DNA Methylation: An Epigenetic Risk Factor in Preterm Birth

Science.gov (United States)

Menon, Ramkumar; Conneely, Karen N.; Smith, Alicia K.

2012-01-01

Spontaneous preterm birth (PTB; birth prior to 37 weeks of gestation) is a complex phenotype with multiple risk factors that complicate our understanding of its etiology. A number of recent studies have supported the hypothesis that epigenetic modifications such as DNA methylation induced by pregnancy-related risk factors may influence the risk of PTB or result in changes that predispose a neonate to adult-onset diseases. The critical role of timing of gene expression in the etiology of PTB makes it a highly relevant disorder in which to examine the potential role of epigenetic changes. Because changes in DNA methylation patterns can result in long-term consequences, it is of critical interest to identify the epigenetic patterns associated with adverse pregnancy outcomes. This review examines the potential role of DNA methylation as a risk factor for PTB and discusses several issues and limitations that should be considered when planning DNA methylation studies. PMID:22228737

Proteopedia: 3D Visualization and Annotation of Transcription Factor-DNA Readout Modes

Science.gov (United States)

Dantas Machado, Ana Carolina; Saleebyan, Skyler B.; Holmes, Bailey T.; Karelina, Maria; Tam, Julia; Kim, Sharon Y.; Kim, Keziah H.; Dror, Iris; Hodis, Eran; Martz, Eric; Compeau, Patricia A.; Rohs, Remo

2012-01-01

3D visualization assists in identifying diverse mechanisms of protein-DNA recognition that can be observed for transcription factors and other DNA binding proteins. We used Proteopedia to illustrate transcription factor-DNA readout modes with a focus on DNA shape, which can be a function of either nucleotide sequence (Hox proteins) or base pairing…

Regulation of DNA repair processes in mammalian cell

International Nuclear Information System (INIS)

Bil'din, V.N.; Sergina, T.B.; Zhestyanikov, V.D.

1992-01-01

A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase α and β (aphidicolin) and DNA polymerase β (2', 3'-dideoxythjymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gy) or by γ-ray irradiation (150 Gy) is more sensitive to aphidicolin and mitogen-simulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase α

[Biomarkers of radiation-induced DNA repair processes].

Science.gov (United States)

Vallard, Alexis; Rancoule, Chloé; Guy, Jean-Baptiste; Espenel, Sophie; Sauvaigo, Sylvie; Rodriguez-Lafrasse, Claire; Magné, Nicolas

2017-11-01

The identification of DNA repair biomarkers is of paramount importance. Indeed, it is the first step in the process of modulating radiosensitivity and radioresistance. Unlike tools of detection and measurement of DNA damage, DNA repair biomarkers highlight the variations of DNA damage responses, depending on the dose and the dose rate. The aim of the present review is to describe the main biomarkers of radiation-induced DNA repair. We will focus on double strand breaks (DSB), because of their major role in radiation-induced cell death. The most important DNA repair biomarkers are DNA damage signaling proteins, with ATM, DNA-PKcs, 53BP1 and γ-H2AX. They can be analyzed either using immunostaining, or using lived cell imaging. However, to date, these techniques are still time and money consuming. The development of "omics" technologies should lead the way to new (and usable in daily routine) DNA repair biomarkers. Copyright © 2017 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

Science.gov (United States)

Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

2003-08-01

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

RPA homologs and ssDNA processing during meiotic recombination.

Science.gov (United States)

Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

2016-06-01

Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

Directory of Open Access Journals (Sweden)

Janet L Smith

2015-05-01

Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

Genomic signal processing for DNA sequence clustering.

Science.gov (United States)

Mendizabal-Ruiz, Gerardo; Román-Godínez, Israel; Torres-Ramos, Sulema; Salido-Ruiz, Ricardo A; Vélez-Pérez, Hugo; Morales, J Alejandro

2018-01-01

Genomic signal processing (GSP) methods which convert DNA data to numerical values have recently been proposed, which would offer the opportunity of employing existing digital signal processing methods for genomic data. One of the most used methods for exploring data is cluster analysis which refers to the unsupervised classification of patterns in data. In this paper, we propose a novel approach for performing cluster analysis of DNA sequences that is based on the use of GSP methods and the K-means algorithm. We also propose a visualization method that facilitates the easy inspection and analysis of the results and possible hidden behaviors. Our results support the feasibility of employing the proposed method to find and easily visualize interesting features of sets of DNA data.

RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

Science.gov (United States)

Manfrini, Nicola; Trovesi, Camilla; Wery, Maxime; Martina, Marina; Cesena, Daniele; Descrimes, Marc; Morillon, Antonin; d'Adda di Fagagna, Fabrizio; Longhese, Maria Pia

2015-02-01

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. © 2014 The Authors.

True Lies: The Double Life of the Nucleotide Excision Repair Factors in Transcription and DNA Repair

Directory of Open Access Journals (Sweden)

Nicolas Le May

2010-01-01

Full Text Available Nucleotide excision repair (NER is a major DNA repair pathway in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation or bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to three genetic disorders that result in predisposition to cancers, accelerated aging, neurological and developmental defects. During NER, more than 30 polypeptides cooperate to recognize, incise, and excise a damaged oligonucleotide from the genomic DNA. Recent papers reveal an additional and unexpected role for the NER factors. In the absence of a genotoxic attack, the promoters of RNA polymerases I- and II-dependent genes recruit XPA, XPC, XPG, and XPF to initiate gene expression. A model that includes the growth arrest and DNA damage 45α protein (Gadd45α and the NER factors, in order to maintain the promoter of active genes under a hypomethylated state, has been proposed but remains controversial. This paper focuses on the double life of the NER factors in DNA repair and transcription and describes the possible roles of these factors in the RNA synthesis process.

Signatures of DNA target selectivity by ETS transcription factors.

Science.gov (United States)

Poon, Gregory M K; Kim, Hye Mi

2017-05-27

The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. DNA target specificity derives from combinatorial interactions with other proteins as well as intrinsic heterogeneity among ETS domains. Emerging evidence suggests molecular hydration as a fundamental feature that defines the intrinsic heterogeneity in DNA target selection and susceptibility to epigenetic DNA modification. This perspective invokes novel hypotheses in the regulation of ETS proteins in physiologic osmotic stress, their pioneering potential in heterochromatin, and the effects of passive and pharmacologic DNA demethylation on ETS regulation.

An assay system for factors involved in mammalian DNA replication

International Nuclear Information System (INIS)

Reinhard, P.; Maillart, P.; Schluchter, M.; Gautschi, J.R.; Schindler, R.

1979-01-01

An assay for cellular factors stimulating DNA synthesis by partially lysed CHO cells is presented. The assay is based on the observation that in highly lysed cells, DNA synthesis, as determined by [ 3 H]dTTP incorporation, was only 2-5% of that in gently lysed cells, and that this low level of DNA synthesis could be increased by a factor of approx. 50 by the addition of CHO cell extract (i.e. supernatant of a cell homogenate subjected to high-speed centrifugation.) (Auth.)

Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites

International Nuclear Information System (INIS)

Lenz, J.; Okenquist, S.A.; LoSardo, J.E.; Hamilton, K.K.; Doetsch, P.W.

1990-01-01

Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of β-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins

Factors influencing parents' decision to donate their healthy infant's DNA for minimal-risk genetic research.

Science.gov (United States)

Hatfield, Linda A; Pearce, Margaret M

2014-11-01

To examine factors that influence a parent's decision to donate their healthy infant's DNA for minimal-risk genetic research. Grounded theory, using semi-structured interviews conducted with 35 postpartum mother or mother-father dyads in an urban teaching hospital. Data were collected from July 2011 to January 2012. Audiorecorded semistructured interviews were conducted in private rooms with mothers or mother-father dyads 24 to 48 hr after the birth of their healthy, full-term infant. Data-driven content analysis using selected principles of grounded theory was performed. Parents' willingness to donate their healthy infant's DNA for minimal-risk pediatric genetic research emerged as a process involving three interacting components: the parents, the scientist, and the comfort of the child embedded within the context of benefit to the child. The purpose of the study and parents' perception of their commitment of time and resources determined their willingness to participate. The scientist's ability to communicate trust in the research process influenced parents' decisions. Physical discomfort of the child shaped parents' decision to donate DNA. Parental perception of a direct benefit to their child affected their willingness to discuss genetic research and its outcomes. Significant gaps and misunderstandings in parental knowledge of pediatric genetic research may affect parental willingness to donate their healthy child's DNA. Nurses knowledgeable about the decision-making process parents utilize to donate their healthy infant's DNA for minimal-risk genetic research and the factors influencing that decision are well positioned to educate parents about the role of genetics in health and illness and reassure potential research participants of the value and safeguards in pediatric genetic research. © 2014 Sigma Theta Tau International.

DNA replication initiator Cdc6 also regulates ribosomal DNA transcription initiation.

Science.gov (United States)

Huang, Shijiao; Xu, Xiaowei; Wang, Guopeng; Lu, Guoliang; Xie, Wenbing; Tao, Wei; Zhang, Hongyin; Jiang, Qing; Zhang, Chuanmao

2016-04-01

RNA-polymerase-I-dependent ribosomal DNA (rDNA) transcription is fundamental to rRNA processing, ribosome assembly and protein synthesis. However, how this process is initiated during the cell cycle is not fully understood. By performing a proteomic analysis of transcription factors that bind RNA polymerase I during rDNA transcription initiation, we identified that the DNA replication initiator Cdc6 interacts with RNA polymerase I and its co-factors, and promotes rDNA transcription in G1 phase in an ATPase-activity-dependent manner. We further showed that Cdc6 is targeted to the nucleolus during late mitosis and G1 phase in a manner that is dependent on B23 (also known as nucleophosmin, NPM1), and preferentially binds to the rDNA promoter through its ATP-binding domain. Overexpression of Cdc6 increases rDNA transcription, whereas knockdown of Cdc6 results in a decreased association of both RNA polymerase I and the RNA polymerase I transcription factor RRN3 with rDNA, and a reduction of rDNA transcription. Furthermore, depletion of Cdc6 impairs the interaction between RRN3 and RNA polymerase I. Taken together, our data demonstrate that Cdc6 also serves as a regulator of rDNA transcription initiation, and indicate a mechanism by which initiation of rDNA transcription and DNA replication can be coordinated in cells. © 2016. Published by The Company of Biologists Ltd.

Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

OpenAIRE

Davidson, John F.; Fox, Richard; Harris, Dawn D.; Lyons-Abbott, Sally; Loeb, Lawrence A.

2003-01-01

Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20–50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq...

Development of identification process for insect group using radiation marker DNA

International Nuclear Information System (INIS)

Muraji, M.; Tamura, T.

2004-01-01

Detection of a band pattern for insect groups was tried by using radiation marked DNA clone. A rapid segregation process for poly-type DNA segment was investigated. A band pattern of silkworm was detected by analysis using DNA type transposon, K1.4. The exon regions on genes of hemiptera insect were segregated by in vitro cloning. Band patterns of the silkworm and the other insects were detected by identification process of DNA clone and radiation marker. Family singularity mutation existed in the inserted position of transposon. The family of insect was identified easily by the difference of the detection band patterns. Effective band pattern for family discrimination were obtained by analysis for a part of mitochondria DNA and ribosomal DNA. DNA segregation process was investigated by using the enriched library, also. (M. Suetake)

Local dynamics of proteins and DNA evaluated from crystallographic B factors

International Nuclear Information System (INIS)

Schneider, Bohdan; Gelly, Jean-Christophe; Brevern, Alexandre G. de; Černý, Jiří

2014-01-01

Distributions of scaled B factors from 704 protein–DNA complexes reflect primarily the neighbourhood of amino-acid and nucleotide residues: their flexibility grows from the protein core to protein–protein and protein–DNA interfaces, to solvent-exposed residues. Some of the findings clearly observed at higher resolution structures can no longer be observed for structures at low resolution indicating problems in refinement protocols. The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-called B factors. To find out how the crystal-derived B factors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaled B factors from a carefully curated data set of over 700 protein–DNA crystal structures were analyzed [Schneider et al. (2014 ▶), Nucleic Acids Res.42, 3381–3394]. Amino acids and nucleotides were categorized based on their molecular neighbourhood as solvent-accessible, solvent-inaccessible (i.e. forming the protein core) or lying at protein–protein or protein–DNA interfaces; the backbone and side-chain atoms were analyzed separately. The B factors of two types of crystal-ordered water molecules were also analyzed. The analysis confirmed several expected features of protein and DNA dynamics, but also revealed surprising facts. Solvent-accessible amino acids have B factors that are larger than those of residues at the biomolecular interfaces, and core-forming amino acids are the most restricted in their movement. A unique feature of the latter group is that their side-chain and backbone atoms are restricted in their movement to the same extent; in all other amino-acid groups the side chains are more floppy than the backbone. The low values of the B factors of water molecules bridging proteins with DNA and the very large fluctuations of DNA phosphates are

Local dynamics of proteins and DNA evaluated from crystallographic B factors

Energy Technology Data Exchange (ETDEWEB)

