of target DNA to detect L. pneumophila.

  9. Synonymous codon bias and functional constraint on GC3-related DNA backbone dynamics in the prokaryotic nucleoid.

    Science.gov (United States)

    Babbitt, Gregory A; Alawad, Mohammed A; Schulze, Katharina V; Hudson, André O

    2014-01-01

    While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for the optimization of local DNA molecular dynamics via GC content at synonymous sites (≈GC3). We confirm a strong association between GC3-related intrinsic DNA flexibility and codon bias across 24 different prokaryotic multiple whole-genome alignments. We develop a novel test of natural selection targeting synonymous sites and demonstrate that GC3-related DNA backbone dynamics have been subject to moderate selective pressure, perhaps contributing to our observation that many genes possess extreme DNA backbone dynamics for their given protein space. This dual function of codons may impose universal functional constraints affecting the evolution of synonymous and non-synonymous sites. We propose that synonymous sites may have evolved as an 'accessory' during an early expansion of a primordial genetic code, allowing for multiplexed protein coding and structural dynamic information within the same molecular context. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Label-free DNA biosensor based on a peptide nucleic acid-functionalized microstructured optical fiber-Bragg grating

    Science.gov (United States)

    Candiani, Alessandro; Bertucci, Alessandro; Giannetti, Sara; Konstantaki, Maria; Manicardi, Alex; Pissadakis, Stavros; Cucinotta, Annamaria; Corradini, Roberto; Selleri, Stefano

    2013-05-01

    We describe a novel sensing approach based on a functionalized microstructured optical fiber-Bragg grating for specific DNA target sequences detection. The inner surface of a microstructured fiber, where a Bragg grating was previously inscribed, has been functionalized by covalent linking of a peptide nucleic acid probe targeting a DNA sequence bearing a single point mutation implicated in cystic fibrosis (CF) disease. A solution of an oligonucleotide (ON) corresponding to a tract of the CF gene containing the mutated DNA has been infiltrated inside the fiber capillaries and allowed to hybridize to the fiber surface according to the Watson-Crick pairing. In order to achieve signal amplification, ON-functionalized gold nanoparticles were then infiltrated and used in a sandwich-like assay. Experimental measurements show a clear shift of the reflected high order mode of a Bragg grating for a 100 nM DNA solution, and fluorescence measurements have confirmed the successful hybridization. Several experiments have been carried out on the same fiber using the identical concentration, showing the same modulation trend, suggesting the possibility of the reuse of the sensor. Measurements have also been made using a 100 nM mismatched DNA solution, containing a single nucleotide mutation and corresponding to the wild-type gene, and the results demonstrate the high selectivity of the sensor.

  11. Realizing non-close-packed crystal structures through directional binding of DNA-functionalized colloidal clusters

    Science.gov (United States)

    Sinno, Talid; Zanjani, Mehdi; Crocker, John

    A promising approach for engineering assembling entities that exhibit anisotropy is to create small clusters out of spherical colloidal particles. Here, we study computationally the self-assembly of large numbers of colloidal clusters formed by a recently-introduced crystal templating method. We study the assembly of clusters mediated by the addition of spherical `bond' particles. In particular, the `bond' spheres are DNA-functionalized so that they interact attractively with the clusters. In this construct, the differing number of particle-particle contacts available between a cluster and bond particle as a function of orientation creates directional bonding. A variety of simulation techniques are employed to study the thermodynamics and kinetics of the assembly process. Specifically, we compute nucleation barriers for several crystalline configurations using tetrahedral, octahedral, and cubic clusters. We show that some cluster types lead to facile growth of crystalline superstructures, while others lead to structures that are highly susceptible to defect formation. Crystal growth kinetics are probed using molecular dynamics and Brownian dynamics simulations and again demonstrate a wide range of kinetic limitations depending on the cluster geometry.

  12. Novel and Functional DNA Sequence Variants within the GATA6 Gene Promoter in Ventricular Septal Defects

    Directory of Open Access Journals (Sweden)

    Chunyu Li

    2014-07-01

    Full Text Available Congenital heart disease (CHD is the most common birth defect in humans. Genetic causes and underlying molecular mechanisms for isolated CHD remain largely unknown. Studies have demonstrated that GATA transcription factor 6 (GATA6 plays an essential role in the heart development. Mutations in GATA6 gene have been associated with diverse types of CHD. As GATA6 functions in a dosage-dependent manner, we speculated that changed GATA6 levels, resulting from DNA sequence variants (DSVs within the gene regulatory regions, may mediate the CHD development. In the present study, GATA6 gene promoter was genetically and functionally analyzed in large groups of patients with ventricular septal defect (VSD (n = 359 and ethnic-matched healthy controls (n = 365. In total, 11 DSVs, including four SNPs, were identified in VSD patients and controls. Two novel and heterozygous DSVs, g.22169190A>T and g.22169311C>G, were identified in two VSD patients, but in none of controls. In cultured cardiomyocytes, the activities of the GATA6 gene promoter were significantly reduced by the DSVs g.22169190A>T and g.22169311C>G. Therefore, our findings suggested that the DSVs within the GATA6 gene promoter identified in VSD patients may change GATA6 levels, contributing to the VSD development as a risk factor.

  13. Assaying multiple restriction endonucleases functionalities and inhibitions on DNA microarray with multifunctional gold nanoparticle probes.

    Science.gov (United States)

    Ma, Lan; Zhu, Zhijun; Li, Tao; Wang, Zhenxin

    2014-02-15

    Herein, a double-stranded (ds) DNA microarray-based resonance light scattering (RLS) assay with multifunctional gold nanoparticle (GNP) probes has been developed for studying restriction endonuclease functionality and inhibition. Because of decreasing significantly melting temperature, the enzyme-cleaved dsDNAs easily unwind to form single-stranded (ss) DNAs. The ssDNAs are hybridized with multiplex complementary ssDNAs functionalized GNP probes followed by silver enhancement and RLS detection. Three restriction endonucleases (EcoRI, BamHI and EcoRV) and three potential inhibitors (doxorubicin hydrochloride (DOX), ethidium bromide (EB) and an EcoRI-derived helical peptide (α4)) were selected to demonstrate capability of the assay. Enzyme activities of restriction endonucleases are detected simultaneously with high specificity down to the limits of 2.0 × 10(-2)U/mL for EcoRI, 1.1 × 10(-2)U/mL for BamHI and 1.6 × 10(-2)U/mL for EcoRV, respectively. More importantly, the inhibitory potencies of three inhibitors are showed quantitatively, indicating that our approach has great promise for high-throughput screening of restriction endonuclease inhibitors. © 2013 Elsevier B.V. All rights reserved.

  14. Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

    DEFF Research Database (Denmark)

    Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan

    2013-01-01

    V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA......-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs(3A)) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpin-sealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease...

  15. Scientific publications about DNA structure-function and PCR technique in Costa Rica: a historic view (1953-2003).

    Science.gov (United States)

    Albertazzi, Federico J

    2004-09-01

    The spreading of knowledge depends on the access to the information and its immediate use. Models are useful to explain specific phenomena. The scientific community accepts some models in Biology after a period of time, once it has evidence to support it. The model of the structure and function of the DNA proposed by Watson & Crick (1953) was not the exception, since a few years later the DNA model was finally accepted. In Costa Rica, DNA function was first mentioned in 1970, in the magazine Biologia Tropical (Tropical Biology Magazine), more than 15 years after its first publication in a scientific journal. An opposite situation occurs with technical innovations. If the efficiency of a new scientific technique is proved in a compelling way, then the acceptance by the community comes swiftly. This was the case of the polymerase chain reaction, or PCR. The first PCR machine in Costa Rica arrived in 1991, only three years after its publication.

  16. Comparative analysis of different methods of Hedera helix DNA extraction and molecular evidence of the functionality in PCR

    Directory of Open Access Journals (Sweden)

    Danka Bošeľová

    2016-12-01

    Full Text Available The most suitable method of total DNA extraction still remains the crucial step for many plant species, although there are many different protocols and commercial kits for DNA isolation. In this study, five different extraction protocols were analysed to find out the most appropriate method for DNA extraction from Hedera helix L. This species has numerous medical and pharmaceutical uses and is also characterized by antioxidant effects on human body. In spite of its wide medical utilization, it belongs to those plant species, where the genomic information is very limited. Comparing of different protocols resulted in the yield of extracted DNA that has ranged from 6.3 to 487 ng μl-1. The purity of extracted DNA has ranged from 1.4 up to 2.0 A260/A280. All the extraction methods used in this study were evaluated not only in term of quantity and purity of DNA but also its functionality in the restriction endonuclease digestion and polymerase chain reaction based downstream analysis was performed.

  17. A paleogenomic perspective on evolution and gene function: new insights from ancient DNA.

    Science.gov (United States)

    Shapiro, B; Hofreiter, M

    2014-01-24

    The publication of partial and complete paleogenomes within the last few years has reinvigorated research in ancient DNA. No longer limited to short fragments of mitochondrial DNA, inference of evolutionary processes through time can now be investigated from genome-wide data sampled as far back as 700,000 years. Tremendous insights have been made, in particular regarding the hominin lineage. With rare exception, however, a paleogenomic perspective has been mired by the quality and quantity of recoverable DNA. Though conceptually simple, extracting ancient DNA remains challenging, and sequencing ancient genomes to high coverage remains prohibitively expensive for most laboratories. Still, with improvements in DNA isolation and declining sequencing costs, the taxonomic and geographic purview of paleogenomics is expanding at a rapid pace. With improved capacity to screen large numbers of samples for those with high proportions of endogenous ancient DNA, paleogenomics is poised to become a key technology to better understand recent evolutionary events.

  18. Regulation of BRCA1 Function by DNA Damage-Induced Site-Specific Phosphorylation

    Science.gov (United States)

    2007-06-01

    concentration of Mg 2. Interestingly, mammalian cell extracts deficient in Fanconi anemia proteins had a 3–9-fold reduction in DNA end-joining...Mavinakere, M., and Campbell, C. Deficient DNA end joining activity in extracts from fanconi anemia fibroblasts. J. Biol. Chem., 276: 9543–9549...suppressive properties of BRCA1 de - rive, at least in part, from its response to tissue-specific DNA damage. In this regard, certain oxidative

  19. The application of psoralens to the study of DNA structure, function and dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Spielmann, Peter Hans [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1991-04-01

    A series of six nitroxide spin-labeled psoralens were designed, synthesized and tested as probes for DNA dynamics. The synthesis of these spin-labeled psoralen derivatives and their photoreactivity with double-stranded DNA fragments is described. The spin labels (nitroxides) were demonstrated to survive the uv irradiation required to bind the probe to the target DNA. EPR spectra of the photobound spin-labels indicate that they do not wobble with respect to the DNA on the time-scales investigated. The author has used psoralen modified DNA as a model for the study of DNA repair enzyme systems in human cell free extracts. He has shown that damage-induced DNA synthesis is associated with removal of psoralen adducts and therefore is "repair synthesis" and not an aberrant DNA synthesis reaction potentiated by deformation of the DNA by adducts. He has found that all DNA synthesis induced by psoralen monoadducts is the consequence of removal of these adducts. By the same approach he has obtained evidence that this in vitro system is capable of removing psoralen cross-links as well. Reported here are synthetic methods that make use of high intensity lasers coupled with HPLC purification to make homogeneous and very pure micromole quantities of furan-side monoadducted, cross-linked, and pyrone-side monoadducted DNA oligonucleotide. These molecules are currently being studied by NMR and X-ray crystallography. The application of the site-specifically psoralen modified oligonucleotide synthesized by these methods to the construction of substrates for the investigation of DNA repair is also discussed.

  20. Origins of biological function in DNA and RNA hairpin loop motifs from replica exchange molecular dynamics simulation.

    Science.gov (United States)

    Swadling, Jacob B; Ishii, Kunihiko; Tahara, Tahei; Kitao, Akio

    2018-01-31

    Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) have remarkably similar chemical structures, but despite this, they play significantly different roles in modern biology. In this article, we explore the possible conformations of DNA and RNA hairpins to better understand the fundamental differences in structure formation and stability. We use large parallel temperature replica exchange molecular dynamics ensembles to sample the full conformational landscape of these hairpin molecules so that we can identify the stable structures formed by the hairpin sequence. Our simulations show RNA adopts a narrower distribution of folded structures compared to DNA at room temperature, which forms both hairpins and many unfolded conformations. RNA is capable of forming twice as many hydrogen bonds than DNA which results in a higher melting temperature. We see that local chemical differences lead to emergent molecular properties such as increased persistence length in RNA that is weakly temperature dependant. These discoveries provide fundamental insight into how RNA forms complex folded tertiary structures which confer enzymatic-like function in ribozymes, whereas DNA retains structural motifs in order to facilitate function such as translation of sequence.

  1. Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

    DEFF Research Database (Denmark)

    Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia

    2016-01-01

    RNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian......Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose...... RAD52 facilitates repair of collapsed DNA replication forks in cancer cells....

  2. Selective propagation of functional mitochondrial DNA during oogenesis restricts the transmission of a deleterious mitochondrial variant.

    Science.gov (United States)

    Hill, Jahda H; Chen, Zhe; Xu, Hong

    2014-04-01

    Although mitochondrial DNA (mtDNA) is prone to mutation and few mtDNA repair mechanisms exist, crippling mitochondrial mutations are exceedingly rare. Recent studies have demonstrated strong purifying selection in the mouse female germline. However, the mechanisms underlying positive selection of healthy mitochondria remain to be elucidated. We visualized mtDNA replication during Drosophila melanogaster oogenesis, finding that mtDNA replication commenced before oocyte determination during the late germarium stage and was dependent on mitochondrial fitness. We isolated a temperature-sensitive lethal mtDNA allele, mt:CoI(T300I), which resulted in reduced mtDNA replication in the germarium at the restrictive temperature. Additionally, the frequency of the mt:CoI(T300I) allele in heteroplasmic flies was decreased, both during oogenesis and over multiple generations, at the restrictive temperature. Furthermore, we determined that selection against mt:CoI(T300I) overlaps with the timing of selective replication of mtDNA in the germarium. These findings establish a previously uncharacterized developmental mechanism for the selective amplification of wild-type mtDNA, which may be evolutionarily conserved to limit the transmission of deleterious mutations.

  3. DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

    Directory of Open Access Journals (Sweden)

    Yuxin Feng

    Full Text Available Estrogen receptor alpha (ERα, a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

  4. Genomic distribution and possible functions of DNA hydroxymethylation in the brain.

    Science.gov (United States)

    Wen, Lu; Tang, Fuchou

    2014-11-01

    DNA methylation (5-methylcytosine, 5mC) is involved in many cellular processes and emerges as an important epigenetic player in brain development and memory formation. The recent discovery that 5mC can be oxidized to 5-hydroxymethylcytosine (5hmC) by TET (Ten-Eleven-Translocation) proteins provides novel insights into the dynamic character of 5mC in the brain. The content of 5hmC is remarkably high in the brain, adding further complexity. In this review, we discuss how recent advances have improved our understanding of the possible biological roles of 5hmC and TET proteins in the brain. These advances attribute to various approaches, including the genome-wide approach to map 5hmC in different genomic contexts, the gene knockout/knockdown approach to elucidate the functions of TET proteins and 5hmC, and the biochemical approach to uncover potential 5hmC readers. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein–Coupled Receptor

    OpenAIRE

    Feng, Yu; Broder, Christopher C.; Kennedy, Paul E.; Berger, Edward A.

    2011-01-01

    A cofactor for HIV-1 (human immunodeficiency virus-type 1) fusion and entry was identified with the use of a novel functional complementary DNA (cDNA) cloning strategy. This protein, designated “fusin,” is a putative G protein–coupled receptor with seven transmembrane segments. Recombinant fusin enabled CD4-expressing nonhuman cell types to support HIV-1 Env-mediated cell fusion and HIV-1 infection. Antibodies to fusin blocked cell fusion and infection with normal CD4-positive human target ce...

  6. Dynamic stepwise opening of integron attC DNA hairpins by SSB prevents toxicity and ensures functionality.

    Science.gov (United States)

    Grieb, Maj Svea; Nivina, Aleksandra; Cheeseman, Bevan L; Hartmann, Andreas; Mazel, Didier; Schlierf, Michael

    2017-10-13

    Biologically functional DNA hairpins are found in archaea, prokaryotes and eukaryotes, playing essential roles in various DNA transactions. However, during DNA replication, hairpin formation can stall the polymerase and is therefore prevented by the single-stranded DNA binding protein (SSB). Here, we address the question how hairpins maintain their functional secondary structure despite SSB's presence. As a model hairpin, we used the recombinogenic form of the attC site, essential for capturing antibiotic-resistance genes in the integrons of bacteria. We found that attC hairpins have a conserved high GC-content near their apical loop that creates a dynamic equilibrium between attC fully opened by SSB and a partially structured attC-6-SSB complex. This complex is recognized by the recombinase IntI, which extrudes the hairpin upon binding while displacing SSB. We anticipate that this intriguing regulation mechanism using a base pair distribution to balance hairpin structure formation and genetic stability is key to the dissemination of antibiotic resistance genes among bacteria and might be conserved among other functional hairpins. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. SSB functions as a sliding platform that migrates on DNA via reptation.

    Science.gov (United States)

    Zhou, Ruobo; Kozlov, Alexander G; Roy, Rahul; Zhang, Jichuan; Korolev, Sergey; Lohman, Timothy M; Ha, Taekjip

    2011-07-22

    SSB proteins bind to and control the accessibility of single-stranded DNA (ssDNA), likely facilitated by their ability to diffuse on ssDNA. Using a hybrid single-molecule method combining fluorescence and force, we probed how proteins with large binding site sizes can migrate rapidly on DNA and how protein-protein interactions and tension may modulate the motion. We observed force-induced progressive unraveling of ssDNA from the SSB surface between 1 and 6 pN, followed by SSB dissociation at ∼10 pN, and obtained experimental evidence of a reptation mechanism for protein movement along DNA wherein a protein slides via DNA bulge formation and propagation. SSB diffusion persists even when bound with RecO and at forces under which the fully wrapped state is perturbed, suggesting that even in crowded cellular conditions SSB can act as a sliding platform to recruit and carry its interacting proteins for use in DNA replication, recombination and repair. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. DNA binding by the Rev1 BRCT region : implications for biological and structural function

    NARCIS (Netherlands)

    Groote, Frederik Hendrik de

    2011-01-01

    Rev1, a protein belonging to the Y-family of polymerases, is a key protein in the process of DNA Translesion Synthesis, and other DNA damage tolerance pathways. Recent studies have shown that its BRCT region is essential for the organisation of TLS events by Rev1. However, the molecular mechanism

  9. Preparation of DNA biosensor application from fuel oil waste by functionalization and characterization of MWCNT

    Directory of Open Access Journals (Sweden)

    Ahmed Mishaal Mohammed

    2017-11-01

    Full Text Available The potential of using a multi-wall carbon nanotube (MWCNT synthesized from a fuel oil waste of power plants has discovered for the first time for DNA biosensors application. The MWCNT surface morphologies were examined by field emission scanning electron microscopy (FE-SEM and atomic force microscopy (AFM. The thickness of the MWCNT was found 203nm and confirmed by FESEM. The electrochemical DNA biosensor was successfully developed using a MWCNT modified on SiO2 thin films. The capacitance measurements were performed to detect the sensitivity of DNA detection. The change in capacitance before and after immobilization of the DNA was measured in the frequency range of 1Hz to 1MHz. The results indicate that bare device exhibited the lowest capacitance value, which was 32.7μF. The capacitance value of the DNA immobilization increase to 52μF. The permittivity and conductivity also were examined to study the effect of the DNA immobilization toward the MWCNT modified surface. This present demonstrated that the MWCNT modified SiO2 a thin film was successfully fabricated for DNA biosensor detection. Keywords: Carbon nanotubes, Sensors, Thin films, Electrochemical DNA

  10. Blood DNA methylation age is not associated with cognitive functioning in middle-aged monozygotic twins

    DEFF Research Database (Denmark)

    Starnawska, A; Tan, Q; Lenart, A

    2016-01-01

    The epigenetic clock, also known as DNA methylation age (DNAmAge), represents age-related changes of DNA methylation at multiple sites of the genome and is suggested to be a biomarker for biological age. Elevated blood DNAmAge is associated with all-cause mortality, with the strongest effects...

  11. Functional organization of mammalian mitochondrial DNA in nucleoids: history, recent developments, and future challenges.

    NARCIS (Netherlands)

    Spelbrink, J.N.

    2010-01-01

    Various proteins involved in replication, repair, and the structural organization of mitochondrial DNA (mtDNA) have been characterized in detail over the past 25 or so years. In addition, in recent years, many proteins were identified with a role in the dynamics of the mitochondrial network. Using

  12. Linking Structure and Function for the DNA Repair Complex Mre11-Rad50-Nbs1

    NARCIS (Netherlands)

    E. Kinoshita (Eri)

    2014-01-01

    markdownabstract__Abstract__ Repair of DNA damage is an essential process in all cells and an important mechanism to avoid cancer development in animals. The repair of DNA double strand breaks (DSB) requires many component proteins including the Mre11-Rad50-Nbs1 (MRN) complex that serves

  13. Antiviral immunity in fish – functional analysis using DNA vaccination as a tool

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lorenzen, Ellen; Einer-Jensen, Katja

    2013-01-01

    In fish, DNA vaccines encoding the glycoproteins (G proteins) of the salmonid rhabdoviruses VHSV and IHNV have proved very efficient under experimental conditions. Nano-gram amounts of plasmid DNA can induce long-lasting protective immunity when delivered by intramuscular injection in rainbow tro...

  14. Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

    DEFF Research Database (Denmark)

    Trujillo, L E; Pupo, E; Miranda, F

    1996-01-01

    We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assay...

  15. Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

    Directory of Open Access Journals (Sweden)

    Maher A Noureddine

    2009-05-01

    Full Text Available The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs. Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

  16. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  17. ERCC6L2 mutations link a distinct bone-marrow-failure syndrome to DNA repair and mitochondrial function.

    Science.gov (United States)

    Tummala, Hemanth; Kirwan, Michael; Walne, Amanda J; Hossain, Upal; Jackson, Nicholas; Pondarre, Corinne; Plagnol, Vincent; Vulliamy, Tom; Dokal, Inderjeet

    2014-02-06

    Exome sequencing was performed in three index cases with bone marrow failure and neurological dysfunction and whose parents are first-degree cousins. Homozygous truncating mutations were identified in ERCC6L2 in two of the individuals. Both of these mutations affect the subcellular localization and stability of ERCC6L2. We show here that knockdown of ERCC6L2 in human A549 cells significantly reduced their viability upon exposure to the DNA-damaging agents mitomycin C and Irofulven, but not etoposide and camptothecin, suggesting a role in nucleotide excision repair. ERCC6L2-knockdown cells also displayed H2AX phosphorylation, which significantly increased upon genotoxic stress, suggesting an early DNA-damage response. Intriguingly, ERCC6L2 was seen to translocate to the mitochondria and the nucleus in response to DNA damage, and ERCC6L2 knockdown induced intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger N-acetyl cysteine attenuated the Irofulven-induced cytotoxicity in ERCC6L2-knockdown cells and abolished ERCCGL2 traffic to the mitochondria and nucleus in response to this DNA-damaging agent. Collectively, these observations identify a distinct bone-marrow-failure syndrome due to mutations in ERCC6L2, a gene implicated in DNA repair and mitochondrial function. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  18. RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications.

    Science.gov (United States)

    Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

    2015-01-01

    Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress toward understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation.

  19. Embryonic caffeine exposure acts via A1 adenosine receptors to alter adult cardiac function and DNA methylation in mice.

    Directory of Open Access Journals (Sweden)

    Daniela L Buscariollo

    Full Text Available Evidence indicates that disruption of normal prenatal development influences an individual's risk of developing obesity and cardiovascular disease as an adult. Thus, understanding how in utero exposure to chemical agents leads to increased susceptibility to adult diseases is a critical health related issue. Our aim was to determine whether adenosine A1 receptors (A1ARs mediate the long-term effects of in utero caffeine exposure on cardiac function and whether these long-term effects are the result of changes in DNA methylation patterns in adult hearts. Pregnant A1AR knockout mice were treated with caffeine (20 mg/kg or vehicle (0.09% NaCl i.p. at embryonic day 8.5. This caffeine treatment results in serum levels equivalent to the consumption of 2-4 cups of coffee in humans. After dams gave birth, offspring were examined at 8-10 weeks of age. A1AR+/+ offspring treated in utero with caffeine were 10% heavier than vehicle controls. Using echocardiography, we observed altered cardiac function and morphology in adult mice exposed to caffeine in utero. Caffeine treatment decreased cardiac output by 11% and increased left ventricular wall thickness by 29% during diastole. Using DNA methylation arrays, we identified altered DNA methylation patterns in A1AR+/+ caffeine treated hearts, including 7719 differentially methylated regions (DMRs within the genome and an overall decrease in DNA methylation of 26%. Analysis of genes associated with DMRs revealed that many are associated with cardiac hypertrophy. These data demonstrate that A1ARs mediate in utero caffeine effects on cardiac function and growth and that caffeine exposure leads to changes in DNA methylation.

  20. Embryonic Caffeine Exposure Acts via A1 Adenosine Receptors to Alter Adult Cardiac Function and DNA Methylation in Mice

    Science.gov (United States)

    Greenwood, Victoria; Xue, Huiling; Rivkees, Scott A.; Wendler, Christopher C.

    2014-01-01

    Evidence indicates that disruption of normal prenatal development influences an individual's risk of developing obesity and cardiovascular disease as an adult. Thus, understanding how in utero exposure to chemical agents leads to increased susceptibility to adult diseases is a critical health related issue. Our aim was to determine whether adenosine A1 receptors (A1ARs) mediate the long-term effects of in utero caffeine exposure on cardiac function and whether these long-term effects are the result of changes in DNA methylation patterns in adult hearts. Pregnant A1AR knockout mice were treated with caffeine (20 mg/kg) or vehicle (0.09% NaCl) i.p. at embryonic day 8.5. This caffeine treatment results in serum levels equivalent to the consumption of 2–4 cups of coffee in humans. After dams gave birth, offspring were examined at 8–10 weeks of age. A1AR+/+ offspring treated in utero with caffeine were 10% heavier than vehicle controls. Using echocardiography, we observed altered cardiac function and morphology in adult mice exposed to caffeine in utero. Caffeine treatment decreased cardiac output by 11% and increased left ventricular wall thickness by 29% during diastole. Using DNA methylation arrays, we identified altered DNA methylation patterns in A1AR+/+ caffeine treated hearts, including 7719 differentially methylated regions (DMRs) within the genome and an overall decrease in DNA methylation of 26%. Analysis of genes associated with DMRs revealed that many are associated with cardiac hypertrophy. These data demonstrate that A1ARs mediate in utero caffeine effects on cardiac function and growth and that caffeine exposure leads to changes in DNA methylation. PMID:24475304

  1. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity.

    Science.gov (United States)

    Cruz-Bermúdez, Alberto; Vicente-Blanco, Ramiro J; Hernández-Sierra, Rosana; Montero, Mayte; Alvarez, Javier; González Manrique, Mar; Blázquez, Alberto; Martín, Miguel Angel; Ayuso, Carmen; Garesse, Rafael; Fernández-Moreno, Miguel A

    2016-01-01

    The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

  2. Rap1 is indispensable for TRF2 function in etoposide-induced DNA damage response in gastric cancer cell line.

    Science.gov (United States)

    Li, X; Liu, W; Wang, H; Yang, L; Li, Y; Wen, H; Ning, H; Wang, J; Zhang, L; Li, J; Fan, D

    2015-03-30

    The telomeric protein TRF2, involving in telomeric and extratelomeric DNA damage response, has been previously reported to facilitate multidrug resistance (MDR) in gastric cancer cells by interfering ATM-dependent DNA damage response induced by anticancer drugs. Rap1 is the TRF2-interacting protein in the shelterin complex. Complex formation between Rap1 and TRF2 is essential for their function in telomere and end protection. Here we focus on the effects of Rap1 on TRF2 function in DNA damage response induced by anticancer drugs. Both Rap1 and TRF2 expression were upregulated in SGC7901 and its MDR variant SGC7901/VCR after etoposide treatment, which was more marked in SGC7901/VCR than in SGC7901. Rap1 silencing by siRNA in SGC7901/VCR partially reversed the etoposide resistance. And Rap1 silencing partially reversed the TRF2-mediated resistance to etoposide in SGC7901. Rap1 silencing did not affect the TRF2 upregulation induced by etoposide, but eliminated the inhibition effect of TRF2 on ATM expression and ATM phosphorylation at serine 1981 (ATM pS1981). Furthermore, phosphorylation of ATM targets, including γH2AX and serine 15 (S15) on p53, were increased in Rap1 silencing cells in response to etoposide. Thus, we confirm that Rap1, interacting with TRF2 in the shelterin complex, also has an important role in TRF2-mediated DNA damage response in gastric cancer cells treated by etoposide.

  3. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity.

    Directory of Open Access Journals (Sweden)

    Alberto Cruz-Bermúdez

    Full Text Available The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids. Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

  4. Two potential Petunia hybrida mitochondrial DNA replication origins show structural and in vitro functional homology with the animal mitochondrial DNA heavy and light strand replication origins

    NARCIS (Netherlands)

    Haas, Jan M. de; Hille, Jacques; Kors, Frank; Meer, Bert van der; Kool, Ad J.; Folkerts, Otto; Nijkamp, H. John J.

    1991-01-01

    Four Petunia hybrida mitochondrial (mt) DNA fragments have been isolated, sequenced, localized on the physical map and analyzed for their ability to initiate specific DNA synthesis. When all four mtDNA fragments were tested as templates in an in vitro DNA synthesizing lysate system, developed from

  5. Oral contraceptives modify DNA methylation and monocyte-derived macrophage function

    Directory of Open Access Journals (Sweden)

    Campesi Ilaria

    2012-01-01

    were lower in FOCA- than in FOCA+. Importantly, FOCs had a lower global DNA methylation, indicating that OC may have epigenetic effects on gene expression. OC did not modify the expression of androgen receptor but increased estrogen receptor α expression, more considerably in FOCA+, and decreased estrogen receptor β, more considerably in FOCA-. Importantly, the activation state of estrogen receptor β in FOCs was decreased, while estrogen receptor α was not active in either Fs or FOCs. Unstimulated MDMs obtained from FOCs showed higher release of TNFα in comparison with Fs. After lipopolysaccharide stimulation, the release of TNFα was significantly higher in Fs than in FOCs. Conclusions OC use induced many changes in hematological and plasmatic markers, modifying hormonal levels, endothelial function, inflammation index and some redox state parameters, producing a perturbation of the internal milieu that impacted macrophagic function. In fact, different levels of estrogen receptor expression and release of TNFα were observed in macrophages derived from OC users. Some of the above activities were linked to the androgenic properties of progestin. Even though it is not known whether these effects are reversible, the results indicate that to avoid potential skewing of results only a single type of OC should be used during a single clinical trial.

  6. Assembly and function of DNA double-strand break repair foci in mammalian cells

    DEFF Research Database (Denmark)

    Bekker-Jensen, Simon; Mailand, Niels

    2010-01-01

    DNA double-strand breaks (DSBs) are among the most cytotoxic types of DNA damage, which if left unrepaired can lead to mutations or gross chromosomal aberrations, and promote the onset of diseases associated with genomic instability such as cancer. One of the most discernible hallmarks...... of the cellular response to DSBs is the accumulation and local concentration of a plethora of DNA damage signaling and repair proteins in the vicinity of the lesion, initiated by ATM-mediated phosphorylation of H2AX (¿-H2AX) and culminating in the generation of distinct nuclear compartments, so-called Ionizing...... of such DNA repair foci still remains limited. In this review, we focus on recent discoveries on the mechanisms that govern the formation of IRIF, and discuss the implications of such findings in light of our understanding of the physiological importance of these structures....

  7. The mammalian transcriptome and the function of non-coding DNA sequences

    National Research Council Canada - National Science Library

    Shabalina, Svetlana A; Spiridonov, Nikolay A

    2004-01-01

    .... With the completion of the human and mouse genomes and the accumulation of data on the mammalian transcriptome, the focus now shifts to non-coding DNA sequences, RNA-coding genes and their transcripts...

  8. ORC function in late G1: maintaining the license for DNA replication.

    Science.gov (United States)

    Da-Silva, Lance F; Duncker, Bernard P

    2007-01-15

    The origin recognition complex (ORC) is essential as a scaffold for the assembly of prereplicative complexes (pre-RCs) in G(1) phase of the cell cycle. Some models have proposed that once origins have been licensed for DNA replication, ORC is dispensable for MCM protein association, and ensuing DNA replication. Although budding yeast Orc6 is not needed for origin recognition or binding in vitro, we have recently shown that this ORC subunit is required in late G(1) phase for maintenance of MCMs, and subsequent DNA replication. Further investigation shows that depletion of Orc6 results in displacement of MCM proteins from both early- and late-firing origins, and eventually results in the activation of the Rad53 checkpoint kinase, consistent with incomplete DNA replication. Loss of MCM association at origins may be mediated by the displacement of Mcm10 and/or Orc2 as a consequence of late G(1) Orc6 depletion.

  9. DNA Recombinase Proteins, their Function and Structure in the Active Form, a Computational Study

    Science.gov (United States)

    Carra, Claudio; Cucinotta, Francis A.

    2007-01-01

    Homologous recombination is a crucial sequence of reactions in all cells for the repair of double strand DNA (dsDNA) breaks. While it was traditionally considered as a means for generating genetic diversity, it is now known to be essential for restart of collapsed replication forks that have met a lesion on the DNA template (Cox et al., 2000). The central stage of this process requires the presence of the DNA recombinase protein, RecA in bacteria, RadA in archaea, or Rad51 in eukaryotes, which leads to an ATP-mediated DNA strand-exchange process. Despite many years of intense study, some aspects of the biochemical mechanism, and structure of the active form of recombinase proteins are not well understood. Our theoretical study is an attempt to shed light on the main structural and mechanistic issues encountered on the RecA of the e-coli, the RecA of the extremely radio resistant Deinococcus Radiodurans (promoting an inverse DNA strand-exchange repair), and the homolog human Rad51. The conformational changes are analyzed for the naked enzymes, and when they are linked to ATP and ADP. The average structures are determined over 2ns time scale of Langevian dynamics using a collision frequency of 1.0 ps(sup -1). The systems are inserted in an octahedron periodic box with a 10 Angstrom buffer of water molecules explicitly described by the TIP3P model. The corresponding binding free energies are calculated in an implicit solvent using the Poisson-Boltzmann solvent accessible surface area, MM-PBSA model. The role of the ATP is not only in stabilizing the interaction RecA-DNA, but its hydrolysis is required to allow the DNA strand-exchange to proceed. Furthermore, we extended our study, using the hybrid QM/MM method, on the mechanism of this chemical process. All the calculations were performed using the commercial code Amber 9.

  10. Preparation of DNA biosensor application from fuel oil waste by functionalization and characterization of MWCNT

    OpenAIRE

    Ahmed Mishaal Mohammed; Ismail K. Al-Khateeb; Adawiya J. Haider; Ruslinda A. Rahim; U. Hashim

    2017-01-01

    The potential of using a multi-wall carbon nanotube (MWCNT) synthesized from a fuel oil waste of power plants has discovered for the first time for DNA biosensors application. The MWCNT surface morphologies were examined by field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). The thickness of the MWCNT was found 203nm and confirmed by FESEM. The electrochemical DNA biosensor was successfully developed using a MWCNT modified on SiO2 thin films. The capacitanc...

  11. Better estimation of protein-DNA interaction parameters improve prediction of functional sites

    Directory of Open Access Journals (Sweden)

    O'Flanagan Ruadhan A

    2008-12-01

    Full Text Available Abstract Background Characterizing transcription factor binding motifs is a common bioinformatics task. For transcription factors with variable binding sites, we need to get many suboptimal binding sites in our training dataset to get accurate estimates of free energy penalties for deviating from the consensus DNA sequence. One procedure to do that involves a modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment method designed to produce many such sequences. Results We analyzed low stringency SELEX data for E. coli Catabolic Activator Protein (CAP, and we show here that appropriate quantitative analysis improves our ability to predict in vitro affinity. To obtain large number of sequences required for this analysis we used a SELEX SAGE protocol developed by Roulet et al. The sequences obtained from here were subjected to bioinformatic analysis. The resulting bioinformatic model characterizes the sequence specificity of the protein more accurately than those sequence specificities predicted from previous analysis just by using a few known binding sites available in the literature. The consequences of this increase in accuracy for prediction of in vivo binding sites (and especially functional ones in the E. coli genome are also discussed. We measured the dissociation constants of several putative CAP binding sites by EMSA (Electrophoretic Mobility Shift Assay and compared the affinities to the bioinformatics scores provided by methods like the weight matrix method and QPMEME (Quadratic Programming Method of Energy Matrix Estimation trained on known binding sites as well as on the new sites from SELEX SAGE data. We also checked predicted genome sites for conservation in the related species S. typhimurium. We found that bioinformatics scores based on SELEX SAGE data does better in terms of prediction of physical binding energies as well as in detecting functional sites. Conclusion We think that training binding site detection

  12. Genome DNA Sequence Variation, Evolution, and Function in Bacteria and Archaea.

    Science.gov (United States)

    Nishida, Hiromi

    2013-01-01

    Comparative genomics has revealed that variations in bacterial and archaeal genome DNA sequences cannot be explained by only neutral mutations. Virus resistance and plasmid distribution systems have resulted in changes in bacterial and archaeal genome sequences during evolution. The restriction-modification system, a virus resistance system, leads to avoidance of palindromic DNA sequences in genomes. Clustered, regularly interspaced, short palindromic repeats (CRISPRs) found in genomes represent yet another virus resistance system. Comparative genomics has shown that bacteria and archaea have failed to gain any DNA with GC content higher than the GC content of their chromosomes. Thus, horizontally transferred DNA regions have lower GC content than the host chromosomal DNA does. Some nucleoid-associated proteins bind DNA regions with low GC content and inhibit the expression of genes contained in those regions. This form of gene repression is another type of virus resistance system. On the other hand, bacteria and archaea have used plasmids to gain additional genes. Virus resistance systems influence plasmid distribution. Interestingly, the restriction-modification system and nucleoid-associated protein genes have been distributed via plasmids. Thus, GC content and genomic signatures do not reflect bacterial and archaeal evolutionary relationships.

  13. Age-related formaldehyde interferes with DNA methyltransferase function, causing memory loss in Alzheimer's disease.

    Science.gov (United States)

    Tong, Zhiqian; Han, Chanshuai; Qiang, Min; Wang, Weishan; Lv, Jihui; Zhang, Shouzi; Luo, Wenhong; Li, Hui; Luo, Hongjun; Zhou, Jiangning; Wu, Beibei; Su, Tao; Yang, Xu; Wang, Xiaomin; Liu, Ying; He, Rongqiao

    2015-01-01

    Hippocampus-related topographic amnesia is the most common symptom of memory disorders in Alzheimer's disease (AD) patients. Recent studies have revealed that experience-mediated DNA methylation, which is regulated by enzymes with DNA methyltransferase (DNMT) activity, is required for the formation of recent memory as well as the maintenance of remote memory. Notably, overexpression of DNMT3a in the hippocampus can reverse spatial memory deficits in aged mice. However, a decline in global DNA methylation was found in the autopsied hippocampi of patients with AD. Exactly, what endogenous factors that affect DNA methylation still remain to be elucidated. Here, we report a marked increase in endogenous formaldehyde levels is associated with a decline in global DNA methylation in the autopsied hippocampus from AD patients. In vitro and in vivo results show that formaldehyde in excess of normal physiological levels reduced global DNA methylation by interfering DNMTs. Interestingly, intrahippocampal injection of excess formaldehyde before spatial learning in healthy adult rats can mimic the learning difficulty of early stage of AD. Moreover, injection of excess formaldehyde after spatial learning can mimic the loss of remote spatial memory observed in late stage of AD. These findings suggest that aging-associated formaldehyde contributes to topographic amnesia in AD patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Plasmodium falciparum origin recognition complex subunit 5: functional characterization and role in DNA replication foci formation.

    Science.gov (United States)

    Gupta, Ashish; Mehra, Parul; Dhar, Suman Kumar

    2008-08-01

    The mechanism of DNA replication initiation and progression is poorly understood in the parasites, including human malaria parasite Plasmodium falciparum. Using bioinformatics tools and yeast complementation assay, we identified a putative homologue of Saccharomyces cerevisiaeorigin recognition complex subunit 5 in P. falciparum (PfORC5). PfORC5 forms distinct nuclear foci colocalized with the replication foci marker proliferating cell nuclear antigen (PfPCNA) and co-immunoprecipitates with PCNA during early-to-mid trophozoite stage replicating parasites. Interestingly, these proteins separate from each other at the non-replicating late schizont stage, citing the evidence of the presence of both PCNA and ORC components in replication foci during eukaryotic DNA replication. PfORC1, another ORC subunit, colocalizes with PfPCNA and PfORC5 at the beginning of DNA replication, but gets degraded at the late schizont stage, ensuring the regulation of DNA replication in the parasites. Further, we have identified putative PCNA-interacting protein box in PfORC1 that may explain in part the colocalization of PfORC and PfPCNA. Additionally, use of specific DNA replication inhibitor hydroxyurea affects ORC5/PCNA foci formation and parasitic growth. These results strongly favour replication factory model in the parasites and confer great potential to understand the co-ordination between ORC and PCNA during eukaryotic DNA replication in general.

  15. Genotoxic stress and DNA repair in plants: emerging functions and tools for improving crop productivity.

    Science.gov (United States)

    Balestrazzi, Alma; Confalonieri, Massimo; Macovei, Anca; Donà, Mattia; Carbonera, Daniela

    2011-03-01

    Crop productivity is strictly related to genome stability, an essential requisite for optimal plant growth/development. Genotoxic agents (e.g., chemical agents, radiations) can cause both chemical and structural damage to DNA. In some cases, they severely affect the integrity of plant genome by inducing base oxidation, which interferes with the basal processes of replication and transcription, eventually leading to cell death. The cell response to oxidative stress includes several DNA repair pathways, which are activated to remove the damaged bases and other lesions. Information concerning DNA repair in plants is still limited, although results from gene profiling and mutant analysis suggest possible differences in repair mechanisms between plants and other eukaryotes. The present review focuses on the base- and nucleotide excision repair (BER, NER) pathways, which operate according to the most common DNA repair rule (excision of damaged bases and replacement by the correct nucleotide), highlighting the most recent findings in plants. An update on DNA repair in organelles, chloroplasts and mitochondria is also provided. Finally, it is generally acknowledged that DNA repair plays a critical role during seed imbibition, preserving seed vigor. Despite this, only a limited number of studies, described here, dedicated to seeds are currently available.

  16. Synonymous codon bias and functional constraint on GC3-related DNA backbone dynamics in the prokaryotic nucleoid

    OpenAIRE

    Babbitt, Gregory A.; Alawad, Mohammed A.; Schulze, Katharina V.; Hudson, André O.

    2014-01-01

    While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for ...

  17. Oxidative DNA damage caused by pulsed discharge with cavitation on the bactericidal function

    Science.gov (United States)

    Kudo, Ken-ichi; Ito, Hironori; Ihara, Satoshi; Terato, Hiroaki

    2015-09-01

    Plasma-based techniques are expected to have practical use for wastewater purification with a potential for killing contaminated microorganisms and degrading recalcitrant materials. In the present study, we analysed oxidative DNA damage in bacterial cells treated by the plasma to unveil its mechanisms in the bactericidal process. Escherichia coli cell suspension was exposed to the plasma induced by applying an alternating-current voltage of about 1 kV with bubbling formed by water-cavitation, termed pulsed discharge with cavitation. Chromosomal DNA damage, such as double strand break (DSB) and oxidative base lesions, increased proportionally with the applied energy, as determined by electrophoretic and mass spectrometric analyses. Among the base lesions identified, the yields of 8-hydroxyguanine (8-OH-G) and 5-hydroxycytosine (5-OH-C) in chromosomal DNA increased by up to 4- and 15-fold, respectively, compared to untreated samples. The progeny DNA sequences, derived from plasmid DNA exposed to the plasma, indicated that the production rate of 5-OH-C exceeded that of 8-OH-G, as G:C to A:T transitions accounted for 65% of all base changes, but only a few G:C to T:A transversions were observed. The cell viabilities of E. coli cells decreased in direct proportion to increases in the applied energy. Therefore, the plasma-induced bactericidal mechanism appears to relate to oxidative damage caused to bacterial DNA. These results were confirmed by observing the generation of hydroxyl radicals and hydrogen peroxide molecules following the plasma exposure. We also compared our results with the plasma to those obtained with 137Cs γ-rays, as a well-known ROS generator to confirm the DNA-damaging mechanism involved.

  18. Sex-specific influences of mtDNA mitotype and diet on mitochondrial functions and physiological traits in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Wen C Aw

    Full Text Available Here we determine the sex-specific influence of mtDNA type (mitotype and diet on mitochondrial functions and physiology in two Drosophila melanogaster lines. In many species, males and females differ in aspects of their energy production. These sex-specific influences may be caused by differences in evolutionary history and physiological functions. We predicted the influence of mtDNA mutations should be stronger in males than females as a result of the organelle's maternal mode of inheritance in the majority of metazoans. In contrast, we predicted the influence of diet would be greater in females due to higher metabolic flexibility. We included four diets that differed in their protein: carbohydrate (P:C ratios as they are the two-major energy-yielding macronutrients in the fly diet. We assayed four mitochondrial function traits (Complex I oxidative phosphorylation, reactive oxygen species production, superoxide dismutase activity, and mtDNA copy number and four physiological traits (fecundity, longevity, lipid content, and starvation resistance. Traits were assayed at 11 d and 25 d of age. Consistent with predictions we observe that the mitotype influenced males more than females supporting the hypothesis of a sex-specific selective sieve in the mitochondrial genome caused by the maternal inheritance of mitochondria. Also, consistent with predictions, we found that the diet influenced females more than males.

  19. Sex-specific influences of mtDNA mitotype and diet on mitochondrial functions and physiological traits in Drosophila melanogaster.

    Science.gov (United States)

    Aw, Wen C; Garvin, Michael R; Melvin, Richard G; Ballard, J William O

    2017-01-01

    Here we determine the sex-specific influence of mtDNA type (mitotype) and diet on mitochondrial functions and physiology in two Drosophila melanogaster lines. In many species, males and females differ in aspects of their energy production. These sex-specific influences may be caused by differences in evolutionary history and physiological functions. We predicted the influence of mtDNA mutations should be stronger in males than females as a result of the organelle's maternal mode of inheritance in the majority of metazoans. In contrast, we predicted the influence of diet would be greater in females due to higher metabolic flexibility. We included four diets that differed in their protein: carbohydrate (P:C) ratios as they are the two-major energy-yielding macronutrients in the fly diet. We assayed four mitochondrial function traits (Complex I oxidative phosphorylation, reactive oxygen species production, superoxide dismutase activity, and mtDNA copy number) and four physiological traits (fecundity, longevity, lipid content, and starvation resistance). Traits were assayed at 11 d and 25 d of age. Consistent with predictions we observe that the mitotype influenced males more than females supporting the hypothesis of a sex-specific selective sieve in the mitochondrial genome caused by the maternal inheritance of mitochondria. Also, consistent with predictions, we found that the diet influenced females more than males.

  20. Functional link between DNA damage responses and transcriptional regulation by ATM in response to a histone deacetylase inhibitor TSA.

    Science.gov (United States)

    Lee, Jong-Soo

    2007-09-01

    Mutations in the ATM (ataxia-telangiectasia mutated) gene, which encodes a 370 kd protein with a kinase catalytic domain, predisposes people to cancers, and these mutations are also linked to ataxia-telangiectasia (A-T). The histone acetylaion/deacetylation- dependent chromatin remodeling can activate the ATM kinase-mediated DNA damage signal pathway (in an accompanying work, Lee, 2007). This has led us to study whether this modification can impinge on the ATM-mediated DNA damage response via transcriptional modulation in order to understand the function of ATM in the regulation of gene transcription. To identify the genes whose expression is regulated by ATM in response to histone deaceylase (HDAC) inhibition, we performed an analysis of oligonucleotide microarrays with using the appropriate cell lines, isogenic A-T (ATM(-)) and control (ATM(+)) cells, following treatment with a HDAC inhibitor TSA. Treatment with TSA reprograms the differential gene expression profile in response to HDAC inhibition in ATM(-) cells and ATM(+) cells. We analyzed the genes that are regulated by TSA in the ATM-dependent manner, and we classified these genes into different functional categories, including those involved in cell cycle/DNA replication, DNA repair, apoptosis, growth/differentiation, cell- cell adhesion, signal transduction, metabolism and transcription. We found that while some genes are regulated by TSA without regard to ATM, the patterns of gene regulation are differentially regulated in an ATM-dependent manner. Taken together, these finding indicate that ATM can regulate the transcription of genes that play critical roles in the molecular response to DNA damage, and this response is modulated through an altered HDAC inhibition-mediated gene expression.

  1. Quantum dot-functionalized porous ZnO nanosheets as a visible light induced photoelectrochemical platform for DNA detection

    Science.gov (United States)

    Wang, Wenjing; Hao, Qing; Wang, Wei; Bao, Lei; Lei, Jianping; Wang, Quanbo; Ju, Huangxian

    2014-02-01

    This work reports the synthesis of novel CdTe quantum dot (QD)-functionalized porous ZnO nanosheets via a covalent binding method with (3-aminopropyl)triethoxysilane as a linker. The functional nanosheets showed an excellent visible-light absorbency and much higher photoelectrochemical activity than both CdTe QDs and ZnO nanosheets due to the porous structure and appropriate band alignment between the CdTe QDs and ZnO nanosheets. Using hydrogen peroxide as an electron acceptor the nanosheet-modified electrode showed a sensitive photocurrent response. This speciality led to a novel methodology for the design of hydrogen peroxide-related biosensors by the formation or consumption of hydrogen peroxide. Using biotin-labeled DNA as capture probe, a model biosensor was proposed by immobilizing the probe on a nanosheet-modified electrode to recognize target DNA in the presence of an assistant DNA, which produced a ``Y'' junction structure to trigger a restriction endonuclease-aided target recycling. The target recycling resulted in the release of biotin labeled to the immobilized DNA from the nanosheet-modified electrode, thus decreased the consumption of hydrogen peroxide by horseradish peroxidase-mediated electrochemical reduction after binding the left biotin with horseradish peroxidase-labeled streptavidin, which produced an increasing photoelectrochemical response. The `signal on' strategy for photoelectrochemical detection of DNA showed a low detection limit down to the subfemtomole level and good specificity to single-base mismatched oligonucleotides. The sensitized porous ZnO nanosheets are promising for applications in both photovoltaic devices and photoelectrochemical biosensing.

  2. Ball with hair: modular functionalization of highly stable G-quadruplex DNA nano-scaffolds through N2-guanine modification.

    Science.gov (United States)

    Lech, Christopher Jacques; Phan, Anh Tuân

    2017-06-20

    Functionalized nanoparticles have seen valuable applications, particularly in the delivery of therapeutic and diagnostic agents in biological systems. However, the manufacturing of such nano-scale systems with the consistency required for biological application can be challenging, as variation in size and shape have large influences in nanoparticle behavior in vivo. We report on the development of a versatile nano-scaffold based on the modular functionalization of a DNA G-quadruplex. DNA sequences are functionalized in a modular fashion using well-established phosphoramidite chemical synthesis with nucleotides containing modification of the amino (N2) position of the guanine base. In physiological conditions, these sequences fold into well-defined G-quadruplex structures. The resulting DNA nano-scaffolds are thermally stable, consistent in size, and functionalized in a manner that allows for control over the density and relative orientation of functional chemistries on the nano-scaffold surface. Various chemistries including small modifications (N2-methyl-guanine), bulky aromatic modifications (N2-benzyl-guanine), and long chain-like modifications (N2-6-amino-hexyl-guanine) are tested and are found to be generally compatible with G-quadruplex formation. Furthermore, these modifications stabilize the G-quadruplex scaffold by 2.0-13.3 °C per modification in the melting temperature, with concurrent modifications producing extremely stable nano-scaffolds. We demonstrate the potential of this approach by functionalizing nano-scaffolds for use within the biotin-avidin conjugation approach. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer.

    Science.gov (United States)

    Ju, Young Seok; Alexandrov, Ludmil B; Gerstung, Moritz; Martincorena, Inigo; Nik-Zainal, Serena; Ramakrishna, Manasa; Davies, Helen R; Papaemmanuil, Elli; Gundem, Gunes; Shlien, Adam; Bolli, Niccolo; Behjati, Sam; Tarpey, Patrick S; Nangalia, Jyoti; Massie, Charles E; Butler, Adam P; Teague, Jon W; Vassiliou, George S; Green, Anthony R; Du, Ming-Qing; Unnikrishnan, Ashwin; Pimanda, John E; Teh, Bin Tean; Munshi, Nikhil; Greaves, Mel; Vyas, Paresh; El-Naggar, Adel K; Santarius, Tom; Collins, V Peter; Grundy, Richard; Taylor, Jack A; Hayes, D Neil; Malkin, David; Foster, Christopher S; Warren, Anne Y; Whitaker, Hayley C; Brewer, Daniel; Eeles, Rosalind; Cooper, Colin; Neal, David; Visakorpi, Tapio; Isaacs, William B; Bova, G Steven; Flanagan, Adrienne M; Futreal, P Andrew; Lynch, Andy G; Chinnery, Patrick F; McDermott, Ultan; Stratton, Michael R; Campbell, Peter J

    2014-10-01

    Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication.

  4. Molecular and Functional Characterization of ssDNA Aptamers that Specifically Bind Leishmania infantum PABP

    Science.gov (United States)

    Guerra-Pérez, Natalia; Ramos, Edurne; García-Hernández, Marta; Pinto, Celia; Soto, Manuel; Martín, M. Elena; González, Víctor M.

    2015-01-01

    Summary A poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study. Results We have selected a ssDNA aptamer population against a recombinant 6xHIS–LiPABP protein (rLiPABP) that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP) protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A) in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes. Conclusion Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be

  5. Total viable molds and fungal DNA in classrooms and association with respiratory health and pulmonary function of European schoolchildren.

    Science.gov (United States)

    Simoni, Marzia; Cai, Gui-Hong; Norback, Dan; Annesi-Maesano, Isabella; Lavaud, François; Sigsgaard, Torben; Wieslander, Gunilla; Nystad, Wenche; Canciani, Mario; Viegi, Giovanni; Sestini, Piersante

    2011-12-01

    Indoor molds are associated with adverse respiratory effects in children. Although schools are important exposure sources of molds, objective measurements were more often taken in homes. Our aim was to assess indoor molds in schools and related effects on schoolchildren health. The Health Effects of the School Environment study (HESE) included 21 schools (46 classrooms) in Italy, Denmark, Sweden, Norway, and France and 654 schoolchildren (mean age 10 yr). Information on schoolchildren was collected by standardized questionnaires. Measurements of total viable molds (VM, colony-forming units, cfu/m(3)) and total/specific fungal DNA (cell equivalents, CE/g dust) were taken inside all classrooms in the cold season during normal activities, using the same standardized methodology. Pulmonary function tests were performed on 244 pupils. VM (mean, 320,cfu/m(3)) and total fungal DNA (geometric mean, 2.2 × 10(5) ± 2.1 CE/g dust) were detectable in all classrooms. The levels were significantly higher in buildings with mold/dampness problems. VM, but not fungal DNA, were inversely related to ventilation rate. VM exceeded the maximum standard of 300 cfu/m(3) in 33% of the classrooms. In the past 12 months, dry cough at night (34%) and rhinitis (32%) were the mostly reported. Children exposed to VM levels ≥ 300 cfu/m(3), compared with those exposed to lower levels, showed higher risk for past year dry cough at night (odds ratio, OR: 3.10, 95% confidence interval, CI: 1.61-5.98) and rhinitis (OR: 2.86, 95% CI: 1.65-4.95), as well as for persistent cough (OR: 3.79, 95% CI: 2.40-5.60). Aspergillus/Penicillium DNA was significantly positively associated with wheeze, and Aspergillus versicolor DNA with wheeze, rhinitis, and cough. There were significant inverse associations of Aspergillus versicolor DNA with forced vitality capacity (FVC) and Streptomyces DNA with both FEV(1) and FVC. In conclusion, indoor VM and fungal DNA were commonly found in monitored European schools and

  6. Improved DNA condensation, stability, and transfection with alkyl sulfonyl-functionalized PAMAM G2

    Energy Technology Data Exchange (ETDEWEB)

    Rata-Aguilar, Azahara, E-mail: azahara@ugr.es; Maldonado-Valderrama, Julia; Jódar-Reyes, Ana Belén; Ortega-Vinuesa, Juan Luis [University of Granada, Biocolloid and Fluid Physics Group, Department of Applied Physics (Spain); Santoyo-Gonzalez, Francisco [University of Granada, Organic Chemistry Department, Institute of Biotechnology (Spain); Martín-Rodríguez, Antonio [University of Granada, Biocolloid and Fluid Physics Group, Department of Applied Physics (Spain)

    2015-04-15

    In this work, we have used a second-generation PAMAM grafted with octadecyl sulfonyl chains to condense plasmid DNA. The influence of this modification at different levels was investigated by comparison with original PAMAM G2. The condensation process and temporal stability of the complexes was studied with DLS, finding that the aliphatic chains influence DNA compaction via hydrophobic forces and markedly improve the formation and temporal stability of a single populated system with a hydrodynamic diameter below 100 nm. Interaction with a cell membrane model was also evaluated with a pendant drop tensiometer, resulting in further incorporation of the C18-PAMAM dendriplexes onto the interface. The improvement observed in transfection with our C18 grafted PAMAM is ascribed to the size, stability, and interfacial behavior of the complexes, which in turn are consequence of the DNA condensation process and the interactions involved.

  7. Exploring function of conserved non-coding DNA in its chromosomal context

    Directory of Open Access Journals (Sweden)

    Delores J. Grant

    2015-11-01

    Full Text Available There is renewed interest in understanding expression of vertebrate genes in their chromosomal context because regulatory sequences that confer tissue-specific expression are often distributed over large distances along the DNA from the gene. One approach inserts a universal sensor/reporter-gene into the mouse or zebrafish genome to identify regulatory sequences in highly conserved non-coding DNA in the vicinity of the integrated reporter-gene. However detailed mechanisms of interaction of these regulatory elements among themselves and/or with the genes they influence remain elusive with the strategy. The inability to associate distant regulatory elements with the genes they regulate makes it difficult to examine the contribution of sequence changes in regulatory DNA to human disease. Such associations have been obtained in favorable circumstances by testing the regulatory potential of highly conserved non-coding DNA individually in small reporter-gene-containing plasmids. Alternative approaches use tiny fragments of chromosomes in Bacterial Artificial Chromosomes, BACs, where the gene of interest is tagged in vitro with a reporter/sensor gene and integrated into the germ-line of animals for expression. Mutational analysis of the BAC DNA identifies regulatory sequences. A recent approach inserts a sensor/reporter-gene into a BAC that is also truncated progressively from an end of genomic insert, and the end-deleted BAC carrying the sensor is then integrated into the genome of a developing animal for expression. The approach allows mechanisms of tissue-specific gene expression to be explored in much greater detail, although the chromosomal context of such mechanisms is limited to the length of the BAC. Here we discuss the relative strengths of the various approaches and explore how the integrated-sensor in the BACs method applied to a contig of BACs spanning a chromosomal region is likely to address mechanistic questions on interactions between

  8. Functional analysis of an acid adaptive DNA adenine methyltransferase from Helicobacter pylori 26695.

    Directory of Open Access Journals (Sweden)

    Arun Banerjee

    Full Text Available HP0593 DNA-(N(6-adenine-methyltransferase (HP0593 MTase is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5'-GCAG-3'and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co(2+, Mn(2+, and also Mg(2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.

  9. Human MLH1 protein participates in genomic damage checkpoint signaling in response to DNA interstrand crosslinks, while MSH2 functions in DNA repair.

    Directory of Open Access Journals (Sweden)

    Qi Wu

    2008-09-01

    Full Text Available DNA interstrand crosslinks (ICLs are among the most toxic types of damage to a cell. For this reason, many ICL-inducing agents are effective therapeutic agents. For example, cisplatin and nitrogen mustards are used for treating cancer and psoralen plus UVA (PUVA is useful for treating psoriasis. However, repair mechanisms for ICLs in the human genome are not clearly defined. Previously, we have shown that MSH2, the common subunit of the human MutSalpha and MutSbeta mismatch recognition complexes, plays a role in the error-free repair of psoralen ICLs. We hypothesized that MLH1, the common subunit of human MutL complexes, is also involved in the cellular response to psoralen ICLs. Surprisingly, we instead found that MLH1-deficient human cells are more resistant to psoralen ICLs, in contrast to the sensitivity to these lesions displayed by MSH2-deficient cells. Apoptosis was not as efficiently induced by psoralen ICLs in MLH1-deficient cells as in MLH1-proficient cells as determined by caspase-3/7 activity and binding of annexin V. Strikingly, CHK2 phosphorylation was undetectable in MLH1-deficient cells, and phosphorylation of CHK1 was reduced after PUVA treatment, indicating that MLH1 is involved in signaling psoralen ICL-induced checkpoint activation. Psoralen ICLs can result in mutations near the crosslinked sites; however, MLH1 function was not required for the mutagenic repair of these lesions, and so its signaling function appears to have a role in maintaining genomic stability following exposure to ICL-induced DNA damage. Distinguishing the genetic status of MMR-deficient tumors as MSH2-deficient or MLH1-deficient is thus potentially important in predicting the efficacy of treatment with psoralen and perhaps with other ICL-inducing agents.

  10. Functional intron+ and intron- rDNA in the same macronucleus of the ciliate Tetrahymena pigmentosa

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Engberg, J

    1985-01-01

    alleles was followed in the total culture and in single cells during their vegetative segregation and it was observed that replication was non-preferential with respect to the two alleles. The diallelic clones were also used to demonstrate that intron-containing rDNA was transcribed and the transcript......Diallelic clones of Tetrahymena pigmentosa containing equal amounts of intron+ and intron- rDNA in the macronucleus were constructed. The macronucleus of the resulting strains divides amitotically during vegetative growth and the diallelic genotype is therefore unstable. The coexistence of the two...

  11. Functionalization of Fatty Acid Vesicles through Newly Synthesized Bolaamphiphile-DNA Conjugates

    DEFF Research Database (Denmark)

    Wamberg, M. C.; Wieczorek, R.; Brier, S. B.

    2014-01-01

    of these structures, only one novel bola-amphiphile DNA conjugate could interact efficiently with or spontaneously pierce into the vesicle bilayers without jeopardizing their self-assembly or stability. This molecule was synthesized via a Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (click reaction......), and consists of a single hydrocarbon chain of 20 carbons having on one end a triazole group linked to the S'-phosphate of the nucleic acid and on the other side a hydroxyl-group. Its insertion was so effective that a fluorescent label on the DNA complementary to the conjugate could be used to visualize fatty...

  12. Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2-or 4-pyrenyl-functionalized O2 '-alkylated RNA monomers

    DEFF Research Database (Denmark)

    Karmakar, Saswata; Madsen, Andreas Stahl; Guenther, Dale C.

    2014-01-01

    and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2......'-alkylated uridine monomers X-Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA......-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure...

  13. Actin polymerization negatively regulates p53 function by impairing its nuclear import in response to DNA damage.

    Directory of Open Access Journals (Sweden)

    Ling Wang

    Full Text Available Actin, one of the most evolutionarily conservative proteins in eukaryotes, is distributed both in the cytoplasm and the nucleus, and its dynamics plays important roles in numerous cellular processes. Previous evidence has shown that actin interacts with p53 and this interaction increases in the process of p53 responding to DNA damage, but the physiological significance of their interaction remains elusive. Here, we show that DNA damage induces both actin polymerization and p53 accumulation. To further understand the implication of actin polymerization in p53 function, cells were treated with actin aggregation agent. We find that the protein level of p53 decrease. The change in p53 is a consequence of the polymeric actin anchoring p53 in the cytoplasm, thus impairing p53 nuclear import. Analysis of phosphorylation and ubiquitination of p53 reveals that actin polymerization promotes the p53 phosphorylation at Ser315 and reduces the stabilization of p53 by recruiting Aurora kinase A. Taken together, our results suggest that the actin polymerization serves as a negative modulator leading to the impairment of nuclear import and destabilization of p53. On the basis of our results, we propose that actin polymerization might be a factor participating in the process of orchestrating p53 function in response to DNA damage.

  14. NBS1 Functions as a Multifaceted Protein in DNA Damage Repair and Gametogenesis

    NARCIS (Netherlands)

    L.J.L. Brugmans (Linda)

    2009-01-01

    markdownabstract__Abstract__ Proper maintenance of the genome is crucial for survival of all organisms. It is of major importance for reproduction and development, that the information encoded in the genome is replicated correctly. However, endogenous and exogenous DNA-damaging agents

  15. DNA-binding specificity and molecular functions of NAC transcription factors

    DEFF Research Database (Denmark)

    Olsen, Addie Nina; Ernst, Heidi Asschenfeldt; Lo Leggio, Leila

    2005-01-01

    . The ability of NAC proteins to dimerize and to bind DNAwas analysed by structure-based mutagenesis. This identified two salt bridge-forming residues essential for NAC protein dimerization. Alteration of basic residues in a loop region containing several highly conserved residues abolished DNA binding. Thus...

  16. Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks.

    Science.gov (United States)

    Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia; Evangelou, Konstantinos; Da-Ré, Caterina; Huber, Florian; Padayachy, Laura; Tardy, Sebastien; Nicati, Noemie L; Barriot, Samia; Ochs, Fena; Lukas, Claudia; Lukas, Jiri; Gorgoulis, Vassilis G; Scapozza, Leonardo; Halazonetis, Thanos D

    2016-12-15

    Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. Reproductive aging is associated with changes in oocyte mitochondrial dynamics, function, and mtDNA quantity.

    Science.gov (United States)

    Babayev, Elnur; Wang, Tianren; Szigeti-Buck, Klara; Lowther, Katie; Taylor, Hugh S; Horvath, Tamas; Seli, Emre

    2016-11-01

    Mitochondria affect numerous aspects of mammalian reproduction. We investigated whether the decrease in oocyte quality associated with aging is related to altered mitochondria. Oocytes from old (12 months) and young (9 weeks) C57BL/6J mice were compared in relation to: mitochondria morphology and dynamics (mitochondria density, coverage, size and shape) throughout folliculogenesis; levels of mitochondrial DNA (mtDNA); mitochondrial stress reflected in the expression of mitochondrial unfolded protein response (mt-UPR) genes; and levels of reactive oxygen species (ROS) under baseline conditions and following H 2 O 2 treatment. In old mice, mitochondria of primary follicle-enclosed oocytes were smaller, with lower mitochondria coverage (total mitochondria μm 2 /μm 2 cytosol area) (pchanges were not significant. Mature oocytes (Metaphase II-MII) from old mice had significantly less mtDNA (paged MII oocytes were also higher following pretreatment with H 2 O 2 (pAging is associated with altered mitochondrial morphological parameters and decreased mtDNA levels in oocytes, as well as an increase in ROS under stressful conditions and elevated expression of mitochondrial stress response gene Hspd1. Delineation of the mechanisms underlying mitochondrial changes associated with ageing may help in the development of diagnostic and therapeutic tools in reproductive medicine. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. A closed parameterization of DNA-damage by charged particles, as a function of energy - a geometrical approach.

    Directory of Open Access Journals (Sweden)

    Frank Van den Heuvel

    Full Text Available PURPOSE: To present a closed formalism calculating charged particle radiation damage induced in DNA. The formalism is valid for all types of charged particles and due to its closed nature is suited to provide fast conversion of dose to DNA-damage. METHODS: The induction of double strand breaks in DNA-strings residing in irradiated cells is quantified using a single particle model. This leads to a proposal to use the cumulative Cauchy distribution to express the mix of high and low LET type damage probability generated by a single particle. A microscopic phenomenological Monte Carlo code is used to fit the parameters of the model as a function of kinetic energy related to the damage to a DNA molecule embedded in a cell. The model is applied for four particles: electrons, protons, alpha-particles, and carbon ions. A geometric interpretation of this observation using the impact ionization mean free path as a quantifier, allows extension of the model to very low energies. RESULTS: The mathematical expression describes the model adequately using a chi-square test ([Formula: see text]. This applies to all particle types with an almost perfect fit for protons, while the other particles seem to result in some discrepancies at very low energies. The implementation calculating a strict version of the RBE based on complex damage alone is corroborated by experimental data from the measured RBE. The geometric interpretation generates a unique dimensionless parameter [Formula: see text] for each type of charged particle. In addition, it predicts a distribution of DNA damage which is different from the current models.

  19. Improved method for Mica functionalization used in single molecule imaging of DNA with atomic force microscopy

    Directory of Open Access Journals (Sweden)

    Hana Zapletalová

    2016-07-01

    Full Text Available The modified procedure of 1-(3-aminopropylsilatrane (APS compound synthesis based on a new derivative (3‑aminopropyltrimethoxysilane for the purpose of DNA immobilization for AFM single imaging is described. New reaction pathway based on kinetically driven reaction approach is described. Necessity of two‑step purification process is proved; ability of purified APS to provide four times smoother surfaces in comparison with a crude product is demonstrated. Various analytical methods such mass spectroscopy and 1H NMR were used to show structure and enhanced purity of the APS product. APS mediates fixation of DNA molecules to mica substrates to be used for DNA imaging under Atomic Force Microscope. The use of an APS compound for simple and rapid silanization of mica surface is demonstrated. The advantages of APS‑based method are based mainly on low roughness of modified mica and homogeneous surface coverage by short sequence dsDNA (246 bp. The product obtained by the condensation reaction was purified in a two step process whose effectiveness was demonstrated not only by reduction of the silanized surface roughness, but also by mass spectroscopy (MS‑ESi, MALDI‑TOF method and proton magnetic resonance spectroscopy. Experiments demonstrate that 1‑(3‑aminopropylsilatrane can be used to fix dsDNA molecules to a mica surface to be visualized by either the tapping mode or the force‑volume mode of AFM microscopy, as demonstrated by experiments. Moreover, necessity of advanced purification protocol is demonstrated by AFM based roughness measurements – pure vs crude APS product. The kinetics of APS‑layer aging, caused by silicon oxide growth on silanized layers, was studied by water contact angle measurements and is discussed.