Schneider, Bohdan, E-mail: bohdan.schneider@gmail.com [Institute of Biotechnology AS CR, Videnska 1083, 142 20 Prague (Czech Republic); Gelly, Jean-Christophe; Brevern, Alexandre G. de [INSERM, U1134, DSIMB, 75739 Paris (France); Université Paris Diderot, Sorbonne Paris Cité, UMR-S 1134, 75739 Paris (France); Institut National de la Transfusion Sanguine (INTS), 75739 Paris (France); Laboratoire d’Excellence GR-Ex, 75739 Paris (France); Černý, Jiří [Institute of Biotechnology AS CR, Videnska 1083, 142 20 Prague (Czech Republic)

2014-09-01

Distributions of scaled B factors from 704 protein–DNA complexes reflect primarily the neighbourhood of amino-acid and nucleotide residues: their flexibility grows from the protein core to protein–protein and protein–DNA interfaces, to solvent-exposed residues. Some of the findings clearly observed at higher resolution structures can no longer be observed for structures at low resolution indicating problems in refinement protocols. The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-called B factors. To find out how the crystal-derived B factors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaled B factors from a carefully curated data set of over 700 protein–DNA crystal structures were analyzed [Schneider et al. (2014 ▶), Nucleic Acids Res.42, 3381–3394]. Amino acids and nucleotides were categorized based on their molecular neighbourhood as solvent-accessible, solvent-inaccessible (i.e. forming the protein core) or lying at protein–protein or protein–DNA interfaces; the backbone and side-chain atoms were analyzed separately. The B factors of two types of crystal-ordered water molecules were also analyzed. The analysis confirmed several expected features of protein and DNA dynamics, but also revealed surprising facts. Solvent-accessible amino acids have B factors that are larger than those of residues at the biomolecular interfaces, and core-forming amino acids are the most restricted in their movement. A unique feature of the latter group is that their side-chain and backbone atoms are restricted in their movement to the same extent; in all other amino-acid groups the side chains are more floppy than the backbone. The low values of the B factors of water molecules bridging proteins with DNA and the very large fluctuations of DNA phosphates are

Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA

International Nuclear Information System (INIS)

Chu, G.; Chang, E.

1990-01-01

Cancer treatment with the drug cisplatin is often thwarted by the emergence of drug-resistant cells. To study this phenomenon, the authors identified two independent cellular factors that recognize cisplatin-damaged DNA. One of the two factors, designated XPE binding factor, is deficient in complementation group E of xeroderma pigmentosum, an inherited disease characterized by defective repair of DNA damaged by ultraviolet radiation, cisplatin, and other agents. Human tumor cell lines selected for resistance to cisplatin showed more efficient DNA repair and increased expression of XPE binding factor. These results suggest that XPE binding factor may be responsible, at least in part, for the development of cisplatin resistance in human tumors and that the mechanism may be increased DNA repair

The dynamic interplay between DNA topoisomerases and DNA topology.

Science.gov (United States)

Seol, Yeonee; Neuman, Keir C

2016-11-01

Topological properties of DNA influence its structure and biochemical interactions. Within the cell, DNA topology is constantly in flux. Transcription and other essential processes, including DNA replication and repair, not only alter the topology of the genome but also introduce additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases is a pervasive factor that influences DNA metabolism in vivo. Building on the extensive structural and biochemical characterization over the past four decades that has established the fundamental mechanistic basis of topoisomerase activity, scientists have begun to explore the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases. In this review we survey established and emerging DNA topology-dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

DNA methylation as a dynamic regulator of development and disease processes: spotlight on the prostate.

Science.gov (United States)

Keil, Kimberly P; Vezina, Chad M

2015-01-01

Prostate development, benign hyperplasia and cancer involve androgen and growth factor signaling as well as stromal-epithelial interactions. We review how DNA methylation influences these and related processes in other organ systems such as how proliferation is restricted to specific cell populations during defined temporal windows, how androgens elicit their actions and how cells establish, maintain and remodel DNA methylation in a time and cell specific fashion. We also discuss mechanisms by which hormones and endocrine disrupting chemicals reprogram DNA methylation in the prostate and elsewhere and examine evidence for a reawakening of developmental epigenetic pathways as drivers of prostate cancer and benign prostate hyperplasia.

Using TESS to predict transcription factor binding sites in DNA sequence.

Science.gov (United States)

Schug, Jonathan

2008-03-01

This unit describes how to use the Transcription Element Search System (TESS). This Web site predicts transcription factor binding sites (TFBS) in DNA sequence using two different kinds of models of sites, strings and positional weight matrices. The binding of transcription factors to DNA is a major part of the control of gene expression. Transcription factors exhibit sequence-specific binding; they form stronger bonds to some DNA sequences than to others. Identification of a good binding site in the promoter for a gene suggests the possibility that the corresponding factor may play a role in the regulation of that gene. However, the sequences transcription factors recognize are typically short and allow for some amount of mismatch. Because of this, binding sites for a factor can typically be found at random every few hundred to a thousand base pairs. TESS has features to help sort through and evaluate the significance of predicted sites.

DNA methylation in metabolic disorders

DEFF Research Database (Denmark)

Barres, Romain; Zierath, Juleen R

2011-01-01

DNA methylation is a major epigenetic modification that controls gene expression in physiologic and pathologic states. Metabolic diseases such as diabetes and obesity are associated with profound alterations in gene expression that are caused by genetic and environmental factors. Recent reports...... have provided evidence that environmental factors at all ages could modify DNA methylation in somatic tissues, which suggests that DNA methylation is a more dynamic process than previously appreciated. Because of the importance of lifestyle factors in metabolic disorders, DNA methylation provides...... a mechanism by which environmental factors, including diet and exercise, can modify genetic predisposition to disease. This article considers the current evidence that defines a role for DNA methylation in metabolic disorders....

Both genetic and dietary factors underlie individual differences in DNA damage levels and DNA repair capacity

Czech Academy of Sciences Publication Activity Database

Slyšková, Jana; Lorenzo, Y.; Karlsen, A.; Carlsen, M. H.; Novosadová, Vendula; Blomhoff, R.; Vodička, Pavel; Collins, A. R.

2014-01-01

Roč. 16, APR 2014 (2014), s. 66-73 ISSN 1568-7864 R&D Projects: GA ČR(CZ) GAP304/12/1585 Institutional support: RVO:68378041 ; RVO:86652036 Keywords : DNA damage * DNA repair capacity * diet Subject RIV: EB - Genetics ; Molecular Biology; EI - Biotechnology ; Bionics (BTO-N) Impact factor: 3.111, year: 2014

Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

NARCIS (Netherlands)

Platteel, Anouk C M; Marit de Groot, A; Andersen, Peter; Ovaa, Huib; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M

2016-01-01

Most vaccines are based on protective humoral responses while for intracellular pathogens CD8(+) T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8(+) T cell priming, hampering vaccine efficacy. The multistage cDNA

One-pot synthesis of strongly fluorescent DNA-CuInS2 quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

International Nuclear Information System (INIS)

Liu, Ziping; Su, Xingguang

2016-01-01

Herein, high quality DNA-CuInS 2 QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS 2 QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS 2 QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS 2 QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I 0 (I 0 is the original fluorescence intensity of DNA-CuInS 2 QDs, and I is the fluorescence intensity of DNA-CuInS 2 QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L −1 with a detection limit of 0.013 nmol L −1 . The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS 2 QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were obtained. • This method was utilized to detect

ZRBA1, a Mixed EGFR/DNA Targeting Molecule, Potentiates Radiation Response Through Delayed DNA Damage Repair Process in a Triple Negative Breast Cancer Model

Energy Technology Data Exchange (ETDEWEB)

Heravi, Mitra [Department of Human Genetics, McGill University, Montreal (Canada); Department of Radiation Oncology, McGill University, Montreal (Canada); Segal Cancer Center, Jewish General Hospital, Montreal (Canada); Kumala, Slawomir [Department of Radiation Oncology, McGill University, Montreal (Canada); Segal Cancer Center, Jewish General Hospital, Montreal (Canada); Rachid, Zakaria; Jean-Claude, Bertrand J. [Cancer Drug Research Laboratory, McGill University Health Center, Montreal (Canada); Radzioch, Danuta [Department of Human Genetics, McGill University, Montreal (Canada); Muanza, Thierry M., E-mail: tmuanza@yahoo.com [Department of Radiation Oncology, McGill University, Montreal (Canada); Segal Cancer Center, Jewish General Hospital, Montreal (Canada)

2015-06-01

Purpose: ZRBA1 is a combi-molecule designed to induce DNA alkylating lesions and to block epidermal growth factor receptor (EGFR) TK domain. Inasmuch as ZRBA1 downregulates the EGFR TK-mediated antisurvival signaling and induces DNA damage, we postulated that it might be a radiosensitizer. The aim of this study was to further investigate the potentiating effect of ZRBA1 in combination with radiation and to elucidate the possible mechanisms of interaction between these 2 treatment modalities. Methods and Materials: The triple negative human breast MDA-MB-468 cancer cell line and mouse mammary cancer 4T1 cell line were used in this study. Clonogenic assay, Western blot analysis, and DNA damage analysis were performed at multiple time points after treatment. To confirm our in vitro findings, in vivo tumor growth delay assay was performed. Results: Our results show that a combination of ZRBA1 and radiation increases the radiation sensitivity of both cell lines significantly with a dose enhancement factor of 1.56, induces significant numbers of DNA strand breaks, prolongs higher DNA damage up to 24 hours after treatment, and significantly increases tumor growth delay in a syngeneic mouse model. Conclusions: Our data suggest that the higher efficacy of this combination could be partially due to increased DNA damage and delayed DNA repair process and to the inhibition of EGFR. The encouraging results of this combination demonstrated a significant improvement in treatment efficiency and therefore could be applicable in early clinical trial settings.

Using Ancient DNA to Understand Evolutionary and Ecological Processes

DEFF Research Database (Denmark)

Orlando, Ludovic Antoine Alexandre; Cooper, Alan

2014-01-01

Ancient DNA provides a unique means to record genetic change through time and directly observe evolutionary and ecological processes. Although mostly based on mitochondrial DNA, the increasing availability of genomic sequences is leading to unprecedented levels of resolution. Temporal studies of ...

A DNA Mini-Barcoding System for Authentication of Processed Fish Products.

Science.gov (United States)

Shokralla, Shadi; Hellberg, Rosalee S; Handy, Sara M; King, Ian; Hajibabaei, Mehrdad

2015-10-30

Species substitution is a form of seafood fraud for the purpose of economic gain. DNA barcoding utilizes species-specific DNA sequence information for specimen identification. Previous work has established the usability of short DNA sequences-mini-barcodes-for identification of specimens harboring degraded DNA. This study aims at establishing a DNA mini-barcoding system for all fish species commonly used in processed fish products in North America. Six mini-barcode primer pairs targeting short (127-314 bp) fragments of the cytochrome c oxidase I (CO1) DNA barcode region were developed by examining over 8,000 DNA barcodes from species in the U.S. Food and Drug Administration (FDA) Seafood List. The mini-barcode primer pairs were then tested against 44 processed fish products representing a range of species and product types. Of the 44 products, 41 (93.2%) could be identified at the species or genus level. The greatest mini-barcoding success rate found with an individual primer pair was 88.6% compared to 20.5% success rate achieved by the full-length DNA barcode primers. Overall, this study presents a mini-barcoding system that can be used to identify a wide range of fish species in commercial products and may be utilized in high throughput DNA sequencing for authentication of heavily processed fish products.

Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq

DEFF Research Database (Denmark)

Jakobsen, Janus S; Bagger, Frederik O; Hasemann, Marie S

2015-01-01

BACKGROUND: Chromatin-Immunoprecipitation coupled with deep sequencing (ChIP-seq) is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low...... transcription factor (CEBPA) and histone mark (H3K4me3) ChIP. We further demonstrate that genomic profiles are highly resilient to changes in carrier DNA to ChIP DNA ratios. CONCLUSIONS: This represents a significant advance compared to existing technologies, which involve either complex steps of pre...... cell numbers. RESULTS: We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, verified down to 10,000 cells. By employing non-mammalian genome mapping bacterial carrier DNA during amplification, we reliably amplify down to 50 pg of ChIP DNA from...

Demonstrating Interactions of Transcription Factors with DNA by Electrophoretic Mobility Shift Assay.

Science.gov (United States)

Yousaf, Nasim; Gould, David

2017-01-01

Confirming the binding of a transcription factor with a particular DNA sequence may be important in characterizing interactions with a synthetic promoter. Electrophoretic mobility shift assay is a powerful approach to demonstrate the specific DNA sequence that is bound by a transcription factor and also to confirm the specific transcription factor involved in the interaction. In this chapter we describe a method we have successfully used to demonstrate interactions of endogenous transcription factors with sequences derived from endogenous and synthetic promoters.

Human tissue factor: cDNA sequence and chromosome localization of the gene

International Nuclear Information System (INIS)

Scarpati, E.M.; Wen, D.; Broze, G.J. Jr.; Miletich, J.P.; Flandermeyer, R.R.; Siegel, N.R.; Sadler, J.E.