  20. Rapid generation of long tandem DNA repeat arrays by homologous recombination in yeast to study their function in mammalian genomes

    Directory of Open Access Journals (Sweden)

    Kouprina Natalay

    2011-10-01

    Full Text Available Abstract We describe here a method to rapidly convert any desirable DNA fragment, as small as 100 bp, into long tandem DNA arrays up to 140 kb in size that are inserted into a microbe vector. This method includes rolling-circle phi29 amplification (RCA of the sequence in vitro and assembly of the RCA products in vivo by homologous recombination in the yeast Saccharomyces cerevisiae. The method was successfully used for a functional analysis of centromeric and pericentromeric repeats and construction of new vehicles for gene delivery to mammalian cells. The method may have general application in elucidating the role of tandem repeats in chromosome organization and dynamics. Each cycle of the protocol takes ~ two weeks to complete.

  1. DNA replication origin function is promoted by H3K4 di-methylation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rizzardi, Lindsay F; Dorn, Elizabeth S; Strahl, Brian D; Cook, Jeanette Gowen

    2012-10-01

    DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.

  2. The PD-(D/EXK superfamily revisited: identification of new members among proteins involved in DNA metabolism and functional predictions for domains of (hitherto unknown function

    Directory of Open Access Journals (Sweden)

    Bujnicki Janusz M

    2005-07-01

    Full Text Available Abstract Background The PD-(D/EXK nuclease superfamily, initially identified in type II restriction endonucleases and later in many enzymes involved in DNA recombination and repair, is one of the most challenging targets for protein sequence analysis and structure prediction. Typically, the sequence similarity between these proteins is so low, that most of the relationships between known members of the PD-(D/EXK superfamily were identified only after the corresponding structures were determined experimentally. Thus, it is tempting to speculate that among the uncharacterized protein families, there are potential nucleases that remain to be discovered, but their identification requires more sensitive tools than traditional PSI-BLAST searches. Results The low degree of amino acid conservation hampers the possibility of identification of new members of the PD-(D/EXK superfamily based solely on sequence comparisons to known members. Therefore, we used a recently developed method HHsearch for sensitive detection of remote similarities between protein families represented as profile Hidden Markov Models enhanced by secondary structure. We carried out a comparison of known families of PD-(D/EXK nucleases to the database comprising the COG and PFAM profiles corresponding to both functionally characterized as well as uncharacterized protein families to detect significant similarities. The initial candidates for new nucleases were subsequently verified by sequence-structure threading, comparative modeling, and identification of potential active site residues. Conclusion In this article, we report identification of the PD-(D/EXK nuclease domain in numerous proteins implicated in interactions with DNA but with unknown structure and mechanism of action (such as putative recombinase RmuC, DNA competence factor CoiA, a DNA-binding protein SfsA, a large human protein predicted to be a DNA repair enzyme, predicted archaeal transcription regulators, and the head

  3. Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Dräger, Carolin

    2015-01-01

    RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high...... the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral......-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from...

  4. Carboxyl-functionalized magnetic microparticle carrier for isolation and identification of DNA in dairy products

    Energy Technology Data Exchange (ETDEWEB)

    Horak, Daniel [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Sq. 2, 162 06 Prague 6 (Czech Republic)]. E-mail: horak@imc.cas.cz; Rittich, Bohuslav [Masaryk University Brno, Tvrdeho 14, 602 00 Brno (Czech Republic)]. E-mail: rittich@sci.muni.cz; Spanova, Alena [Masaryk University Brno, Tvrdeho 14, 602 00 Brno (Czech Republic)]. E-mail: spanova@sci.muni.cz

    2007-04-15

    Magnetite nanoparticles about 14nm in diameter were obtained by chemical coprecipitation of Fe(II) and Fe(III) salts with aqueous ammonia in the presence of poly(ethylene glycol) (PEG). Magnetic poly(glycidyl methacrylate) (PGMA) microspheres about 1{mu}m in diameter were prepared by dispersion polymerization of GMA in aqueous ethanol in the presence of PEG-coated magnetite nanoparticles. The microspheres were hydrolyzed and carboxyl groups introduced by oxidation with KMnO{sub 4}. The particles reversibly bound bacterial DNA of Bifidobacterium and Lactobacillus genera in the presence of high concentrations of PEG 6000 and sodium chloride from crude cell lysates of various dairy products (butter milk, cheese, yoghurt, probiotic tablets) or from cell lyophilisates. The presence of Bifidobacterium and Lactobacillus DNA in samples was confirmed by PCR amplification.

  5. Carboxyl-functionalized magnetic microparticle carrier for isolation and identification of DNA in dairy products

    Science.gov (United States)

    Horák, Daniel; Rittich, Bohuslav; Španová, Alena

    2007-04-01

    Magnetite nanoparticles about 14 nm in diameter were obtained by chemical coprecipitation of Fe(II) and Fe(III) salts with aqueous ammonia in the presence of poly(ethylene glycol) (PEG). Magnetic poly(glycidyl methacrylate) (PGMA) microspheres about 1 μm in diameter were prepared by dispersion polymerization of GMA in aqueous ethanol in the presence of PEG-coated magnetite nanoparticles. The microspheres were hydrolyzed and carboxyl groups introduced by oxidation with KMnO4. The particles reversibly bound bacterial DNA of Bifidobacterium and Lactobacillus genera in the presence of high concentrations of PEG 6000 and sodium chloride from crude cell lysates of various dairy products (butter milk, cheese, yoghurt, probiotic tablets) or from cell lyophilisates. The presence of Bifidobacterium and Lactobacillus DNA in samples was confirmed by PCR amplification.

  6. Lung function discordance in monozygotic twins and associated differences in blood DNA methylation

    DEFF Research Database (Denmark)

    Bolund, Anneli C S; Starnawska, Anna; Miller, Martin R

    2017-01-01

    anatomical structure and combination of various environmental factors; however, the exact molecular mechanisms contributing to this decline are not fully understood. DNA methylation is an epigenetic modification that changes across individual's lifetime, as well as allows for interplay between environmental...... and tumour-suppressor/pro-oncogenic mechanisms. Change in FEV1 during the 11-year follow-up period was associated with blood DNA methylation level in TRIM27 gene (p value = 1.55 × 10-6), a negative regulator of CD4 T cells, and also involved in cancer development. Several enriched pathways were identified......, especially for FEV1, with one being "TGFBR" (Benjamini-Hochbergadj p value = 0.045), the receptor for TGFβ, a growth factor involved in normal lung tissue repair through pro-fibrotic effects. Conclusions: Our findings suggest that epigenetic regulation of immunological- and cancer-related genes, as well...

  7. DNA Microarray Assay Helps to Identify Functional Genes Specific for Leukemia Stem Cells

    Directory of Open Access Journals (Sweden)

    Haojian Zhang

    2013-01-01

    Full Text Available Chronic myeloid leukemia (CML is a myeloproliferative disease derived from an abnormal hematopoietic stem cell (HSC and is consistently associated with the formation of Philadelphia (Ph chromosome. Tyrosine kinase inhibitors (TKIs are highly effective in treating chronic phase CML but do not eliminate leukemia stem cells (LSCs, which are believed to be related to disease relapse. Therefore, one major challenge in the current CML research is to understand the biology of LSCs and to identify the molecular difference between LSCs and its normal stem cell counterparts. Comparing the gene expression profiles between LSCs and normal HSCs by DNA microarray assay is a systematic and unbiased approach to address this issue. In this paper, we present a DNA microarray dataset for CML LSCs and normal HSCs to show that the microarray assay will benefit the current and future studies of the biology of CML stem cells.

  8. Two-dimensional self-assembly of DNA-functionalized gold nanoparticles

    Science.gov (United States)

    Wang, Wenjie; Zhang, Honghu; Hagen, Noah; Kuzmenko, Ivan; Akinc, Mufit; Travesset, Alex; Mallapragada, Surya; Vaknin, David

    2D superlattices of nanoparticles (NPs) are promising candidates for nano-devices. It is still challenging to develop a simple yet efficient protocol to assemble NPs in a controlled manner. Here, we report on formation of 2D Gibbs monolayers of single-stranded DNA-coated gold nanoparticles (ssDNA-AuNPs) at the air-water interface by manipulation of salts contents. MgCl2 and CaCl2 in solutions facilitate the accumulation of the non-complementary ssDNA-AuNPs on aqueous surfaces. Grazing-incidence small-angle X-ray scattering (GISAXS) and X-ray reflectivity show that the surface AuNPs assembly forms a mono-particle layer and undergoes a transformation from short-range to long-range (hexagonal) order above a threshold of [MgCl2] or [CaCl2]. For solutions that include two kinds of ssDNA-AuNPs with complementary base-pairing, the surface AuNPs form a thicker film and only in-plane short-range order is observed. By using other salts (NaCl or LaCl3) at concentrations of similar ionic strength to those of MgCl2 or CaCl2, we find that surface adsorbed NPs lack any orders. X-ray fluorescence measurements provide direct evidence of surface enrichment of AuNPs and divalent ions (Ca2 +) . The work was supported by the Office of Basic Energy Sciences, USDOE under Contract No. DE-AC02-07CH11358 and DE-AC02-06CH11357.

  9. One-step synthesis of DNA functionalized cadmium-free quantum dots and its application in FRET-based protein sensing

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Cuiling, E-mail: clzhang@chem.ecnu.edu.cn [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Ding, Caiping [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Zhou, Guohua [School of Chemistry and Chemical Engineering, Lingnan Normal University, Zhanjiang, 524048 (China); Xue, Qin [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Xian, Yuezhong, E-mail: yzxian@chem.ecnu.edu.cn [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China)

    2017-03-08

    DNA functionalized quantum dots (QDs) are promising nanoprobes for the fluorescence resonance energy transfer (FRET)-based biosensing. Herein, cadmium-free DNA functionalized Mn-doped ZnS (DNA-ZnS:Mn{sup 2+}) QDs were successfully synthesized by one-step route. As-synthesized QDs show excellent photo-stability with the help of PAA and DNA. Then, we constructed a novel FRET model based on the QDs and WS{sub 2} nanosheets as the energy donor-acceptor pairs, which was successfully applied for the protein detection through the terminal protection of small molecule-linked DNA assay. This work not only explores the potential bioapplication of the DNA-ZnS:Mn{sup 2+} QDs, but also provides a platform for the investigation of small molecule-protein interaction. - Highlights: • The stable and cadmium-free DNA functionalized ZnS:Mn{sup 2+} QDs were successfully synthesized through a facile one-step route. • We constructed a novel FRET system based on one-step synthesized DNA-ZnS:Mn{sup 2+} QDs (donor) and WS{sub 2} nanosheets (acceptor). • The FRET-based strategy was applied for the detection of streptavidin and folate receptor by combining TPSMLD and Exo III.

  10. Bacteriophage Nf DNA region controlling late transcription: structural and functional homology with bacteriophage phi 29.

    Science.gov (United States)

    Nuez, B; Salas, M

    1993-06-25

    The putative region for the control of late transcription of the Bacillus subtilis phage Nf has been identified by DNA sequence homology with the equivalent region of the evolutionary related phage phi 29. A similar arrangement of early and late promoters has been detected in the two phages, suggesting that viral transcription could be regulated in a similar way at late times of the infection. Transcription of late genes requires the presence of a viral early protein, gpF in phage Nf and p4 in phage phi 29, being the latter known to bind to a DNA region located upstream from the phage phi 29 late promoter. We have identified a DNA region located upstream from the putative late promoter of phage Nf that is probably involved in binding protein gpF. Furthermore, we show that the phage phi 29 protein p4 is able to bind to this region and activate transcription from the phage Nf putative late promoter. Sequence alignment has also revealed the existence of significant internal homology between the two early promoters contained in this region of each phage.

  11. Thermodynamic, structural and fluorescent characteristics of DNA hairpins containing functionalized pyrrolo-2'-deoxycytidines.

    Science.gov (United States)

    Jahnz-Wechmann, Zofia; Lisowiec-Wachnicka, Jolanta; Framski, Grzegorz; Kosman, Joanna; Boryski, Jerzy; Pasternak, Anna

    2017-04-01

    Herein, we present comprehensive physicochemical and structural analysis of various DNA hairpins modified with pyrrolo-2'-deoxycytidine (Py-dC) derivatives. The introduction of modified Py-dC in most cases causes minor decrease of hairpin thermodynamic stability. The energetically unfavorable effect is more pronounced when modified residue is present within hairpin loop. Our studies indicate that thermodynamic effects induced by all Py-dC derivatives are net results of increased stacking interactions caused by larger surface of pyrrolo-2'-deoxycytidine aromatic ring and unfavorable effect implied by the presence of additional side chains. The CD spectra of all modified hairpins are similar to unmodified hairpin indicating that the presence of Py-dC derivatives does not disrupt the secondary structure of DNA. Interestingly, the presence of various side chains can increase fluorescent discrimination of paired and unpaired regions of DNA. The fluorescence observed for hairpins modified within loop is significantly quenched when Py-dC derivative is present in the stem region. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. A Rationally Designed Connector for Assembly of Protein-Functionalized DNA Nanostructures.

    Science.gov (United States)

    Koßmann, Katja J; Ziegler, Cornelia; Angelin, Alessandro; Meyer, Rebecca; Skoupi, Marc; Rabe, Kersten S; Niemeyer, Christof M

    2016-06-16

    We report on the rational engineering of the binding interface of the self-ligating HaloTag protein to generate an optimized linker for DNA nanostructures. Five amino acids positioned around the active-site entry channel for the chlorohexyl ligand (CH) of the HaloTag protein were exchanged for positively charged lysine amino acids to produce the HOB (halo-based oligonucleotide binder) protein. HOB was genetically fused with the enzyme cytochrome P450 BM3, as well as with BMR, the separated reductase domain of BM3. The resulting HOB-fusion proteins revealed significantly improved rates in ligation with CH-modified oligonucleotides and DNA origami nanostructures. These results suggest that the efficient self-assembly of protein-decorated DNA structures can be greatly improved by fine-tuning of the electrostatic interactions between proteins and the negatively charged nucleic acid nanostructures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

    Directory of Open Access Journals (Sweden)

    Halkier Barbara A

    2006-10-01

    Full Text Available Abstract Background We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector. Results Injection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized. Conclusion Expression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified.

  14. Assessment of DNA contamination from dried blood spots and determination of DNA yield and function using archival newborn dried blood spots.

    Science.gov (United States)

    Cordovado, Suzanne K; Earley, Marie C; Hendrix, Miyono; Driscoll-Dunn, Rena; Glass, Michael; Mueller, Patricia W; Hannon, W Harry

    2009-04-01

    Residual dried blood spots (DBS) from newborn screening programs are often stored for years and are sometimes used for epidemiological studies. Because there is potential for DNA cross-contamination from specimen-to-specimen contact, we determined contamination levels following intentional contact and assessed archival DBS DNA degradation after storage in an uncontrolled environment. DBS from healthy adult females were rubbed with DBS from healthy or cystic fibrosis (CF)-affected adult males. Total human and male DNA was measured from the female DBS. Contamination levels were assessed using short tandem repeats (STRs). Female DBS contaminated with CF male DNA containing the F508del were analyzed for presence of this mutation. Archival DBS DNA amplification efficiency was determined using STR analysis. Most female DBS were contaminated, however only one specimen showed an incomplete STR profile consistent with contaminating CF-affected male DNA. Further testing by CF mutation screening was negative. DNA extracted from archival DBS showed robust amplification (range 100 bp-320 bp). Lightly abrasive contact between DBS resulted in DNA cross-contamination. The contaminating DNA did not interfere in CF-mutation tests; however this should be determined for individual assays. Since DNA from archival DBS robustly amplifies, newborn DBS could provide an invaluable resource for public health studies.

  15. Functionalized gold nanoparticles as additive to form polymer/metal composite matrix for improved DNA sequencing by capillary electrophoresis.

    Science.gov (United States)

    Zhou, Dan; Yang, Liping; Yang, Runmiao; Song, Weihua; Peng, Shuhua; Wang, Yanmei

    2009-11-15

    A new matrix additive, poly (N,N-dimethylacrylamide)-functionalized gold nanoparticle (GNP-PDMA), was prepared by "grafting-to" approach, and then incorporated into quasi-interpenetrating network (quasi-IPN) composed of linear polyacrylamide (LPA, 3.3 MDa) and PDMA to form novel polymer/metal composite sieving matrix (quasi-IPN/GNP-PDMA) for DNA sequencing by capillary electrophoresis. Without complete optimization, quasi-IPN/GNP-PDMA yielded a readlength of 801 bases at 98% accuracy in about 64 min by using the ABI 310 Genetic Analyzer at 50 degrees C and 150 V/cm. Compared with previous quasi-IPN/GNPs, quasi-IPN/GNP-PDMA can further improve DNA sequencing performances. This is because the presence of GNP-PDMA can improve the compatibility of GNPs with the whole sequencing system, enhance the entanglement degree of networks, and increase the GNP concentration in system, which consequently lead to higher restriction and stability, higher apparent molecular weight (MW), and smaller pore size of the total sieving networks. Furthermore, the composite matrix was also compared with quasi-IPN containing higher-MW LPA and commercial POP-6. The results indicate that the composite matrix is a promising one for DNA sequencing to achieve full automation due to the separation provided with high resolution, speediness, excellent reproducibility, and easy loading in the presence of GNP-PDMA.

  16. Gestational exposure to diethylstilbestrol alters cardiac structure/function, protein expression and DNA methylation in adult male mice progeny

    Energy Technology Data Exchange (ETDEWEB)

    Haddad, Rami, E-mail: rami.haddad@mail.mcgill.ca [Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montréal, Québec, Canada H3T 1E2 (Canada); Division of Experimental Medicine, Department of Medicine, McGill University, 850 Sherbrooke Street, Montréal, Québec, Canada H3A 1A2 (Canada); Kasneci, Amanda, E-mail: amanda.kasneci@mail.mcgill.ca [Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montréal, Québec, Canada H3T 1E2 (Canada); Mepham, Kathryn, E-mail: katherine.mepham@mail.mcgill.ca [Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montréal, Québec, Canada H3T 1E2 (Canada); Division of Experimental Medicine, Department of Medicine, McGill University, 850 Sherbrooke Street, Montréal, Québec, Canada H3A 1A2 (Canada); Sebag, Igal A., E-mail: igal.sebag@mcgill.ca [Division of Cardiology, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montréal, Québec, Canada H3T 1E2 (Canada); and others

    2013-01-01

    Pregnant women, and thus their fetuses, are exposed to many endocrine disruptor compounds (EDCs). Fetal cardiomyocytes express sex hormone receptors making them potentially susceptible to re-programming by estrogenizing EDCs. Diethylstilbestrol (DES) is a proto-typical, non-steroidal estrogen. We hypothesized that changes in adult cardiac structure/function after gestational exposure to the test compound DES would be a proof in principle for the possibility of estrogenizing environmental EDCs to also alter the fetal heart. Vehicle (peanut oil) or DES (0.1, 1.0 and 10.0 μg/kg/da.) was orally delivered to pregnant C57bl/6n dams on gestation days 11.5–14.5. At 3 months, male progeny were left sedentary or were swim trained for 4 weeks. Echocardiography of isoflurane anesthetized mice revealed similar cardiac structure/function in all sedentary mice, but evidence of systolic dysfunction and increased diastolic relaxation after swim training at higher DES doses. The calcium homeostasis proteins, SERCA2a, phospholamban, phospho-serine 16 phospholamban and calsequestrin 2, are important for cardiac contraction and relaxation. Immunoblot analyses of ventricle homogenates showed increased expression of SERCA2a and calsequestrin 2 in DES mice and greater molecular remodeling of these proteins and phospho-serine 16 phospholamban in swim trained DES mice. DES increased cardiac DNA methyltransferase 3a expression and DNA methylation in the CpG island within the calsequestrin 2 promoter in heart. Thus, gestational DES epigenetically altered ventricular DNA, altered cardiac function and expression, and reduced the ability of adult progeny to cardiac remodel when physically challenged. We conclude that gestational exposure to estrogenizing EDCs may impact cardiac structure/function in adult males. -- Highlights: ► Gestational DES changes cardiac SERCA2a and CASQ2 expression. ► Echocardiography identified systolic dysfunction and increased diastolic relaxation. ► DES

  17. Transient oxidative stress and inflammation after intraperitoneal administration of multiwalled carbon nanotubes functionalized with single strand DNA in rats

    Energy Technology Data Exchange (ETDEWEB)

    Clichici, Simona, E-mail: simonaclichici@yahoo.com [Department of Physiology, University of Medicine and Pharmacy, Cluj-Napoca (Romania); Biris, Alexandru Radu [National R and D Institute of Isotopic and Molecular Technologies, Cluj-Napoca (Romania); Tabaran, Flaviu [University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca (Romania); Filip, Adriana [Department of Physiology, University of Medicine and Pharmacy, Cluj-Napoca (Romania)

    2012-03-15

    Multi-walled carbon nanotubes (MWCNTs) are widely used for nanotechnology. Their impact on living organisms is, however, not entirely clarified. Oxidative stress and inflammation seem to be the key mechanisms involved in MWCNTs' cytotoxicity. Until present, pulmonary and skin models were the main tested experimental designs to assess carbon nanotubes' toxicity. The systemic administration of MWCNTs is essential, with respect for future medical applications. Our research is performed on Wistar rats and is focused on the dynamics of oxidative stress parameters in blood and liver and pro-inflammatory cytokines in liver, after single dose (270 mg l{sup −1}) ip administration of MWCNTs (exterior diameter 15–25 nm, interior diameter 10–15 nm, surface 88 m{sup 2} g{sup −1}) functionalized with single strand DNA (ss-DNA). The presence of MWCNTs in blood was assessed by Raman spectroscopy, while in liver histological examination and confocal microscopy were used. It was found that ss-DNA-MWCNTs induce oxidative stress in plasma and liver, with the return of the tested parameters to normal values, 6 h after ip injection of nanotubes, with the exception of reduced glutathione in plasma. The inflammatory cytokines (TNF-α, IL-1β) had a similar pattern of evolution. We also assessed the level of ERK1/2 and the phosphorylation of p65 subunit of NF-kB in liver that had a transient increase and returned to normal at the end of the tested period. Our results demonstrate that ss-DNA-MWCNTs produce oxidative stress and inflammation, but with a transient pattern. Given the fact that antioxidants modify the profile not only for oxidative stress, but also of inflammation, the dynamics of these alterations may be of practical importance for future protective strategies. -- Highlights: ► ss-DNA-MWCNTs ip administration induce oxidative stress in plasma and liver. ► ss-DNA-MWCNTs ip administration determine liver inflammation. ► ERK1/2 and p65 phosphorylated NF

  18. Crystal structure of family 4 uracil-DNA glycosylase from Sulfolobus tokodaii and a function of tyrosine 170 in DNA binding.

    Science.gov (United States)

    Kawai, Akito; Higuchi, Shigesada; Tsunoda, Masaru; Nakamura, Kazuo T; Yamagata, Yuriko; Miyamoto, Shuichi

    2015-09-14

    Uracil-DNA glycosylases (UDGs) excise uracil from DNA by catalyzing the N-glycosidic bond hydrolysis. Here we report the first crystal structures of an archaeal UDG (stoUDG). Compared with other UDGs, stoUDG has a different structure of the leucine-intercalation loop, which is important for DNA binding. The stoUDG-DNA complex model indicated that Leu169, Tyr170, and Asn171 in the loop are involved in DNA intercalation. Mutational analysis showed that Tyr170 is critical for substrate DNA recognition. These results indicate that Tyr170 occupies the intercalation site formed after the structural change of the leucine-intercalation loop required for the catalysis. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Identification of a high-efficiency baculovirus DNA replication origin that functions in insect and mammalian cells.

    Science.gov (United States)

    Wu, Yueh-Lung; Wu, Carol-P; Huang, Yu-Hui; Huang, Sheng-Ping; Lo, Huei-Ru; Chang, Hao-Shuo; Lin, Pi-Hsiu; Wu, Ming-Cheng; Chang, Chia-Jung; Chao, Yu-Chan

    2014-11-01

    The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143-3 fragment showed that subfragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143-3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori. Further study showed that the p143-3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, the p143-3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells. Baculovirus DNA replication relies on both hr and non-hr oris; however, so far very little is known about the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143 ori. This ori contains

  20. 8-Oxoguanine DNA glycosylase 1 (ogg1) maintains the function of cardiac progenitor cells during heart formation in zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Lifeng [State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029 (China); Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029 (China); Zhou, Yong [Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Yu, Shanhe [Shanghai Institute of Hematology, RuiJin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200025 (China); Ji, Guixiang [Nanjing Institute of Environmental Sciences/Key Laboratory of Pesticide Environmental Assessment and Pollution Control, Ministry of Environmental Protection, Nanjing 210042 (China); Wang, Lei [Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Liu, Wei [State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029 (China); Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029 (China); Gu, Aihua, E-mail: aihuagu@njmu.edu.cn [State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029 (China); Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029 (China)

    2013-11-15

    Genomic damage may devastate the potential of progenitor cells and consequently impair early organogenesis. We found that ogg1, a key enzyme initiating the base-excision repair, was enriched in the embryonic heart in zebrafish. So far, little is known about DNA repair in cardiogenesis. Here, we addressed the critical role of ogg1 in cardiogenesis for the first time. ogg1 mainly expressed in the anterior lateral plate mesoderm (ALPM), the primary heart tube, and subsequently the embryonic myocardium by in situ hybridisation. Loss of ogg1 resulted in severe cardiac morphogenesis and functional abnormalities, including the short heart length, arrhythmia, decreased cardiomyocytes and nkx2.5{sup +} cardiac progenitor cells. Moreover, the increased apoptosis and repressed proliferation of progenitor cells caused by ogg1 deficiency might contribute to the heart phenotype. The microarray analysis showed that the expression of genes involved in embryonic heart tube morphogenesis and heart structure were significantly changed due to the lack of ogg1. Among those, foxh1 is an important partner of ogg1 in the cardiac development in response to DNA damage. Our work demonstrates the requirement of ogg1 in cardiac progenitors and heart development in zebrafish. These findings may be helpful for understanding the aetiology of congenital cardiac deficits. - Highlights: • A key DNA repair enzyme ogg1 is expressed in the embryonic heart in zebrafish. • We found that ogg1 is essential for normal cardiac morphogenesis in zebrafish. • The production of embryonic cardiomyocytes requires appropriate ogg1 expression. • Ogg1 critically regulated proliferation of cardiac progenitor cells in zebrafish. • foxh1 is a partner of ogg1 in the cardiac development in response to DNA damage.

  1. Molecular Cloning and Functional Expression of Chitinase-Encoding cDNA from the Cabbage Moth, Mamestra brassicae

    Science.gov (United States)

    Paek, Aron; Park, Hee Yun; Jeong, Seong Eun

    2012-01-01

    Chitinase is a rate-limiting and endo-splitting enzyme involved in the bio-degradation of chitin, an important component of the cuticular exoskeleton and peritrophic matrix in insects. We isolated a cDNA-encoding chitinase from the last larval integument of the cabbage moth, Mamestra brassicae (Lepidoptera; Noctuidae), cloned the ORF cDNA into E. coli to confirm its functionality, and analyzed the deduced amino acid sequence in comparison with previously described lepidopteran chitinases. M. brassicae chitinase expressed in the transformed E. coli cells with the chitinase-encoding cDNA enhanced cell proliferation to about 1.6 times of the untransformed wild type strain in a colloidal chitin-including medium with only a very limited amount of other nutrients. Compared with the wild type strain, the intracellular levels of chitin degradation derivatives, glucosamine and N-acetylglucosamine were about 7.2 and 2.3 times higher, respectively, while the extracellular chitinase activity was about 2.2 times higher in the transformed strain. The ORF of M. brassicae chitinase-encoding cDNA consisted of 1686 nucleotides (562 amino acid residues) except for the stop codon, and its deduced amino acid composition revealed a calculated molecular weight of 62.7 and theoretical pI of 5.3. The ORF was composed of N-terminal leading signal peptide (AA 1-20), catalytic domain (AA 21–392), linker region (AA 393–498), and C-terminal chitin-binding domain (AA 499–562) showing its characteristic structure as a molting fluid chitinase. In phylogenetic analysis, the enzymes from 6 noctuid species were grouped together, separately from a group of 3 bombycid and 1 tortricid enzymes, corresponding to their taxonomic relationships at both the family and genus levels. PMID:22124732

  2. Novel base-functionalized DNA. Efficient methodology for construction and bioanalytical applications

    Czech Academy of Sciences Publication Activity Database

    Hocek, Michal; Vrábel, Milan; Cahová, Hana; Riedl, Jan; Kalachová, Lubica; Pivoňková, Hana; Horáková Brázdilová, Petra; Havran, Luděk; Fojta, Miroslav

    -, č. 52 (2008), s. 53-54 ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA ČR GA203/07/1195; GA MŠk LC512; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507 Keywords : cross-coupling reactions * nucleoside triphosphates * DNA Subject RIV: CC - Organic Chemistry

  3. Addressing RNA Integrity to Determine the Impact of Mitochondrial DNA Mutations on Brain Mitochondrial Function with Age

    OpenAIRE

    Wei Wang; Katja Scheffler; Ying Esbensen; Strand, Janne M.; Stewart, James B.; Magnar Bjørås; Lars Eide

    2014-01-01

    Mitochondrial DNA (mtDNA) mutations can result in mitochondrial dysfunction, but emerging experimental data question the fundamental role of mtDNA mutagenesis in age-associated mitochondrial impairment. The multicopy nature of mtDNA renders the impact of a given mtDNA mutation unpredictable. In this study, we compared mtDNA stability and mtRNA integrity during normal aging. Seven distinct sites in mouse brain mtDNA and corresponding mtRNA were analyzed. Accumulation of mtDNA mutations during ...

  4. MRE11–RAD50–NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair

    Science.gov (United States)

    Roques, Céline; Coulombe, Yan; Delannoy, Mathieu; Vignard, Julien; Grossi, Simona; Brodeur, Isabelle; Rodrigue, Amélie; Gautier, Jean; Stasiak, Alicja Z; Stasiak, Andrzej; Constantinou, Angelos; Masson, Jean-Yves

    2009-01-01

    Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response. PMID:19609304

  5. High Functional Stability of a Low-cost HBV DNA qPCR Primer Pair and Plasmid Standard

    Science.gov (United States)

    García, Gerardo; León, Yamila; Canales, Eduardo; Angel Silva, José; Gell, Omar; Estrada, Regla; Morán, Ivis; Muzio, Verena; Guillén, Gerardo; Pentón, Eduardo; Aguilar, Julio Cesar

    2016-01-01

    ABSTRACT Aim We studied the functional stability of a primer pair and the standard curve based on a plasmid carrying full-length HBV genome, from a novel low-cost real-time quantitative polymerase chain reaction (qPCR) assay. The assay was developed at the Center for Genetic Engineering and Biotechnology (CIGB) in Havana, to quantify the serum hepatitis B virus (HBV) DNA from chronic HBV-infected (CHB) patients. Materials and methods In-house generated oligonucleotides and plasmids were incubated at 37°C during 1 month and compared with the same materials incubated at –20, 4, and 25°C during the same time in qPCR experiments. Results This work shows that the oligonucleotide pair and the plasmid for the quantitative standard curve are functionally stable in severe temperature conditions during 1 month. Polymerase chain reaction amplification with both materials after its incubation 30 days at 37°C produced similar cycle threshold (CT) values and similar degree of sample quantifications compared with the same materials preserved using the conventional storage conditions at –20°C. Conclusion These results are indicative of the robustness of this low-cost qPCR system for HBV DNA quantification. These results also support that this qPCR assay can be used as a low-cost technology in clinical studies to monitor the viral load changes of serum HBV DNA of CHB patients, which could be used by poor people of third world countries, where there are frequent blackouts and temperature changes that can hinder the primer and plasmid stability. How to cite this article Aguiar J, García G, León Y, Canales E, Silva JA, Gell O, Estrada R, Morán I, Muzio V, Guillén G, Pentón E, Aguilar JC. High Functional Stability of a Low-cost HBV DNA qPCR Primer Pair and Plasmid Standard. Euroasian J Hepato-Gastroenterol 2016;6(1):19-24. PMID:29201719

  6. Phylogenetic and Functional Diversity of Total (DNA) and Expressed (RNA) Bacterial Communities in Urban Green Infrastructure Bioswale Soils.

    Science.gov (United States)

    Gill, Aman S; Lee, Angela; McGuire, Krista L

    2017-08-15

    New York City (NYC) is pioneering green infrastructure with the use of bioswales and other engineered soil-based habitats to provide stormwater infiltration and other ecosystem functions. In addition to avoiding the environmental and financial costs of expanding traditional built infrastructure, green infrastructure is thought to generate cobenefits in the form of diverse ecological processes performed by its plant and microbial communities. Yet, although plant communities in these habitats are closely managed, we lack basic knowledge about how engineered ecosystems impact the distribution and functioning of soil bacteria. We sequenced amplicons of the 16S ribosomal subunit, as well as seven genes associated with functional pathways, generated from both total (DNA-based) and expressed (RNA) soil communities in the Bronx, NYC, NY, in order to test whether bioswale soils host characteristic bacterial communities with evidence for enriched microbial functioning, compared to nonengineered soils in park lawns and tree pits. Bioswales had distinct, phylogenetically diverse bacterial communities, including taxa associated with nutrient cycling and metabolism of hydrocarbons and other pollutants. Bioswale soils also had a significantly greater diversity of genes involved in several functional pathways, including carbon fixation ( cbbL-R [ cbbL gene, red-like subunit] and apsA ), nitrogen cycling ( noxZ and amoA ), and contaminant degradation ( bphA ); conversely, no functional genes were significantly more abundant in nonengineered soils. These results provide preliminary evidence that urban land management can shape the diversity and activity of soil communities, with positive consequences for genetic resources underlying valuable ecological functions, including biogeochemical cycling and degradation of common urban pollutants. IMPORTANCE Management of urban soil biodiversity by favoring taxa associated with decontamination or other microbial metabolic processes is a

  7. ssDNA-Functionalized Nanoceria: A Redox-Active Aptaswitch for Biomolecular Recognition.

    Science.gov (United States)

    Bülbül, Gonca; Hayat, Akhtar; Andreescu, Silvana

    2016-04-06

    Quantification of biomolecular binding events is a critical step for the development of biorecognition assays for diagnostics and therapeutic applications. This paper reports the design of redox-active switches based on aptamer conjugated nanoceria for detection and quantification of biomolecular recognition. It is shown that the conformational transition state of the aptamer on nanoceria, combined with the redox properties of these particles can be used to create surface based structure switchable aptasensing platforms. Changes in the redox properties at the nanoceria surface upon binding of the ssDNA and its target analyte enables rapid and highly sensitive measurement of biomolecular interactions. This concept is demonstrated as a general applicable method to the colorimetric detection of DNA binding events. An example of a nanoceria aptaswitch for the colorimetric sensing of Ochratoxin A (OTA) and applicability to other targets is provided. The system can sensitively and selectivity detect as low as 0.15 × 10(-9) m OTA. This novel assay is simple in design and does not involve oligonucleotide labeling or elaborate nanoparticle modification steps. The proposed mechanism discovered here opens up a new way of designing optical sensing methods based on aptamer recognition. This approach can be broadly applicable to many bimolecular recognition processes and related applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments.

    Science.gov (United States)

    Villbrandt, B; Sagner, G; Schomburg, D

    1997-11-01

    The thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) has been truncated to molecular regions essential for polymerase activity. Two truncated forms of the full-length 832 amino acid Taq polymerase have been constructed according to sequence alignments and the known domain structure of the homologous Escherichia coli DNA polymerase I (E.coli pol I): variant delta288 (lacking the N-terminal 288 amino acid portion) and variant delta413 (lacking the N-terminal 413 amino acid portion). Both protein fragments were stable and showed polymerase activity, albeit specific activity and thermostability of the variant delta413 were significantly decreased compared with the full length Taq polymerase. In order to increase the thermostability of the variant delta413, a three-dimensional model of the polymerase domain of Taq polymerase was built by homology with a model of the Klenow fragment of the E.coli pol I based on the available Calpha coordinates. Consequently two variants were designed and constructed using site-directed mutagenesis. The strategies used were deletion of 10 flexible amino acids and replacement of two hydrophobic amino acids on the surface by more hydrophilic ones. Compared with the initial protein fragment, both variant enzymes showed an increase in polymerase activity and thermostability. After the completion of this work, X-ray coordinates of the Taq polymerase became available from the protein structure data bank. A comparison between the homology model and the experimental three-dimensional structure proved the quality of the model.

  9. Ultrasensitive electrochemical DNA biosensor based on functionalized gold clusters/graphene nanohybrids coupling with exonuclease III-aided cascade target recycling.

    Science.gov (United States)

    Wang, Wei; Bao, Ting; Zeng, Xi; Xiong, Huayu; Wen, Wei; Zhang, Xiuhua; Wang, Shengfu

    2017-05-15

    In this work, a novel and ultrasensitive electrochemical biosensor was constructed for DNA detection based on functionalized gold clusters/graphene nanohybrids (AuNCs/GR nanobybrids) and exonuclease III (Exo III)-aided cascade target recycling. By utilizing the capacity of GR as universal template, different metal nanoclusters including AuNCs/GR nanobybrids and PtNCs/GR nanohybrids were synthesized through convenient ultrasonic method. Exo III-aided cascade recycling was initiated by target DNA, generating the final cleavage product (S2), which acted as a linkage between capture probe and the functionalized metal nanoclusters/GR conjugates in the construction of the biosensor. The AuNCs/GR-DNA-enzyme conjugates acted as interfaces of enzyme-catalyzed silver deposition reaction, achieving DNA detection ranging from 0.02 fM to 20 pM with a detection limit of 0.057 fM. In addition, PtNCs/GR-DNA conjugates presented peroxidase-like activity and the functionalized PtNCs/GR nanohybrids-based electrochemical biosensor also realized DNA detection by catalyzing the 3,3',5,5'-tetramethylbenzidine-hydrogen peroxide (TMB-H2O2) system to produce electrochemical signal. This metal clusters/GR-based multiple-amplified electrochemical biosensor provided an universal method for DNA detection. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    Energy Technology Data Exchange (ETDEWEB)

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  11. Identifying gene-independent noncoding functional elements in the yeast ribosomal DNA by phylogenetic footprinting.

    Science.gov (United States)

    Ganley, Austen R D; Hayashi, Kouji; Horiuchi, Takashi; Kobayashi, Takehiko

    2005-08-16

    Sequences involved in the regulation of genetic and genomic processes are primarily located in noncoding regions. Identifying such cis-acting sequences from sequence data is difficult because their patterns are not readily apparent, and, to date, identification has concentrated on regions associated with gene-coding functions. Here, we used phylogenetic footprinting to look for gene-independent noncoding elements in the ribosomal RNA gene repeats from several Saccharomyces species. Similarity plots of ribosomal intergenic spacer alignments from six closely related Saccharomyces species allowed the identification of previously characterized functional elements, such as the origin-of-replication and replication-fork barrier sites, demonstrating that this method is a powerful predictor of noncoding functional elements. Seventeen previously uncharacterized elements, showing high levels of conservation, were also discovered. The conservation of these elements suggests that they are functional, and we demonstrate the functionality of two classes of these elements, a putative bidirectional noncoding promoter and a series of conserved peaks with matches to the origin-of-replication core consensus. Our results paint a comprehensive picture of the functionality of the Saccharomyces ribosomal intergenic region and demonstrate that functional elements not involved in gene-coding function can be identified by using comparative genomics based on sequence conservation.

  12. Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts.

    Science.gov (United States)

    Ershova, E S; Sergeeva, V A; Chausheva, A I; Zheglo, D G; Nikitina, V A; Smirnova, T D; Kameneva, L V; Porokhovnik, L N; Kutsev, S I; Troshin, P A; Voronov, I I; Khakina, E A; Veiko, N N; Kostyuk, S V

    2016-07-01

    Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25μM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-β, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Structural and functional analysis of DNA sequences with potential for forming G-quadruplex

    OpenAIRE

    Luciana Souto Mofatto

    2013-01-01

    Resumo: Os G-quadruplexes são estruturas secundárias de DNA altamente organizadas, constituídas por sequências ricas em guaninas capazes de formar tétrades ligadas por pontes de hidrogênio. Essas sequências são capazes de modular a transcrição gênica e o splicing alternativo de éxons. Além disso, estudos também mostraram que os G-quadruplexes estão presentes na região promotora de oncogenes (como c-MYC) e nas regiões terminais dos telômeros, indicando que o G-quadruplex pode ser um possível a...

  14. Functions of Burkholderia virulence factors: Input from proteomics and DNA microarray analyses

    Directory of Open Access Journals (Sweden)

    Kumutha Malar Vellasamy1

    2012-03-01

    Full Text Available Burkholderia spp. consists of organisms that are extremely diverse and versatile with a natural habitat in the soil. Members of this genus, which include B. pseudomallei, B. mallei, B. thailandensis and B. cepacia, are capable of causing severe, life threatening opportunistic infection in patients who are immunocompromised. The underlying virulence mechanisms of the bacteria, their interactions with the host and the host defense mechanisms may be reflected by changes of the expression of proteins of both the pathogen as well as the host. In this article, we reviewed the current knowledge on interactions of Burkholderia spp. pathogens with their host mainly from the perspective of data that was generated from recent proteomics and DNA microarray investigations.

  15. Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein

    DEFF Research Database (Denmark)

    Bendtsen, Jannick Dyrløv; Nilsson, A.S.; Lehnherr, H.

    2002-01-01

    Bacteriophage P1 encodes a single-stranded DNA-binding protein (SSB-P1), which shows 66% amino acid sequence identity to the SSB protein of the host bacterium Escherichia coli. A phylogenetic analysis indicated that the P1 ssb gene coexists with its E. coli counterpart as an independent unit....... Expression studies showed that the P1 ssb gene is transcribed only, in an rpoS-independent fashion, during stationary-phase growth in E. coli. Mixed infection experiments demonstrated that a wild-type phage has a selective advantage over an ssb-null mutant when exposed to a bacterial host in the stationary...... phase. These results reconciled the observed evolutionary conservation with the seemingly redundant presence of ssb genes in many bacteriophages and conjugative plasmids....

  16. Isolation and functional characterization of DNA damage repair protein (DRT) from Lepidium latifolium L.

    Science.gov (United States)

    Sinha, Vimlendu Bhushan; Grover, Atul; Ahmed, Zakwan; Pande, Veena

    2014-05-01

    We have isolated and in silico characterized a cold regulated plastocyanin encoding gene from Lepidium latifolium L designated as LlaDRT. Its cDNA sequence (JN214346) consists of a 504 bp ORF, 48 and 205 bp of 5' and 3' UTR regions, respectively encoding a protein of 17.07 KDa and pI 4.95. In silico and phylogenetic analysis of LlaDRT suggested that the protein has features of a typical plastocyanin family member and of a nearest relative of the predominant isoform of Arabidopsis (PETE2) plastocyanin. Validation of stress response of LlaDRT by qPCR under different abiotic stress regulators viz salicylic acid, jasmonic acid, calcium chloride, ethylene and abscisic acid revealed its possible regulation and crosstalk amongst different pathways. Copyright © 2014 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  17. Cohesin Function in Cohesion, Condensation, and DNA Repair Is Regulated by Wpl1p via a Common Mechanism inSaccharomyces cerevisiae.

    Science.gov (United States)

    Bloom, Michelle S; Koshland, Douglas; Guacci, Vincent

    2018-01-01

    Cohesin tethers DNA to mediate sister chromatid cohesion, chromosome condensation, and DNA repair. How the cell regulates cohesin to perform these distinct functions remains to be elucidated. One cohesin regulator, Wpl1p, was characterized in Saccharomyces cerevisiae as a promoter of efficient cohesion and an inhibitor of condensation. Wpl1p is also required for resistance to DNA-damaging agents. Here, we provide evidence that Wpl1p promotes the timely repair of DNA damage induced during S-phase. Previous studies have indicated that Wpl1p destabilizes cohesin's binding to DNA by modulating the interface between the cohesin subunits Mcd1p and Smc3p Our results suggest that Wpl1p likely modulates this interface to regulate all of cohesin's biological functions. Furthermore, we show that Wpl1p regulates cohesion and condensation through the formation of a functional complex with another cohesin-associated factor, Pds5p In contrast, Wpl1p regulates DNA repair independently of its interaction with Pds5p Together, these results suggest that Wpl1p regulates distinct biological functions of cohesin by Pds5p-dependent and -independent modulation of the Smc3p/Mcd1p interface. Copyright © 2018 by the Genetics Society of America.

  18. CDK2 and PKA mediated-sequential phosphorylation is critical for p19INK4d function in the DNA damage response.

    Directory of Open Access Journals (Sweden)

    Mariela C Marazita

    Full Text Available DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with (32P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage.

  19. SVD identifies transcript length distribution functions from DNA microarray data and reveals evolutionary forces globally affecting GBM metabolism.

    Directory of Open Access Journals (Sweden)

    Nicolas M Bertagnolli

    Full Text Available To search for evolutionary forces that might act upon transcript length, we use the singular value decomposition (SVD to identify the length distribution functions of sets and subsets of human and yeast transcripts from profiles of mRNA abundance levels across gel electrophoresis migration distances that were previously measured by DNA microarrays. We show that the SVD identifies the transcript length distribution functions as "asymmetric generalized coherent states" from the DNA microarray data and with no a-priori assumptions. Comparing subsets of human and yeast transcripts of the same gene ontology annotations, we find that in both disparate eukaryotes, transcripts involved in protein synthesis or mitochondrial metabolism are significantly shorter than typical, and in particular, significantly shorter than those involved in glucose metabolism. Comparing the subsets of human transcripts that are overexpressed in glioblastoma multiforme (GBM or normal brain tissue samples from The Cancer Genome Atlas, we find that GBM maintains normal brain overexpression of significantly short transcripts, enriched in transcripts that are involved in protein synthesis or mitochondrial metabolism, but suppresses normal overexpression of significantly longer transcripts, enriched in transcripts that are involved in glucose metabolism and brain activity. These global relations among transcript length, cellular metabolism and tumor development suggest a previously unrecognized physical mode for tumor and normal cells to differentially regulate metabolism in a transcript length-dependent manner. The identified distribution functions support a previous hypothesis from mathematical modeling of evolutionary forces that act upon transcript length in the manner of the restoring force of the harmonic oscillator.

  20. Adaptation and Impairment of DNA Repair Function in Pollen of Betula verrucosa and Seeds of Oenothera biennis from Differently Radionuclide-contaminated Sites of Chernobyl

    Science.gov (United States)

    Boubriak, I. I.; Grodzinsky, D. M.; Polischuk, V. P.; Naumenko, V. D.; Gushcha, N. P.; Micheev, A. N.; McCready, S. J.; Osborne, D. J.

    2008-01-01

    Background and Aims The plants that have remained in the contaminated areas around Chernobyl since 1986 encapsulate the effects of radiation. Such plants are chronically exposed to radionuclides that they have accumulated internally as well as to α-, β- and γ-emitting radionuclides from external sources and from the soil. This radiation leads to genetic damage that can be countered by DNA repair systems. The objective of this study is to follow DNA repair and adaptation in haploid cells (birch pollen) and diploid cells (seed embryos of the evening primrose) from plants that have been growing in situ in different radionuclide fall-out sites in monitored regions surrounding the Chernobyl explosion of 1986. Methods Radionuclide levels in soil were detected using gamma-spectroscopy and radiochemistry. DNA repair assays included measurement of unscheduled DNA synthesis, electrophoretic determination of single-strand DNA breaks and image analysis of rDNA repeats after repair intervals. Nucleosome levels were established using an ELISA kit. Key Results Birch pollen collected in 1987 failed to perform unscheduled DNA synthesis, but pollen at γ/β-emitter sites has now recovered this ability. At a site with high levels of combined α- and γ/β-emitters, pollen still exhibits hidden damage, as shown by reduced unscheduled DNA synthesis and failure to repair lesions in rDNA repeats properly. Evening primrose seed embryos generated on plants at the same γ/β-emitter sites now show an improved DNA repair capacity and ability to germinate under abiotic stresses (salinity and accelerated ageing). Again those from combined α- and γ/β-contaminated site do not show this improvement. Conclusions Chronic irradiation at γ/β-emitter sites has provided opportunities for plant cells (both pollen and embryo cells) to adapt to ionizing irradiation and other environmental stresses. This may be explained by facilitation of DNA repair function. PMID:17981881

  1. Visual and on-site detection of mercury(II) ions on lateral flow strips using DNA-functionalized gold nanoparticles.

    Science.gov (United States)

    Yang, Fei; Duan, Jing; Li, Min; Wang, Zebo; Guo, Zhiyong

    2012-01-01

    A test strip for detection of Hg(2+) in aqueous solution based on the DNA-functionalized gold nanoparticles (DNA-AuNPs) was developed and evaluated. When Hg(2+) ions were introduced, the biotinylated DNA(2) hybridized with thiolated DNA(1) functionalized on the AuNPs (DNA(1)-AuNPs) to form mismatch complexes through thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination. The formed mismatch complexes and excess DNA(1)-AuNPs could be captured on the test line formed by streptavidin and the control line formed by DNA(3)-BSA, respectively. Two red lines appeared due to the accumulation of AuNPs, enabling visual detection of Hg(2+) with a detection limit of about 6 nM. The assay results can be obtained within 5 min. The results show that the test strip has excellent sensitivity and selectivity for detection of Hg(2+); thus it holds a great potential for rapid, on-site and real time detection of Hg(2+).

  2. Interaction of electrons with cisplatin and the subsequent effect on DNA damage: a density functional theory study.

    Science.gov (United States)

    Chen, Hsing-Yin; Chen, Hui-Fen; Kao, Chai-Lin; Yang, Po-Yu; Hsu, Sodio C N

    2014-09-28

    Cisplatin, Pt(NH3)2Cl2, is a leading chemotherapeutic agent that has been widely used for various cancers. Recent experiments show that combining cisplatin and electron sources can dramatically enhance DNA damage and the cell-killing rate and, therefore, is a promising way to overcome the side effects and the resistance of cisplatin. However, the molecular mechanisms underlying this phenomenon are not clear yet. By using density functional theory calculations, we confirm that cisplatin can efficiently capture the prehydrated electrons and then undergo dissociation. The first electron attachment triggers a spontaneous departure of the chloride ion, forming a T-shaped [Pt(NH3)2Cl]˙ neutral radical, whereas the second electron attachment leads to a spontaneous departure of ammine, forming a linear [Pt(NH3)Cl](-) anion. We further recognize that the one-electron reduced product [Pt(NH3)2Cl]˙ is extremely harmful to DNA. It can abstract hydrogen atoms from the C-H bonds of the ribose moiety and the methyl group of thymine, which in turn leads to DNA strand breaks and cross-link lesions. The activation energies of these hydrogen abstraction reactions are relatively small compared to the hydrolysis of cisplatin, a prerequisite step in the normal mechanism of action of cisplatin. These results rationalize the improved cytotoxicity of cisplatin by supplying electrons. Although the biological effects of the two-electron reduced product [Pt(NH3)Cl](-) are not clear at this stage, our calculations indicate that it might be protonated by the surrounding water.

  3. Relationship of structure and function of DNA-binding domain in vitamin D receptor.

    Science.gov (United States)

    Wan, Lin-Yan; Zhang, Yan-Qiong; Chen, Meng-Di; Liu, Chang-Bai; Wu, Jiang-Feng

    2015-07-07

    While the structure of the DNA-binding domain (DBD) of the vitamin D receptor (VDR) has been determined in great detail, the roles of its domains and how to bind the motif of its target genes are still under debate. The VDR DBD consists of two zinc finger modules and a C-terminal extension (CTE), at the end of the C-terminal of each structure presenting α-helix. For the first zinc finger structure, N37 and S-box take part in forming a dimer with 9-cis retinoid X receptor (RXR), while V26, R50, P-box and S-box participate in binding with VDR response elements (VDRE). For the second zinc finger structure, P61, F62 and H75 are essential in the structure of the VDR homodimer with the residues N37, E92 and F93 of the downstream of partner VDR, which form the inter-DBD interface. T-box of the CTE, especially the F93 and I94, plays a critical role in heterodimerization and heterodimers-VDRE binding. Six essential residues (R102, K103, M106, I107, K109, and R110) of the CTE α-helix of VDR construct one interaction face, which packs against the DBD core of the adjacent symmetry mate. In 1,25(OH)2D3-activated signaling, the VDR-RXR heterodimer may bind to DR3-type VDRE and ER9-type VDREs of its target gene directly resulting in transactivation and also bind to DR3-liked nVDRE of its target gene directly resulting in transrepression. Except for this, 1α,25(OH)2D3 ligand VDR-RXR may bind to 1αnVDRE indirectly through VDIR, resulting in transrepression of the target gene. Upon binding of 1α,25(OH)2D3, VDR can transactivate and transrepress its target genes depending on the DNA motif that DBD binds.

  4. Relationship of Structure and Function of DNA-Binding Domain in Vitamin D Receptor

    Directory of Open Access Journals (Sweden)

    Lin-Yan Wan

    2015-07-01

    Full Text Available While the structure of the DNA-binding domain (DBD of the vitamin D receptor (VDR has been determined in great detail, the roles of its domains and how to bind the motif of its target genes are still under debate. The VDR DBD consists of two zinc finger modules and a C-terminal extension (CTE, at the end of the C-terminal of each structure presenting α-helix. For the first zinc finger structure, N37 and S-box take part in forming a dimer with 9-cis retinoid X receptor (RXR, while V26, R50, P-box and S-box participate in binding with VDR response elements (VDRE. For the second zinc finger structure, P61, F62 and H75 are essential in the structure of the VDR homodimer with the residues N37, E92 and F93 of the downstream of partner VDR, which form the inter-DBD interface. T-box of the CTE, especially the F93 and I94, plays a critical role in heterodimerization and heterodimers–VDRE binding. Six essential residues (R102, K103, M106, I107, K109, and R110 of the CTE α-helix of VDR construct one interaction face, which packs against the DBD core of the adjacent symmetry mate. In 1,25(OH2D3-activated signaling, the VDR-RXR heterodimer may bind to DR3-type VDRE and ER9-type VDREs of its target gene directly resulting in transactivation and also bind to DR3-liked nVDRE of its target gene directly resulting in transrepression. Except for this, 1α,25(OH2D3 ligand VDR-RXR may bind to 1αnVDRE indirectly through VDIR, resulting in transrepression of the target gene. Upon binding of 1α,25(OH2D3, VDR can transactivate and transrepress its target genes depending on the DNA motif that DBD binds.

  5. Identification of DNA-binding protein target sequences by physical effective energy functions: free energy analysis of lambda repressor-DNA complexes.

    Directory of Open Access Journals (Sweden)

    Caselle Michele

    2007-09-01

    Full Text Available Abstract Background Specific binding of proteins to DNA is one of the most common ways gene expression is controlled. Although general rules for the DNA-protein recognition can be derived, the ambiguous and complex nature of this mechanism precludes a simple recognition code, therefore the prediction of DNA target sequences is not straightforward. DNA-protein interactions can be studied using computational methods which can complement the current experimental methods and offer some advantages. In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the λ repressor-DNA complex for which structural and thermodynamic experimental data are available. Results The binding free energy of two molecules can be expressed as the sum of an intermolecular energy (evaluated using a molecular mechanics forcefield, a solvation free energy term and an entropic term. Different solvation models are used including distance dependent dielectric constants, solvent accessible surface tension models and the Generalized Born model. The effect of conformational sampling by Molecular Dynamics simulations on the computed binding energy is assessed; results show that this effect is in general negative and the reproducibility of the experimental values decreases with the increase of simulation time considered. The free energy of binding for non-specific complexes, estimated using the best energetic model, agrees with earlier theoretical suggestions. As a results of these analyses, we propose a protocol for the prediction of DNA-binding target sequences. The possibility of searching regulatory elements within the bacteriophage λ genome using this protocol is explored. Our analysis shows good prediction capabilities, even in absence of any thermodynamic data and information on the naturally recognized sequence. Conclusion This study supports the conclusion that physics-based methods can offer a completely complementary

  6. Plasmid origin of replication of herpesvirus papio: DNA sequence and enhancer function.

    Science.gov (United States)

    Loeb, D D; Sung, N S; Pesano, R L; Sexton, C J; Hutchison, C; Pagano, J S

    1990-01-01

    Herpesvirus papio (HVP) is a lymphotropic virus of baboons which is related to Epstein-Barr virus (EBV) and produces latent infection. The nucleotide sequence of the 5,775-base-pair (bp) EcoRI K fragment of HVP, which has previously been shown to confer the ability to replicate autonomously, has been determined. Within this DNA fragment is a region which bears structural and sequence similarity to the ori-P region of EBV. The HVP ori-P region has a 10- by 26-bp tandem array which is related to the 20- by 30-bp tandem array from the EBV ori-P region. In HVP there is an intervening region of 764 bp followed by five partial copies of the 26-bp monomer. Both the EBV and HVP 3' regions have the potential to form dyad structures which, however, differ in arrangement. We also demonstrate that a transcriptional enhancer which requires transactivation by a virus-encoded factor is present in the HVP ori-P. Images PMID:2159548

  7. Cloning, Characterization, and Functional Expression of Phospholipase Dα cDNA from Banana (Musa acuminate L.

    Directory of Open Access Journals (Sweden)

    Li Li

    2017-01-01

    Full Text Available Phospholipase D (PLD plays a key role in adaptive responses of postharvest fruits. A cDNA clone of banana (Musa acuminate L. PLDα (MaPLDα was obtained by RT-PCR in this study. The MaPLDα gene contains a complete open reading frame (ORF encoding a 92-kDa protein composed of 832 amino acid residues and possesses a characteristic C2 domain and two catalytic H×K×××D (abbr. HKD motifs. The two HKD motifs are separated by 341 amino acid residues in the primary structure. Relatively higher PLD activity and expression of MaPLDα mRNA were detected in developing tissues compared to senescent or mature tissues in individual leaves, flower, stem, and fruit organs, respectively. The expression profile of PLDα mRNA in postharvest banana fruits at different temperatures was determined, and the MaPLDα mRNA reached the highest expression peak on day 5 at 25°C and on day 7 at 12°C. The results provide useful information for maintaining postharvest quality and extending the storage life of banana fruit.

  8. A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

    Science.gov (United States)

    Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

    2016-12-01

    In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

  9. 14-3-3 proteins function in the initiation and elongation steps of DNA replication in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yahyaoui, Wafaa; Zannis-Hadjopoulos, Maria

    2009-12-15

    14-3-3s are highly conserved abundant eukaryotic proteins essential for viability, at least in lower eukaryotes. We previously showed that they associate with mammalian and yeast replication origins in a cell-cycle-dependent manner, and are involved in the initiation of DNA replication. Here, we present evidence that 14-3-3 proteins are novel regulators of the initiation and elongation steps of DNA replication in Saccharomyces cerevisiae. The results show that the Bmh2 protein, one of the two 14-3-3 homologues in S. cerevisiae, interacts with Mcm2 and Orc2 proteins, binds to ARS1 maximally at the G1 phase, is essential for plasmid stability, and is required for normal S-phase entry and progression. Furthermore, during G1 phase, the Bmh2 protein is required for the association of MCM proteins with chromatin and their maintenance at replication origins. The results reveal that 14-3-3 proteins function as essential factors for the assembly and maintenance of the pre-replication complex during G1 phase.

  10. Functional characterization of an acidic SK(3) dehydrin isolated from an Opuntia streptacantha cDNA library.

    Science.gov (United States)

    Ochoa-Alfaro, A E; Rodríguez-Kessler, M; Pérez-Morales, M B; Delgado-Sánchez, P; Cuevas-Velazquez, C L; Gómez-Anduro, G; Jiménez-Bremont, J F

    2012-03-01

    Cactus pears are succulent plants of the Cactaceae family adapted to extremely arid, hot and cold environments, making them excellent models for the study of molecular mechanisms underlying abiotic stress tolerance. Herein, we report a directional cDNA library from 12-month-old cladodes of Opuntia streptacantha plants subjected to abiotic stresses. A total of 442 clones were sequenced, representing 329 cactus pear unigenes, classified into eleven functional categories. The most abundant EST (unigen 33) was characterized under abiotic stress. This cDNA of 905 bp encodes a SK(3)-type acidic dehydrin of 248 amino acids. The OpsDHN1 gene contains an intron inserted within the sequence encoding the S-motif. qRT-PCR analysis shows that the OpsDHN1 transcript is specifically accumulated in response to cold stress, and induced by abscisic acid. Over-expression of the OpsDHN1 gene in Arabidopsis thaliana leads to enhanced tolerance to freezing treatment, suggesting that OpsDHN1 participates in freezing stress responsiveness. Generation of the first EST collection for the characterization of cactus pear genes constitutes a useful platform for the understanding of molecular mechanisms of stress tolerance in Opuntia and other CAM plants.

  11. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B.

    Science.gov (United States)

    Chénard, Caroline; Wirth, Jennifer F; Suttle, Curtis A

    2016-06-14

    Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. Filamentous cyanobacteria belonging to the genus Nostoc are widespread and ecologically important in freshwater, yet little is known about the genomic content of their viruses. Here we report the first genomic analysis of cyanophages infecting

  12. Nucleobase-functionalized grapheme nanoribbons for accurate high-speed DNA sequencing

    NARCIS (Netherlands)

    Paulechka, Eugene; Wassenaar, Tsjerk; Kroenlein, Kenneth; Kazakov, Andrei; Smolyanitsky, Alex

    2016-01-01

    We propose a water-immersed nucleobase-functionalized suspended graphene nanoribbon as an intrinsically selective device for nucleotide detection. The proposed sensing method combines Watson–Crick selective base pairing with graphene's capacity for converting anisotropic lattice strain to changes in

  13. A hotspot in the glucocorticoid receptor DNA-binding domain susceptible to loss of function mutation.

    Science.gov (United States)

    Banuelos, Jesus; Shin, Soon Cheon; Lu, Nick Z

    2015-04-01

    Glucocorticoids (GCs) are used to treat a variety of inflammatory disorders and certain cancers. However, GC resistance occurs in subsets of patients. We found that EL4 cells, a GC-resistant mouse thymoma cell line, harbored a point mutation in their GC receptor (GR) gene, resulting in the substitution of arginine 493 by a cysteine in the second zinc finger of the DNA-binding domain. Allelic discrimination analyses revealed that the R493C mutation occurred on both alleles. In the absence of GCs, the GR in EL4 cells localized predominantly in the cytoplasm and upon dexamethasone treatment underwent nuclear translocation, suggesting that the ligand binding ability of the GR in EL4 cells was intact. In transient transfection assays, the R493C mutant could not transactivate the MMTV-luciferase reporter. Site-directed mutagenesis to revert the R493C mutation restored the transactivation activity. Cotransfection experiments showed that the R493C mutant did not inhibit the transcriptional activities of the wild-type GR. In addition, the R493C mutant did not repress either the AP-1 or NF-κB reporters as effectively as WT GR. Furthermore, stable expression of the WT GR in the EL4 cells enabled GC-mediated gene regulation, specifically upregulation of IκBα and downregulation of interferon γ and interleukin 17A. Arginine 493 is conserved among multiple species and all human nuclear receptors and its mutation has also been found in the human GR, androgen receptor, and mineralocorticoid receptor. Thus, R493 is necessary for the transcriptional activity of the GR and a hotspot for mutations that result in GC resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Delineation of VEGF-regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis

    Directory of Open Access Journals (Sweden)

    Papka Raymond E

    2008-12-01

    Full Text Available Abstract Background VEGF-regulated genes in the cervices of pregnant and non-pregnant rodents (rats and mice were delineated by DNA microarray and Real Time PCR, after locally altering levels of or action of VEGF using VEGF agents, namely siRNA, VEGF receptor antagonist and mouse VEGF recombinant protein. Methods Tissues were analyzed by genome-wide DNA microarray analysis, Real-time and gel-based PCR, and SEM, to decipher VEGF function during cervical remodeling. Data were analyzed by EASE score (microarray and ANOVA (Real Time PCR followed by Scheffe's F-test for multiple comparisons. Results Of the 30,000 genes analyzed, about 4,200 genes were altered in expression by VEGF, i.e., expression of about 2,400 and 1,700 genes were down- and up-regulated, respectively. Based on EASE score, i.e., grouping of genes according to their biological process, cell component and molecular functions, a number of vascular- and non-vascular-related processes were found to be regulated by VEGF in the cervix, including immune response (including inflammatory, cell proliferation, protein kinase activity, and cell adhesion molecule activity. Of interest, mRNA levels of a select group of genes, known to or with potential to influence cervical remodeling were altered. For example, real time PCR analysis showed that levels of VCAM-1, a key molecule in leukocyte recruitment, endothelial adhesion, and subsequent trans-endothelial migration, were elevated about 10 folds by VEGF. Further, VEGF agents also altered mRNA levels of decorin, which is involved in cervical collagen fibrillogenesis, and expression of eNO, PLC and PKC mRNA, critical downstream mediators of VEGF. Of note, we show that VEGF may regulate cervical epithelial proliferation, as revealed by SEM. Conclusion These data are important in that they shed new insights in VEGF's possible roles and mechanisms in cervical events near-term, including cervical remodeling.

  15. Loss of MLL5 results in pleiotropic hematopoietic defects, reduced neutrophil immune function, and extreme sensitivity to DNA demethylation.

    Science.gov (United States)

    Heuser, Michael; Yap, Damian B; Leung, Malina; de Algara, Teresa Ruiz; Tafech, Alaeddin; McKinney, Steven; Dixon, John; Thresher, Rosemary; Colledge, Bill; Carlton, Mark; Humphries, R Keith; Aparicio, Samuel A

    2009-02-12

    MLL5 is a divergent member of the Drosophila Trithorax-related (SET) domain and plant homeodomain (PHD) domain-containing chromatin regulators that are involved in the regulation of transcriptional "memory" during differentiation. Human MLL5 is located on chromosome 7q22, which frequently is deleted in myeloid leukemias, suggesting a possible role in hemopoiesis. To address this question, we generated a loss-of-function allele (Mll5(tm1Apa)) in the murine Mll5 locus. Unlike other Mll genes, Mll5(tm1Apa) homozygous mice are viable but display defects in immunity and hematopoiesis. First, Mll5(tm1Apa) homozygous mice show increased susceptibility to spontaneous eye infections, associated with a cell-autonomous impairment of neutrophil function. Second, Mll5(tm1Apa/tm1Apa) mice exhibit a mild impairment of erythropoiesis. Third, Mll5(tm1Apa/tm1Apa) hematopoietic stem cells (HSCs) have impaired competitive repopulating capacity both under normal conditions and when subjected to self-renewal stimulation by NUP98-HOXA10. Fourth, Mll5(tm1Apa) homozygous HSCs show a dramatic sensitivity to DNA demethylation-induced differentiation (5-azadeoxycytidine). Taken together, our data show that MLL5 is involved in terminal myeloid differentiation and the regulation of HSC self-renewal by a mechanism that involves DNA methylation. These data warrant investigation of MLL5 expression levels as a predictive marker of demethylating-agent response in patients with myelodysplastic syndromes and leukemias and identify MLL5 as a key regulator of normal hematopoiesis.

  16. Functional Analysis of BARD1 Missense Variants in Homology-Directed Repair of DNA Double Strand Breaks.

    Science.gov (United States)

    Lee, Cindy; Banerjee, Tapahsama; Gillespie, Jessica; Ceravolo, Amanda; Parvinsmith, Matthew R; Starita, Lea M; Fields, Stanley; Toland, Amanda E; Parvin, Jeffrey D

    2015-12-01

    Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous because of the detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology-directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. Twenty-three of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N- and C- termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc-coordinating amino acids and to the BRCA1-BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance. © 2015 WILEY PERIODICALS, INC.

  17. DNA damage-induced transcriptional program in CLL: biological and diagnostic implications for functional p53 testing.

    Science.gov (United States)

    Mohr, Julia; Helfrich, Hanne; Fuge, Maxi; Eldering, Eric; Bühler, Andreas; Winkler, Dirk; Volden, Matthias; Kater, Arnon P; Mertens, Daniel; Te Raa, Doreen; Döhner, Hartmut; Stilgenbauer, Stephan; Zenz, Thorsten

    2011-02-03

    The DNA damage pathway plays a central role in chemoresistance in chronic lymphocytic leukemia (CLL), as indicated by the prognostic impact of TP53 and ATM loss/mutations. We investigated the function of the p53 axis in primary CLL samples by studying p53 and p21 responses to irradiation by FACS and RT-PCR. We observed a distinct response pattern for most cases with a 17p deletion (n = 16) or a sole TP53 mutation (n = 8), but not all cases with a p53 aberration were detected based on a number of different assays used. Samples with a small clone with a TP53 mutation remained undetected in all assays. Only 1 of 123 cases showed high expression of p53, which is suggestive of p53 aberration without proof of mutation of TP53. Samples with an 11q deletion showed a heterogeneous response, with only 13 of 30 showing an abnormal response based on cutoff. Nevertheless, the overall induction of p53 and p21 was impaired, suggesting a gene-dosage effect for ATM in the 11q-deleted samples. The detectability of p53 defects is influenced by clonal heterogeneity and sample purity. Functional assays of p53 defects will detect a small number of cases not detectable by FISH or TP53 mutational analysis. The clinical utility of functional p53 testing will need to be derived from clinical trials.