1987-01-01

A human placenta cDNA library in λgt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, λHTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of λHTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of ∼ 3.2 kilobases in poly(A) + RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased several fold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of λHTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(a) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser. The 3'-noncoding region contains an inverted Alu family repetitive sequence. The tissue factor gene was localized to chromosome 1 by hybridization of the cDNA insert of λHTF8 to flow-sorted human chromosomes

The effect of low radiation doses on DNA repair processes

International Nuclear Information System (INIS)

Tuschl, H.

1978-08-01

Error free DNA repair processes are an important preprequisite for the maintenance of genetic integrity of cells. They are of special importance for persons therapeutically or occupationally exposed to radiation. Therefore the effect of radiation therapy and elevated natural background radiation on unscheduled DNA synthesis was tested in peripheral lymphocytes of exposed persons. Both, autoradiographic studies of unscheduled DNA synthesis and measurement of 3 H-thymidine uptake into double stranded and single-strand containing DNA fractions revealed an increase of capacity for DNA repair. (author)

Processing of free radical damaged DNA bases

International Nuclear Information System (INIS)

Wallace, S.

2003-01-01

Free radicals produced during the radiolysis of water gives rise to a plethora of DNA damages including single strand breaks, sites of base loss and a wide variety of purine and pyrimidine base lesions. All these damages are processed in cells by base excision repair. The oxidative DNA glycosylases which catalyze the first step in the removal of a base damage during base excision repair evolved primarily to protect the cells from the deleterious mutagenic effects of single free radical-induced DNA lesions arising during oxidative metabolism. This is evidenced by the high spontaneous mutation rate in bacterial mutants lacking the oxidative DNA glycosylases. However, when a low LET photon transverses the DNA molecule, a burst of free radicals is produced during the radiolysis of water that leads to the formation of clustered damages in the DNA molecule, that are recognized by the oxidative DNA glycosylases. When substrates containing two closely opposed sugar damages or base and sugar damages are incubated with the oxidative DNA glycosylases in vitro, one strand is readily incised by the lyase activity of the DNA glycosylase. Whether or not the second strand is incised depends on the distance between the strand break resulting from the incised first strand and the remaining DNA lesion on the other strand. If the lesions are more than two or three base pairs apart, the second strand is readily cleaved by the DNA glycosylase, giving rise to a double strand break. Even if the entire base excision repair system is reconstituted in vitro, whether or not a double strand break ensues depends solely upon the ability of the DNA glycosylase to cleave the second strand. These data predicted that cells deficient in the oxidative DNA glycosylases would be radioresistant while those that overproduce an oxidative DNA glycosylase would be radiosensitive. This prediction was indeed borne in Escherichia coli that is, mutants lacking the oxidative DNA glycosylases are radioresistant

One-pot synthesis of strongly fluorescent DNA-CuInS{sub 2} quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

Energy Technology Data Exchange (ETDEWEB)

Liu, Ziping; Su, Xingguang, E-mail: suxg@jlu.edu.cn

2016-10-26

Herein, high quality DNA-CuInS{sub 2} QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS{sub 2} QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS{sub 2} QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS{sub 2} QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I{sub 0} (I{sub 0} is the original fluorescence intensity of DNA-CuInS{sub 2} QDs, and I is the fluorescence intensity of DNA-CuInS{sub 2} QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L{sup −1} with a detection limit of 0.013 nmol L{sup −1}. The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS{sub 2} QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were

Quantification of DNA fragmentation in processed foods using real-time PCR.

Science.gov (United States)

Mano, Junichi; Nishitsuji, Yasuyuki; Kikuchi, Yosuke; Fukudome, Shin-Ichi; Hayashida, Takuya; Kawakami, Hiroyuki; Kurimoto, Youichi; Noguchi, Akio; Kondo, Kazunari; Teshima, Reiko; Takabatake, Reona; Kitta, Kazumi

2017-07-01

DNA analysis of processed foods is performed widely to detect various targets, such as genetically modified organisms (GMOs). Food processing often causes DNA fragmentation, which consequently affects the results of PCR analysis. In order to assess the effects of DNA fragmentation on the reliability of PCR analysis, we investigated a novel methodology to quantify the degree of DNA fragmentation. We designed four real-time PCR assays that amplified 18S ribosomal RNA gene sequences common to various plants at lengths of approximately 100, 200, 400, and 800 base pairs (bp). Then, we created an indicator value, "DNA fragmentation index (DFI)", which is calculated from the Cq values derived from the real-time PCR assays. Finally, we demonstrated the efficacy of this method for the quality control of GMO detection in processed foods by evaluating the relationship between the DFI and the limit of detection. Copyright © 2017 Elsevier Ltd. All rights reserved.

Capturing Snapshots of APE1 Processing DNA Damage

Science.gov (United States)

Freudenthal, Bret D.; Beard, William A.; Cuneo, Matthew J.; Dyrkheeva, Nadezhda S.; Wilson, Samuel H.

2015-01-01

DNA apurinic-apyrimidinic (AP) sites are prevalent non-coding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive due in part to limited structural information. We report multiple high-resolution human APE1:DNA structures that divulge novel features of the APE1 reaction, including the metal binding site, nucleophile, and arginine clamps that mediate product release. We also report APE1:DNA structures with a T:G mismatch 5′ to the AP-site, representing a clustered lesion occurring in methylated CpG dinucleotides. These reveal that APE1 molds the T:G mismatch into a unique Watson-Crick like geometry that distorts the active site reducing incision. These snapshots provide mechanistic clarity for APE1, while affording a rational framework to manipulate biological responses to DNA damage. PMID:26458045

TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA targeting proteins

Science.gov (United States)

Doyle, Erin L.; Stoddard, Barry L.; Voytas, Daniel F.; Bogdanove, Adam J.

2013-01-01

Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria in the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenues for engineering disease resistance. It has also enabled TAL effector customization for targeted gene control, genome editing, and other applications. This article reviews the structural basis for TAL effector-DNA specificity, the impact of the TAL effector-DNA code on plant pathology and engineered resistance, and recent accomplishments and future challenges in TAL effector-based DNA targeting. PMID:23707478

G =  MAT: linking transcription factor expression and DNA binding data.

Science.gov (United States)

Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

2011-01-31

Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/.

G = MAT: Linking Transcription Factor Expression and DNA Binding Data

Science.gov (United States)

Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

2011-01-01

Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/. PMID:21297945

Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

Science.gov (United States)

Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

2015-03-31

Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

International Nuclear Information System (INIS)

Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çaglar; Koc, Ahmet

2014-01-01

Highlights: • Aging process increases ROS accumulation. • Aging process increases DNA damage levels. • Absence of SOD activity does not cause DNA damage in young cells. • Absence of SOD activity accelerate aging and increase oxidative DNA damages during the aging process. - Abstract: Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

Energy Technology Data Exchange (ETDEWEB)

Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çaglar; Koc, Ahmet, E-mail: ahmetkoc@iyte.edu.tr

2014-02-07

Highlights: • Aging process increases ROS accumulation. • Aging process increases DNA damage levels. • Absence of SOD activity does not cause DNA damage in young cells. • Absence of SOD activity accelerate aging and increase oxidative DNA damages during the aging process. - Abstract: Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.

Photochemical Acceleration of DNA Strand Displacement by Using Ultrafast DNA Photo-crosslinking.

Science.gov (United States)

Nakamura, Shigetaka; Hashimoto, Hirokazu; Kobayashi, Satoshi; Fujimoto, Kenzo

2017-10-18

DNA strand displacement is an essential reaction in genetic recombination, biological processes, and DNA nanotechnology. In particular, various DNA nanodevices enable complicated calculations. However, it takes time before the output is obtained, so acceleration of DNA strand displacement is required for a rapid-response DNA nanodevice. Herein, DNA strand displacement by using DNA photo-crosslinking to accelerate this displacement is evaluated. The DNA photo-crosslinking of 3-cyanovinylcarbazole ( CNV K) was accelerated at least 20 times, showing a faster DNA strand displacement. The rate of photo-crosslinking is a key factor and the rate of DNA strand displacement is accelerated through ultrafast photo-crosslinking. The rate of DNA strand displacement was regulated by photoirradiation energy. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vitro fluorescence studies of transcription factor IIB-DNA interaction.

Science.gov (United States)

Górecki, Andrzej; Figiel, Małgorzata; Dziedzicka-Wasylewska, Marta

2015-01-01

General transcription factor TFIIB is one of the basal constituents of the preinitiation complex of eukaryotic RNA polymerase II, acting as a bridge between the preinitiation complex and the polymerase, and binding promoter DNA in an asymmetric manner, thereby defining the direction of the transcription. Methods of fluorescence spectroscopy together with circular dichroism spectroscopy were used to observe conformational changes in the structure of recombinant human TFIIB after binding to specific DNA sequence. To facilitate the exploration of the structural changes, several site-directed mutations have been introduced altering the fluorescence properties of the protein. Our observations showed that binding of specific DNA sequences changed the protein structure and dynamics, and TFIIB may exist in two conformational states, which can be described by a different microenvironment of W52. Fluorescence studies using both intrinsic and exogenous fluorophores showed that these changes significantly depended on the recognition sequence and concerned various regions of the protein, including those interacting with other transcription factors and RNA polymerase II. DNA binding can cause rearrangements in regions of proteins interacting with the polymerase in a manner dependent on the recognized sequences, and therefore, influence the gene expression.

G =  MAT: linking transcription factor expression and DNA binding data.

Directory of Open Access Journals (Sweden)

Konstantin Tretyakov

Full Text Available Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/.

Two-step interrogation then recognition of DNA binding site by Integration Host Factor: an architectural DNA-bending protein.

Science.gov (United States)

Velmurugu, Yogambigai; Vivas, Paula; Connolly, Mitchell; Kuznetsov, Serguei V; Rice, Phoebe A; Ansari, Anjum

2018-02-28

The dynamics and mechanism of how site-specific DNA-bending proteins initially interrogate potential binding sites prior to recognition have remained elusive for most systems. Here we present these dynamics for Integration Host factor (IHF), a nucleoid-associated architectural protein, using a μs-resolved T-jump approach. Our studies show two distinct DNA-bending steps during site recognition by IHF. While the faster (∼100 μs) step is unaffected by changes in DNA or protein sequence that alter affinity by >100-fold, the slower (1-10 ms) step is accelerated ∼5-fold when mismatches are introduced at DNA sites that are sharply kinked in the specific complex. The amplitudes of the fast phase increase when the specific complex is destabilized and decrease with increasing [salt], which increases specificity. Taken together, these results indicate that the fast phase is non-specific DNA bending while the slow phase, which responds only to changes in DNA flexibility at the kink sites, is specific DNA kinking during site recognition. Notably, the timescales for the fast phase overlap with one-dimensional diffusion times measured for several proteins on DNA, suggesting that these dynamics reflect partial DNA bending during interrogation of potential binding sites by IHF as it scans DNA.

DNA recognition by synthetic constructs.

Science.gov (United States)

Pazos, Elena; Mosquera, Jesús; Vázquez, M Eugenio; Mascareñas, José L

2011-09-05

The interaction of transcription factors with specific DNA sites is key for the regulation of gene expression. Despite the availability of a large body of structural data on protein-DNA complexes, we are still far from fully understanding the molecular and biophysical bases underlying such interactions. Therefore, the development of non-natural agents that can reproduce the DNA-recognition properties of natural transcription factors remains a major and challenging goal in chemical biology. In this review we summarize the basics of double-stranded DNA recognition by transcription factors, and describe recent developments in the design and preparation of synthetic DNA binders. We mainly focus on synthetic peptides that have been designed by following the DNA interaction of natural proteins, and we discuss how the tools of organic synthesis can be used to make artificial constructs equipped with functionalities that introduce additional properties to the recognition process, such as sensing and controllability. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

International Nuclear Information System (INIS)

Lutwyche, Jodi K.; Keough, Rebecca A.; Hunter, Julie; Coles, Leeanne S.; Gonda, Thomas J.

2006-01-01

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor

Correlation of bistranded clustered abasic DNA lesion processing with structural and dynamic DNA helix distortion

Science.gov (United States)

Bignon, Emmanuelle; Gattuso, Hugo; Morell, Christophe; Dehez, François; Georgakilas, Alexandros G.; Monari, Antonio; Dumont, Elise

2016-01-01

Clustered apurinic/apyrimidinic (AP; abasic) DNA lesions produced by ionizing radiation are by far more cytotoxic than isolated AP lesion entities. The structure and dynamics of a series of seven 23-bp oligonucleotides featuring simple bistranded clustered damage sites, comprising of two AP sites, zero, one, three or five bases 3′ or 5′ apart from each other, were investigated through 400 ns explicit solvent molecular dynamics simulations. They provide representative structures of synthetically engineered multiply damage sites-containing oligonucleotides whose repair was investigated experimentally (Nucl. Acids Res. 2004, 32:5609-5620; Nucl. Acids Res. 2002, 30: 2800–2808). The inspection of extrahelical positioning of the AP sites, bulge and non Watson–Crick hydrogen bonding corroborates the experimental measurements of repair efficiencies by bacterial or human AP endonucleases Nfo and APE1, respectively. This study provides unprecedented knowledge into the structure and dynamics of clustered abasic DNA lesions, notably rationalizing the non-symmetry with respect to 3′ to 5′ position. In addition, it provides strong mechanistic insights and basis for future studies on the effects of clustered DNA damage on the recognition and processing of these lesions by bacterial or human DNA repair enzymes specialized in the processing of such lesions. PMID:27587587

pH Modulates the Binding of EGR1 Transcription Factor to DNA

Science.gov (United States)

Mikles, David C.; Bhat, Vikas; Schuchardt, Brett J.; Deegan, Brian J.; Seldeen, Kenneth L.; McDonald, Caleb B.; Farooq, Amjad

2013-01-01

EGR1 transcription factor orchestrates a plethora of signaling cascades involved in cellular homeostasis and its down-regulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with increasing pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as H382 by virtue of the fact that its substitution to non-ionizable residues abolishes pH-dependence of the binding of EGR1 to DNA. Notably, H382 inserts into the major groove of DNA and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, H382 is predominantly conserved across other members of EGR1 family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating protein-DNA interactions central to this family of transcription factors. Collectively, our findings uncover an unexpected but a key step in the molecular recognition of EGR1 family of transcription factors and suggest that they may act as sensors of pH within the intracellular environment. PMID:23718776

Eukaryotic DNA Replicases

KAUST Repository

Zaher, Manal S.; Oke, Muse; Hamdan, Samir

2014-01-01

The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

Eukaryotic DNA Replicases

KAUST Repository

Zaher, Manal S.

2014-11-21

The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

Involvement of DNA methylation in memory processing in the honey bee.

Science.gov (United States)

Lockett, Gabrielle A; Helliwell, Paul; Maleszka, Ryszard

2010-08-23

DNA methylation, an important and evolutionarily conserved epigenetic mechanism, is implicated in learning and memory processes in vertebrates, but its role in behaviour in invertebrates is unknown. We examined the role of DNA methylation in memory in the honey bee using an appetitive Pavlovian olfactory discrimination task, and by assessing the expression of DNA methyltransferase3, a key driver of epigenetic reprogramming. Here we report that DNA methyltransferase inhibition reduces acquisition retention and alters the extinction depending on treatment time, and DNA methyltransferase3 is upregulated after training. Our findings add to the understanding of epigenetic mechanisms in learning and memory, extending known roles of DNA methylation to appetitive and extinction memory, and for the first time implicate DNA methylation in memory in invertebrates.

Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review.

Science.gov (United States)

Gryson, Nicolas

2010-03-01

The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials.

Quantification of transcription factor-DNA binding affinity in a living cell.

Science.gov (United States)

Belikov, Sergey; Berg, Otto G; Wrange, Örjan

2016-04-20

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [(3)H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Amplified Self-replication of DNA Origami Nanostructures through Multi-cycle Fast-annealing Process

Science.gov (United States)

Zhou, Feng; Zhuo, Rebecca; He, Xiaojin; Sha, Ruojie; Seeman, Nadrian; Chaikin, Paul

We have developed a non-biological self-replication process using templated reversible association of components and irreversible linking with annealing and UV cycles. The current method requires a long annealing time, up to several days, to achieve the specific self-assembly of DNA nanostructures. In this work, we accomplished the self-replication with a shorter time and smaller replication rate per cycle. By decreasing the ramping time, we obtained the comparable replication yield within 90 min. Systematic studies show that the temperature and annealing time play essential roles in the self-replication process. In this manner, we can amplify the self-replication process to a factor of 20 by increasing the number of cycles within the same amount of time.

Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors

Directory of Open Access Journals (Sweden)

Heyman Yvan

2005-11-01

Full Text Available Abstract Background Recent work has shown that mitochondrial biogenesis and mitochondrial functions are critical determinants of embryonic development. However, the expression of the factors controlling mitochondrial biogenesis in early embryogenesis has received little attention so far. Methods We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA in bovine oocytes and in various stages of in vitro produced embryos. To investigate the molecular mechanisms responsible for the replication and the transcriptional activation of mtDNA, we quantified the mRNA corresponding to the mtDNA-encoded cytochrome oxidase 1 (COX1, and two nuclear-encoded factors, i.e. the Nuclear Respiratory Factor 1 (NRF1, and the nuclear-encoded Mitochondrial Transcription Factor A (mtTFA. Results Unlike findings reported in mouse embryos, the mtDNA content was not constant during early bovine embryogenesis. We found a sharp, 60% decrease in mtDNA content between the 2-cell and the 4/8-cell stages. COX1 mRNA was constant until the morula stage after which it increased dramatically. mtTFA mRNA was undetectable in oocytes and remained so until the 8/16-cell stage; it began to appear only at the morula stage, suggesting de novo synthesis. In contrast, NRF1 mRNA was detectable in oocytes and the quantity remained constant until the morula stage. Conclusion Our results revealed a reduction of mtDNA content in early bovine embryos suggesting an active process of mitochondrial DNA degradation. In addition, de novo mtTFA expression associated with mitochondrial biogenesis activation and high levels of NRF1 mRNA from the oocyte stage onwards argue for the essential function of these factors during the first steps of bovine embryogenesis.

The herpes viral transcription factor ICP4 forms a novel DNA recognition complex

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Tunnicliffe, Richard B.; Lockhart-Cairns, Michael P.; Levy, Colin; Mould, A. Paul; Jowitt, Thomas A.; Sito, Hilary; Baldock, Clair; Sandri-Goldin, Rozanne M.

2017-01-01

Abstract The transcription factor ICP4 from herpes simplex virus has a central role in regulating the gene expression cascade which controls viral infection. Here we present the crystal structure of the functionally essential ICP4 DNA binding domain in complex with a segment from its own promoter, revealing a novel homo-dimeric fold. We also studied the complex in solution by small angle X-Ray scattering, nuclear magnetic resonance and surface-plasmon resonance which indicated that, in addition to the globular domain, a flanking intrinsically disordered region also recognizes DNA. Together the data provides a rationale for the bi-partite nature of the ICP4 DNA recognition consensus sequence as the globular and disordered regions bind synergistically to adjacent DNA motifs. Therefore in common with its eukaryotic host, the viral transcription factor ICP4 utilizes disordered regions to enhance the affinity and tune the specificity of DNA interactions in tandem with a globular domain. PMID:28505309

Studying DNA Looping by Single-Molecule FRET

OpenAIRE

Le, Tung T.; Kim, Harold D.

2014-01-01

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can als...

Processing of radiation-induced clustered DNA damage generates DSB in mammalian cells

International Nuclear Information System (INIS)

Gulston, M.K.; De Lara, C.M.; Davis, E.L.; Jenner, T.J.; O'Neill, P.

2003-01-01

Full text: Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cell. With 60 Co-radiation, the abundance of clustered DNA damage induced in CHO cells is ∼4x that of prompt double strand breaks (DSB) determined by PFGE. Less is known about the processing of non-DSB clustered DNA damage induced in cells. To optimize observation of any additional DSB formed during processing of DNA damage at 37 deg C, xrs-5 cells deficient in non-homologous end joining were used. Surprisingly, ∼30% of the DSB induced by irradiation at 37 deg C are rejoined within 4 minutes in both mutant and wild type cells. No significant mis-repair of these apparent DSB was observed. It is suggested that a class of non-DSB clustered DNA damage is formed which repair correctly within 4 min but, if 'trapped' prior to repair, are converted into DSB during the lysis procedure of PFGE. However at longer times, a proportion of non-DSB clustered DNA damage sites induced by γ-radiation are converted into DSB within ∼30 min following post-irradiation incubation at 37 deg C. The corresponding formation of additional DSB was not apparent in wild type CHO cells. From these observations, it is estimated that only ∼10% of the total yield of non DSB clustered DNA damage sites are converted into DSB through cellular processing. The biological consequences that the majority of non-DSB clustered DNA damage sites are not converted into DSBs may be significant even at low doses, since a finite chance exists of these clusters being formed in a cell by a single radiation track

Role of Chromatin assembly factor 1 in DNA replication of Plasmodium falciparum.

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Gupta, Mohit Kumar; Agarawal, Meetu; Banu, Khadija; Reddy, K Sony; Gaur, Deepak; Dhar, Suman Kumar

2018-01-01

Nucleosome assembly in P. falciparum could be the key process in maintaining its genomic integrity as DNA replicates more than once per cell cycle during several stages of its life cycle. Here, we report the functional characterization of P. falciparum chromatin assembly factor 1 (CAF1), which interacts with several proteins namely PfCAF2, Histones, PfHP1 and others. Consistent with the above findings, we demonstrate the presence of PfCAF1 at the telomeric repeat regions, central and subtelomeric var genes of multiple var gene family along with PfHP1. Further, we report the upregulation of PfCAF1 after treatment with genotoxic agents like MMS and HU. Together, these findings establish role of PfCAF1 in heterochromatin maintenance and as histone chaperone in nucleosome assembly and DNA damage repair. Copyright © 2017 Elsevier Inc. All rights reserved.

The positive transcription factor of the 5S RNA gene proteolyses during direct exchange between 5S DNA sites

OpenAIRE

1986-01-01

We have examined the association, dissociation, and exchange of the 5S specific transcription factor (TFIIIA) with somatic- and oocyte-type 5S DNA. The factor associates faster with somatic than with oocyte 5S DNA, and the rate of complex formation is accelerated by vector DNA. Once formed, the TFIIIA-5S DNA complex is stable for greater than 4 h in the absence of free 5S DNA, and its dissociation is identical for somatic and for oocyte 5S DNA. In the presence of free 5S DNA, the factor trans...

Model studies of radiation induced oxidation and reduction processes in DNA

International Nuclear Information System (INIS)

Hole, E.O.

1992-01-01

The papers presented in this thesis represent the major part of a systematic study of primary and secondary radiation induced damages in DNA. The magnetic resonance techniques EPR, ENDOR and FSE have been the experimental methods used. The study of radical formation in isolated DNA components under different environmental conditions demonstrates certain characteristics of the DNA components which are important in the study of DNA. It has been clearly demonstrated that the electrostatic environment, in particular the hydrogen bond pattern, is a vital factor for the secondary reaction scheme. Even radicals which are found in all related systems seem to be formed by different reaction pathways, depending upon the specific matrix. 92 refs., 2 figs., 6 tabs

Poxvirus uracil-DNA glycosylase-An unusual member of the family I uracil-DNA glycosylases: Poxvirus Uracil-DNA Glycosylase

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Schormann, Norbert [Department of Medicine, University of Alabama at Birmingham, Birmingham Alabama 35294; Zhukovskaya, Natalia [Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia Pennsylvania 19104; Bedwell, Gregory [Department of Microbiology, University of Alabama at Birmingham, Birmingham Alabama 35294; Nuth, Manunya [Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia Pennsylvania 19104; Gillilan, Richard [MacCHESS (Macromolecular Diffraction Facility at CHESS) Cornell University, Ithaca New York 14853; Prevelige, Peter E. [Department of Microbiology, University of Alabama at Birmingham, Birmingham Alabama 35294; Ricciardi, Robert P. [Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia Pennsylvania 19104; Abramson Cancer Center, School of Medicine, University of Pennsylvania, Philadelphia Pennsylvania 19104; Banerjee, Surajit [Department of Chemistry and Chemical Biology, Cornell University, and NE-CAT Argonne Illinois 60439; Chattopadhyay, Debasish [Department of Medicine, University of Alabama at Birmingham, Birmingham Alabama 35294

2016-11-02

We report that uracil-DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil-DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non-enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus-specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA-binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. In conclusion, the adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three-dimensional structure of D4. We provide an overview of the current state of the knowledge on the structure-function relationship of D4.

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.

A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity

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Rodríguez, Irene; Lázaro, José M.; Blanco, Luis; Kamtekar, Satwik; Berman, Andrea J.; Wang, Jimin; Steitz, Thomas A.; Salas, Margarita; de Vega, Miguel

2005-01-01

Recent crystallographic studies of φ29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a φ29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of φ29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity. PMID:15845765

Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

Science.gov (United States)

Liu, Jun; Lai, Ting; Mu, Kejie; Zhou, Zheng

2014-10-07

We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-β). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-β antibody to the target protein. Polyclonal anti-NGF-β antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-β, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-β. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

Modeling DNA structure and processes through animation and kinesthetic visualizations

Science.gov (United States)

Hager, Christine

There have been many studies regarding the effectiveness of visual aids that go beyond that of static illustrations. Many of these have been concentrated on the effectiveness of visual aids such as animations and models or even non-traditional visual aid activities like role-playing activities. This study focuses on the effectiveness of three different types of visual aids: models, animation, and a role-playing activity. Students used a modeling kit made of Styrofoam balls and toothpicks to construct nucleotides and then bond nucleotides together to form DNA. Next, students created their own animation to depict the processes of DNA replication, transcription, and translation. Finally, students worked in teams to build proteins while acting out the process of translation. Students were given a pre- and post-test that measured their knowledge and comprehension of the four topics mentioned above. Results show that there was a significant gain in the post-test scores when compared to the pre-test scores. This indicates that the incorporated visual aids were effective methods for teaching DNA structure and processes.

Epigenetic and genetic factors in the cellular response to radiations and DNA-damaging chemicals

International Nuclear Information System (INIS)

Williams, J.R.; D'Arpa, P.