  18. Scientific publications about DNA structure-function and PCR technique in Costa Rica: A historic view (1953-2003

    Directory of Open Access Journals (Sweden)

    Federico J Albertazzi

    2004-09-01

    Full Text Available The spreading of knowledge depends on the access to the information and its immediate use. Models are useful to explain specific phenomena. The scientific community accepts some models in Biology after a period of time, once it has evidence to support it. The model of the structure and function of the DNA proposed by Watson & Crick (1953 was not the exception, since a few years later the DNA model was finally accepted. In Costa Rica, DNA function was first mentioned in 1970, in the magazine Biología Tropical (Tropical Biology Magazine, more than 15 years after its first publication in a scientific journal. An opposite situation occurs with technical innovations. If the efficiency of a new scientific technique is proved in a compelling way, then the acceptance by the community comes swiftly. This was the case of the polymerase chain reaction, or PCR. The first PCR machine in Costa Rica arrived in 1991, only three years after its publication. Rev. Biol. Trop. 52(3: 417-421. Epub 2004 Dic 15.La diseminación del conocimiento depende de la disponibilidad de la información y aplicar dicha información para resolver una problema. Los modelos sirvan para explicar fenómenos determinados. En Biología los modelos son aceptados por la comunidad científica después de cierto tiempo si ha probado su validez y reconocido la evidencia para apoyar dicho modelo. El modelo estructural y función de la molécula de ADN propuesto por Watson y Crick (1953 no fue la excepción pues tardó varios años en ser completamente aceptado por la comunidad científica. En Costa Rica la primera publicación relacionada con la función del ADN fue en la Revista Biología Tropical fue en 1970, más de 15 años después de ser propuesta. La situación contraria se presenta cuando son innovaciones técnicas. Si la eficiencia es demostrada, rápidamente se incorpora dentro de la comunidad. Este fue el caso de la reacción en cadena de la polimerasa, abreviado en inglés como

  19. Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment

    Directory of Open Access Journals (Sweden)

    Oliver I. Fregoso

    2016-09-01

    Full Text Available There has been extraordinary progress in understanding the roles of lentiviral accessory proteins in antagonizing host antiviral defense proteins. However, the precise primary function of the accessory gene Vpr remains elusive. Here we suggest that engagement with the DNA damage response is an important function of primate lentiviral Vpr proteins because of its conserved function among diverse lentiviral lineages. In contrast, we show that, for HIV-1, HIV-2, and related Vpr isolates and orthologs, there is a lack of correlation between DNA damage response activation and interaction with the host SLX4 protein complex of structure specific endonucleases; some Vpr proteins are able to interact with SLX4, but the majority are not. Using the clustered regularly interspaced short palindromic repeat (CRISPR/Cas9 method to knock out SLX4, we formally showed that HIV-1 and HIV-2 Vpr orthologs can still activate the DNA damage response and cell cycle arrest in the absence of SLX4. Together, our data suggest that activation of the DNA damage response, but not SLX4 interaction, is conserved and therefore indicative of an important function of Vpr. Our data also indicate that Vpr activates the DNA damage response through an SLX4-independent mechanism that remains uncharacterized.

  20. Stimuli-Responsive DNA-Functionalized Metal-Organic Frameworks (MOFs).

    Science.gov (United States)

    Kahn, Jason S; Freage, Lina; Enkin, Natalie; Garcia, Miguel Angel Aleman; Willner, Itamar

    2017-02-01

    The synthesis of nucleic acid-functionalized metal-organic frameworks (MOFs) is described. The metal-organic frameworks are loaded with a dye being locked in the structures by means of stimuli-responsive nucleic acid caps. The pH and K(+) -ion-triggered release, and switchable release, are demonstrated. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Hybridization State Detection of DNA-Functionalized Gold Nanoparticles Using Hyperspectral Imaging

    Directory of Open Access Journals (Sweden)

    Richard C. Murdock

    2017-01-01

    Full Text Available Hyperspectral imaging has the unique ability of capturing spectral data for multiple wavelengths at each pixel in an image. This gives the ability to distinguish, with certainty, different nanomaterials and/or distinguish nanomaterials from biological materials. In this study, 4 nm and 13 nm gold nanoparticles (Au NPs were synthesized, functionalized with complimentary oligonucleotides, and hybridized to form large networks of NPs. Scattering spectra were collected from each sample (unfunctionalized, functionalized, and hybridized and evaluated. The spectra showed unique peaks for each size of Au NP sample and also exhibited narrowing and intensifying of the spectra as the NPs were functionalized and then subsequently hybridized. These spectra are different from normal aggregation effects where the LSPR and reflected spectrum broaden and are red-shifted. Rather, this appears to be dependent on the ability to control the interparticle distance through oligonucleotide length, which is also investigated through the incorporation of a poly-A spacer. Also, hybridized Au NPs were exposed to cells with no adverse effects and retained their unique spectral signatures. With the ability to distinguish between hybridization states at nearly individual NP levels, this could provide a new method of tracking the intracellular actions of nanomaterials as well as extracellular biosensing applications.

  2. Repair of DNA strand breaks by the overlapping functions of lesion-specific and non-lesion-specific DNA 3' phosphatases.

    Science.gov (United States)

    Vance, J R; Wilson, T E

    2001-11-01

    In Saccharomyces cerevisiae, the apurinic/apyrimidinic (AP) endonucleases Apn1 and Apn2 act as alternative pathways for the removal of various 3'-terminal blocking lesions from DNA strand breaks and in the repair of abasic sites, which both result from oxidative DNA damage. Here we demonstrate that Tpp1, a homologue of the 3' phosphatase domain of polynucleotide kinase, is a third member of this group of redundant 3' processing enzymes. Unlike Apn1 and Apn2, Tpp1 is specific for the removal of 3' phosphates at strand breaks and does not possess more general 3' phosphodiesterase, exonuclease, or AP endonuclease activities. Deletion of TPP1 in an apn1 apn2 mutant background dramatically increased the sensitivity of the double mutant to DNA damage caused by H2O2 and bleomycin but not to damage caused by methyl methanesulfonate. The triple mutant was also deficient in the repair of 3' phosphate lesions left by Tdp1-mediated cleavage of camptothecin-stabilized Top1-DNA covalent complexes. Finally, the tpp1 apn1 apn2 triple mutation displayed synthetic lethality in combination with rad52, possibly implicating postreplication repair in the removal of unrepaired 3'-terminal lesions resulting from endogenous damage. Taken together, these results demonstrate a clear role for the lesion-specific enzyme, Tpp1, in the repair of a subset of DNA strand breaks.

  3. Repair of DNA Strand Breaks by the Overlapping Functions of Lesion-Specific and Non-Lesion-Specific DNA 3′ Phosphatases

    Science.gov (United States)

    Vance, John R.; Wilson, Thomas E.

    2001-01-01

    In Saccharomyces cerevisiae, the apurinic/apyrimidinic (AP) endonucleases Apn1 and Apn2 act as alternative pathways for the removal of various 3′-terminal blocking lesions from DNA strand breaks and in the repair of abasic sites, which both result from oxidative DNA damage. Here we demonstrate that Tpp1, a homologue of the 3′ phosphatase domain of polynucleotide kinase, is a third member of this group of redundant 3′ processing enzymes. Unlike Apn1 and Apn2, Tpp1 is specific for the removal of 3′ phosphates at strand breaks and does not possess more general 3′ phosphodiesterase, exonuclease, or AP endonuclease activities. Deletion of TPP1 in an apn1 apn2 mutant background dramatically increased the sensitivity of the double mutant to DNA damage caused by H2O2 and bleomycin but not to damage caused by methyl methanesulfonate. The triple mutant was also deficient in the repair of 3′ phosphate lesions left by Tdp1-mediated cleavage of camptothecin-stabilized Top1-DNA covalent complexes. Finally, the tpp1 apn1 apn2 triple mutation displayed synthetic lethality in combination with rad52, possibly implicating postreplication repair in the removal of unrepaired 3′-terminal lesions resulting from endogenous damage. Taken together, these results demonstrate a clear role for the lesion-specific enzyme, Tpp1, in the repair of a subset of DNA strand breaks. PMID:11585902

  4. DNA in a Tunnel: A Comfy Spot for Recognition - or -The Structure of BsoBI Complexed with DNA. What can we Learn about Function via Structure Determination and how can this be Applied to Bone or Muscle Biology?

    Science.gov (United States)

    vanderWoerd, Mark

    2004-01-01

    The structure and function of a biologically active molecule are related. To understand its function, it is necessary (but not always sufficient) to know the structure of the molecule. There are many ways of relating the molecular function with the structure. Mutation analysis can identify pertinent amino acids of an enzyme, or alternatively structure comparison of the of two similar molecules with different function may lead to understanding which parts are responsible for a functional aspect, or a series of "structural cartoons" - enzyme structure, enzyme plus substrate, enzyme with transition state analog, and enzyme with product - may give insight in the function of a molecule. As an example we will discuss the structure and function of the restriction enzyme BsoBI from Bacillus stearothemzophilus in complex with its cognate DNA. The enzyme forms a unique complex with DNA in that it completely encircles the DNA. The structure reveals the enzyme-DNA contacts, how the DNA is distorted compared with the canonical forms, and elegantly shows how two distinct DNA sequences can be recognized with the same efficiency. Based on the structure we may also propose a hypothesis how the enzymatic mechanism works. The knowledge gained thru studies such as this one can be used to alter the function by changing the molecular structure. Usually this is done by design of inhibitors specifically active against and fitting into an active site of the enzyme of choice. In the case of BsoBI one of the objectives of the study was to alter the enzyme specificity. In bone biology there are many candidates available for molecular study in order to explain, alter, or (temporarily) suspend activity. For example, the understanding of a pathway that negatively regulates bone formation may be a good target for drug design to stimulate bone formation and have good potential as the basis for new countermeasures against bone loss. In principle the same approach may aid muscle atrophy, radiation

  5. Repair of DNA Strand Breaks by the Overlapping Functions of Lesion-Specific and Non-Lesion-Specific DNA 3′ Phosphatases

    OpenAIRE

    Vance, John R.; Wilson, Thomas E.

    2001-01-01

    In Saccharomyces cerevisiae, the apurinic/apyrimidinic (AP) endonucleases Apn1 and Apn2 act as alternative pathways for the removal of various 3′-terminal blocking lesions from DNA strand breaks and in the repair of abasic sites, which both result from oxidative DNA damage. Here we demonstrate that Tpp1, a homologue of the 3′ phosphatase domain of polynucleotide kinase, is a third member of this group of redundant 3′ processing enzymes. Unlike Apn1 and Apn2, Tpp1 is specific for the removal o...

  6. A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

    DEFF Research Database (Denmark)

    Syed, Salahuddin; Madsen, Claus Desler; Rasmussen, Lene J.

    2016-01-01

    hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5......, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.......In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N...

  7. Functional centromeres determine the activation time of pericentric origins of DNA replication in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Thomas J Pohl

    Full Text Available The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.

  8. A Decade of Biochemical and Structural Studies of the DNA Repair Machinery of Deinococcus radiodurans: Major Findings, Functional and Mechanistic Insight and Challenges

    Directory of Open Access Journals (Sweden)

    Joanna Timmins

    2016-01-01

    Full Text Available The Deinococcus radiodurans bacterium is extremely resistant to ionising radiation and desiccation and can withstand a 200-fold higher radiation dose than most other bacteria with no loss of viability. The mechanisms behind this extreme resistance are not fully understood, but it is clear that several factors contribute to this phenotype. Efficient scavenging of reactive oxygen species and repair of damaged DNA are two of these. In this review, we summarise the results from a decade of structural and functional studies of the DNA repair machinery of Deinococcus radiodurans and discuss how these studies have contributed to an improved understanding of the molecular mechanisms underlying DNA repair and to the outstanding resistance of Deinococcus radiodurans to DNA damaging agents.

  9. A Decade of Biochemical and Structural Studies of the DNA Repair Machinery of Deinococcus radiodurans: Major Findings, Functional and Mechanistic Insight and Challenges.

    Science.gov (United States)

    Timmins, Joanna; Moe, Elin

    2016-01-01

    The Deinococcus radiodurans bacterium is extremely resistant to ionising radiation and desiccation and can withstand a 200-fold higher radiation dose than most other bacteria with no loss of viability. The mechanisms behind this extreme resistance are not fully understood, but it is clear that several factors contribute to this phenotype. Efficient scavenging of reactive oxygen species and repair of damaged DNA are two of these. In this review, we summarise the results from a decade of structural and functional studies of the DNA repair machinery of Deinococcus radiodurans and discuss how these studies have contributed to an improved understanding of the molecular mechanisms underlying DNA repair and to the outstanding resistance of Deinococcus radiodurans to DNA damaging agents.

  10. A Viral Satellite DNA Vector (TYLCCNV) for Functional Analysis of miRNAs and siRNAs in Plants.

    Science.gov (United States)

    Ju, Zheng; Cao, Dongyan; Gao, Chao; Zuo, Jinhua; Zhai, Baiqiang; Li, Shan; Zhu, Hongliang; Fu, Daqi; Luo, Yunbo; Zhu, Benzhong

    2017-04-01

    With experimental and bioinformatical methods, numerous small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), have been found in plants, and they play vital roles in various biological regulation processes. However, most of these small RNAs remain to be functionally characterized. Until now, only several viral vectors were developed to overexpress miRNAs with limited application in plants. In this study, we report a new small RNA overexpression system via viral satellite DNA associated with Tomato yellow leaf curl China virus (TYLCCNV) vector, which could highly overexpress not only artificial and endogenous miRNAs but also endogenous siRNAs in Nicotiana benthamiana First, we constructed basic TYLCCNV-amiRPDS(319L) vector with widely used AtMIR319a backbone, but the expected photobleaching phenotype was very weak. Second, through comparing the effect of backbones (AtMIR319a, AtMIR390a, and SlMIR159) on specificity and significance of generating small RNAs, the AtMIR390a backbone was optimally selected to construct the small RNA overexpression system. Third, through sRNA-Seq and Degradome-Seq, the small RNAs from AtMIR390a backbone in TYLCCNV-amiRPDS(390) vector were confirmed to highly overexpress amiRPDS and specifically silence targeted PDS gene. Using this system, rapid functional analysis of endogenous miRNAs and siRNAs was carried out, including miR156 and athTAS3a 5'D8(+). Meanwhile, through designing corresponding artificial miRNAs, this system could also significantly silence targeted endogenous genes and show specific phenotypes, including PDS, Su, and PCNA These results demonstrated that this small RNA overexpression system could contribute to investigating not only the function of endogenous small RNAs, but also the functional genes in plants. © 2017 American Society of Plant Biologists. All Rights Reserved.

  11. PprA Protein Is Involved in Chromosome Segregation via Its Physical and Functional Interaction with DNA Gyrase in Irradiated Deinococcus radiodurans Bacteria

    Science.gov (United States)

    Devigne, Alice; Guérin, Philippe; Lisboa, Johnny; Quevillon-Cheruel, Sophie; Armengaud, Jean; Sommer, Suzanne; Bouthier de la Tour, Claire

    2016-01-01

    ABSTRACT PprA, a radiation-induced Deinococcus-specific protein, was previously shown to be required for cell survival and accurate chromosome segregation after exposure to ionizing radiation. Here, we used an in vivo approach to determine, by shotgun proteomics, putative PprA partners coimmunoprecipitating with PprA when cells were exposed to gamma rays. Among them, we found the two subunits of DNA gyrase and, thus, chose to focus our work on characterizing the activities of the deinococcal DNA gyrase in the presence or absence of PprA. Loss of PprA rendered cells hypersensitive to novobiocin, an inhibitor of the B subunit of DNA gyrase. We showed that treatment of bacteria with novobiocin resulted in induction of the radiation desiccation response (RDR) regulon and in defects in chromosome segregation that were aggravated by the absence of PprA. In vitro, the deinococcal DNA gyrase, like other bacterial DNA gyrases, possesses DNA negative supercoiling and decatenation activities. These two activities are inhibited in vitro by novobiocin and nalidixic acid, whereas PprA specifically stimulates the decatenation activity of DNA gyrase. Together, these results suggest that PprA plays a major role in chromosome decatenation via its interaction with the deinococcal DNA gyrase when D. radiodurans cells are recovering from exposure to ionizing radiation. IMPORTANCE D. radiodurans is one of the most radiation-resistant organisms known. This bacterium is able to cope with high levels of DNA lesions generated by exposure to extreme doses of ionizing radiation and to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Here, we identified partners of PprA, a radiation-induced Deinococcus-specific protein, previously shown to be required for radioresistance. Our study leads to three main findings: (i) PprA interacts with DNA gyrase after irradiation, (ii) treatment of cells with novobiocin results in defects in chromosome segregation

  12. PprA Protein Is Involved in Chromosome Segregation via Its Physical and Functional Interaction with DNA Gyrase in Irradiated Deinococcus radiodurans Bacteria.

    Science.gov (United States)

    Devigne, Alice; Guérin, Philippe; Lisboa, Johnny; Quevillon-Cheruel, Sophie; Armengaud, Jean; Sommer, Suzanne; Bouthier de la Tour, Claire; Servant, Pascale

    2016-01-01

    PprA, a radiation-induced Deinococcus-specific protein, was previously shown to be required for cell survival and accurate chromosome segregation after exposure to ionizing radiation. Here, we used an in vivo approach to determine, by shotgun proteomics, putative PprA partners coimmunoprecipitating with PprA when cells were exposed to gamma rays. Among them, we found the two subunits of DNA gyrase and, thus, chose to focus our work on characterizing the activities of the deinococcal DNA gyrase in the presence or absence of PprA. Loss of PprA rendered cells hypersensitive to novobiocin, an inhibitor of the B subunit of DNA gyrase. We showed that treatment of bacteria with novobiocin resulted in induction of the radiation desiccation response (RDR) regulon and in defects in chromosome segregation that were aggravated by the absence of PprA. In vitro, the deinococcal DNA gyrase, like other bacterial DNA gyrases, possesses DNA negative supercoiling and decatenation activities. These two activities are inhibited in vitro by novobiocin and nalidixic acid, whereas PprA specifically stimulates the decatenation activity of DNA gyrase. Together, these results suggest that PprA plays a major role in chromosome decatenation via its interaction with the deinococcal DNA gyrase when D. radiodurans cells are recovering from exposure to ionizing radiation. IMPORTANCE D. radiodurans is one of the most radiation-resistant organisms known. This bacterium is able to cope with high levels of DNA lesions generated by exposure to extreme doses of ionizing radiation and to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Here, we identified partners of PprA, a radiation-induced Deinococcus-specific protein, previously shown to be required for radioresistance. Our study leads to three main findings: (i) PprA interacts with DNA gyrase after irradiation, (ii) treatment of cells with novobiocin results in defects in chromosome segregation that are

  13. Unbiased mutagenesis of MHV68 LANA reveals a DNA-binding domain required for LANA function in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Clinton R Paden

    2012-09-01

    Full Text Available The Latency-Associated Nuclear Antigen (LANA, encoded by ORF73, is a conserved gene among the γ2-herpesviruses (rhadinoviruses. The Kaposi's Sarcoma-Associated Herpesvirus (KSHV LANA is consistently expressed in KSHV-associated malignancies. In the case of the rodent γ2-herpesvirus, murine gammaherpesvirus 68 (MHV68, the LANA homolog (mLANA is required for efficient virus replication, reactivation from latency and immortalization of murine fetal liver-derived B cells. To gain insights into mLANA function(s, knowing that KSHV LANA binds DNA and can modulate transcription of a variety of promoters, we sought out and identified a mLANA-responsive promoter which maps to the terminal repeat (TR of MHV68. Notably, mLANA strongly repressed activity from this promoter. We extended these analyses to demonstrate direct, sequence-specific binding of recombinant mLANA to TR DNA by DNase I footprinting. To assess whether the DNA-binding and/or transcription modulating function is important in the known mLANA phenotypes, we generated an unbiased library of mLANA point mutants using error-prone PCR, and screened a large panel of mutants for repression of the mLANA-responsive promoter to identify loss of function mutants. Notably, among the mutant mLANA proteins recovered, many of the mutations are in a predicted EBNA-1-like DNA-binding domain. Consistent with this prediction, those tested displayed loss of DNA binding activity. We engineered six of these mLANA mutants into the MHV68 genome and tested the resulting mutant viruses for: (i replication fitness; (ii efficiency of latency establishment; and (iii reactivation from latency. Interestingly, each of these mLANA-mutant viruses exhibited phenotypes similar to the mLANA-null mutant virus, indicating that DNA-binding is critical for mLANA function.

  14. Dual-colored graphene quantum dots-labeled nanoprobes/graphene oxide: functional carbon materials for respective and simultaneous detection of DNA and thrombin

    Science.gov (United States)

    Qian, Zhao Sheng; Shan, Xiao Yue; Chai, Lu Jing; Chen, Jian Rong; Feng, Hui

    2014-10-01

    Convenient and simultaneous detection of multiple biomarkers such as DNA and proteins with biocompatible materials and good analytical performance still remains a challenge. Herein, we report the respective and simultaneous detection of DNA and bovine α-thrombin (thrombin) entirely based on biocompatible carbon materials through a specially designed fluorescence on-off-on process. Colorful fluorescence, high emission efficiency, good photostability and excellent compatibility enables graphene quantum dots (GQDs) as the best choice for fluorophores in bioprobes, and thus two-colored GQDs as labeling fluorophores were chemically bonded with specific oligonucleotide sequence and aptamer to prepare two probes targeting the DNA and thrombin, respectively. Each probe can be assembled on the graphene oxide (GO) platform spontaneously by π-π stacking and electrostatic attraction; as a result, fast electron transfer in the assembly efficiently quenches the fluorescence of probe. The presence of DNA or thrombin can trigger the self-recognition between capturing a nucleotide sequence and its target DNA or between thrombin and its aptamer due to their specific hybridization and duplex DNA structures or the formation of apatamer-substrate complex, which is taken advantage of in order to achieve a separate quantitative analysis of DNA and thrombin. A dual-functional biosensor for simultaneous detection of DNA and thrombin was also constructed by self-assembly of two probes with distinct colors and GO platform, and was further evaluated with the presence of various concentrations of DNA and thrombin. Both biosensors serving as a general detection model for multiple species exhibit outstanding analytical performance, and are expected to be applied in vivo because of the excellent biocompatibility of their used materials.

  15. A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase - Structural and modeling insight into its functions

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hui-Guang [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Huang, Philip L. [American Biosciences, Boston, MA 02114 (United States); Zhang, Dawei; Sun, Yongtao [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Chen, Hao-Chia [Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892 (United States); Zhang, John [Department of Chemistry, New York University, New York, NY 10003 (United States); Huang, Paul L. [Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114 (United States); Kong, Xiang-Peng, E-mail: xiangpeng.kong@med.nyu.edu [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Lee-Huang, Sylvia, E-mail: sylvia.lee-huang@med.nyu.edu [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States)

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  16. A New Activity of Anti-HIV and Anti-tumor Protein GAP31: DNA Adenosine Glycosidase – Structural and Modeling Insight into its Functions

    Energy Technology Data Exchange (ETDEWEB)

    Li, H.; Huang, P; Zhang, D; Sun, Y; Chen, H; Zhang, J; Huang, P; Kong, X; Lee-Huang, S

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  17. [Cloning and functional characterization of a cDNA encoding isopentenyl diphosphate isomerase involved in taxol biosynthesis in Taxus media].

    Science.gov (United States)

    Shen, Tian; Qiu, Fei; Chen, Min; Lan, Xiao-zhong; Liao, Zhi-hua

    2015-05-01

    Taxol is one of the most potent anti-cancer agents, which is extracted from the plants of Taxus species. Isopentenyl diphosphate isomerase (IPI) catalyzes the reversible transformation between IPP and DMAPP, both of which are the general 5-carbon precursors for taxol biosynthesis. In the present study, a new gene encoding IPI was cloned from Taxus media (namely TmIPI with the GenBank Accession Number KP970677) for the first time. The full-length cDNA of TmIPI was 1 232 bps encoding a polypeptide with 233 amino acids, in which the conserved domain Nudix was found. Bioinformatic analysis indicated that the sequence of TmIPI was highly similar to those of other plant IPI proteins, and the phylogenetic analysis showed that there were two clades of plant IPI proteins, including IPIs of angiosperm plants and IPIs of gymnosperm plants. TmIPI belonged to the clade of gymnosperm plant IPIs, and this was consistent with the fact that Taxus media is a plant species of gymnosperm. Southern blotting analysis demonstrated that there was a gene family of IPI in Taxus media. Finally, functional verification was applied to identify the function of TmIPI. The results showed that biosynthesis of β-carotenoid was enhanced by overexpressing TmIPI in the engineered E. coli strain, and this suggested that TmIPI might be a key gene involved in isoprenoid/terpenoid biosynthesis.

  18. Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillus subtilis PriA initiator

    Science.gov (United States)

    Polard, Patrice; Marsin, Stéphanie; McGovern, Stephen; Velten, Marion; Wigley, Dale B.; Ehrlich, S. Dusko; Bruand, Claude

    2002-01-01

    The PriA protein was identified in Escherichia coli as a factor involved in the replication of extrachromosomal elements such as bacteriophage φX174 and plasmid pBR322. Recent data show that PriA plays an important role in chromosomal replication, by promoting reassembly of the replication machinery during reinitiation of inactivated forks. A gene encoding a product 32% identical to the E.coli PriA protein has been identified in Bacillus subtilis. To characterise this protein, designated PriABs, we constructed priABs mutants. These mutants are poorly viable, filamentous and sensitive to rich medium and UV irradiation. Replication of pAMβ1-type plasmids, which is initiated through the formation of a D-loop structure, and the activity of the primosome assembly site ssiA of plasmid pAMβ1 are strongly affected in the mutants. The purified PriABs protein binds preferentially to the active strand of ssiA, even in the presence of B.subtilis SSB protein (SSBBs). PriABs also binds stably and specifically to an artificial D-loop structure in vitro. These data show that PriABs recognises two specific substrates, ssiA and D-loops, and suggest that it triggers primosome assembly on them. PriABs also displays a single-stranded DNA-dependent ATPase activity, which is reduced in the presence of SSBBs, unless the ssiA sequence is present on the ssDNA substrate. Finally, PriABs is shown to be an active helicase. Altogether, these results demonstrate a clear functional identity between PriAEc and PriABs. However, PriABs does not complement an E.coli priA null mutant strain. This host specificity may be due to the divergence between the proteins composing the E.coli and B.subtilis PriA-dependent primosomes. PMID:11917020

  19. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp. Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B

    Directory of Open Access Journals (Sweden)

    Caroline Chénard

    2016-06-01

    Full Text Available Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages.

  20. Regions of common inter-individual DNA methylation differences in human monocytes: genetic basis and potential function.

    Science.gov (United States)

    Schröder, Christopher; Leitão, Elsa; Wallner, Stefan; Schmitz, Gerd; Klein-Hitpass, Ludger; Sinha, Anupam; Jöckel, Karl-Heinz; Heilmann-Heimbach, Stefanie; Hoffmann, Per; Nöthen, Markus M; Steffens, Michael; Ebert, Peter; Rahmann, Sven; Horsthemke, Bernhard

    2017-07-26

    There is increasing evidence for inter-individual methylation differences at CpG dinucleotides in the human genome, but the regional extent and function of these differences have not yet been studied in detail. For identifying regions of common methylation differences, we used whole genome bisulfite sequencing data of monocytes from five donors and a novel bioinformatic strategy. We identified 157 differentially methylated regions (DMRs) with four or more CpGs, almost none of which has been described before. The DMRs fall into different chromatin states, where methylation is inversely correlated with active, but not repressive histone marks. However, methylation is not correlated with the expression of associated genes. High-resolution single nucleotide polymorphism (SNP) genotyping of the five donors revealed evidence for a role of cis-acting genetic variation in establishing methylation patterns. To validate this finding in a larger cohort, we performed genome-wide association studies (GWAS) using SNP genotypes and 450k array methylation data from blood samples of 1128 individuals. Only 30/157 (19%) DMRs include at least one 450k CpG, which shows that these arrays miss a large proportion of DNA methylation variation. In most cases, the GWAS peak overlapped the CpG position, and these regions are enriched for CREB group, NF-1, Sp100 and CTCF binding motifs. In two cases, there was tentative evidence for a trans-effect by KRAB zinc finger proteins. Allele-specific DNA methylation occurs in discrete chromosomal regions and is driven by genetic variation in cis and trans, but in general has little effect on gene expression.

  1. A Conserved Tripeptide Sequence at the C Terminus of the Poxvirus DNA Processivity Factor D4 Is Essential for Protein Integrity and Function.

    Science.gov (United States)

    Nuth, Manunya; Guan, Hancheng; Ricciardi, Robert P

    2016-12-30

    Vaccinia virus (VACV) is a poxvirus, and the VACV D4 protein serves both as a uracil-DNA glycosylase and as an essential component required for processive DNA synthesis. The VACV A20 protein has no known catalytic function itself but associates with D4 to form the D4-A20 heterodimer that functions as the poxvirus DNA processivity factor. The heterodimer enables the DNA polymerase to efficiently synthesize extended strands of DNA. Upon characterizing the interaction between D4 and A20, we observed that the C terminus of D4 is susceptible to perturbation. Further analysis demonstrated that a conserved hexapeptide stretch at the extreme C terminus of D4 is essential for maintaining protein integrity, as assessed by its requirement for the production of soluble recombinant protein that is functional in processive DNA synthesis. From the known crystal structures of D4, the C-terminal hexapeptide is shown to make intramolecular contact with residues spanning the inner core of the protein. Our mutational analysis revealed that a tripeptide motif ((215)GFI(217)) within the hexapeptide comprises apparent residues necessary for the contact. Prediction of protein disorder identified the hexapeptide and several regions upstream of Gly(215) that comprise residues of the interface surfaces of the D4-A20 heterodimer. Our study suggests that (215)GFI(217) anchors these potentially dynamic upstream regions of the protein to maintain protein integrity. Unlike uracil-DNA glycosylases from diverse sources, where the C termini are disordered and do not form comparable intramolecular contacts, this feature may be unique to orthopoxviruses. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. DNA shuffling of adeno-associated virus yields functionally diverse viral progeny.

    Science.gov (United States)

    Koerber, James T; Jang, Jae-Hyung; Schaffer, David V

    2008-10-01

    Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (10(7)), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4-6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.

  3. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  4. A single portion of blueberry (Vaccinium corymbosum L) improves protection against DNA damage but not vascular function in healthy male volunteers.

    Science.gov (United States)

    Del Bó, Cristian; Riso, Patrizia; Campolo, Jonica; Møller, Peter; Loft, Steffen; Klimis-Zacas, Dorothy; Brambilla, Ada; Rizzolo, Anna; Porrini, Marisa

    2013-03-01

    It has been suggested that anthocyanin-rich foods may exert antioxidant effects and improve vascular function as demonstrated mainly in vitro and in the animal model. Blueberries are rich sources of anthocyanins and we hypothesized that their intake could improve cell protection against oxidative stress and affect endothelial function in humans. The aim of the study was to investigate the effect of one portion (300 g) of blueberries on selected markers of oxidative stress and antioxidant protection (endogenous and oxidatively induced DNA damage) and of vascular function (changes in peripheral arterial tone and plasma nitric oxide levels) in male subjects. In a randomized cross-over design, separated by a wash out period ten young volunteers received one portion of blueberries ground by blender or one portion of a control jelly. Before and after consumption (at 1, 2, and 24 hours), blood samples were collected and used to evaluate anthocyanin absorption (through mass spectrometry), endogenous and H(2)O(2)-induced DNA damage in blood mononuclear cells (through the comet assay), and plasma nitric oxide concentrations (through a fluorometric assay). Peripheral arterial function was assessed by means of Endo-PAT 2000. Blueberries significantly reduced (P < .01) H(2)O(2)-induced DNA damage (-18%) 1 hour after blueberry consumption compared to control. No significant differences were observed for endogenous DNA damage, peripheral arterial function and nitric oxide levels after blueberry intake. In conclusion, one portion of blueberries seems sufficient to improve cell antioxidant defense against DNA damage, but further studies are necessary to understand their role on vascular function. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. A functional IFN-λ4-generating DNA polymorphism could protect older asthmatic women from aeroallergen sensitization and associate with clinical features of asthma

    DEFF Research Database (Denmark)

    Chinnaswamy, Sreedhar; Wardzynska, Aleksandra; Pawelczyk, Malgorzata

    2017-01-01

    Lambda interferons (IFNLs) have immunomodulatory functions at epithelial barrier surfaces. IFN-λ4, a recent member of this family is expressed only in a subset of the population due to a frameshift-causing DNA polymorphism rs368234815. We examined the association of this polymorphism with atopy (...

  6. A paper/polymer hybrid CD-like microfluidic SpinChip integrated with DNA-functionalized graphene oxide nanosensors for multiplex qLAMP detection.

    Science.gov (United States)

    Dou, Maowei; Sanjay, Sharma T; Dominguez, Delfina C; Zhan, Sihui; Li, XiuJun

    2017-10-03

    A paper/poly(methyl methacrylate) (PMMA) hybrid CD-like microfluidic SpinChip integrated with DNA probe-functionalized graphene oxide (GO) nanosensors was developed for multiplex quantitative LAMP detection (mqLAMP). This approach can simply and effectively address a major challenging problem of multiplexing in current LAMP methods.

  7. Functional activity of plasmid DNA after entry into the atmosphere of earth investigated by a new biomarker stability assay for ballistic spaceflight experiments.

    Science.gov (United States)

    Thiel, Cora S; Tauber, Svantje; Schütte, Andreas; Schmitz, Burkhard; Nuesse, Harald; Moeller, Ralf; Ullrich, Oliver

    2014-01-01

    Sounding rockets represent an excellent platform for testing the influence of space conditions during the passage of Earth's atmosphere and re-entry on biological, physical and chemical experiments for astrobiological purposes. We designed a robust functionality biomarker assay to analyze the biological effects of suborbital spaceflights prevailing during ballistic rocket flights. During the TEXUS-49 rocket mission in March 2011, artificial plasmid DNA carrying a fluorescent marker (enhanced green fluorescent protein: EGFP) and an antibiotic resistance cassette (kanamycin/neomycin) was attached on different positions of rocket exterior; (i) circular every 90 degree on the outer surface concentrical of the payload, (ii) in the grooves of screw heads located in between the surface application sites, and (iii) on the surface of the bottom side of the payload. Temperature measurements showed two major peaks at 118 and 130 °C during the 780 seconds lasting flight on the inside of the recovery module, while outer gas temperatures of more than 1000 °C were estimated on the sample application locations. Directly after retrieval and return transport of the payload, the plasmid DNA samples were recovered. Subsequent analyses showed that DNA could be recovered from all application sites with a maximum of 53% in the grooves of the screw heads. We could further show that up to 35% of DNA retained its full biological function, i.e., mediating antibiotic resistance in bacteria and fluorescent marker expression in eukaryotic cells. These experiments show that our plasmid DNA biomarker assay is suitable to characterize the environmental conditions affecting DNA during an atmospheric transit and the re-entry and constitute the first report of the stability of DNA during hypervelocity atmospheric transit indicating that sounding rocket flights can be used to model the high-speed atmospheric entry of organics-laden artificial meteorites.