1981-01-01

DNA-damaging agents are widely used as therapeutic tools for a variety of disease states. Many such agents are considered to produce detrimental side effects. Thus, it is important to evaluate both therapeutic efficacy and potential risk. DNA-damaging agents can be so evaluated by comparison to agents whose therapeutic benefit and potential hazards are better known. We propose a framework for such comparison, demonstrating that a simple transformation of cytotoxicity-dose response patterns permits a facile comparison of variation between cells exposed to a single DNA-damaging agent or to different cytotoxic agents. Further, by transforming data from experiments which compare responses of 2 cell populations to an effects ratio, different patterns for the changes in cytotoxicity produced by epigenetic and genetic factors were compared. Using these transformations, we found that there is a wide variation (a factor of 4) between laboratories for a single agent (UVC) and only a slightly larger variation (factor of 6) between normal cell response for different types of DNA-damaging agents (x-ray, UVC, alkylating agents, crosslinking agents). Epigenetic factors such as repair and recovery appear to be a factor only at higher dose levels. Comparison in the cytotoxic effect of a spectrum of DNA-damaging agents in xeroderma pigmentosum, ataxia telangiectasia, and Fanconi's anemia cells indicates significantly different patterns, implying that the effect, and perhaps the nature, of these genetic conditions are quite different

Studying DNA looping by single-molecule FRET.

Science.gov (United States)

Le, Tung T; Kim, Harold D

2014-06-28

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

The shear flow processing of controlled DNA tethering and stretching for organic molecular electronics.

Science.gov (United States)

Yu, Guihua; Kushwaha, Amit; Lee, Jungkyu K; Shaqfeh, Eric S G; Bao, Zhenan

2011-01-25

DNA has been recently explored as a powerful tool for developing molecular scaffolds for making reproducible and reliable metal contacts to single organic semiconducting molecules. A critical step in the process of exploiting DNA-organic molecule-DNA (DOD) array structures is the controlled tethering and stretching of DNA molecules. Here we report the development of reproducible surface chemistry for tethering DNA molecules at tunable density and demonstrate shear flow processing as a rationally controlled approach for stretching/aligning DNA molecules of various lengths. Through enzymatic cleavage of λ-phage DNA to yield a series of DNA chains of various lengths from 17.3 μm down to 4.2 μm, we have investigated the flow/extension behavior of these tethered DNA molecules under different flow strengths in the flow-gradient plane. We compared Brownian dynamic simulations for the flow dynamics of tethered λ-DNA in shear, and found our flow-gradient plane experimental results matched well with our bead-spring simulations. The shear flow processing demonstrated in our studies represents a controllable approach for tethering and stretching DNA molecules of various lengths. Together with further metallization of DNA chains within DOD structures, this bottom-up approach can potentially enable efficient and reliable fabrication of large-scale nanoelectronic devices based on single organic molecules, therefore opening opportunities in both fundamental understanding of charge transport at the single molecular level and many exciting applications for ever-shrinking molecular circuits.

Colloidal Au-enhanced surface plasmon resonance imaging: application in a DNA hybridization process

International Nuclear Information System (INIS)

Manera, M G; Spadavecchia, J; Taurino, A; Rella, R

2010-01-01

The detection of the DNA hybridization mechanism using monodispersed gold nanoparticles as labels is an interesting alternative to increase the sensitivity of the SPR imaging technique. DNA-modified Au nanoparticles (DNA-Au NPs) containing single-stranded (ss) portions of DNA were prepared by monitoring their monolayer formation by UV–vis spectroscopy. The hybridization process between specific thio-oligonucleotides immobilized on the DNA–Au NPs and the corresponding complementary strands is reported and compared with the traditional hybridization process on properly self-assembled thin gold films deposited on glass substrates. A remarkable signal amplification is observed, following the incorporation of colloidal Au into a SPR biosensing experiment, resulting in an increased SPR response to DNA–DNA interactions. In particular Fusarium thiolated DNA (5'HS poly(T) 15 ATC CCT CAA AAA CTG CCG CT-3) and trichothecenes complementary DNA (5'-AGC GGC AGT TTT TGA GGG AT-3') sequences have been explored due to their possible application to agro-industry for the control of food quality

Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

Science.gov (United States)

Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

2012-03-13

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

Science.gov (United States)

Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

2012-01-01

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

A DNA-binding-site landscape and regulatory network analysis for NAC transcription factors in Arabidopsis thaliana

DEFF Research Database (Denmark)

Lindemose, Søren; Jensen, Michael Krogh; de Velde, Jan Van

2014-01-01

regulatory networks of 12 NAC transcription factors. Our data offer specific single-base resolution fingerprints for most TFs studied and indicate that NAC DNA-binding specificities might be predicted from their DNA-binding domain's sequence. The developed methodology, including the application......Target gene identification for transcription factors is a prerequisite for the systems wide understanding of organismal behaviour. NAM-ATAF1/2-CUC2 (NAC) transcription factors are amongst the largest transcription factor families in plants, yet limited data exist from unbiased approaches to resolve...... the DNA-binding preferences of individual members. Here, we present a TF-target gene identification workflow based on the integration of novel protein binding microarray data with gene expression and multi-species promoter sequence conservation to identify the DNA-binding specificities and the gene...

Automatic DNA Diagnosis for 1D Gel Electrophoresis Images using Bio-image Processing Technique.

Science.gov (United States)

Intarapanich, Apichart; Kaewkamnerd, Saowaluck; Shaw, Philip J; Ukosakit, Kittipat; Tragoonrung, Somvong; Tongsima, Sissades

2015-01-01

DNA gel electrophoresis is a molecular biology technique for separating different sizes of DNA fragments. Applications of DNA gel electrophoresis include DNA fingerprinting (genetic diagnosis), size estimation of DNA, and DNA separation for Southern blotting. Accurate interpretation of DNA banding patterns from electrophoretic images can be laborious and error prone when a large number of bands are interrogated manually. Although many bio-imaging techniques have been proposed, none of them can fully automate the typing of DNA owing to the complexities of migration patterns typically obtained. We developed an image-processing tool that automatically calls genotypes from DNA gel electrophoresis images. The image processing workflow comprises three main steps: 1) lane segmentation, 2) extraction of DNA bands and 3) band genotyping classification. The tool was originally intended to facilitate large-scale genotyping analysis of sugarcane cultivars. We tested the proposed tool on 10 gel images (433 cultivars) obtained from polyacrylamide gel electrophoresis (PAGE) of PCR amplicons for detecting intron length polymorphisms (ILP) on one locus of the sugarcanes. These gel images demonstrated many challenges in automated lane/band segmentation in image processing including lane distortion, band deformity, high degree of noise in the background, and bands that are very close together (doublets). Using the proposed bio-imaging workflow, lanes and DNA bands contained within are properly segmented, even for adjacent bands with aberrant migration that cannot be separated by conventional techniques. The software, called GELect, automatically performs genotype calling on each lane by comparing with an all-banding reference, which was created by clustering the existing bands into the non-redundant set of reference bands. The automated genotype calling results were verified by independent manual typing by molecular biologists. This work presents an automated genotyping tool from DNA

Automatic DNA Diagnosis for 1D Gel Electrophoresis Images using Bio-image Processing Technique

Science.gov (United States)

2015-01-01

Background DNA gel electrophoresis is a molecular biology technique for separating different sizes of DNA fragments. Applications of DNA gel electrophoresis include DNA fingerprinting (genetic diagnosis), size estimation of DNA, and DNA separation for Southern blotting. Accurate interpretation of DNA banding patterns from electrophoretic images can be laborious and error prone when a large number of bands are interrogated manually. Although many bio-imaging techniques have been proposed, none of them can fully automate the typing of DNA owing to the complexities of migration patterns typically obtained. Results We developed an image-processing tool that automatically calls genotypes from DNA gel electrophoresis images. The image processing workflow comprises three main steps: 1) lane segmentation, 2) extraction of DNA bands and 3) band genotyping classification. The tool was originally intended to facilitate large-scale genotyping analysis of sugarcane cultivars. We tested the proposed tool on 10 gel images (433 cultivars) obtained from polyacrylamide gel electrophoresis (PAGE) of PCR amplicons for detecting intron length polymorphisms (ILP) on one locus of the sugarcanes. These gel images demonstrated many challenges in automated lane/band segmentation in image processing including lane distortion, band deformity, high degree of noise in the background, and bands that are very close together (doublets). Using the proposed bio-imaging workflow, lanes and DNA bands contained within are properly segmented, even for adjacent bands with aberrant migration that cannot be separated by conventional techniques. The software, called GELect, automatically performs genotype calling on each lane by comparing with an all-banding reference, which was created by clustering the existing bands into the non-redundant set of reference bands. The automated genotype calling results were verified by independent manual typing by molecular biologists. Conclusions This work presents an

Multiple phosphorylation sites at the C-terminus regulate nuclear import of HCMV DNA polymerase processivity factor ppUL44

International Nuclear Information System (INIS)

Alvisi, Gualtiero; Marin, Oriano; Pari, Gregory; Mancini, Manuela; Avanzi, Simone; Loregian, Arianna; Jans, David A.; Ripalti, Alessandro

2011-01-01

The processivity factor of human cytomegalovirus DNA polymerase, phosphoprotein ppUL44, is essential for viral replication. During viral infection ppUL44 is phosphorylated by the viral kinase pUL97, but neither the target residues on ppUL44 nor the effect of phosphorylation on ppUL44's activity are known. We report here that ppUL44 is phosphorylated when transiently expressed in mammalian cells and coimmunoprecipitates with cellular kinases. Of three potential phosphorylation sites (S413, S415, S418) located upstream of ppUL44's nuclear localization signal (NLS) and one (T427) within the NLS itself, protein kinase CK2 (CK2) specifically phosphorylates S413, to trigger a cascade of phosphorylation of S418 and S415 by CK1 and CK2, respectively. Negative charge at the CK2/CK1 target serine residues facilitates optimal nuclear accumulation of ppUL44, whereas negative charge on T427, a potential cyclin-dependent 1 phosphorylation site, strongly decreases nuclear accumulation. Thus, nuclear transport of ppUL44 is finely tuned during viral infection through complex phosphorylation events.

Degradation of transgene DNA in genetically modified herbicide-tolerant rice during food processing.

Science.gov (United States)

Song, Shangxin; Zhou, Guanghong; Gao, Feng; Zhang, Wei; Qiu, Liangyan; Dai, Sifa; Xu, Xinglian; Xiao, Hongmei

2011-12-01

In order to assess the effect of food processing on the degradation of exogenous DNA components in sweet rice wine and rice crackers made from genetically modified (GM) rice (Oryza sativa L.), we developed genomic DNA extraction methods and compared the effect of different food processing procedures on DNA degradation. It was found that the purity, quantity and quality of DNA by alkaline lysis method were higher than by CTAB (cetyltrimethylammonium bromide) method. For sweet rice wine, CAMV35S (cauliflower mosaic virus 35S) promoter and NOS (nopaline synthase) terminator were degraded by the third day, whereas the exogenous gene Bar (bialaphos resistance) remained unaffected. For rice crackers, boiling, drying and microwaving contributed to the initial degradations of DNA. Baking resulted in further degradations, and frying led to the most severe changes. These results indicated that the stability of DNA in GM rice was different under different processing conditions. For sweet rice wine, Bar was most stable, followed by NOS, CAMV35S, and SPS. For rice crackers, CAMV35S was most stable, followed by SPS, NOS, and Bar. Copyright © 2011 Elsevier Ltd. All rights reserved.

DNA methylation in obesity

Directory of Open Access Journals (Sweden)

Małgorzata Pokrywka

2014-11-01

Full Text Available The number of overweight and obese people is increasing at an alarming rate, especially in the developed and developing countries. Obesity is a major risk factor for diabetes, cardiovascular disease, and cancer, and in consequence for premature death. The development of obesity results from the interplay of both genetic and environmental factors, which include sedentary life style and abnormal eating habits. In the past few years a number of events accompanying obesity, affecting expression of genes which are not directly connected with the DNA base sequence (e.g. epigenetic changes, have been described. Epigenetic processes include DNA methylation, histone modifications such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, as well as non-coding micro-RNA (miRNA synthesis. In this review, the known changes in the profile of DNA methylation as a factor affecting obesity and its complications are described.

Dietary restriction delays the secretion of senescence associated secretory phenotype by reducing DNA damage response in the process of renal aging.

Science.gov (United States)

Wang, Wenjuan; Cai, Guangyan; Chen, Xiangmei

2017-09-13

Dietary restriction (DR) has multiple and essential effects in protecting against DNA damage in model organisms. Persistent DNA damage plays a central role in the process of aging. Senescence-associated secretory phenotype (SASP), as a product of cellular aging, can accelerate the process of cellular senescence as a feedback. In this study, we directly observed whether a DR of 30% for 6months in aged rats could retard SASP by delaying the progression of DNA damage and also found the specific mechanism. The results revealed that a 30% DR could significantly improve renal pathology and some metabolic characteristics. The biomarkers and products of DNA damage were decreased in the process of renal aging on a 30% DR. A series of SASP, notably cytokine, chemokine, and growth factor, were obviously reduced by DR during renal aging. The phosphorylation levels of NF-κB and IκBα in aged kidneys of DR group were markedly reduced. These findings suggest that a 30% DR for 6months can delay renal aging and reduce the accumulation of SASP by retarding the progression of DNA damage and decreasing the transcription activity of NF-κB, thus providing a target to delay renal aging. Copyright © 2017 Elsevier Inc. All rights reserved.

Factors influencing detection of eDNA from a stream-dwelling amphibian

Science.gov (United States)

Pilliod, David S.; Goldberg, Caren S.; Arkle, Robert S.; Waits, Lisette P.

2013-01-01

Environmental DNA (eDNA) methods for detecting and estimating abundance of aquatic species are emerging rapidly, but little is known about how processes such as secretion rate, environmental degradation, and time since colonization or extirpation from a given site affect eDNA measurements. Using stream-dwelling salamanders and quantitative PCR (qPCR) analysis, we conducted three experiments to assess eDNA: (i) production rate; (ii) persistence time under different temperature and light conditions; and (iii) detectability and concentration through time following experimental introduction and removal of salamanders into previously unoccupied streams. We found that 44–50 g individuals held in aquaria produced 77 ng eDNA/h for 2 h, after which production either slowed considerably or began to equilibrate with degradation. eDNA in both full-sun and shaded treatments degraded exponentially to 2) and when samples were collected within 5 m of the animals. Concentrations of eDNA detected were very low and increased steadily from 6–24 h after introduction, reaching 0.0022 ng/L. Within 1 h of removing salamanders from the stream, eDNA was no longer detectable. These results suggest that eDNA detectability and concentration depend on production rates of individuals, environmental conditions, density of animals, and their residence time.

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

Science.gov (United States)

Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

2003-03-01

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Automated Processing of 2-D Gel Electrophoretograms of Genomic DNA for Hunting Pathogenic DNA Molecular Changes.

Science.gov (United States)

Takahashi; Nakazawa; Watanabe; Konagaya

1999-01-01

We have developed the automated processing algorithms for 2-dimensional (2-D) electrophoretograms of genomic DNA based on RLGS (Restriction Landmark Genomic Scanning) method, which scans the restriction enzyme recognition sites as the landmark and maps them onto a 2-D electrophoresis gel. Our powerful processing algorithms realize the automated spot recognition from RLGS electrophoretograms and the automated comparison of a huge number of such images. In the final stage of the automated processing, a master spot pattern, on which all the spots in the RLGS images are mapped at once, can be obtained. The spot pattern variations which seemed to be specific to the pathogenic DNA molecular changes can be easily detected by simply looking over the master spot pattern. When we applied our algorithms to the analysis of 33 RLGS images derived from human colon tissues, we successfully detected several colon tumor specific spot pattern changes.

Real sequence effects on the search dynamics of transcription factors on DNA

DEFF Research Database (Denmark)

Bauer, Maximilian; Rasmussen, Emil S.; Lomholt, Michael A.

2015-01-01

Recent experiments show that transcription factors (TFs) indeed use the facilitated diffusion mechanism to locate their target sequences on DNA in living bacteria cells: TFs alternate between sliding motion along DNA and relocation events through the cytoplasm. From simulations and theoretical...... analysis we study the TF-sliding motion for a large section of the DNA-sequence of a common E. coli strain, based on the two-state TF-model with a fast-sliding search state and a recognition state enabling target detection. For the probability to detect the target before dissociating from DNA the TF...... on the underlying nucleotide sequence is varied. A moderate dependence maximises the capability to distinguish between the main operator and similar sequences. Moreover, these auxiliary operators serve as starting points for DNA looping with the main operator, yielding a spectrum of target detection times spanning...

Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms

Directory of Open Access Journals (Sweden)

Olga A. Kladova

2018-03-01

Full Text Available Endonuclease III (Endo III or Nth is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tCO, a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5–37 °C. Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van’t Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU recognition and desolvation-accompanied entropy-driven adjustment of the enzyme–substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme–DNA complex formation.

A ChIP-chip approach reveals a novel role for transcription factor IRF1 in the DNA damage response.

Science.gov (United States)

Frontini, Mattia; Vijayakumar, Meeraa; Garvin, Alexander; Clarke, Nicole

2009-03-01

IRF1 is a transcription factor that regulates key processes in the immune system and in tumour suppression. To gain further insight into IRF1's role in these processes, we searched for new target genes by performing chromatin immunoprecipitation coupled to a CpG island microarray (ChIP-chip). Using this approach we identified 202 new IRF1-binding sites with high confidence. Functional categorization of the target genes revealed a surprising cadre of new roles that can be linked to IRF1. One of the major functional categories was the DNA damage response pathway. In order to further validate our findings, we show that IRF1 can regulate the mRNA expression of a number of the DNA damage response genes in our list. In particular, we demonstrate that the mRNA and protein levels of the DNA repair protein BRIP1 [Fanconi anemia gene J (FANC J)] are upregulated after IRF1 over-expression. We also demonstrate that knockdown of IRF1 by siRNA results in loss of BRIP1 expression, abrogation of BRIP1 foci after DNA interstrand crosslink (ICL) damage and hypersensitivity to the DNA crosslinking agent, melphalan; a characteristic phenotype of FANC J cells. Taken together, our data provides a more complete understanding of the regulatory networks controlled by IRF1 and reveals a novel role for IRF1 in regulating the ICL DNA damage response.

A ChIP–chip approach reveals a novel role for transcription factor IRF1 in the DNA damage response

Science.gov (United States)

Frontini, Mattia; Vijayakumar, Meeraa; Garvin, Alexander; Clarke, Nicole

2009-01-01

IRF1 is a transcription factor that regulates key processes in the immune system and in tumour suppression. To gain further insight into IRF1's role in these processes, we searched for new target genes by performing chromatin immunoprecipitation coupled to a CpG island microarray (ChIP–chip). Using this approach we identified 202 new IRF1-binding sites with high confidence. Functional categorization of the target genes revealed a surprising cadre of new roles that can be linked to IRF1. One of the major functional categories was the DNA damage response pathway. In order to further validate our findings, we show that IRF1 can regulate the mRNA expression of a number of the DNA damage response genes in our list. In particular, we demonstrate that the mRNA and protein levels of the DNA repair protein BRIP1 [Fanconi anemia gene J (FANC J)] are upregulated after IRF1 over-expression. We also demonstrate that knockdown of IRF1 by siRNA results in loss of BRIP1 expression, abrogation of BRIP1 foci after DNA interstrand crosslink (ICL) damage and hypersensitivity to the DNA crosslinking agent, melphalan; a characteristic phenotype of FANC J cells. Taken together, our data provides a more complete understanding of the regulatory networks controlled by IRF1 and reveals a novel role for IRF1 in regulating the ICL DNA damage response. PMID:19129219

Cloning of the DNA-binding subunit of human nuclear factor κB: The level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor α

International Nuclear Information System (INIS)

Meyer, R.; Hatada, E.N.; Bartsch, C.; Scheidereit, C.; Hohmann, H.P.; Haiker, M.; Roethlisberger, U.; Lahm, H.W.; Schlaeger, E.J.; van Loon, A.P.G.M.

1991-01-01

The DNA binding subunit of nuclear factor κB (NF-κB), a B-cell protein that interacts with the immunoglobulin κ light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor α (TNFα), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-κB protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-κB binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNFα or phorbol ester. Thus, both factors not only activate NF-κB protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-κB

A biological inspired fuzzy adaptive window median filter (FAWMF) for enhancing DNA signal processing.

Science.gov (United States)

Ahmad, Muneer; Jung, Low Tan; Bhuiyan, Al-Amin

2017-10-01

Digital signal processing techniques commonly employ fixed length window filters to process the signal contents. DNA signals differ in characteristics from common digital signals since they carry nucleotides as contents. The nucleotides own genetic code context and fuzzy behaviors due to their special structure and order in DNA strand. Employing conventional fixed length window filters for DNA signal processing produce spectral leakage and hence results in signal noise. A biological context aware adaptive window filter is required to process the DNA signals. This paper introduces a biological inspired fuzzy adaptive window median filter (FAWMF) which computes the fuzzy membership strength of nucleotides in each slide of window and filters nucleotides based on median filtering with a combination of s-shaped and z-shaped filters. Since coding regions cause 3-base periodicity by an unbalanced nucleotides' distribution producing a relatively high bias for nucleotides' usage, such fundamental characteristic of nucleotides has been exploited in FAWMF to suppress the signal noise. Along with adaptive response of FAWMF, a strong correlation between median nucleotides and the Π shaped filter was observed which produced enhanced discrimination between coding and non-coding regions contrary to fixed length conventional window filters. The proposed FAWMF attains a significant enhancement in coding regions identification i.e. 40% to 125% as compared to other conventional window filters tested over more than 250 benchmarked and randomly taken DNA datasets of different organisms. This study proves that conventional fixed length window filters applied to DNA signals do not achieve significant results since the nucleotides carry genetic code context. The proposed FAWMF algorithm is adaptive and outperforms significantly to process DNA signal contents. The algorithm applied to variety of DNA datasets produced noteworthy discrimination between coding and non-coding regions contrary

Replication protein A (RPA hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

Directory of Open Access Journals (Sweden)

Artem G Lada

Full Text Available Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G, restricts retroviruses, and Activation Induced Deaminase (AID generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA, the eukaryotic single-stranded DNA (ssDNA binding protein, severely inhibits the deamination activity and processivity of A3G.We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast.Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

Science.gov (United States)

Lada, Artem G; Waisertreiger, Irina S-R; Grabow, Corinn E; Prakash, Aishwarya; Borgstahl, Gloria E O; Rogozin, Igor B; Pavlov, Youri I

2011-01-01

Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast. Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.

Directory of Open Access Journals (Sweden)

Yonghe Qi

2016-10-01

Full Text Available Hepatitis B virus (HBV infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP, followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK, a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.

Genomic signal processing methods for computation of alignment-free distances from DNA sequences.

Science.gov (United States)

Borrayo, Ernesto; Mendizabal-Ruiz, E Gerardo; Vélez-Pérez, Hugo; Romo-Vázquez, Rebeca; Mendizabal, Adriana P; Morales, J Alejandro

2014-01-01

Genomic signal processing (GSP) refers to the use of digital signal processing (DSP) tools for analyzing genomic data such as DNA sequences. A possible application of GSP that has not been fully explored is the computation of the distance between a pair of sequences. In this work we present GAFD, a novel GSP alignment-free distance computation method. We introduce a DNA sequence-to-signal mapping function based on the employment of doublet values, which increases the number of possible amplitude values for the generated signal. Additionally, we explore the use of three DSP distance metrics as descriptors for categorizing DNA signal fragments. Our results indicate the feasibility of employing GAFD for computing sequence distances and the use of descriptors for characterizing DNA fragments.

DNA Binding Drugs Targeting the Regulatory DNA Binding Site of the ETS Domain Family Transcription Factor Associated With Human Breast Cancer

National Research Council Canada - National Science Library

Wang, Yong-Dong

1999-01-01

.... The key approach is to prevent the binding of two transcription factors, ESX and AP-2, to the consensus DNA binding sites contained within the Her2/neu promoter resulting in inhibition of transcription factor function...

DNA topology and transcription

Science.gov (United States)

Kouzine, Fedor; Levens, David; Baranello, Laura

2014-01-01

Chromatin is a complex assembly that compacts DNA inside the nucleus while providing the necessary level of accessibility to regulatory factors conscripted by cellular signaling systems. In this superstructure, DNA is the subject of mechanical forces applied by variety of molecular motors. Rather than being a rigid stick, DNA possesses dynamic structural variability that could be harnessed during critical steps of genome functioning. The strong relationship between DNA structure and key genomic processes necessitates the study of physical constrains acting on the double helix. Here we provide insight into the source, dynamics, and biology of DNA topological domains in the eukaryotic cells and summarize their possible involvement in gene transcription. We emphasize recent studies that might inspire and impact future experiments on the involvement of DNA topology in cellular functions. PMID:24755522

PFGE analysis of DNA double-strand breaks and DNA repair process in human osteosarcoma cells irradiated by X-ray

International Nuclear Information System (INIS)

Cao Jianping; Majima, H.; Yamaguchi, C.

2000-01-01

Objective: To study the induction of DNA double-strand breaks (DSBs) in human osteosarcoma cells irradiated by X-ray, the DNA DSBs repair process and the tumour cell radiosensitivity. Methods: Two cell lines of human osteosarcoma, Rho0 and 143. B were used. Initial DNA damage of DSBs by X-ray irradiation was measured using clamped homogeneous electrical field (CHEF) electrophoresis. Results: X-ray-induced DNA DSBs of human osteosarcoma cells after CHEF-electrophoresis increased linearly with the irradiation dose between 0 and 50 Gy. The repair of DNA DSBs in human osteosarcoma cells increased with the post-irradiation incubation time. In contrast to 14.3B cell line at the same dose point, much more DNA DSBs were induced in Rho0 cell line after X-ray irradiation. Conclusion: CHEF pulsed-field gel electrophoresis (PEGE) is a sensitive method for the determination of radiation-induced DNA DSBs in high molecular weight DNA of human osteosarcoma cells. Radiation-induced DNA DSBs of osteosarcoma increase with the dose in a linear manner. After incubation, both Rho0 cell line and 143. B cell line can repair the DNA DSBs. Between two cell lines of human osteosarcoma, Rho0 and 143.B, Rho0 cell line is more sensitive to ionizing radiation than 143.B line

DNA scanning mechanism of T4 endonuclease V. Effect of NaCl concentration on processive nicking activity

International Nuclear Information System (INIS)

Gruskin, E.A.; Lloyd, R.S.