  8. Functional activity of plasmid DNA after entry into the atmosphere of earth investigated by a new biomarker stability assay for ballistic spaceflight experiments.

    Directory of Open Access Journals (Sweden)

    Cora S Thiel

    Full Text Available Sounding rockets represent an excellent platform for testing the influence of space conditions during the passage of Earth's atmosphere and re-entry on biological, physical and chemical experiments for astrobiological purposes. We designed a robust functionality biomarker assay to analyze the biological effects of suborbital spaceflights prevailing during ballistic rocket flights. During the TEXUS-49 rocket mission in March 2011, artificial plasmid DNA carrying a fluorescent marker (enhanced green fluorescent protein: EGFP and an antibiotic resistance cassette (kanamycin/neomycin was attached on different positions of rocket exterior; (i circular every 90 degree on the outer surface concentrical of the payload, (ii in the grooves of screw heads located in between the surface application sites, and (iii on the surface of the bottom side of the payload. Temperature measurements showed two major peaks at 118 and 130 °C during the 780 seconds lasting flight on the inside of the recovery module, while outer gas temperatures of more than 1000 °C were estimated on the sample application locations. Directly after retrieval and return transport of the payload, the plasmid DNA samples were recovered. Subsequent analyses showed that DNA could be recovered from all application sites with a maximum of 53% in the grooves of the screw heads. We could further show that up to 35% of DNA retained its full biological function, i.e., mediating antibiotic resistance in bacteria and fluorescent marker expression in eukaryotic cells. These experiments show that our plasmid DNA biomarker assay is suitable to characterize the environmental conditions affecting DNA during an atmospheric transit and the re-entry and constitute the first report of the stability of DNA during hypervelocity atmospheric transit indicating that sounding rocket flights can be used to model the high-speed atmospheric entry of organics-laden artificial meteorites.

  9. ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

    DEFF Research Database (Denmark)

    Zhao, A.; Gray, F. C; MacNeill, S. A.

    2006-01-01

    DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile....... volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that Lig...

  10. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  11. Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

    Science.gov (United States)

    Arias, María Elena; Sánchez-Villalba, Esther; Delgado, Andrea; Felmer, Ricardo

    2017-02-01

    Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

  12. Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway as a scaffold protein in the fission yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Yue, Ming; Zeng, Li; Singh, Amanpreet; Xu, Yongjie

    2014-01-01

    Rad4/Cut5 is a scaffold protein in the Chk1-mediated DNA damage checkpoint in S. pombe. However, whether it contains a robust ATR-activation domain (AAD) required for checkpoint signaling like its orthologs TopBP1 in humans and Dpb11 in budding yeast has been incompletely clear. To identify the putative AAD in Rad4, we carried out an extensive genetic screen looking for novel mutants with an enhanced sensitivity to replication stress or DNA damage in which the function of the AAD can be eliminated by the mutations. Two new mutations near the N-terminus were identified that caused significantly higher sensitivities to DNA damage or chronic replication stress than all previously reported mutants, suggesting that most of the checkpoint function of the protein is eliminated. However, these mutations did not affect the activation of Rad3 (ATR in humans) yet eliminated the scaffolding function of the protein required for the activation of Chk1. Several mutations were also identified in or near the recently reported AAD in the C-terminus of Rad4. However, all mutations in the C-terminus only slightly sensitized the cells to DNA damage. Interestingly, a mutant lacking the whole C-terminus was found resistant to DNA damage and replication stress almost like the wild type cells. Consistent with the resistance, all known Rad3 dependent phosphorylations of checkpoint proteins remained intact in the C-terminal deletion mutant, indicating that unlike that in Dpb11, the C-terminus of Rad4 does not contain a robust AAD. These results, together with those from the biochemical studies, show that Rad4 mainly functions as a scaffold protein in the Chk1, not the Cds1(CHK2 in humans), checkpoint pathway. It plays a minor role or is functionally redundant with an unknown factor in Rad3 activation.

  13. Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway as a scaffold protein in the fission yeast Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Ming Yue

    Full Text Available Rad4/Cut5 is a scaffold protein in the Chk1-mediated DNA damage checkpoint in S. pombe. However, whether it contains a robust ATR-activation domain (AAD required for checkpoint signaling like its orthologs TopBP1 in humans and Dpb11 in budding yeast has been incompletely clear. To identify the putative AAD in Rad4, we carried out an extensive genetic screen looking for novel mutants with an enhanced sensitivity to replication stress or DNA damage in which the function of the AAD can be eliminated by the mutations. Two new mutations near the N-terminus were identified that caused significantly higher sensitivities to DNA damage or chronic replication stress than all previously reported mutants, suggesting that most of the checkpoint function of the protein is eliminated. However, these mutations did not affect the activation of Rad3 (ATR in humans yet eliminated the scaffolding function of the protein required for the activation of Chk1. Several mutations were also identified in or near the recently reported AAD in the C-terminus of Rad4. However, all mutations in the C-terminus only slightly sensitized the cells to DNA damage. Interestingly, a mutant lacking the whole C-terminus was found resistant to DNA damage and replication stress almost like the wild type cells. Consistent with the resistance, all known Rad3 dependent phosphorylations of checkpoint proteins remained intact in the C-terminal deletion mutant, indicating that unlike that in Dpb11, the C-terminus of Rad4 does not contain a robust AAD. These results, together with those from the biochemical studies, show that Rad4 mainly functions as a scaffold protein in the Chk1, not the Cds1(CHK2 in humans, checkpoint pathway. It plays a minor role or is functionally redundant with an unknown factor in Rad3 activation.

  14. Identification and functional analysis of a new glyphosate resistance gene from a fungus cDNA library.

    Science.gov (United States)

    Tao, Bo; Shao, Bai-Hui; Qiao, Yu-Xin; Wang, Xiao-Qin; Chang, Shu-Jun; Qiu, Li-Juan

    2017-08-01

    Glyphosate is a widely used broad spectrum herbicide; however, this limits its use once crops are planted. If glyphosate-resistant crops are grown, glyphosate can be used for weed control in crops. While several glyphosate resistance genes are used in commercial glyphosate tolerant crops, there is interest in identifying additional genes for glyphosate tolerance. This research constructed a high-quality cDNA library form the glyphosate-resistant fungus Aspergillus oryzae RIB40 to identify genes that may confer resistance to glyphosate. Using a medium containing glyphosate (120mM), we screened several clones from the library. Based on a nucleotide sequence analysis, we identified a gene of unknown function (GenBank accession number: XM_001826835.2) that encoded a hypothetical 344-amino acid protein. The gene was named MFS40. Its ORF was amplified to construct an expression vector, pGEX-4T-1-MFS40, to express the protein in Escherichia coli BL21. The gene conferred glyphosate tolerance to E. coli ER2799 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Functional genomics indicates yeast requires Golgi/ER transport, chromatin remodeling, and DNA repair for low dose DMSO tolerance

    Directory of Open Access Journals (Sweden)

    Brandon David Gaytán

    2013-08-01

    Full Text Available Dimethyl sulfoxide (DMSO is frequently utilized as a solvent in toxicological and pharmaceutical investigations. It is therefore important to establish the cellular and molecular targets of DMSO in order to differentiate its intrinsic effects from those elicited by a compound of interest. We performed a genome-wide functional screen in Saccharomyces cerevisiae to identify deletion mutants exhibiting sensitivity to 1% DMSO, a concentration standard to yeast chemical profiling studies. We report that mutants defective in Golgi/ER transport are sensitive to DMSO, including those lacking components of the conserved oligomeric Golgi (COG complex. Moreover, strains deleted for members of the SWR1 histone exchange complex are hypersensitive to DMSO, with additional chromatin remodeling mutants displaying a range of growth defects. We also identify DNA repair genes important for DMSO tolerance. Finally, we demonstrate that overexpression of histone H2A.Z, which replaces chromatin-associated histone H2A in a SWR1-catalyzed reaction, confers resistance to DMSO. Many yeast genes described in this study have homologs in more complex organisms, and the data provided is applicable to future investigations into the cellular and molecular mechanisms of DMSO toxicity.

  16. Engineering of DNA templated tri-functional nano-chain of Fecore–Aushell and a preliminary study for cancer cell labeling and treatment

    Directory of Open Access Journals (Sweden)

    Madhuri Mandal

    2012-10-01

    Full Text Available Here DNA has been used as templating and self-assembling reagent to grow the chain like nanostructure. We have designed the composite in such a fashion that we obtained optical and magnetic properties together in a single biological material. Optical properties characterized by UV–visible absorption, Circular Dichroism (CD and their analysis show no denaturization of DNA. Transmission electron micrographs (TEM indicate formation of chain like structure of the nanoparticles. Particles were functionalized with folic acid for labeling and treatment of cancer cell.

  17. Molecular cloning and functional identification of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Tripterygium wilfordii

    Directory of Open Access Journals (Sweden)

    Qiqing Cheng

    2017-03-01

    Full Text Available The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR is the last step key enzyme of the methylerythritol phosphate (MEP pathway, synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which is important for regulation of isoprenoid biosynthesis. Here the full-length cDNA of HDR, designated TwHDR (GenBank Accession No. KJ933412.1, was isolated from Tripterygium wilfordii for the first time. TwHDR has an open reading frame (ORF of 1386 bp encoding 461 amino acids. TwHDR exhibits high homology with HDRs of other plants, with an N-terminal conserved domain and three conserved cysteine residues. TwHDR cDNA was cloned into an expression vector and transformed into an Escherichia coli hdr mutant. Since loss-of-function E.coli hdr mutant is lethal, the result showed that transformation of TwHDR cDNA rescued the E.coli hdr mutant. This complementation assay suggests that the TwHDR cDNA encodes a functional HDR enzyme. The expression of TwHDR was induced by methyl-jasmonate (MJ in T. wilfordii suspension cells. The expression of TwHDR reached the highest level after 1 h of MJ treatment. These results indicate that we have identified a functional TwHDR enzyme, which may play a pivotal role in the biosynthesis of diterpenoid triptolide in T. wilfordii.

  18. Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced DNA damage depends on functional TFIIH

    NARCIS (Netherlands)

    A. Zotter (Angelika); A.B. Houtsmuller (Adriaan); M.S. Luijsterburg (Martijn); D.O. Warmerdam (Daniël); S.M. Ibrahim (Shehu); A.L. Nigg (Alex); W.A. van Cappellen (Gert); J.H.J. Hoeijmakers (Jan); R. van Driel; W. Vermeulen (Wim)

    2006-01-01

    textabstractThe structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the

  19. Towards intelligent bioreactor systems: triggering the release and mixing of compounds based on DNA-functionalized hybrid hydrogel.

    Science.gov (United States)

    Zhou, Li; Chen, Cuie; Ren, Jinsong; Qu, Xiaogang

    2014-09-14

    We have designed and synthesized an intelligent mesoporous silica nanoparticle-DNA hydrogel bioreactor system that can be controlled by external stimuli. The system allowed the simultaneous incorporation of multiple components, and the separation between the components can be destroyed by a structural change of the DNA to initiate a reaction.

  20. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Directory of Open Access Journals (Sweden)

    Morgan L Henderson

    Full Text Available Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs, located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs. Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation: RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA. In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

  1. Structural and Functional Analysis of the H19Y Mutated Primary DNA Recognition Subdomain of Sleeping Beauty Transposase

    Science.gov (United States)

    Leighton, Gage O.

    The Sleeping Beauty transposon system, consisting of the transposon DNA and the transposase enzyme, is a member of the Tc1/mariner family of DNA transposons. Although it is an important tool in genetic applications and has been adapted for human gene therapy, its molecular mechanism remains obscure. Here, we use an experimental biophysics approach to study the molecular mechanism of the Sleeping Beauty transposon. We investigate the folding of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain. We show that only the folded PAI binds to DNA, however the amount of unfolded conformation is significant at close to physiologic conditions. We identify amino acid substitutions that result in stabilization of its folded conformation. Overall, our results provide a mechanistic insight into DNA recognition by the Sleeping Beauty transposase and suggest modifications to improve its activity.

  2. Functional studies of ssDNA binding ability of MarR family protein TcaR from Staphylococcus epidermidis.

    Science.gov (United States)

    Chang, Yu-Ming; Chen, Cammy K-M; Chang, Yuan-Chih; Jeng, Wen-Yih; Hou, Ming-Hon; Wang, Andrew H-J

    2012-01-01

    The negative transcription regulator of the ica locus, TcaR, regulates proteins involved in the biosynthesis of poly-N-acetylglucosamine (PNAG). Absence of TcaR increases PNAG production and promotes biofilm formation in Staphylococci. Previously, the 3D structure of TcaR in its apo form and its complex structure with several antibiotics have been analyzed. However, the detailed mechanism of multiple antibiotic resistance regulator (MarR) family proteins such as TcaR is unclear and only restricted on the binding ability of double-strand DNA (dsDNA). Here we show by electrophoretic mobility shift assay (EMSA), electron microscopy (EM), circular dichroism (CD), and Biacore analysis that TcaR can interact strongly with single-stranded DNA (ssDNA), thereby identifying a new role in MarR family proteins. Moreover, we show that TcaR preferentially binds 33-mer ssDNA over double-stranded DNA and inhibits viral ssDNA replication. In contrast, such ssDNA binding properties were not observed for other MarR family protein and TetR family protein, suggesting that the results from our studies are not an artifact due to simple charge interactions between TcaR and ssDNA. Overall, these results suggest a novel role for TcaR in regulation of DNA replication. We anticipate that the results of this work will extend our understanding of MarR family protein and broaden the development of new therapeutic strategies for Staphylococci.

  3. RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates

    Science.gov (United States)

    Polevoda, Bogdan; McDougall, William M.; Tun, Bradley N.; Cheung, Michael; Salter, Jason D.; Friedman, Alan E.; Smith, Harold C.

    2015-01-01

    APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181–194 in the N-terminus and aa 314–320 and 345–374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15–29, 41–52 and 83–99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states. PMID:26424853

  4. Functional studies of ssDNA binding ability of MarR family protein TcaR from Staphylococcus epidermidis.

    Directory of Open Access Journals (Sweden)

    Yu-Ming Chang

    Full Text Available The negative transcription regulator of the ica locus, TcaR, regulates proteins involved in the biosynthesis of poly-N-acetylglucosamine (PNAG. Absence of TcaR increases PNAG production and promotes biofilm formation in Staphylococci. Previously, the 3D structure of TcaR in its apo form and its complex structure with several antibiotics have been analyzed. However, the detailed mechanism of multiple antibiotic resistance regulator (MarR family proteins such as TcaR is unclear and only restricted on the binding ability of double-strand DNA (dsDNA. Here we show by electrophoretic mobility shift assay (EMSA, electron microscopy (EM, circular dichroism (CD, and Biacore analysis that TcaR can interact strongly with single-stranded DNA (ssDNA, thereby identifying a new role in MarR family proteins. Moreover, we show that TcaR preferentially binds 33-mer ssDNA over double-stranded DNA and inhibits viral ssDNA replication. In contrast, such ssDNA binding properties were not observed for other MarR family protein and TetR family protein, suggesting that the results from our studies are not an artifact due to simple charge interactions between TcaR and ssDNA. Overall, these results suggest a novel role for TcaR in regulation of DNA replication. We anticipate that the results of this work will extend our understanding of MarR family protein and broaden the development of new therapeutic strategies for Staphylococci.

  5. Insights into the functional characteristics of geminivirus rolling-circle replication initiator protein and its interaction with host factors affecting viral DNA replication.

    Science.gov (United States)

    Rizvi, Irum; Choudhury, Nirupam Roy; Tuteja, Narendra

    2015-02-01

    Geminiviruses are DNA viruses that infect several economically important crops, resulting in a reduction in their overall yield. These plant viruses have circular, single-stranded DNA genomes that replicate mainly by a rolling-circle mechanism. Geminivirus infection results in crosstalk between viral and cellular factors to complete the viral life cycle or counteract the infection as part of defense mechanisms of host plants. The geminiviral replication initiator protein Rep is the only essential viral factor required for replication. It is multifunctional and is known to interact with a number of host factors to modulate the cellular environment or to function as a part of the replication machinery. This review provides a holistic view of the research related to the viral Rep protein and various host factors involved in geminiviral DNA replication. Studies on the promiscuous nature of geminiviral satellite DNAs are also reviewed.

  6. Biochemical studies on the DNA binding function of the cyclic-amp reactor protein of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Angulo, J.A.

    1986-01-01

    The cAMP receptor protein (CRP) is an allosteric protein in which binding of cAMP effects a conformational change with a consequent increased affinity for DNA. Binding of double-stranded deoxyribopolynucleotides and calf thymus DNA by cAMP-CRP confers protection against attack by trypsin, subtilisin, Staph. aureus V8 protease and clostripain. Of the single-stranded deoxy- and ribopolynucleotides tested, only r(I)/sub n/ and r(A)/sub n/ gave significant protection against attack by these proteases. In the absence of cAMP, CRP is resistant to proteolysis. Incubation of CRP-DNA with trypsin results in the accumulation of two novel fragments. CRP-DNA is partially sensitive to digestion by chymotrypsin but resistant to attack by subtilisin, the Staph. aureus V8 protease and clostripain. Cleavage of CRP-DNA to fragments is accompanied by the loss of /sup 3/H-cAMP binding activity. Modification of the arginines with phenylglyoxal or butanedione results in loss of DNA binding activity. cAMP-CRP incorporates more /sup 14/C-phenylglyoxal than unliganded CRP. Titration of the arginines with /sup 14/C-phenylglyoxal to where over 90% of the DNA binding activity is lost results in incorporation of one mole of reagent per mole of subunit.

  7. The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Trego, Kelly S.; Chernikova, Sophia B.; Davalos, Albert R.; Perry, J. Jefferson P.; Finger, L. David; Ng, Cliff; Tsai, Miaw-Sheue; Yannone, Steven M.; Tainer, John A.; Campisi, Judith; Cooper, Priscilla K.

    2011-04-20

    XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar sub-nuclear redistribution in S-phase and co-localize in nuclear foci. The co-localization was observed in mid- to late-S-phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain protein markers of both stalled replication forks and telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains, but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S-phase that is at least in part carried out coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.

  8. K-mer Content, Correlation, and Position Analysis of Genome DNA Sequences for the Identification of Function and Evolutionary Features.

    Science.gov (United States)

    Sievers, Aaron; Bosiek, Katharina; Bisch, Marc; Dreessen, Chris; Riedel, Jascha; Froß, Patrick; Hausmann, Michael; Hildenbrand, Georg

    2017-04-19

    In genome analysis, k-mer-based comparison methods have become standard tools. However, even though they are able to deliver reliable results, other algorithms seem to work better in some cases. To improve k-mer-based DNA sequence analysis and comparison, we successfully checked whether adding positional resolution is beneficial for finding and/or comparing interesting organizational structures. A simple but efficient algorithm for extracting and saving local k-mer spectra (frequency distribution of k-mers) was developed and used. The results were analyzed by including positional information based on visualizations as genomic maps and by applying basic vector correlation methods. This analysis was concentrated on small word lengths (1 ≤ k ≤ 4) on relatively small viral genomes of Papillomaviridae and Herpesviridae, while also checking its usability for larger sequences, namely human chromosome 2 and the homologous chromosomes (2A, 2B) of a chimpanzee. Using this alignment-free analysis, several regions with specific characteristics in Papillomaviridae and Herpesviridae formerly identified by independent, mostly alignment-based methods, were confirmed. Correlations between the k-mer content and several genes in these genomes have been found, showing similarities between classified and unclassified viruses, which may be potentially useful for further taxonomic research. Furthermore, unknown k-mer correlations in the genomes of Human Herpesviruses (HHVs), which are probably of major biological function, are found and described. Using the chromosomes of a chimpanzee and human that are currently known, identities between the species on every analyzed chromosome were reproduced. This demonstrates the feasibility of our approach for large data sets of complex genomes. Based on these results, we suggest k-mer analysis with positional resolution as a method for closing a gap between the effectiveness of alignment-based methods (like NCBI BLAST) and the high pace of

  9. Improved description of the structural and optoelectronic properties of DNA/RNA nucleobase anhydrous crystals: Experiment and dispersion-corrected density functional theory calculations

    Science.gov (United States)

    da Silva, M. B.; Francisco, T. S.; Maia, F. F.; Caetano, E. W. S.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.

    2017-08-01

    The development of low cost and environmentally friendly organic electronic/optoelectronic devices has attracted a lot of interest. The integration of DNA and RNA nucleobases to improve the performance of organic light-emitting diodes has been proposed recently [Gomez et al., Sci. Rep. 4, 7105 (2014), 10.1038/srep07105], notwithstanding limited experimental and theoretical information on the optoelectronic properties of DNA/RNA thin films. As a contribution to an improved understanding of DNA/RNA-based devices in the solid state, we have performed in this paper dispersion corrected density functional theory (DFT) and time-dependent DFT (TDDFT) calculations to obtain the optimized geometries, Kohn-Sham band structures and orbitals, charge distribution, optical absorption, Frenkel exciton binding energies, and complex dielectric functions of the five DNA/RNA nucleobase anhydrous crystals, namely cytosine, guanine, adenine, thymine, and uracil. Optical absorption measurements on DNA/RNA nucleobase powders were also performed for comparison with the simulations. An improvement on the local density approximation (LDA) description of the lattice parameter estimates was achieved considering the generalized gradient approach (GGA) with a semiempirical dispersion correction scheme in comparison with structural x-ray data found in the literature. Energy gap correction using the Δ-sol methodology provided a good agreement between theory and experimental estimates from our optical absorption data, greatly surpassing the quality of previous simulations. Effective masses for the carriers were also found, indicating that the guanine crystal as well as the cytosine one (although with some drawbacks) has potential applications in optoelectronics as a direct gap semiconductor, with the other nucleobases presenting either a semiconductor or an insulator character depending on the carrier type. The complex dielectric function exhibits a high degree of anisotropy for different states

  10. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    Directory of Open Access Journals (Sweden)

    Alamar Santiago

    2009-09-01

    Full Text Available Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new

  11. Multiple genes for functional 6 fatty acyl desaturases (Fad) in Atlantic salmon (Salmo salar L.): gene and cDNA characterization, functional expression, tissue distribution and nutritional regulation.

    Science.gov (United States)

    Monroig, Oscar; Zheng, Xiaozhong; Morais, Sofia; Leaver, Michael J; Taggart, John B; Tocher, Douglas R

    2010-09-01

    Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids, eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs encoding functionally distinct Delta5 and Delta6 Fads were isolated, but screening of a genomic DNA library revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes, Delta6fad_a and Delta5fad, corresponded to the previously cloned Delta6 and Delta5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, Delta6fad_b and Delta6fad_c, had only Delta6 activity, converting 47 % and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of 18:2n-6 to 18:3n-6, for 6Fad_b and Delta6fad_c, respectively. Both 6fad_a and 6fad_b genes were highly expressed in intestine (pyloric caeca), liver and brain, with 6fad_b also highly expressed in gill, whereas 6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The expression levels of the 6fad_a gene in liver and the 6fad_b gene in intestine were significantly higher in fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for 6fad_a in intestine, 6fad_b in liver, or 6fad_c in liver or intestine of fish fed vegetable oil compared to fish fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes are discussed in relation to gene structures and fish

  12. DNA damage response

    NARCIS (Netherlands)

    G. Giglia-Mari (Giuseppina); A. Zotter (Angelika); W. Vermeulen (Wim)

    2011-01-01

    textabstractStructural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network ofDNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance

  13. Protein Degradation Pathways Regulate the Functions of Helicases in the DNA Damage Response and Maintenance of Genomic Stability

    Directory of Open Access Journals (Sweden)

    Joshua A. Sommers

    2015-04-01

    Full Text Available Degradation of helicases or helicase-like proteins, often mediated by ubiquitin-proteasomal pathways, plays important regulatory roles in cellular mechanisms that respond to DNA damage or replication stress. The Bloom’s syndrome helicase (BLM provides an example of how helicase degradation pathways, regulated by post-translational modifications and protein interactions with components of the Fanconi Anemia (FA interstrand cross-link (ICL repair pathway, influence cell cycle checkpoints, DNA repair, and replication restart. The FANCM DNA translocase can be targeted by checkpoint kinases that exert dramatic effects on FANCM stability and chromosomal integrity. Other work provides evidence that degradation of the F-box DNA helicase (FBH1 helps to balance translesion synthesis (TLS and homologous recombination (HR repair at blocked replication forks. Degradation of the helicase-like transcription factor (HLTF, a DNA translocase and ubiquitylating enzyme, influences the choice of post replication repair (PRR pathway. Stability of the Werner syndrome helicase-nuclease (WRN involved in the replication stress response is regulated by its acetylation. Turning to transcription, stability of the Cockayne Syndrome Group B DNA translocase (CSB implicated in transcription-coupled repair (TCR is regulated by a CSA ubiquitin ligase complex enabling recovery of RNA synthesis. Collectively, these studies demonstrate that helicases can be targeted for degradation to maintain genome homeostasis.

  14. Organisms posses enzymes that function in the repair of DNA damaged by radiations, chemicals and metabolic events

    Energy Technology Data Exchange (ETDEWEB)

    Mizuma, Nagayo [Kyoto Univ., Kumatori, Osaka (Japan). Research Reactor Inst.

    1998-01-01

    This report briefly describes the studies on the mechanism of in vivo DNA repairing by the author in Research Reactor Institute, Kyoto Univ. for the past 30 years. First, the ability of UV radiation to induce transformation was investigated with viral DNA. The formation of thymine-thymine dimer was found harmful to organisms and such dimers were removable by UV-radiation at a low frequency. The mutability was determined in three different E.coli strains with mutator gene, mutT, mutS or mutL. The ability to excise 8-oxoguanin developed in primer DNA was deficient in mutT and miss-pairing left after DNA replication could not be recovered in mutL and mutS strains. Further, DNA repairing mechanism was investigated in other microorganisms; single-strand cleavage caused by exposure to BNCB radiation (boron-neutron-captured beam) could not be repaired in E. coli. Whereas for Deinococcus radiodurans, of which survival rate was not decreased by {gamma}-ray radiation at 5 kGy or less, it was found that its single-strand DNA was damaged by {gamma}-radiation to smaller molecules, but it was mended to the similar size to that in the non-irradiated cells during incubation. In addition, the transformation frequency was also recovered in the actinomycetes. Thus, it was demonstrated that de novo protein synthesis is necessary for the repairing system of recombination. (M.N.)

  15. Structure-function analysis of Escherichia coli DNA helicase I reveals non-overlapping transesterase and helicase domains.

    Science.gov (United States)

    Byrd, Devon R; Sampson, Juliana K; Ragonese, Heather M; Matson, Steven W

    2002-11-08

    TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.

  16. Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA).

    Science.gov (United States)

    Miller, Daniel E; Patel, Zubin H; Lu, Xiaoming; Lynch, Arthur T; Weirauch, Matthew T; Kottyan, Leah C

    2016-08-21

    Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

  17. Structure-function study of deinococcal serine/threonine protein kinase implicates its kinase activity and DNA repair protein phosphorylation roles in radioresistance of Deinococcus radiodurans.

    Science.gov (United States)

    Rajpurohit, Yogendra S; Misra, Hari S

    2013-11-01

    The DR2518 (RqkA) a eukaryotic type serine/threonine protein kinase in Deinococcus radiodurans was characterized for its role in bacterial response to oxidative stress and DNA damage. The K42A, S162A, T169A and S171A mutation in RqkA differentially affected its kinase activity and functional complementation for γ radiation resistance in Δdr2518 mutant. For example, K42A mutant was completely inactive and showed no complementation while S171A, T169A and T169A/S171A mutants were less active and complemented proportionally to different levels as compared to wild type. Amongst, different DNA binding proteins that purified RqkA could phosphorylate, PprA a DNA repair protein, phosphorylation had improved its affinity to DNA by 4 fold and could enhance its supportive role in intermolecular ligation by T4 DNA ligase. RqkA phosphorylates PprA at threonine 72 (T72), serine 112 (S112) and threonine 144 (T144) in vitro with the majority of it goes to T72 site. Unlike wild type PprA and single mutants of T72, S112 and T144 residues, the T72AS112A double and T72AS112AT144A triple mutant derivatives of PprA did not phosphorylate in vivo and also failed to complement PprA loss in D. radiodurans. Deletion of rqkA in pprA::cat background enhanced radiosensitivity of pprA mutant, which became nearly similar to ΔrqkA resistance to γ radiation. These results suggested that K42 of RqkA is essential for catalytic functions and the kinase activity of RqkA as well as phosphorylation of PprA have roles in γ radiation resistance of D. radiodurans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function.

    Science.gov (United States)

    Ciolkowski, Ingo; Wanke, Dierk; Birkenbihl, Rainer P; Somssich, Imre E

    2008-09-01

    WRKY transcription factors have been shown to play a major role in regulating, both positively and negatively, the plant defense transcriptome. Nearly all studied WRKY factors appear to have a stereotypic binding preference to one DNA element termed the W-box. How specificity for certain promoters is accomplished therefore remains completely unknown. In this study, we tested five distinct Arabidopsis WRKY transcription factor subfamily members for their DNA binding selectivity towards variants of the W-box embedded in neighboring DNA sequences. These studies revealed for the first time differences in their binding site preferences, which are partly dependent on additional adjacent DNA sequences outside of the TTGACY-core motif. A consensus WRKY binding site derived from these studies was used for in silico analysis to identify potential target genes within the Arabidopsis genome. Furthermore, we show that even subtle amino acid substitutions within the DNA binding region of AtWRKY11 strongly impinge on its binding activity. Additionally, all five factors were found localized exclusively to the plant cell nucleus and to be capable of trans-activating expression of a reporter gene construct in vivo.

  19. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

    Science.gov (United States)

    Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

    2017-05-15

    5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Higher-order organisation of extremely amplified, potentially functional and massively methylated 5S rDNA in European pikes (Esox sp.).

    Science.gov (United States)

    Symonová, Radka; Ocalewicz, Konrad; Kirtiklis, Lech; Delmastro, Giovanni Battista; Pelikánová, Šárka; Garcia, Sonia; Kovařík, Aleš

    2017-05-18

    Pikes represent an important genus (Esox) harbouring a pre-duplication karyotype (2n = 2x = 50) of economically important salmonid pseudopolyploids. Here, we have characterized the 5S ribosomal RNA genes (rDNA) in Esox lucius and its closely related E. cisalpinus using cytogenetic, molecular and genomic approaches. Intragenomic homogeneity and copy number estimation was carried out using Illumina reads. The higher-order structure of rDNA arrays was investigated by the analysis of long PacBio reads. Position of loci on chromosomes was determined by FISH. DNA methylation was analysed by methylation-sensitive restriction enzymes. The 5S rDNA loci occupy exclusively (peri)centromeric regions on 30-38 acrocentric chromosomes in both E. lucius and E. cisalpinus. The large number of loci is accompanied by extreme amplification of genes (>20,000 copies), which is to the best of our knowledge one of the highest copy number of rRNA genes in animals ever reported. Conserved secondary structures of predicted 5S rRNAs indicate that most of the amplified genes are potentially functional. Only few SNPs were found in genic regions indicating their high homogeneity while intergenic spacers were more heterogeneous and several families were identified. Analysis of 10-30 kb-long molecules sequenced by the PacBio technology (containing about 40% of total 5S rDNA) revealed that the vast majority (96%) of genes are organised in large several kilobase-long blocks. Dispersed genes or short tandems were less common (4%). The adjacent 5S blocks were directly linked, separated by intervening DNA and even inverted. The 5S units differing in the intergenic spacers formed both homogeneous and heterogeneous (mixed) blocks indicating variable degree of homogenisation between the loci. Both E. lucius and E. cisalpinus 5S rDNA was heavily methylated at CG dinucleotides. Extreme amplification of 5S rRNA genes in the Esox genome occurred in the absence of significant pseudogenisation

  1. Crystal structure of the UvrB dimer: insights into the nature and functioning of the UvrAB damage engagement and UvrB–DNA complexes

    Science.gov (United States)

    Webster, Matthew P. J.; Jukes, Rachael; Zamfir, Vlad S.; Kay, Christopher W. M.; Bagnéris, Claire; Barrett, Tracey

    2012-01-01

    UvrB has a central role in the highly conserved UvrABC pathway functioning not only as a damage recognition element but also as an essential component of the lesion tracking machinery. While it has been recently confirmed that the tracking assembly comprises a UvrA2B2 heterotetramer, the configurations of the damage engagement and UvrB–DNA handover complexes remain obscure. Here, we present the first crystal structure of a UvrB dimer whose biological significance has been verified using both chemical cross-linking and electron paramagnetic resonance spectroscopy. We demonstrate that this dimeric species stably associates with UvrA and forms a UvrA2B2–DNA complex. Our studies also illustrate how signals are transduced between the ATP and DNA binding sites to generate the helicase activity pivotal to handover and formation of the UvrB2–DNA complex, providing key insights into the configurations of these important repair intermediates. PMID:22753105

  2. Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase

    OpenAIRE

    Bibillo, Arkadiusz; Lener, Daniela; Klarmann, George J.; Le Grice, Stuart F. J.

    2005-01-01

    Aspartic acid residues comprising the -D-(aa) n -Y-L-D-D- DNA polymerase active site triad of reverse transcriptase from the Saccharomyces cerevisiae long terminal repeat-retrotransposon Ty3 (Asp151, Asp213 and Asp214) were evaluated via site-directed mutagenesis. An Asp151→Glu substitution showed a dramatic decrease in catalytic efficiency and a severe translocation defect following initiation of DNA synthesis. In contrast, enzymes harboring the equivalent alteration at Asp213 and Asp214 ret...

  3. Biochip functionalization using electrowetting-on-dielectric digital microfluidics for surface plasmon resonance imaging detection of DNA hybridization.

    Science.gov (United States)

    Malic, Lidija; Veres, Teodor; Tabrizian, Maryam

    2009-03-15

    This work reports on a dynamically configurable micro-array surface plasmon resonance biochip platform. The platform comprises a digital electrowetting-on-dielectric (EWOD) microfluidic device tailored to surface plasmon resonance imaging (SPRi). We demonstrate its application for simultaneous immobilization of different DNA probes at the designated detection sites on-chip from sub-microL volume solutions in combination with multichannel label-free real-time detection of subsequent hybridization reactions. Successful on-chip DNA probe dilution and immobilization is also demonstrated using SPRi hybridization detection. Furthermore, active control of the immobilized probe density and orientation is achieved under an applied potential using the electric interface of the EWOD device. For low probe densities, under negative applied potential, the DNA hybridization efficiency is enhanced compared to passive probe immobilization, yielding a two-fold SPR signal increase within only 8min of hybridization. EWOD microfluidic platform coupled with SPRi promises to dramatically increase the speed of detection and quantification of biomolecular interactions while reducing reagent consumption. The proposed system would enable the development of high-throughput, rapid and ultrasensitive detection of biomolecules beyond DNA microarray applications.

  4. Optimal functional levels of activation-induced deaminase specifically require the Hsp40 DnaJa1

    OpenAIRE

    Orthwein, Alexandre; Zahn, Astrid; Methot, Stephen P; Godin, David; Conticello, Silvestro G; Terada, Kazutoyo; Di Noia, Javier M

    2011-01-01

    AID deaminates deoxycytidine at immunoglobulin genes to generate an antibody response. AID misregulation can contribute to cancer and autoimmune disease. Here, the chaperone DnaJa1 is shown to determine AID protein levels and biological activity during the murine immune response.

  5. Cloning and functional characterization of a testicular TSH receptor cDNA from the African catfish (Clarias gariepinus)

    NARCIS (Netherlands)

    Vischer, H F; Bogerd, J.

    A cDNA encoding a putative thyroid-stimulating hormone receptor (cfTSH-R) was cloned from the testis of the African catfish (Clarias gariepinus). The cfTSH-R showed the highest amino acid sequence identity with the TSH-Rs of other fish species. In addition, an insertion of approximately 50 amino

  6. Systematic functional comparative analysis of four single-stranded DNA-binding proteins and their affection on viral RNA metabolism.

    Directory of Open Access Journals (Sweden)

    Haiyan Shi

    Full Text Available The accumulation of single-stranded DNA-binding (SSB proteins is essential for organisms and has various applications. However, no study has simultaneously and systematically compared the characteristics of SSB proteins. In addition, SSB proteins may bind RNA and play an unknown biological role in RNA metabolism. Here, we expressed a novel species of SSB protein derived from Thermococcus kodakarensis KOD1 (KOD, as well as SSB proteins from Thermus thermophilus (TTH, Escherichia coli, and Sulfolobus Solfataricus P2 (SSOB, abbreviated kod, tth, bl21, and ssob, respectively. These SSB proteins could bind ssDNA and viral RNA. bl21 resisted heat treatment for more than 9 h, Ssob and kod could withstand 95°C for 10 h and retain its ssDNA- and RNA-binding ability. Four SSB proteins promoted the specificity of the DNA polymerase in PCR-based 5- and 9-kb genome fragment amplification. kod also increased the amplification of a 13-kb PCR product, and SSB protein-bound RNA resisted Benzonase digestion. The SSB proteins could also enter the host cell bound to RNA, which resulted in modulation of viral RNA metabolism, particularly ssob and bl21.

  7. Fumarase is involved in DNA double-strand break resection through a functional interaction with Sae2

    DEFF Research Database (Denmark)

    Leshets, Michael; Ramamurthy, Dharanidharan; Lisby, Michael

    2018-01-01

    One of the most severe forms of DNA damage is the double-strand break (DSB). Failure to properly repair the damage can cause mutation, gross chromosomal rearrangements and lead to the development of cancer. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are the...

  8. Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases

    NARCIS (Netherlands)

    Lücker, J.; Tamer, El M.K.; Schwab, W.; Verstappen, F.W.A.; Plas, van der L.H.W.; Bouwmeester, H.J.; Verhoeven, H.A.

    2002-01-01

    Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the

  9. Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase.

    Science.gov (United States)

    Bibillo, Arkadiusz; Lener, Daniela; Klarmann, George J; Le Grice, Stuart F J

    2005-01-01

    Aspartic acid residues comprising the -D-(aa) n -Y-L-D-D- DNA polymerase active site triad of reverse transcriptase from the Saccharomyces cerevisiae long terminal repeat-retrotransposon Ty3 (Asp151, Asp213 and Asp214) were evaluated via site-directed mutagenesis. An Asp151-->Glu substitution showed a dramatic decrease in catalytic efficiency and a severe translocation defect following initiation of DNA synthesis. In contrast, enzymes harboring the equivalent alteration at Asp213 and Asp214 retained DNA polymerase activity. Asp151-->Asn and Asp213-->Asn substitutions eliminated both polymerase activities. However, while Asp214 of the triad could be replaced by either Asn or Glu, introducing Gln seriously affected processivity. Mutants of the carboxylate triad at positions 151 and 213 also failed to catalyze pyrophosphorolysis. Finally, alterations to the DNA polymerase active site affected RNase H activity, suggesting a close spatial relationship between these N- and C-terminal catalytic centers. Taken together, our data reveal a critical role for Asp151 and Asp213 in catalysis. In contrast, the second carboxylate of the Y-L-D-D motif (Asp214) is not essential for catalysis, and possibly fulfills a structural role. Although Asp214 was most insensitive to substitution with respect to activity of the recombinant enzyme, all alterations at this position were lethal for Ty3 transposition.

  10. An SGS3-like protein functions in RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Zheng, Zhimin

    2010-01-06

    RNA-directed DNA methylation (RdDM) is an important epigenetic mechanism for silencing transgenes and endogenous repetitive sequences such as transposons. The RD29A promoter-driven LUCIFERASE transgene and its corresponding endogenous RD29A gene are hypermethylated and silenced in the Arabidopsis DNA demethylase mutant ros1. By screening for second-site suppressors of ros1, we identified the RDM12 locus. The rdm12 mutation releases the silencing of the RD29A-LUC transgene and the endogenous RD29A gene by reducing the promoter DNA methylation. The rdm12 mutation also reduces DNA methylation at endogenous RdDM target loci, including transposons and other repetitive sequences. In addition, the rdm12 mutation affects the levels of small interfering RNAs (siRNAs) from some of the RdDM target loci. RDM12 encodes a protein with XS and coiled-coil domains, and is similar to SGS3, which is a partner protein of RDR6 and can bind to double-stranded RNAs with a 5′ overhang, and is required for several post-transcriptional gene silencing pathways. Our results show that RDM12 is a component of the RdDM pathway, and suggest that RdDM may involve double-stranded RNAs with a 5′ overhang and the partnering between RDM12 and RDR2. © 2010 Blackwell Publishing Ltd.

  11. Impact of bacterial infections on aging and cancer: impairment of DNA repair and mitochondrial function of host cells.

    Science.gov (United States)

    Strickertsson, Jesper A B; Desler, Claus; Rasmussen, Lene Juel

    2014-08-01

    The commensal floras that inhabit the gastrointestinal tract play critical roles in immune responses, energy metabolism, and even cancer prevention. Pathogenic and out of place commensal bacteria, can however have detrimental effects on the host, by introducing genomic instability and mitochondrial dysfunction, which are hallmarks of both aging and cancer. Helicobacter pylori and Enterococcus faecalis are bacteria of the gastrointestinal tract that have been demonstrated to affect these two hallmarks. These, and other bacteria, have been shown to decrease the transcription and translation of essential DNA repair subunits of major DNA repair pathways and increase production of reactive oxygen species (ROS). Defects in DNA repair cause mutations and genomic instability and are found in several cancers as well as in progeroid syndromes. This review describes our contemporary view on how bacterial infections impact DNA repair and damage, and the consequence on the mitochondrial and nuclear genomes. We argue that in the gastrointestinal tract, these mechanisms can contribute to tumorigenesis as well as cellular aging of the digestive system. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Biophysics of DNA

    CERN Document Server

    Vologodskii, Alexander

    2015-01-01

    Surveying the last sixty years of research, this book describes the physical properties of DNA in the context of its biological functioning. It is designed to enable both students and researchers of molecular biology, biochemistry and physics to better understand the biophysics of DNA, addressing key questions and facilitating further research. The chapters integrate theoretical and experimental approaches, emphasising throughout the importance of a quantitative knowledge of physical properties in building and analysing models of DNA functioning. For example, the book shows how the relationship between DNA mechanical properties and the sequence specificity of DNA-protein binding can be analyzed quantitatively by using our current knowledge of the physical and structural properties of DNA. Theoretical models and experimental methods in the field are critically considered to enable the reader to engage effectively with the current scientific literature on the physical properties of DNA.

  13. Functional Analysis of the Interdependence between DNA Uptake Sequence and Its Cognate ComP Receptor during Natural Transformation in Neisseria Species

    Science.gov (United States)

    Berry, Jamie-Lee; Cehovin, Ana; McDowell, Melanie A.; Lea, Susan M.; Pelicic, Vladimir

    2013-01-01

    Natural transformation is the widespread biological process by which “competent” bacteria take up free DNA, incorporate it into their genomes, and become genetically altered or “transformed”. To curb often deleterious transformation by foreign DNA, several competent species preferentially take up their own DNA that contains specific DUS (DNA uptake sequence) watermarks. Our recent finding that ComP is the long sought DUS receptor in Neisseria species paves the way for the functional analysis of the DUS-ComP interdependence which is reported here. By abolishing/modulating ComP levels in Neisseria meningitidis, we show that the enhancement of transformation seen in the presence of DUS is entirely dependent on ComP, which also controls transformation in the absence of DUS. While peripheral bases in the DUS were found to be less important, inner bases are essential since single base mutations led to dramatically impaired interaction with ComP and transformation. Strikingly, naturally occurring DUS variants in the genomes of human Neisseria commensals differing from DUS by only one or two bases were found to be similarly impaired for transformation of N. meningitidis. By showing that ComPsub from the N. subflava commensal specifically binds its cognate DUS variant and mediates DUS-enhanced transformation when expressed in a comP mutant of N. meningitidis, we confirm that a similar mechanism is used by all Neisseria species to promote transformation by their own, or closely related DNA. Together, these findings shed new light on the molecular events involved in the earliest step in natural transformation, and reveal an elegant mechanism for modulating horizontal gene transfer between competent species sharing the same niche. PMID:24385921

  14. Functional analysis of the interdependence between DNA uptake sequence and its cognate ComP receptor during natural transformation in Neisseria species.

    Directory of Open Access Journals (Sweden)

    Jamie-Lee Berry

    Full Text Available Natural transformation is the widespread biological process by which "competent" bacteria take up free DNA, incorporate it into their genomes, and become genetically altered or "transformed". To curb often deleterious transformation by foreign DNA, several competent species preferentially take up their own DNA that contains specific DUS (DNA uptake sequence watermarks. Our recent finding that ComP is the long sought DUS receptor in Neisseria species paves the way for the functional analysis of the DUS-ComP interdependence which is reported here. By abolishing/modulating ComP levels in Neisseria meningitidis, we show that the enhancement of transformation seen in the presence of DUS is entirely dependent on ComP, which also controls transformation in the absence of DUS. While peripheral bases in the DUS were found to be less important, inner bases are essential since single base mutations led to dramatically impaired interaction with ComP and transformation. Strikingly, naturally occurring DUS variants in the genomes of human Neisseria commensals differing from DUS by only one or two bases were found to be similarly impaired for transformation of N. meningitidis. By showing that ComPsub from the N. subflava commensal specifically binds its cognate DUS variant and mediates DUS-enhanced transformation when expressed in a comP mutant of N. meningitidis, we confirm that a similar mechanism is used by all Neisseria species to promote transformation by their own, or closely related DNA. Together, these findings shed new light on the molecular events involved in the earliest step in natural transformation, and reveal an elegant mechanism for modulating horizontal gene transfer between competent species sharing the same niche.

  15. Potent functional antibody responses elicited by HIV-I DNA priming and boosting with heterologous HIV-1 recombinant MVA in healthy Tanzanian adults.

    Directory of Open Access Journals (Sweden)

    Agricola Joachim

    Full Text Available Vaccine-induced HIV antibodies were evaluated in serum samples collected from healthy Tanzanian volunteers participating in a phase I/II placebo-controlled double blind trial using multi-clade, multigene HIV-DNA priming and recombinant modified vaccinia Ankara (HIV-MVA virus boosting (HIVIS03. The HIV-DNA vaccine contained plasmids expressing HIV-1 gp160 subtypes A, B, C, Rev B, Gag A, B and RTmut B, and the recombinant HIV-MVA boost expressed CRF01_AE HIV-1 Env subtype E and Gag-Pol subtype A. While no neutralizing antibodies were detected using pseudoviruses in the TZM-bl cell assay, this prime-boost vaccination induced neutralizing antibodies in 83% of HIVIS03 vaccinees when a peripheral blood mononuclear cell (PBMC assay using luciferase reporter-infectious molecular clones (LucR-IMC was employed. The serum neutralizing activity was significantly (but not completely reduced upon depletion of natural killer (NK cells from PBMC (p=0.006, indicating a role for antibody-mediated Fcγ-receptor function. High levels of antibody-dependent cellular cytotoxicity (ADCC-mediating antibodies against CRF01_AE and/or subtype B were subsequently demonstrated in 97% of the sera of vaccinees. The magnitude of ADCC-mediating antibodies against CM235 CRF01_AE IMC-infected cells correlated with neutralizing antibodies against CM235 in the IMC/PBMC assay. In conclusion, HIV-DNA priming, followed by two HIV-MVA boosts elicited potent ADCC responses in a high proportion of Tanzanian vaccinees. Our findings highlight the potential of HIV-DNA prime HIV-MVA boost vaccines for induction of functional antibody responses and suggest this vaccine regimen and ADCC studies as potentially important new avenues in HIV vaccine development.Controlled-Trials ISRCTN90053831 The Pan African Clinical Trials Registry ATMR2009040001075080 (currently PACTR2009040001075080.

  16. The complexity of DNA double strand break is a crucial factor for activating ATR signaling pathway for G2/M checkpoint regulation regardless of ATM function.

    Science.gov (United States)

    Xue, Lian; Furusawa, Yoshiya; Okayasu, Ryuichi; Miura, Masahiko; Cui, Xing; Liu, Cuihua; Hirayama, Ryoichi; Matsumoto, Yoshitaka; Yajima, Hirohiko; Yu, Dong

    2015-01-01

    DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function

    OpenAIRE

    Nitharwal, Ram Gopal; Paul, Subhankar; Dar, Ashraf; Choudhury, Nirupam Roy; Soni, Rajesh K; Prusty, Dhaneswar; Sinha, Sukrat; Kashav, Tara; Mukhopadhyay, Gauranga; Chaudhuri, Tapan Kumar; Gourinath, Samudrala; Dhar, Suman Kumar

    2007-01-01

    Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase...

  18. A comparative approach for the investigation of biological information processing: An examination of the structure and function of computer hard drives and DNA

    Directory of Open Access Journals (Sweden)

    D'Onofrio David J

    2010-01-01

    Full Text Available Abstract Background The robust storage, updating and utilization of information are necessary for the maintenance and perpetuation of dynamic systems. These systems can exist as constructs of metal-oxide semiconductors and silicon, as in a digital computer, or in the "wetware" of organic compounds, proteins and nucleic acids that make up biological organisms. We propose that there are essential functional properties of centralized information-processing systems; for digital computers these properties reside in the computer's hard drive, and for eukaryotic cells they are manifest in the DNA and associated structures. Methods Presented herein is a descriptive framework that compares DNA and its associated proteins and sub-nuclear structure with the structure and function of the computer hard drive. We identify four essential properties of information for a centralized storage and processing system: (1 orthogonal uniqueness, (2 low level formatting, (3 high level formatting and (4 translation of stored to usable form. The corresponding aspects of the DNA complex and a computer hard drive are categorized using this classification. This is intended to demonstrate a functional equivalence between the components of the two systems, and thus the systems themselves. Results Both the DNA complex and the computer hard drive contain components that fulfill the essential properties of a centralized information storage and processing system. The functional equivalence of these components provides insight into both the design process of engineered systems and the evolved solutions addressing similar system requirements. However, there are points where the comparison breaks down, particularly when there are externally imposed information-organizing structures on the computer hard drive. A specific example of this is the imposition of the File Allocation Table (FAT during high level formatting of the computer hard drive and the subsequent loading of an operating

  19. Multi-step surface functionalization of polyimide based evanescent wave photonic biosensors and application for DNA hybridization by Mach-Zehnder interferometer

    Energy Technology Data Exchange (ETDEWEB)

    Melnik, Eva [Health and Environment Department, Nano Systems, AIT Austrian Institute of Technology GmbH, Donau-City-Strasse 1, 1220 Vienna (Austria); Department of Analytical Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna (Austria); Bruck, Roman [Health and Environment Department, Nano Systems, AIT Austrian Institute of Technology GmbH, Donau-City-Strasse 1, 1220 Vienna (Austria); Hainberger, Rainer, E-mail: rainer.hainberger@ait.ac.at [Health and Environment Department, Nano Systems, AIT Austrian Institute of Technology GmbH, Donau-City-Strasse 1, 1220 Vienna (Austria); Laemmerhofer, Michael, E-mail: michael.laemmerhofer@univie.ac.at [Department of Analytical Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna (Austria)

    2011-08-12

    Highlights: {yields} We realize a biosensing platform for polyimide evanescent photonic wave sensors. {yields} We show that the surface functionalization via silanisation and biotinylation followed by streptavidin immobilization do not destroy or damage the thin polyimide film. {yields} A highly dense streptavidin layer enables the immobilisation of biotinylated ligands such as biotinylated ssDNA for the selective measurement of DNA hybridization. - Abstract: The process of surface functionalization involving silanization, biotinylation and streptavidin bonding as platform for biospecific ligand immobilization was optimized for thin film polyimide spin-coated silicon wafers, of which the polyimide film serves as a wave guiding layer in evanescent wave photonic biosensors. This type of optical sensors make great demands on the materials involved as well as on the layer properties, such as the optical quality, the layer thickness and the surface roughness. In this work we realized the binding of a 3-mercaptopropyl trimethoxysilane on an oxygen plasma activated polyimide surface followed by subsequent derivatization of the reactive thiol groups with maleimide-PEG{sub 2}-biotin and immobilization of streptavidin. The progress of the functionalization was monitored by using different fluorescence labels for optimization of the chemical derivatization steps. Further, X-ray photoelectron spectroscopy and atomic force microscopy were utilized for the characterization of the modified surface. These established analytical methods allowed to derive information like chemical composition of the surface, surface coverage with immobilized streptavidin, as well as parameters of the surface roughness. The proposed functionalization protocol furnished a surface density of 144 fmol mm{sup -2} streptavidin with good reproducibility (13.9% RSD, n = 10) and without inflicted damage to the surface. This surface modification was applied to polyimide based Mach-Zehnder interferometer

  20. Effects of Electroacupuncture on Facial Nerve Function and HSV-1 DNA Quantity in HSV-1 Induced Facial Nerve Palsy Mice

    Directory of Open Access Journals (Sweden)

    Hongzhi Tang

    2014-01-01

    Full Text Available Acupuncture is a common and effective therapeutic method to treat facial nerve palsy (FNP. However, its underlying mechanism remains unclear. This study was aimed to investigate the effects of electroacupuncture on symptoms and content of HSV-1 DNA in FNP mice. Mice were randomized into four groups, an electroacupuncture treatment group, saline group, model animal group, and blank control group. Electroacupuncture was applied at Jiache (ST6 and Hegu (LI4 in electroacupuncture group once daily for 14 days, while electroacupuncture was not applied in model animal group. In electroacupuncture group, mice recovered more rapidly and HSV-1 DNA content also decreased more rapidly, compared with model animal group. We conclude that electroacupuncture is effective to alleviate symptoms and promote the reduction of HSV-1 in FNP.

  1. Effects of Electroacupuncture on Facial Nerve Function and HSV-1 DNA Quantity in HSV-1 Induced Facial Nerve Palsy Mice

    Science.gov (United States)

    Tang, Hongzhi; Feng, Shuwei; Chen, Jiao; Yang, Mingxiao; Zhong, Zhendong; Li, Ying; Liang, Fanrong

    2014-01-01

    Acupuncture is a common and effective therapeutic method to treat facial nerve palsy (FNP). However, its underlying mechanism remains unclear. This study was aimed to investigate the effects of electroacupuncture on symptoms and content of HSV-1 DNA in FNP mice. Mice were randomized into four groups, an electroacupuncture treatment group, saline group, model animal group, and blank control group. Electroacupuncture was applied at Jiache (ST6) and Hegu (LI4) in electroacupuncture group once daily for 14 days, while electroacupuncture was not applied in model animal group. In electroacupuncture group, mice recovered more rapidly and HSV-1 DNA content also decreased more rapidly, compared with model animal group. We conclude that electroacupuncture is effective to alleviate symptoms and promote the reduction of HSV-1 in FNP. PMID:24991226

  2. The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function

    Science.gov (United States)

    Nitharwal, Ram Gopal; Paul, Subhankar; Dar, Ashraf; Choudhury, Nirupam Roy; Soni, Rajesh K; Prusty, Dhaneswar; Sinha, Sukrat; Kashav, Tara; Mukhopadhyay, Gauranga; Chaudhuri, Tapan Kumar; Gourinath, Samudrala; Dhar, Suman Kumar

    2007-01-01

    Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function. PMID:17430964

  3. Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

    Science.gov (United States)

    Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino

    2017-01-01

    Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging' is a DNA-dependent symmetrization of portal protein. PMID:28134243

  4. Two different and functional nuclear rDNA genes in the abalone Haliotis tuberculata: tissue differential expression.

    Science.gov (United States)

    Van Wormhoudt, Alain; Gaume, Béatrice; Le Bras, Yvan; Roussel, Valérie; Huchette, Sylvain

    2011-10-01

    Analysis of the 18S rDNA sequences of Haliotis tuberculata tuberculata and H. t. coccinea subtaxa identified two different types of 18S rDNA genes and ITS1 regions. These two different genes were also detected in H. marmorata, H. rugosa and H. diversicolor that are separated from H. tuberculata by 5-65 mya. The mean divergence value between type I and type II sequences ranged from 7.25% for 18S to 80% for ITS1. ITS1 type II is homologous with the ITS1 consensus sequences published for many abalone species, whereas ITS1 type I presented only minor homology with a unique database entry for H. iris ITS1. A phylogenetic analysis makes a clear separation between type I and type II ITS1 sequences and supports grouping H. t. tuberculata, H. t. coccinea and H. marmorata together. The two subtaxa do not show any significant differences between the homologous 18S rDNA sequences. A general structure of the ITS1 transcript was proposed, with four major helices for the two types. The two genes were expressed and, for the first time, a putative differential expression of ITS1 type I was detected in the gills, digestive gland and gonads whereas ITS1 type II was expressed in all tissues.

  5. PGC-1α Modulates Telomere Function and DNA Damage in Protecting against Aging-Related Chronic Diseases

    Directory of Open Access Journals (Sweden)

    Shiqin Xiong

    2015-09-01

    Full Text Available Cellular senescence and organismal aging predispose age-related chronic diseases, such as neurodegenerative, metabolic, and cardiovascular disorders. These diseases emerge coincidently from elevated oxidative/electrophilic stress, inflammation, mitochondrial dysfunction, DNA damage, and telomere dysfunction and shortening. Mechanistic linkages are incompletely understood. Here, we show that ablation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α accelerates vascular aging and atherosclerosis, coinciding with telomere dysfunction and shortening and DNA damage. PGC-1α deletion reduces expression and activity of telomerase reverse transcriptase (TERT and increases p53 levels. Ectopic expression of PGC-1α coactivates TERT transcription and reverses telomere malfunction and DNA damage. Furthermore, alpha lipoic acid (ALA, a non-dispensable mitochondrial cofactor, upregulates PGC-1α-dependent TERT and the cytoprotective Nrf-2-mediated antioxidant/electrophile-responsive element (ARE/ERE signaling cascades, and counteracts high-fat-diet-induced, age-dependent arteriopathy. These results illustrate the pivotal importance of PGC-1α in ameliorating senescence, aging, and associated chronic diseases, and may inform novel therapeutic approaches involving electrophilic specificity.

  6. Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

    Energy Technology Data Exchange (ETDEWEB)

    Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino (Rutgers); (LBNL); (Connecticut); (TJU); (MSU)

    2017-01-30

    Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid’) built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging’ is a DNA-dependent symmetrization of portal protein.

  7. Taxol biosynthesis: Molecular cloning of a benzoyl- CoA:taxane 2α-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli

    Science.gov (United States)

    Walker, Kevin; Croteau, Rodney

    2000-01-01

    A cDNA clone encoding a taxane 2α-O-benzoyltransferase has been isolated from Taxus cuspidata. The recombinant enzyme catalyzes the conversion of 2-debenzoyl-7,13-diacetylbaccatin III, a semisynthetic substrate, to 7,13-diacetylbaccatin III, and thus appears to function in a late-stage acylation step of the Taxol biosynthetic pathway. By employing a homology-based PCR cloning strategy for generating acyltransferase oligodeoxynucleotide probes, several gene fragments were amplified and used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which several full-length acyltransferases were obtained and individually expressed in Escherichia coli. The functionally expressed benzoyltransferase was confirmed by radio-HPLC, 1H-NMR, and combined HPLC-MS verification of the product, 7,13-diacetylbaccatin III, derived from 2-debenzoyl-7,13-diacetylbaccatin III and benzoyl-CoA as cosubstrates in the corresponding cell-free extract. The full-length cDNA has an open reading frame of 1,320 base pairs and encodes a protein of 440 residues with a molecular weight of 50,089. The recombinant benzoyltransferase has a pH optimum of 8.0, Km values of 0.64 mM and 0.30 mM for the taxoid substrate and benzoyl-CoA, respectively, and is apparently regiospecific for acylation of the 2α-hydroxyl group of the functionalized taxane nucleus. This enzyme may be used to improve the production yields of Taxol and for the semisynthesis of drug analogs bearing modified aroyl groups at the C2 position. PMID:11095755

  8. Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet.

    Science.gov (United States)

    Malyshev, Denis A; Dhami, Kirandeep; Quach, Henry T; Lavergne, Thomas; Ordoukhanian, Phillip; Torkamani, Ali; Romesberg, Floyd E

    2012-07-24

    The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chemical and synthetic biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.

  9. High-throughput sample-to-answer detection of DNA/RNA in crude samples within functionalized micro-pipette tips.

    Science.gov (United States)

    Lu, Wenjing; Wang, Jidong; Wu, Qiong; Sun, Jiashu; Chen, Yiping; Zhang, Lu; Zheng, Chunsheng; Gao, Wenna; Liu, Yi; Jiang, Xingyu

    2016-01-15

    We develop a micro-pipette tip-based nucleic acid test (MTNT) for high-throughput sample-to-answer detection of both DNA and RNA from crude samples including cells, bacteria, and solid plants, without the need of sample pretreatment and complex operation. MTNT consists of micro-pipette tips and embedded solid phase nucleic acid extraction membranes, and fully integrates the functions of nucleic acid extraction from crude samples, loop-mediated isothermal amplification (LAMP) of nucleic acids, and visual readout of assays. The total assaying time for DNA or RNA from a variety of crude samples ranges from 90 to 160 min. The limit of detection (LOD) of MTNT is 2 copies of plasmids containing the target nucleic acid fragments of Ebola virus, and 8 CFU of Escherichia coli carrying Ebola virus-derived plasmids. MTNT can also detect CK-19 mRNA from as few as 2 cancer cells without complicated procedures such as RNA extraction and purification. We further demonstrate MTNT in a high-throughput format using an eight-channel pipette and a homemade mini-heater, with a maximum throughput of 40 samples. Compared with other point-of-care (POC) nucleic acid tests (NAT), MTNT could assay both DNA and RNA directly from liquid (cells/bacteria/blood) or solid (plant) samples in a straightforward, sensitive, high-throughput, and containment-free manner, suggesting a considerable promise for low-cost and POC NAT in remote areas.

  10. Surface functionalized Cu{sub 2}Zn{sub 1-x}Cd{sub x}SnS{sub 4} quinternary alloyed nanostructure for DNA sensing

    Energy Technology Data Exchange (ETDEWEB)

    Ibraheam, A.S.; Voon, C.H.; Foo, K.L.; Azizah, N. [University Malaysia Perlis, Institute of Nano Electronic Engineering, Kangar, Perlis (Malaysia); Al-Douri, Y. [University of Sidi-Bel-Abbes, Physics Department, Faculty of Science, Sidi Bel-Abbes (Algeria); Gopinath, S.C.B. [University Malaysia Perlis, Institute of Nano Electronic Engineering, Kangar, Perlis (Malaysia); Universiti Malaysia Perlis, School of Bioprocess Engineering, Arau, Perlis (Malaysia); Ameri, M. [Universite Djilali Liabes de Sidi Bel-Abbes, Laboratoire Physico-Chimie des Materiaux Avances (LPCMA), Sidi Bel-Abbes (Algeria); Ibrahim, Sattar S. [University of Anbar, Chemisty Department, College of Science, Al Rumadi (Iraq)

    2017-03-15

    A sensing plate of extended Cu{sub 2}Zn{sub 1-x}Cd{sub x}SnS{sub 4} quinternary alloy nanostructures, fabricated on an oxidized silicon substrate by the sol-gel method, is reported in this paper. The fabricated device was characterized and analyzed via field emission-scanning electron microscopy, X-ray diffraction (XRD), and photoluminescence (PL). The XRD peaks shifted towards the lower angle side alongside increasing concentration of cadmium. The average diameter of the Cu{sub 2}Zn{sub 1-x}Cd{sub x}SnS{sub 4} quinternary alloy nanostructures falls between 21.55 and 43.12 nm, while the shift of the PL bandgap was from 1.81 eV (x = 0) to 1.72 eV (x = 1). The resulting Cu{sub 2}Zn{sub 1-x}Cd{sub x}SnS{sub 4} quinternary alloy nanostructures components were functionalized with oligonucleotides probe DNA molecules and interacted with the target, exhibiting good sensing capabilities due to its large surface-to-volume ratio. The fabrication, immobilization, and hybridization processes were analyzed via representative current-voltage (I-V) plots. Its electrical profile shows that the device is capable to distinguish biomolecules. Its high performance was evident from the linear relationship between the probe DNA from cervical cancer and the target DNA, showing its applicability for medical applications. (orig.)

  11. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  12. The role of DNA dependent protein kinase in synapsis of DNA ends

    NARCIS (Netherlands)

    E.P.W.C. Weterings (Eric); N.S. Verkaik (Nicole); H.T. Brüggenwirth (Hennie); D.C. van Gent (Dik); J.H.J. Hoeijmakers (Jan)

    2003-01-01

    textabstractDNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks

  13. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects.

    Directory of Open Access Journals (Sweden)

    Johannes S Gach

    Full Text Available Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5 boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART. Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC. We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir.

  14. DNA methyltransferase 3B (DNMT3B) mutations in ICF syndrome lead to altered epigenetic modifications and aberrant expression of genes regulating development, neurogenesis and immune function.

    Science.gov (United States)

    Jin, Bilian; Tao, Qian; Peng, Jinrong; Soo, Hui Meng; Wu, Wei; Ying, Jianming; Fields, C Robert; Delmas, Amber L; Liu, Xuefeng; Qiu, Jingxin; Robertson, Keith D

    2008-03-01

    Genome-wide DNA methylation patterns are established and maintained by the coordinated action of three DNA methyltransferases (DNMTs), DNMT1, DNMT3A and DNMT3B. DNMT3B hypomorphic germline mutations are responsible for two-thirds of immunodeficiency, centromere instability, facial anomalies (ICF) syndrome cases, a rare recessive disease characterized by immune defects, instability of pericentromeric satellite 2-containing heterochromatin, facial abnormalities and mental retardation. The molecular defects in transcription, DNA methylation and chromatin structure in ICF cells remain relatively uncharacterized. In the present study, we used global expression profiling to elucidate the role of DNMT3B in these processes using cell lines derived from ICF syndrome and normal individuals. We show that there are significant changes in the expression of genes critical for immune function, development and neurogenesis that are highly relevant to the ICF phenotype. Approximately half the upregulated genes we analyzed were marked with low-level DNA methylation in normal cells that was lost in ICF cells, concomitant with loss of repressive histone modifications, particularly H3K27 trimethylation, and gains in transcriptionally active H3K9 acetylation and H3K4 trimethylation marks. In addition, we consistently observed loss of binding of the SUZ12 component of the PRC2 polycomb repression complex and DNMT3B to derepressed genes, including a number of homeobox genes critical for immune system, brain and craniofacial development. We also observed altered global levels of certain histone modifications in ICF cells, particularly ubiquitinated H2AK119. Therefore, this study provides important new insights into the role of DNMT3B in modulating gene expression and chromatin structure and reveals new connections between DNMT3B and polycomb-mediated repression.

  15. HER2 copy number of circulating tumour DNA functions as a biomarker to predict and monitor trastuzumab efficacy in advanced gastric cancer.

    Science.gov (United States)

    Wang, Haixing; Li, Beifang; Liu, Zhentao; Gong, Jifang; Shao, Lin; Ren, Jun; Niu, Yunyun; Bo, Shiping; Li, Zhongwu; Lai, Yumei; Lu, Sijia; Gao, Jing; Shen, Lin

    2018-01-01

    HER2 status is significant to trastuzumab therapy; however, it is difficult to determine HER2 status accurately with few pieces of biopsies from advanced gastric cancer (AGC) due to highly heterogeneity and invasive behaviour, which will be investigated in this study. Fifty-six patients with AGC were included in this study. Primary tumour tissues and matched plasmas before medication from 36 patients were retrospectively collected, and the other 20 patients with primary tumour tissues and paired plasmas were prospectively collected. HER2 expression and amplification in 56 tumour tissues were determined by immunohistochemistry (IHC) and dual in situ hybridisation (DISH), and HER2 copy number in 135 circulating tumour DNAs (ctDNAs) was judged by next-generation sequencing. For tumour tissues, HER2 amplification by DISH was most commonly found in patients with HER2 score 3+by IHC. For plasmas, HER2 amplification defined as HER2 copy number >2.22 was identified in 26 of 56 patients. There was a high concordance of HER2 amplification between ctDNA and tumour tissues, suggesting that ctDNA could function as an alternative to screen HER2-targeted population. Moreover, the changes of HER2 copy number in ctDNA could efficiently monitor trastuzumab efficacy, the power of which was superior to commonly used markers carcinoembryonic antigen (CEA) and CA199, suggesting its potential role in clinical practice. ctDNA for HER2 analysis was strongly recommended to serve as a surrogate to screen trastuzumab-suitable population and monitor trastuzumab efficacy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. ERCC6L2 Mutations Link a Distinct Bone-Marrow-Failure Syndrome to DNA Repair and Mitochondrial Function

    OpenAIRE

    Tummala, Hemanth; Kirwan, Michael; Walne, Amanda J.; Hossain, Upal; Jackson, Nicholas; Pondarre, Corinne; Plagnol, Vincent; Vulliamy, Tom; Dokal, Inderjeet

    2014-01-01

    Exome sequencing was performed in three index cases with bone marrow failure and neurological dysfunction and whose parents are first-degree cousins. Homozygous truncating mutations were identified in ERCC6L2 in two of the individuals. Both of these mutations affect the subcellular localization and stability of ERCC6L2. We show here that knockdown of ERCC6L2 in human A549 cells significantly reduced their viability upon exposure to the DNA-damaging agents mitomycin C and Irofulven, but not et...