1986-01-01

T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism. A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers. The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction. Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl. The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining. Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased. At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism. The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM. The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction. In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed

DNA dynamics play a role as a basal transcription factor in the positioning and regulation of gene transcription initiation

OpenAIRE

Alexandrov, Boian S.; Gelev, Vladimir; Yoo, Sang Wook; Alexandrov, Ludmil B.; Fukuyo, Yayoi; Bishop, Alan R.; Rasmussen, Kim ?.; Usheva, Anny

2009-01-01

We assess the role of DNA breathing dynamics as a determinant of promoter strength and transcription start site (TSS) location. We compare DNA Langevin dynamic profiles of representative gene promoters, calculated with the extended non-linear PBD model of DNA with experimental data on transcription factor binding and transcriptional activity. Our results demonstrate that DNA dynamic activity at the TSS can be suppressed by mutations that do not affect basal transcription factor binding–DNA co...

Efficient representation of DNA data for pattern recognition using failure factor oracles

NARCIS (Netherlands)

Cleophas, Loek; Kourie, Derrick G.; Watson, Bruce W.

2013-01-01

In indexing of and pattern matching on DNA sequences, representing all factors of a sequence is important. One efficient, compact representation is the factor oracle (FO). At the same time, any classical deterministic finite automata (DFA) can be transformed to a so-called failure one (FDFA), which

Diversity and dynamics of the DNA- and cDNA-derived compost fungal communities throughout the commercial cultivation process for Agaricus bisporus.

Science.gov (United States)

McGee, C F; Byrne, H; Irvine, A; Wilson, J

2017-01-01

Commercial cultivation of the button mushroom Agaricus bisporus is performed through the inoculation of a semipasteurized composted material. Pasteurization of the compost material prior to inoculation results in a substrate with a fungal community that becomes dominated by A. bisporus. However, little is known about the composition and activity in the wider fungal community beyond the presence of A. bisporus in compost throughout the mushroom cropping process. In this study, the fungal cropping compost community was characterized by sequencing nuc rDNA ITS1-5.8S-ITS2 amplified from extractable DNA and RNA. The fungal community generated from DNA extracts identified a diverse community containing 211 unique species, although only 51 were identified from cDNA. Agaricus bisporus was found to dominate in the DNA-derived fungal community for the duration of the cropping process. However, analysis of cDNA extracts found A. bisporus to dominate only up to the first crop flush, after which activity decreased sharply and a much broader fungal community became active. This study has highlighted the diverse fungal community that is present in mushroom compost during cropping.

Validation of a DNA IQ-based extraction method for TECAN robotic liquid handling workstations for processing casework.

Science.gov (United States)

Frégeau, Chantal J; Lett, C Marc; Fourney, Ron M

2010-10-01

A semi-automated DNA extraction process for casework samples based on the Promega DNA IQ™ system was optimized and validated on TECAN Genesis 150/8 and Freedom EVO robotic liquid handling stations configured with fixed tips and a TECAN TE-Shake™ unit. The use of an orbital shaker during the extraction process promoted efficiency with respect to DNA capture, magnetic bead/DNA complex washes and DNA elution. Validation studies determined the reliability and limitations of this shaker-based process. Reproducibility with regards to DNA yields for the tested robotic workstations proved to be excellent and not significantly different than that offered by the manual phenol/chloroform extraction. DNA extraction of animal:human blood mixtures contaminated with soil demonstrated that a human profile was detectable even in the presence of abundant animal blood. For exhibits containing small amounts of biological material, concordance studies confirmed that DNA yields for this shaker-based extraction process are equivalent or greater to those observed with phenol/chloroform extraction as well as our original validated automated magnetic bead percolation-based extraction process. Our data further supports the increasing use of robotics for the processing of casework samples. Crown Copyright © 2009. Published by Elsevier Ireland Ltd. All rights reserved.

Tyrosyl-DNA Phosphodiesterase I a critical survival factor for neuronal development and homeostasis.

Science.gov (United States)

van Waardenburg, Robert C A M

2016-01-01

Tyrosyl-DNA phosphodiesterase I (TDP1), like most DNA repair associated proteins, is not essential for cell viability. However, dysfunctioning TDP1 or ATM (ataxia telangiectasia mutated) results in autosomal recessive neuropathology with similar phenotypes, including cerebellar atrophy. Dual inactivation of TDP1 and ATM causes synthetic lethality. A TDP1H 493 R catalytic mutant is associated with spinocerebellar ataxia with axonal neuropathy (SCAN1), and stabilizes the TDP1 catalytic obligatory enzyme-DNA covalent complex. The ATM kinase activates proteins early on in response to DNA damage. Tdp1-/- and Atm-/- mice exhibit accumulation of DNA topoisomerase I-DNA covalent complexes (TOPO1-cc) explicitly in neuronal tissue during development. TDP1 resolves 3'- and 5'-DNA adducts including trapped TOPO1-cc and TOPO1 protease resistant peptide-DNA complex. ATM appears to regulate the response to TOPO1-cc via a noncanonical function by regulating SUMO/ubiquitin-mediated TOPO1 degradation. In conclusion, TDP1 and ATM are critical factors for neuronal cell viability via two independent but cooperative pathways.

Application of the microbiological method DEFT/APC and DNA comet assay to detect ionizing radiation processing of minimally processed vegetables

International Nuclear Information System (INIS)

Araujo, Michel Mozeika

2008-01-01

Marketing of minimally processed vegetables (MPV) are gaining impetus due to its convenience, freshness and apparent healthy. However, minimal processing does not reduce pathogenic microorganisms to safe levels. Food irradiation is used to extend the shelf life and inactivation of food-borne pathogens, Its combination with minimal processing could improve the safety and quality of MPV. Two different food irradiation detection methods, a biological, the DEFT/APC, and another biochemical, the DNA Comet Assay were applied to MPV in order to test its applicability to detect irradiation treatment. DEFT/APC is a microbiological screening method based on the use of the direct epi fluorescent filter technique (DEFT) and the aerobic plate count (APC). DNA Comet Assay detects DNA damage due to ionizing radiation. Samples of lettuce, chard, watercress, dandelion, kale, chicory, spinach, cabbage from retail market were irradiated O.5 kGy and 1.0 kGy using a 60 Co facility. Irradiation treatment guaranteed at least 2 log cycle reduction for aerobic and psychotropic microorganisms. In general, with increasing radiation doses, DEFT counts remained similar independent of irradiation processing while APC counts decreased gradually. The difference of the two counts gradually increased with dose increment in all samples. It could be suggested that a DEFT/APC difference over 2.0 log would be a criteria to judge if a MPV was treated by irradiation. DNA Comet Assay allowed distinguishing non-irradiated samples from irradiated ones, which showed different types of comets owing to DNA fragmentation. Both DEFT/APC method and DNA Comet Assay would be satisfactorily used as a screening method for indicating irradiation processing. (author)

Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

DEFF Research Database (Denmark)

Beli, Petra; Lukashchuk, Natalia; Wagner, Sebastian A

2012-01-01

/ATR/DNA-PK target consensus motif, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes...

DNA fragmentation and cytotoxicity by recombinant human tumor necrosis factor in L929 fibroblast cells

International Nuclear Information System (INIS)

Kosaka, T.; Kuwabara, M.; Koide, F.

1992-01-01

Induction of cell DNA fragmentation by treatment of recombinant human Tumor Necrosis Factor alpha (rhTNF alpha) was examined by using mouse L929 cells derived from mouse fibroblast cells. The amount of DNA fragments derived from rhTNF alpha-treated cells, detected by alkaline elution technique, was smaller than that derived from X-irradiated cells. The rhTNF alpha caused the DNA fragmentation depending on its incubation time and concentration. The DNA damage caused by rhTNF alpha treatment correlated with its cytotoxicity. This result suggested that the DNA fragmentation is one of causes of cell death. The treatment with proteinase K of DNA obtained from rhTNF alpha-treated cells did not increase the amount of DNA fragmentation, which indicates that rhTNF alpha causes DNA-fragmentation but not DNA-protein cross-linking

The electrostatic role of the Zn-Cys2His2 complex in binding of operator DNA with transcription factors: mouse EGR-1 from the Cys2His2 family.

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Chirgadze, Y N; Boshkova, E A; Polozov, R V; Sivozhelezov, V S; Dzyabchenko, A V; Kuzminsky, M B; Stepanenko, V A; Ivanov, V V

2018-01-07

The mouse factor Zif268, known also as early growth response protein EGR-1, is a classical representative for the Cys2His2 transcription factor family. It is required for binding the RNA polymerase with operator dsDNA to initialize the transcription process. We have shown that only in this family of total six Zn-finger protein families the Zn complex plays a significant role in the protein-DNA binding. Electrostatic feature of this complex in the binding of factor Zif268 from Mus musculus with operator DNA has been considered. The factor consists of three similar Zn-finger units which bind with triplets of coding DNA. Essential contacts of the factor with the DNA phosphates are formed by three conservative His residues, one in each finger. We describe here the results of calculations of the electrostatic potentials for the Zn-Cys2His2 complex, Zn-finger unit 1, and the whole transcription factor. The potential of Zif268 has a positive area on the factor surface, and it corresponds exactly to the binding sites of each of Zn-finger units. The main part of these areas is determined by conservative His residues, which form contacts with the DNA phosphate groups. Our result shows that the electrostatic positive potential of this histidine residue is enhanced due to the Zn complex. The other contacts of the Zn-finger with DNA are related to nucleotide bases, and they are responsible for the sequence-specific binding with DNA. This result may be extended to all other members of the Cys2His2 transcription factor family.

Extra-chromosomal DNA maintenance in Bacillus subtilis, dependence on flagellation factor FliF and moonlighting mediator EdmS.

Science.gov (United States)

Hakumai, Yuichi; Shimomoto, Kouko; Ashiuchi, Makoto

2015-05-15

Extra-chromosomal DNA maintenance (EDM) as an important process in the propagation and genetic engineering of microbes. Bacillus subtilis EdmS (formerly PgsE), a protein comprising 55 amino acids, is a mediator of the EDM process. In this study, the effect of mutation of global regulators on B. subtilis EDM was examined. Mutation of the swrA gene abolished EdmS-mediated EDM. It is known that swrA predominantly regulates expression of the fla/che operon in B. subtilis. We therefore performed EDM analysis using fla/che-deletion mutants and identified an EDM-mediated EDM cooperator in the flgB-fliL region. Further genetic investigation identified the flagellation factor FliF is a crucial EDM cooperator. To our knowledge, this is the first observation of the moonlighting function of FliF in DNA maintenance. Copyright © 2015 Elsevier Inc. All rights reserved.

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

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Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

2016-02-01

Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and

Exploring translocation of proteins on DNA by NMR

International Nuclear Information System (INIS)

Marius Clore, G.

2011-01-01

While an extensive body of knowledge has accumulated on the structures of transcription factors, DNA and their complexes from both NMR and crystallography, much less is known at a molecular level regarding the mechanisms whereby transcription factors locate their specific DNA target site within an overwhelming sea of non-specific DNA sites. Indirect kinetic data suggested that three processes are involved in the search procedure: jumping by dissociation of the protein from the DNA followed by re-association at another site, direct transfer from one DNA molecule or segment to another, and one-dimensional sliding. In this brief perspective I summarize recent NMR developments from our laboratory that have permitted direct characterization of the species and molecular mechanisms involved in the target search process, including the detection of highly transient sparsely-populated states. The main tool in these studies involves the application of paramagnetic relaxation enhancement, supplemented by z-exchange spectroscopy, lineshape analysis and residual dipolar couplings. These studies led to the first direct demonstration of rotation-coupled sliding of a protein along the DNA and the direct transfer of a protein from one DNA molecule to another without dissociating into free solution.

Cocoa Consumption Alters the Global DNA Methylation of Peripheral Leukocytes in Humans with Cardiovascular Disease Risk Factors: A Randomized Controlled Trial.

Science.gov (United States)

Crescenti, Anna; Solà, Rosa; Valls, Rosa M; Caimari, Antoni; Del Bas, Josep M; Anguera, Anna; Anglés, Neus; Arola, Lluís

2013-01-01

DNA methylation regulates gene expression and can be modified by different bioactive compounds in foods, such as polyphenols. Cocoa is a rich source of polyphenols, but its role in DNA methylation is still unknown. The objective was to assess the effect of cocoa consumption on DNA methylation and to determine whether the enzymes involved in the DNA methylation process participate in the mechanisms by which cocoa exerts these effects in humans. The global DNA methylation levels in the peripheral blood were evaluated in 214 volunteers who were pre-hypertensive, stage-1 hypertensive or hypercholesterolemic. The volunteers were divided into two groups: 110 subjects who consumed cocoa (6 g/d) for two weeks and 104 control subjects. In addition, the peripheral blood mononuclear cells (PBMCs) from six subjects were treated with a cocoa extract to analyze the mRNA levels of the DNA methyltransferases (DNMTs), methylenetetrahydrofolate reductase (MTHFR), and methionine synthase reductase (MTRR) genes. Cocoa consumption significantly reduced the DNA methylation levels (2.991±0.366 vs. 3.909±0.380, pcocoa effects on DNA methylation and three polymorphisms located in the MTHFR, MTRR, and DNMT3B genes. Furthermore, in PBMCs, the cocoa extract significantly lowered the mRNA levels of the DNMTs, MTHFR, and MTRR. Our study demonstrates for the first time that the consumption of cocoa decreases the global DNA methylation of peripheral leukocytes in humans with cardiovascular risk factors. In vitro experiments with PBMCs suggest that cocoa may exert this effect partially via the down-regulation of DNMTs, MTHFR and MTRR, which are key genes involved in this epigenetic process. Clinicaltrials.govNCT00511420 and NCT00502047.