  17. Molecular cloning and functional expression of a human cDNA encoding translation initiation factor 6

    OpenAIRE

    Si, Kausik; Chaudhuri, Jayanta; Chevesich, Jorge; Maitra, Umadas

    1997-01-01

    Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. In this paper, we devised a procedure for purifying eIF6 from rabbit reticulocyte lysates and immunochemically characterized the protein by using antibodies isolated from egg yolks of laying hens immunized with rabbit eIF6. By using these monospecific antibodies, a 1.096-kb human cDNA that encodes an eIF6 of 245 amino acids (calculated Mr 26,558) ha...

  18. Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks.

    Science.gov (United States)

    Pennisi, Rosa; Antoccia, Antonio; Leone, Stefano; Ascenzi, Paolo; di Masi, Alessandra

    2017-08-01

    The molecular chaperone heat shock protein 90 (Hsp90α) regulates cell proteostasis and mitigates the harmful effects of endogenous and exogenous stressors on the proteome. Indeed, the inhibition of Hsp90α ATPase activity affects the cellular response to ionizing radiation (IR). Although the interplay between Hsp90α and several DNA damage response (DDR) proteins has been reported, its role in the DDR is still unclear. Here, we show that ataxia-telangiectasia-mutated kinase (ATM) and nibrin (NBN), but not 53BP1, RAD50, and MRE11, are Hsp90α clients as the Hsp90α inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) induces ATM and NBN polyubiquitination and proteosomal degradation in normal fibroblasts and lymphoblastoid cell lines. Hsp90α-ATM and Hsp90α-NBN complexes are present in unstressed and irradiated cells, allowing the maintenance of ATM and NBN stability that is required for the MRE11/RAD50/NBN complex-dependent ATM activation and the ATM-dependent phosphorylation of both NBN and Hsp90α in response to IR-induced DNA double-strand breaks (DSBs). Hsp90α forms a complex also with ph-Ser1981-ATM following IR. Upon phosphorylation, NBN dissociates from Hsp90α and translocates at the DSBs, while phThr5/7-Hsp90α is not recruited at the damaged sites. The inhibition of Hsp90α affects nuclear localization of MRE11 and RAD50, impairs DDR signaling (e.g., BRCA1 and CHK2 phosphorylation), and slows down DSBs repair. Hsp90α inhibition does not affect DNA-dependent protein kinase (DNA-PK) activity, which possibly phosphorylates Hsp90α and H2AX after IR. Notably, Hsp90α inhibition causes H2AX phosphorylation in proliferating cells, this possibly indicating replication stress events. Overall, present data shed light on the regulatory role of Hsp90α on the DDR, controlling ATM and NBN stability and influencing the DSBs signaling and repair. © 2017 Federation of European Biochemical Societies.

  19. Time-Dependent and Organ-Specific Changes in Mitochondrial Function, Mitochondrial DNA Integrity, Oxidative Stress and Mononuclear Cell Infiltration in a Mouse Model of Burn Injury.

    Science.gov (United States)

    Szczesny, Bartosz; Brunyánszki, Attila; Ahmad, Akbar; Oláh, Gabor; Porter, Craig; Toliver-Kinsky, Tracy; Sidossis, Labros; Herndon, David N; Szabo, Csaba

    2015-01-01

    Severe thermal injury induces a pathophysiological response that affects most of the organs within the body; liver, heart, lung, skeletal muscle among others, with inflammation and hyper-metabolism as a hallmark of the post-burn damage. Oxidative stress has been implicated as a key component in development of inflammatory and metabolic responses induced by burn. The goal of the current study was to evaluate several critical mitochondrial functions in a mouse model of severe burn injury. Mitochondrial bioenergetics, measured by Extracellular Flux Analyzer, showed a time dependent, post-burn decrease in basal respiration and ATP-turnover but enhanced maximal respiratory capacity in mitochondria isolated from the liver and lung of animals subjected to burn injury. Moreover, we detected a tissue-specific degree of DNA damage, particularly of the mitochondrial DNA, with the most profound effect detected in lungs and hearts of mice subjected to burn injury. Increased mitochondrial biogenesis in lung tissue in response to burn injury was also observed. Burn injury also induced time dependent increases in oxidative stress (measured by amount of malondialdehyde) and neutrophil infiltration (measured by myeloperoxidase activity), particularly in lung and heart. Tissue mononuclear cell infiltration was also confirmed by immunohistochemistry. The amount of poly(ADP-ribose) polymers decreased in the liver, but increased in the heart in later time points after burn. All of these biochemical changes were also associated with histological alterations in all three organs studied. Finally, we detected a significant increase in mitochondrial DNA fragments circulating in the blood immediately post-burn. There was no evidence of systemic bacteremia, or the presence of bacterial DNA fragments at any time after burn injury. The majority of the measured parameters demonstrated a sustained elevation even at 20-40 days post injury suggesting a long-lasting effect of thermal injury on organ

  20. Condensin HEAT subunits required for DNA repair, kinetochore/centromere function and ploidy maintenance in fission yeast.

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    Xingya Xu

    Full Text Available Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance.

  1. Multiple Functions of Long Non-Coding RNAs in Oxidative Stress, DNA Damage Response and Cancer Progression.

    Science.gov (United States)

    Tehrani, Sadra Samavarchi; Karimian, Ansar; Parsian, Hadi; Majidinia, Maryam; Yousefi, Bahman

    2017-06-13

    In addition to aberrant alternation of transcriptome, it is now suggested that dysregulation of the non-coding transcripts, particularly long non-coding RNAs (lncRNAs), which comprise the majority of the genome, is contributed to cancer initiation and progression. As the result of recent huge efforts, the possible roles of numerous lncRNAs in the human cancers were characterized, as well as various strategies with inhibitory effects to target these transcripts on the transformed cells. Moreover, DNA damage response (DDR) pathway is a complex regulatory network responsible for the identification of disruptions in DNA structure, integrity and stability- it is reported to be associated with the up-regulation and down-regulation of lncRNAs. This review explores the involvement of the various lncRNAs in different human cancers, afterwards discusses the association of the lncRNAs expression with the DDR and oxidative stress, which are implicated in a myriad pathophysiological and physiological intra- and extracellular damages. J. Cell. Biochem. 9999: 1-14, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. One-Step Synthesis of Rox-DNA Functionalized CdZnTeS Quantum Dots for the Visual Detection of Hydrogen Peroxide and Blood Glucose.

    Science.gov (United States)

    Mao, Guobin; Cai, Qin; Wang, Fubing; Luo, Changliang; Ji, Xinghu; He, Zhike

    2017-10-26

    As the blood glucose concentration is an important clinical parameter of diabetes, the rapid and effective detection of blood glucose is very significant for monitoring and managing diabetes. Here, a facile method to prepare Rox-DNA functionalized CdZnTeS quantum dots (QDs) was developed. The Rox-DNA functionalized CdZnTeS QDs were prepared by a one-pot hydrothermal method through phosphorothioate DNA bound to QDs, which were employed as a ratiometric fluorescent probe for the rapid and sensitive detection of H2O2 and glucose. Compared with the traditional multistep construction of ratiometric fluorescent probes, this presented approach is simpler and more effective without chemical modification and complicated separation. The CdZnTeS QDs with green fluorescence is specifically sensitive to H2O2, while the red fluorescence of Rox is invariable. H2O2 is the product from the oxidation of glucose catalyzed by glucose oxidase (GOx). Therefore, a facile method to detect H2O2 and glucose with a detection limit of 0.075 μM for H2O2 and 0.042 μM for glucose was developed. In addition, this proposed probe has been employed for the detection of glucose in human serum with a satisfactory result. Moreover, this probe has been used for visual detection, and the health and diabetics can be distinguished by the naked eye. Meanwhile, this nanoprobe is also generalizable and can be extended to the detection of many other H2O2-mediated analytes.

  3. MITOCHONDRIAL DNA- REVOLUTIONARY EVOLUTION

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    Vaidhehi Narayan Nayak

    2017-07-01

    Full Text Available BACKGROUND Mitochondrion, the sausage-shaped organelle residing in the cytoplasm of all eukaryotic cells, apart from being the power house, represents endosymbiotic evolution of a free living organism to intracellular structure. Anthropologically, mitochondrial DNA is the fossilised source to trace the human ancestry particularly of maternal lineage. This article attempts to highlight the various biological functions of mitochondrial DNA (mtDNA with a note on its forensic application.

  4. Novel functional residues in the core domain of histone H2B regulate yeast gene expression and silencing and affect the response to DNA damage.

    Science.gov (United States)

    Kyriss, McKenna N M; Jin, Yi; Gallegos, Isaura J; Sanford, James A; Wyrick, John J

    2010-07-01

    Previous studies have identified novel modifications in the core fold domain of histone H2B, but relatively little is known about the function of these putative histone modification sites. We have mutated core modifiable residues that are conserved in Saccharomyces cerevisiae histone H2B and characterized the effects of the mutants on yeast silencing, gene expression, and the DNA damage response. We identified three histone H2B core modifiable residues as functionally important. We find that mutating H2B K49 in yeast confers a UV sensitivity phenotype, and we confirm that the homologous residue in human histone H2B is acetylated and methylated in human cells. Our results also indicate that mutating H2B K111 impairs the response to methyl methanesulfonate (MMS)-induced DNA lesions and disrupts telomeric silencing and Sir4 binding. In contrast, mutating H2B R102 enhances silencing at yeast telomeres and the HML silent mating loci and increases Sir4 binding to these regions. The H2B R102A mutant also represses the expression of endogenous genes adjacent to yeast telomeres, which is likely due to the ectopic spreading of the Sir complex in this mutant strain. We propose a structural model by which H2B R102 and K111 regulate the binding of the Sir complex to the nucleosome.

  5. One-pot Synthesis of Functional Poly(amino ester sulfide)s and Utility in Delivering pDNA and siRNA.

    Science.gov (United States)

    Yan, Yunfeng; Xue, Lian; Miller, Jason B; Zhou, Kejin; Kos, Petra; Elkassih, Sussana; Liu, Li; Nagai, Atsushi; Xiong, Hu; Siegwart, Daniel J

    2015-08-18

    The development of efficacious carriers is an important long-standing challenge in gene therapy. In the past few decades, tremendous progress has been made toward non-viral vectors for gene delivery including cationic lipids and polymers. However, there continues to be a need for clinically translatable polymer-based delivery carriers because they offer tunable degradation profiles and functional groups, diverse structures/morphologies, and scalability in preparation. Herein, we developed a library of 144 degradable polymers with varying amine and hydrophobic content via a facile method that involves thiobutyrolactone aminolysis and consequent thiol-(meth)acrylate or acrylamide addition in one-pot. The polymer platform was evaluated for pDNA and siRNA delivery to HeLa cells in vitro. Hydrophobically modified 5S, 2E1, 6CY1, 5CY2, and 2M1 grafted HEMATL polymers are capable of delivering pDNA depending on the chemical composition and the size of the polyplexes. Hydrophobically modified 5S and 2B grafted HEMATL and 5S grafted ATL polymers exhibit capability for siRNA delivery that approaches the efficacy of commercially available transfection reagents. Due to tunable functionality and scalable preparation, this synthetic approach may have broad applicability in the design of delivery materials for gene therapy.

  6. Novel Functional Residues in the Core Domain of Histone H2B Regulate Yeast Gene Expression and Silencing and Affect the Response to DNA Damage ▿

    Science.gov (United States)

    Kyriss, McKenna N. M.; Jin, Yi; Gallegos, Isaura J.; Sanford, James A.; Wyrick, John J.

    2010-01-01

    Previous studies have identified novel modifications in the core fold domain of histone H2B, but relatively little is known about the function of these putative histone modification sites. We have mutated core modifiable residues that are conserved in Saccharomyces cerevisiae histone H2B and characterized the effects of the mutants on yeast silencing, gene expression, and the DNA damage response. We identified three histone H2B core modifiable residues as functionally important. We find that mutating H2B K49 in yeast confers a UV sensitivity phenotype, and we confirm that the homologous residue in human histone H2B is acetylated and methylated in human cells. Our results also indicate that mutating H2B K111 impairs the response to methyl methanesulfonate (MMS)-induced DNA lesions and disrupts telomeric silencing and Sir4 binding. In contrast, mutating H2B R102 enhances silencing at yeast telomeres and the HML silent mating loci and increases Sir4 binding to these regions. The H2B R102A mutant also represses the expression of endogenous genes adjacent to yeast telomeres, which is likely due to the ectopic spreading of the Sir complex in this mutant strain. We propose a structural model by which H2B R102 and K111 regulate the binding of the Sir complex to the nucleosome. PMID:20479120

  7. Aging and DNA repair capability. [Review

    Energy Technology Data Exchange (ETDEWEB)

    Tice, R R

    1977-01-01

    A review of the literature on DNA repair processes in relation to aging is presented under the following headings: DNA repair processes; age-related occurrence of unrepaired DNA lesions; DNA repair capability as a function of age; tissue-specific DNA repair capability; acceleration of the aging process by exposure to DNA damaging agents; human genetic syndromes; and longevity and DNA repair processes. (HLW)

  8. Functional mapping of the fission yeast DNA polymerase δ B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis

    Directory of Open Access Journals (Sweden)

    Gray Fiona C

    2009-08-01

    Full Text Available Abstract Background DNA polymerase δ plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol δ is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol δ comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively. Results To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1. Conclusion In the absence of a three-dimensional structure of the entire Pol δ complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.

  9. DNA Polymerase Gamma in Mitochondrial DNA Replication and Repair

    Directory of Open Access Journals (Sweden)

    William C. Copeland

    2003-01-01

    Full Text Available Mutations in mitochondrial DNA (mtDNA are associated with aging, and they can cause tissue degeneration and neuromuscular pathologies known as mitochondrial diseases. Because DNA polymerase γ (pol γ is the enzyme responsible for replication and repair of mitochondrial DNA, the burden of faithful duplication of mitochondrial DNA, both in preventing spontaneous errors and in DNA repair synthesis, falls on pol γ. Investigating the biological functions of pol γ and its inhibitors aids our understanding of the sources of mtDNA mutations. In animal cells, pol γ is composed of two subunits, a larger catalytic subunit of 125–140 kDa and second subunit of 35–55 kDa. The catalytic subunit contains DNA polymerase activity, 3’-5’ exonuclease activity, and a 5’-dRP lyase activity. The accessory subunit is required for highly processive DNA synthesis and increases the affinity of pol gamma to the DNA.

  10. Functional Genomics of 5- to 8-Cell Stage Human Embryos by Blastomere Single-Cell cDNA Analysis

    Science.gov (United States)

    Galán, Amparo; Montaner, David; Póo, M. Eugenia; Valbuena, Diana; Ruiz, Verónica; Aguilar, Cristóbal; Dopazo, Joaquín; Simón, Carlos

    2010-01-01

    Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented. PMID:21049019

  11. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  12. DNA Microarrays

    Science.gov (United States)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  13. DNA origami nanopores.

    Science.gov (United States)

    Bell, Nicholas A W; Engst, Christian R; Ablay, Marc; Divitini, Giorgio; Ducati, Caterina; Liedl, Tim; Keyser, Ulrich F

    2012-01-11

    We demonstrate the assembly of functional hybrid nanopores for single molecule sensing by inserting DNA origami structures into solid-state nanopores. In our experiments, single artificial nanopores based on DNA origami are repeatedly inserted in and ejected from solid-state nanopores with diameters around 15 nm. We show that these hybrid nanopores can be employed for the detection of λ-DNA molecules. Our approach paves the way for future development of adaptable single-molecule nanopore sensors based on the combination of solid-state nanopores and DNA self-assembly. © 2011 American Chemical Society

  14. Bombyx mori DNA/RNA non-specific nuclease: expression of isoforms in insect culture cells, subcellular localization and functional assays.

    Science.gov (United States)

    Liu, Jisheng; Swevers, Luc; Iatrou, Kostas; Huvenne, Hanneke; Smagghe, Guy

    2012-08-01

    A DNA/RNA non-specific alkaline nuclease (BmdsRNase) was isolated from the digestive juice of Bombyx mori. While originally reported to be produced by the midgut only, in this project it was found that the mRNA of this enzyme was also expressed in the epidermis, fat body, gut, thoracic muscles, Malpighian tubules, brain, and silk glands of 5th instar larvae, indicating additional functions to its reported role in nucleic acid digestion in the midgut. In order to study the functional properties of BmdsRNase, three pEA-BmdsRNase expression constructs were generated, characterized by presence or absence of a signal peptide and a propeptide, and used for expression in lepidopteran Hi5 tissue culture cells. Western blot indicated that these different forms of BmdsRNase protein were not secreted into the growth medium, while they were detected in the pellets and supernatants of Hi5 cell extracts. Nucleic acids cleavage experiments indicated that full-length BmdsRNase could digest dsRNA and that the processed form (absence of signal peptide and propeptide) of BmdsRNase could degrade both DNA and dsRNA in Hi5 cell culture. Using a reporter assay targeted by transfected homologous dsRNA, it was shown that the digestive property of the processed form could interfere with the RNAi response. Immunostaining of processed BmdsRNase protein showed asymmetric localization in the cellular cytoplasm and co-localization with Flag-tagged Dicer-2 was also observed. In conclusion, our in vitro studies indicated that intracellular protein isoforms of BmdsRNase can be functional and involved in the regulation of nucleic acid metabolism in the cytoplasm. In particular, because of its propensity to degrade dsRNA, the enzyme might be involved in the innate immune response against invading nucleic acids such as RNA viruses. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. The N terminus of Myxococcus xanthus CarA repressor is an autonomously folding domain that mediates physical and functional interactions with both operator DNA and antirepressor protein.

    Science.gov (United States)

    Pérez-Marín, Mari Cruz; López-Rubio, Jose Juan; Murillo, Francisco Jose; Elías-Arnanz, Montserrat; Padmanabhan, S

    2004-08-06

    Expression of the Myxococcus xanthus carB operon, which encodes the majority of the enzymes involved in light-induced carotenogenesis, is down-regulated in the dark by the CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves repression of carB by physically interacting with CarA to dis-mantle CarA-DNA complexes. Here, we demonstrate that the N- and C-terminal portions of CarA are organized as distinct structural and functional domains. Specifically, we show that the 78 N-terminal residues of CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro, sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter binding, and this is relieved by CarS. In vivo, substitution of the gene carA by that for CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background. However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual function domain that orchestrates specific DNA-protein and protein-protein interactions and, when overexpressed, can be functionally competent in vivo.

  16. A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.

    Science.gov (United States)

    Zhou, Mi; Xue, Ling; Chen, Yaru; Li, Haiying; He, Qiufen; Wang, Bibin; Meng, Feilong; Wang, Meng; Guan, Min-Xin

    2017-12-08

    Defective nucleotide modifications of mitochondrial tRNAs have been associated with several human diseases, but their pathophysiology remains poorly understood. In this report, we investigated the pathogenic molecular mechanism underlying a hypertension-associated 4435A>G mutation in mitochondrial tRNAMet The m.4435A>G mutation affected a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon, which is important for the fidelity of codon recognition and stabilization. We hypothesized that the m.4435A>G mutation introduced an m1G37 modification of tRNAMet, altering its structure and function. Primer extension and methylation activity assays indeed confirmed that the m.4435A>G mutation created a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAMet We found that this mutation altered the tRNAMet structure, indicated by an increased melting temperature and electrophoretic mobility of the mutated tRNA compared with the wild-type molecule. We demonstrated that cybrid cell lines carrying the m.4435A>G mutation exhibited significantly decreased efficiency in aminoacylation and steady-state levels of tRNAMet, as compared with those of control cybrids. The aberrant tRNAMet metabolism resulted in variable decreases in mitochondrial DNA (mtDNA)-encoded polypeptides in the mutant cybrids. Furthermore, we found that the m.4435A>G mutation caused respiratory deficiency, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in mutant cybrids. These results demonstrated that an aberrant m1G37 modification of mitochondrial tRNAMet affected the structure and function of its tRNA and consequently altered mitochondrial function. Our findings provide critical insights into the pathophysiology of maternally inherited hypertension, which is manifested by the deficient tRNA nucleotide modification. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  17. A microcantilever-based silver ion sensor using DNA-functionalized gold nanoparticles as a mass amplifier

    Science.gov (United States)

    You, Juneseok; Song, Yeongjin; Park, Chanho; Jang, Kuewhan; Na, Sungsoo

    2017-06-01

    Silver ions have been used to sterilize many products, however, it has recently been demonstrated that silver ions can be toxic. This toxicity has been studied over many years with the lethal concentration at 10 μM. Silver ions can accumulate through the food chain, causing serious health problems in many species. Hence, there is a need for a commercially available silver ion sensor, with high detection sensitivity. In this work, we develop an ultra-sensitive silver ion sensor platform, using cytosine based DNA and gold nanoparticles as the mass amplifier. We achieve a lower detection limit for silver ions of 10 pM; this detection limit is one million times lower than the toxic concentration. Using our sensor platform we examine highly selective characteristics of other typical ions in water from natural sources. Furthermore, our sensor platform is able to detect silver ions in a real practical sample of commercially available drinking water. Our sensor platform, which we have termed a ‘MAIS’ (mass amplifier ion sensor), with a simple detection procedure, high sensitivity, selectivity and real practical applicability has shown potential as an early toxicity assessment of silver ions in the environment.

  18. EFFECTOR OF TRANSCRIPTION2 is involved in xylem differentiation and includes a functional DNA single strand cutting domain.

    Science.gov (United States)

    Ivanov, Rumen; Tiedemann, Jens; Czihal, Andreas; Schallau, Anna; Diep, Le Hong; Mock, Hans-Peter; Claus, Bernhard; Tewes, Annegret; Bäumlein, Helmut

    2008-01-01

    EFFECTORS OF TRANSCRIPTION2 (ET) are plant-specific regulatory proteins characterized by the presence of two to five C-terminal DNA- and Zn-binding repeats, and a highly conserved cysteine pattern. We describe the structural characterization of the three member Arabidopsis thaliana ET gene family and reveal some allelic sequence polymorphisms. A mutation analysis showed that AtET2 affects the expression of various KNAT genes involved in the maintenance of the undifferentiated state of cambial meristem cells. It also plays a role in the regulation of GA5 (gibberellin 3-beta-dioxygenase) and the cell-cycle-related GASA4. A correlation was established between AtET2 expression and the cellular differentiation state. AtET-GFP fusion proteins shuttle between the cytoplasm and nucleus, with the AtET2 product prevented from entering the nucleus in non-differentiating cells. Within the nucleus, AtET2 probably acts via a single strand cutting domain. A more general regulatory role for ET factors is proposed, governing cell differentiation in cambial meristems, a crucial process for the development of plant vascular tissues.

  19. Gain-of-function mutations of Ptpn11 (Shp2) cause aberrant mitosis and increase susceptibility to DNA damage-induced malignancies.

    Science.gov (United States)

    Liu, Xia; Zheng, Hong; Li, Xiaobo; Wang, Siying; Meyerson, Howard J; Yang, Wentian; Neel, Benjamin G; Qu, Cheng-Kui

    2016-01-26

    Gain-of-function (GOF) mutations of protein tyrosine phosphatase nonreceptor type 11 Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling pathways, are associated with childhood leukemias and solid tumors. The underlying mechanisms are not fully understood. Here, we report that Ptpn11 GOF mutations disturb mitosis and cytokinesis, causing chromosomal instability and greatly increased susceptibility to DNA damage-induced malignancies. We find that Shp2 is distributed to the kinetochore, centrosome, spindle midzone, and midbody, all of which are known to play critical roles in chromosome segregation and cytokinesis. Mouse embryonic fibroblasts with Ptpn11 GOF mutations show a compromised mitotic checkpoint. Centrosome amplification and aberrant mitosis with misaligned or lagging chromosomes are significantly increased in Ptpn11-mutated mouse and patient cells. Abnormal cytokinesis is also markedly increased in these cells. Further mechanistic analyses reveal that GOF mutant Shp2 hyperactivates the Polo-like kinase 1 (Plk1) kinase by enhancing c-Src kinase-mediated tyrosine phosphorylation of Plk1. This study provides novel insights into the tumorigenesis associated with Ptpn11 GOF mutations and cautions that DNA-damaging treatments in Noonan syndrome patients with germ-line Ptpn11 GOF mutations could increase the risk of therapy-induced malignancies.

  20. Electron Resonance Decay into a Biological Function: Decrease in Viability of E. coli Transformed by Plasmid DNA Irradiated with 0.5-18 eV Electrons.

    Science.gov (United States)

    Kouass Sahbani, S; Cloutier, P; Bass, A D; Hunting, D J; Sanche, L

    2015-10-01

    Transient negative ions (TNIs) are ubiquitous in electron-molecule scattering at low electron impact energies (0-20 eV) and are particularly effective in damaging large biomolecules. Because ionizing radiation generates mostly 0-20 eV electrons, TNIs are expected to play important roles in cell mutagenesis and death during radiotherapeutic cancer treatment, although this hypothesis has never been directly verified. Here, we measure the efficiency of transforming E. coli bacteria by inserting into the cells, pGEM-3ZfL(-) plasmid DNA that confers resistance to the antibiotic ampicillin. Before transformation, plasmids are irradiated with electrons of specific energies between 0.5 and 18 eV. The loss of transformation efficiency plotted as a function of irradiation energy reveals TNIs at 5.5 and 9.5 eV, corresponding to similar states observed in the yields of DNA double strand breaks. We show that TNIs are detectable in the electron-energy dependence of a biological process and can decrease cell viability.

  1. The Reliability and Predictive Ability of a Biomarker of Oxidative DNA Damage on Functional Outcomes after Stroke Rehabilitation

    Directory of Open Access Journals (Sweden)

    Yu-Wei Hsieh

    2014-04-01

    Full Text Available We evaluated the reliability of 8-hydroxy-2'-deoxyguanosine (8-OHdG, and determined its ability to predict functional outcomes in stroke survivors. The rehabilitation effect on 8-OHdG and functional outcomes were also assessed. Sixty-one stroke patients received a 4-week rehabilitation. Urinary 8-OHdG levels were determined by liquid chromatography–tandem mass spectrometry. The test-retest reliability of 8-OHdG was good (interclass correlation coefficient = 0.76. Upper-limb motor function and muscle power determined by the Fugl-Meyer Assessment (FMA and Medical Research Council (MRC scales before rehabilitation showed significant negative correlation with 8-OHdG (r = −0.38, r = −0.30; p < 0.05. After rehabilitation, we found a fair and significant correlation between 8-OHdG and FMA (r = −0.34 and 8-OHdG and pain (r = 0.26, p < 0.05. Baseline 8-OHdG was significantly correlated with post-treatment FMA, MRC, and pain scores (r = −0.34, −0.31, and 0.25; p < 0.05, indicating its ability to predict functional outcomes. 8-OHdG levels were significantly decreased, and functional outcomes were improved after rehabilitation. The exploratory study findings conclude that 8-OHdG is a reliable and promising biomarker of oxidative stress and could be a valid predictor of functional outcomes in patients. Monitoring of behavioral indicators along with biomarkers may have crucial benefits in translational stroke research.

  2. The Reliability and Predictive Ability of a Biomarker of Oxidative DNA Damage on Functional Outcomes after Stroke Rehabilitation

    Science.gov (United States)

    Hsieh, Yu-Wei; Lin, Keh-Chung; Korivi, Mallikarjuna; Lee, Tsong-Hai; Wu, Ching-Yi; Wu, Kuen-Yuh

    2014-01-01

    We evaluated the reliability of 8-hydroxy-2′-deoxyguanosine (8-OHdG), and determined its ability to predict functional outcomes in stroke survivors. The rehabilitation effect on 8-OHdG and functional outcomes were also assessed. Sixty-one stroke patients received a 4-week rehabilitation. Urinary 8-OHdG levels were determined by liquid chromatography–tandem mass spectrometry. The test-retest reliability of 8-OHdG was good (interclass correlation coefficient = 0.76). Upper-limb motor function and muscle power determined by the Fugl-Meyer Assessment (FMA) and Medical Research Council (MRC) scales before rehabilitation showed significant negative correlation with 8-OHdG (r = −0.38, r = −0.30; p rehabilitation, we found a fair and significant correlation between 8-OHdG and FMA (r = −0.34) and 8-OHdG and pain (r = 0.26, p rehabilitation. The exploratory study findings conclude that 8-OHdG is a reliable and promising biomarker of oxidative stress and could be a valid predictor of functional outcomes in patients. Monitoring of behavioral indicators along with biomarkers may have crucial benefits in translational stroke research. PMID:24743892

  3. Effect of heavy metals on nitrification activity as measured by RNA- and DNA-based function-specific assays

    Science.gov (United States)

    Heavy metals can inhibit nitrification, a key process for nitrogen removal in wastewater treatment. The transcriptional responses of functional genes (amoA, hao, nirK and norB) were measured in conjunction with specific oxygen uptake rate (sOUR) for nitrifying enrichment cultures...

  4. Molecular and functional characterization of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Dunaliella salina.

    Science.gov (United States)

    Ramos, Ana A; Marques, Ana R; Rodrigues, Marta; Henriques, Nuno; Baumgartner, Alexandra; Castilho, Rita; Brenig, Bertram; Varela, João C

    2009-06-01

    In green algae, the final step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR; EC: 1.17.1.2), an enzyme proposed to play a key role in the regulation of isoprenoid biosynthesis. Here we report the isolation and functional characterization of a 1959-bp Dunaliella salina HDR (DsHDR) cDNA encoding a deduced polypeptide of 474 amino acid residues. Phylogenetic analysis implied a cyanobacterial origin for plant and algal HDR genes. Steady-state DsHDR transcript levels were higher in D. salina cells submitted to nutritional depletion, high salt and/or high light, suggesting that DsHDR may respond to the same environmental cues as genes involved in carotenoid biosynthesis.

  5. Interfacing DNA nanodevices with biology

    DEFF Research Database (Denmark)

    Vinther, Mathias; Kjems, Jørgen

    2016-01-01

    in biology and biomedicine acting as a molecular ‘nanorobot’ or smart drug interacting with the cellular machinery. In this review, we will explore and examine the perspective of DNA nanotechnology for such use. We summarize which requirements DNA nanostructures must fulfil to function in cellular...... environments and inside living organisms. In addition, we highlight recent advances in interfacing DNA nanostructures with biology....

  6. XPF with mutations in its conserved nuclease domain is defective in DNA repair but functions in TRF2-mediated telomere shortening.

    Science.gov (United States)

    Wu, Yili; Zacal, Natalie J; Rainbow, Andrew J; Zhu, Xu-Dong

    2007-02-04

    TRF2, a telomere-binding protein, is a crucial player in telomere length maintenance. Overexpression of TRF2 results in telomere shortening in both normal primary fibroblasts and telomerase-positive cancer cells. TRF2 is found to be associated with XPF-ERCC1, a structure-specific endonuclease involved in nucleotide excision repair, crosslink repair and DNA recombination. XPF-ERCC1 is implicated in TRF2-dependent telomere loss in mouse keratinocytes, however, whether XPF-ERCC1 and its nuclease activity are required for TRF2-mediated telomere shortening in human cells is unknown. Here we report that TRF2-induced telomere shortening is abrogated in human cells deficient in XPF, demonstrating that XPF-ERCC1 is required for TRF2-promoted telomere shortening. To further understand the role of XPF in TRF2-dependent telomere shortening, we generated constructs containing either wild type XPF or mutant XPF proteins carrying amino acid substitutions in its conserved nuclease domain. We show that wild type XPF can complement XPF-deficient cells for repair of UV-induced DNA damage whereas the nuclease-inactive XPF proteins fail to do so, indicating that the nuclease activity of XPF is essential for nucleotide excision repair. In contrast, both wild type XPF and nuclease-inactive XPF proteins, when expressed in XPF-deficient cells, are able to rescue TRF2-mediated telomere shortening. Thus, our results suggest that the function of XPF in TRF2-mediated telomere shortening is conserved between mouse and human. Furthermore, our findings reveal an unanticipated nuclease-independent function of XPF in TRF2-mediated telomere shortening.

  7. Functional and DNA-protein binding studies of WRKY transcription factors and their expression analysis in response to biotic and abiotic stress in wheat (Triticum aestivum L.).

    Science.gov (United States)

    Satapathy, Lopamudra; Kumar, Dhananjay; Kumar, Manish; Mukhopadhyay, Kunal

    2018-01-01

    WRKY, a plant-specific transcription factor family, plays vital roles in pathogen defense, abiotic stress, and phytohormone signalling. Little is known about the roles and function of WRKY transcription factors in response to rust diseases in wheat. In the present study, three TaWRKY genes encoding complete protein sequences were cloned. They belonged to class II and III WRKY based on the number of WRKY domains and the pattern of zinc finger structures. Twenty-two DNA-protein binding docking complexes predicted stable interactions of WRKY domain with W-box. Quantitative real-time-PCR using wheat near-isogenic lines with or without Lr28 gene revealed differential up- or down-regulation in response to biotic and abiotic stress treatments which could be responsible for their functional divergence in wheat. TaWRKY62 was found to be induced upon treatment with JA, MJ, and SA and reduced after ABA treatments. Maximum induction of six out of seven genes occurred at 48 h post inoculation due to pathogen inoculation. Hence, TaWRKY (49, 50, 52, 55, 57, and 62) can be considered as potential candidate genes for further functional validation as well as for crop improvement programs for stress resistance. The results of the present study will enhance knowledge towards understanding the molecular basis of mode of action of WRKY transcription factor genes in wheat and their role during leaf rust pathogenesis in particular.

  8. A Sleeping Beauty DNA transposon-based genetic sensor for functional screening of vitamin D3 analogues

    DEFF Research Database (Denmark)

    Staunstrup, Nicklas H; Sharma, Nynne; Bak, Rasmus O

    2011-01-01

    methods that allow a comparative evaluation of drug properties. As a new tool in functional screening of vitamin D3 analogues, we describe a genomically integratable sensor for sensitive drug detection. This system facilitates assessment of the pharmacokinetic and pharmadynamic properties of vitamin D3...... analogues. The tri-cistronic genetic sensor encodes a drug-sensoring protein, a reporter protein expressed from an activated sensor-responsive promoter, and a resistance marker....

  9. DNA-based Artificial Nanostructures: Fabrication, Properties, and Applications

    OpenAIRE

    Sun, Young; Kiang, Ching-Hwa

    2005-01-01

    Table of Content 1. Introduction 2. DNA fundamentals 3. Attachment of DNA to surface 4. Fabrication of nanostructures using DNA 4.1 Nanostructures of pure DNA 4.2 DNA-based assembly of metal nanoparticles 4.3 Construction of semiconductor particle arrays using DNA 4.4 DNA-directed nanowires 4.5 DNA-functionalized carbon nanotubes 4.6 Field-transistor based on DNA 4.7 Nanofabrication using artificial DNA 5. DNA-based nanostructures as biosensors 6. Properties of DNA-linked gold nanoparticles 6...

  10. Protein and DNA technologies for