DNA replication machinery is required for development in Drosophila.

Science.gov (United States)

Kohzaki, Hidetsugu; Asano, Maki; Murakami, Yota

2018-01-01

 In Drosophila , some factors involved in chromosome replication seem to be involved in gene amplification and endoreplication, which are actively utilized in particular tissue development, but direct evidence has not been shown. Therefore, we examined the effect of depletion of replication factors on these processes. First, we confirmed RNAi knockdown can be used for the depletion of replication factors by comparing the phenotypes of RNAi knockdown and deletion or point mutants of the components of DNA licensing factor, MCM2, MCM4 and Cdt1. Next, we found that tissue-specific RNAi knockdown of replication factors caused tissue-specific defects, probably due to defects in DNA replication. In particular, we found that depletion inhibited gene amplification of the chorion gene in follicle cells and endoreplication in salivary glands, showing that chromosomal DNA replication factors are required for these processes. Finally, using RNAi, we screened the genes for chromosomal DNA replication that affected tissue development. Interestingly, wing specific knockdown of Mcm10 induced wing formation defects. These results suggest that some components of chromosomal replication machinery are directly involved in tissue development.

The Role of XPG in Processing (CAGn/(CTGn DNA Hairpins

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Hou Caixia

2011-03-01

Full Text Available Abstract Background During DNA replication or repair, disease-associated (CAGn/(CTGn expansion can result from formation of hairpin structures in the repeat tract of the newly synthesized or nicked DNA strand. Recent studies identified a nick-directed (CAGn/(CTGn hairpin repair (HPR system that removes (CAGn/(CTGn hairpins from human cells via endonucleolytic incisions. Because the process is highly similar to the mechanism by which XPG and XPF endonucleases remove bulky DNA lesions during nucleotide excision repair, we assessed the potential role of XPG in conducting (CAGn/(CTGn HPR. Results To determine if the XPG endonuclease is involved in (CAGn/(CTGn hairpin removal, two XPG-deficient cell lines (GM16024 and AG08802 were examined for their ability to process (CAGn/(CTGn hairpins in vitro. We demonstrated that the GM16024 cell line processes all hairpin substrates as efficiently as HeLa cells, and that the AG08802 cell line is partially defective in HPR. Analysis of repair intermediates revealed that nuclear extracts from both XPG-deficient lines remove CAG/CTG hairpins via incisions, but the incision products are distinct from those generated in HeLa extracts. We also show that purified recombinant XPG protein greatly stimulates HPR in XPG-deficient extracts by promoting an incision 5' to the hairpin. Conclusions Our results strongly suggest that 1 human cells possess multiple pathways to remove (CAGn/(CTGn hairpins located in newly synthesized (or nicked DNA strand; and 2 XPG, although not essential for (CAGn/(CTGn hairpin removal, stimulates HPR by facilitating a 5' incision to the hairpin. This study reveals a novel role for XPG in genome-maintenance and implicates XPG in diseases caused by trinucleotide repeat expansion.

Chromosomal Replication Complexity: A Novel DNA Metrics and Genome Instability Factor.

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Andrei Kuzminov

2016-10-01

Full Text Available As the ratio of the copy number of the most replicated to the unreplicated regions in the same chromosome, the definition of chromosomal replication complexity (CRC appears to leave little room for variation, being either two during S-phase or one otherwise. However, bacteria dividing faster than they replicate their chromosome spike CRC to four and even eight. A recent experimental inquiry about the limits of CRC in Escherichia coli revealed two major reasons to avoid elevating it further: (i increased chromosomal fragmentation and (ii complications with subsequent double-strand break repair. Remarkably, examples of stable elevated CRC in eukaryotic chromosomes are well known under various terms like "differential replication," "underreplication," "DNA puffs," "onion-skin replication," or "re-replication" and highlight the phenomenon of static replication fork (sRF. To accurately describe the resulting "amplification by overinitiation," I propose a new term: "replification" (subchromosomal overreplication. In both prokaryotes and eukaryotes, replification, via sRF processing, causes double-strand DNA breaks and, with their repair elevating chromosomal rearrangements, represents a novel genome instability factor. I suggest how static replication bubbles could be stabilized and speculate that some tandem duplications represent such persistent static bubbles. Moreover, I propose how static replication bubbles could be transformed into tandem duplications, double minutes, or inverted triplications. Possible experimental tests of these models are discussed.

pH modulates the binding of early growth response protein 1 transcription factor to DNA.

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Mikles, David C; Bhat, Vikas; Schuchardt, Brett J; Deegan, Brian J; Seldeen, Kenneth L; McDonald, Caleb B; Farooq, Amjad

2013-08-01

The transcription factor early growth response protein (EGR)1 orchestrates a plethora of signaling cascades involved in cellular homeostasis, and its downregulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with an increase in pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as His382 by virtue of the fact that its replacement by nonionizable residues abolishes the pH dependence of the binding of EGR1 to DNA. Notably, His382 inserts into the major groove of DNA, and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, His382 is mainly conserved across other members of the EGR family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating the protein-DNA interactions that are central to this family of transcription factors. Collectively, our findings reveal an unexpected but a key step in the molecular recognition of the EGR family of transcription factors, and suggest that they may act as sensors of pH within the intracellular environment. © 2013 FEBS.

Defective processing of methylated single-stranded DNA by E. coli alkB mutants

Science.gov (United States)

Dinglay, Suneet; Trewick, Sarah C.; Lindahl, Tomas; Sedgwick, Barbara

2000-01-01

Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells. PMID:10950872

Diversity, expansion, and evolutionary novelty of plant DNA-binding transcription factor families.

Science.gov (United States)

Lehti-Shiu, Melissa D; Panchy, Nicholas; Wang, Peipei; Uygun, Sahra; Shiu, Shin-Han

2017-01-01

Plant transcription factors (TFs) that interact with specific sequences via DNA-binding domains are crucial for regulating transcriptional initiation and are fundamental to plant development and environmental response. In addition, expansion of TF families has allowed functional divergence of duplicate copies, which has contributed to novel, and in some cases adaptive, traits in plants. Thus, TFs are central to the generation of the diverse plant species that we see today. Major plant agronomic traits, including those relevant to domestication, have also frequently arisen through changes in TF coding sequence or expression patterns. Here our goal is to provide an overview of plant TF evolution by first comparing the diversity of DNA-binding domains and the sizes of these domain families in plants and other eukaryotes. Because TFs are among the most highly expanded gene families in plants, the birth and death process of TFs as well as the mechanisms contributing to their retention are discussed. We also provide recent examples of how TFs have contributed to novel traits that are important in plant evolution and in agriculture.This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer. Copyright © 2016 Elsevier B.V. All rights reserved.

Cocoa Consumption Alters the Global DNA Methylation of Peripheral Leukocytes in Humans with Cardiovascular Disease Risk Factors: A Randomized Controlled Trial.

Directory of Open Access Journals (Sweden)

Anna Crescenti

Full Text Available DNA methylation regulates gene expression and can be modified by different bioactive compounds in foods, such as polyphenols. Cocoa is a rich source of polyphenols, but its role in DNA methylation is still unknown. The objective was to assess the effect of cocoa consumption on DNA methylation and to determine whether the enzymes involved in the DNA methylation process participate in the mechanisms by which cocoa exerts these effects in humans. The global DNA methylation levels in the peripheral blood were evaluated in 214 volunteers who were pre-hypertensive, stage-1 hypertensive or hypercholesterolemic. The volunteers were divided into two groups: 110 subjects who consumed cocoa (6 g/d for two weeks and 104 control subjects. In addition, the peripheral blood mononuclear cells (PBMCs from six subjects were treated with a cocoa extract to analyze the mRNA levels of the DNA methyltransferases (DNMTs, methylenetetrahydrofolate reductase (MTHFR, and methionine synthase reductase (MTRR genes. Cocoa consumption significantly reduced the DNA methylation levels (2.991±0.366 vs. 3.909±0.380, p<0.001. Additionally, we found an association between the cocoa effects on DNA methylation and three polymorphisms located in the MTHFR, MTRR, and DNMT3B genes. Furthermore, in PBMCs, the cocoa extract significantly lowered the mRNA levels of the DNMTs, MTHFR, and MTRR. Our study demonstrates for the first time that the consumption of cocoa decreases the global DNA methylation of peripheral leukocytes in humans with cardiovascular risk factors. In vitro experiments with PBMCs suggest that cocoa may exert this effect partially via the down-regulation of DNMTs, MTHFR and MTRR, which are key genes involved in this epigenetic process.Clinicaltrials.govNCT00511420 and NCT00502047.

Two DNA polymerase sliding clamps from the thermophilic archaeon Sulfolobus solfataricus.

Science.gov (United States)

De Felice, M; Sensen, C W; Charlebois, R L; Rossi, M; Pisani, F M

1999-08-06

Herein, we report the identification and characterization of two DNA polymerase processivity factors from the thermoacidophilic archaeon Sulfolobus solfataricus. They, referred to as 039p (244 amino acid residues, 27 kDa) and 048p (249 amino acid residues, 27 kDa), present significant primary structure similarity to eukaryotic proliferating cell nuclear antigen (PCNA). We demonstrate that both 039p and 048p form oligomers in solution and are able to substantially activate the synthetic activity of the single-subunit family B DNA polymerase from S. solfataricus (Sso DNA pol B1) on poly(dA)-oligo(dT) as a primer-template. This stimulatory effect is the result of enhanced DNA polymerase processivity, as indicated by the analysis of the elongation products on polyacrylamide gels. Activation of Sso DNA pol B1 synthetic activity was also observed on linear primed single-stranded M13 mp18 DNA as a template. By immunoblot analysis using specific rabbit antisera, 039p and 048p were both detected in the logarithmic and stationary phases of S. solfataricus growth curve. This is the first report of the identification and biochemical characterization of two distinct DNA polymerase processivity factors from the same organism. The significance of these findings for the understanding of the DNA replication process in Archaea is discussed. Copyright 1999 Academic Press.

Extracellular DNA metabolism in Haloferax volcanii

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Scott eChimileski

2014-02-01

Full Text Available Extracellular DNA is found in all environments and is a dynamic component of the micro-bial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA con-centrations measured in nature–a potential rich source of carbon, nitrogen and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA sources. Additionally, fluorescence microscopy experiments showed that labeled DNA colocalized with Haloferax volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in extracellular DNA processing at the cell surface, and deletion of Hvo_1477 created an H. volcanii strain deficient in its ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.

Structural modeling and DNA binding autoinhibition analysis of Ergp55, a critical transcription factor in prostate cancer.

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Shanti P Gangwar

Full Text Available BACKGROUND: The Ergp55 protein belongs to Ets family of transcription factor. The Ets proteins are highly conserved in their DNA binding domain and involved in various development processes and regulation of cancer metabolism. To study the structure and DNA binding autoinhibition mechanism of Ergp55 protein, we have produced full length and smaller polypeptides of Ergp55 protein in E. coli and characterized using various biophysical techniques. RESULTS: The Ergp55 polypeptides contain large amount of α-helix and random coil structures as measured by circular dichorism spectroscopy. The full length Ergp55 forms a flexible and elongated molecule as revealed by molecular modeling, dynamics simulation and structural prediction algorithms. The binding analyses of Ergp55 polypeptides with target DNA sequences of E74 and cfos promoters indicate that longer fragments of Ergp55 (beyond the Ets domain showed the evidence of auto-inhibition. This study also revealed the parts of Ergp55 protein that mediate auto-inhibition. SIGNIFICANCE: The current study will aid in designing the compounds that stabilize the inhibited form of Ergp55 and inhibit its binding to promoter DNA. It will contribute in the development of drugs targeting Ergp55 for the prostate cancer treatment.

Formation of double-strand breaks in DNA of γ-irradiated bacteria depending on the function of fast repair processes of DNA single-strand breaks

International Nuclear Information System (INIS)

Petrov, S.I.; Gaziev, A.I.

1980-01-01

The formation of double-strand breaks in DNA of γ-irradiated ( 60 Co)Ex coli bacteria depending on the function of fast repair processes of DNA single-strand breaks, is investigated. The profiles of sedimentation of DNA Ex coli cells, irradiated at 0-2 deg C in the salt medium and in EDTA-borate buffer, are presented. It is shown that when irradiating cells in EDTA-borate buffer, the output of single- and double strand breaks in DNA is much higher than in the case of their irradiation in the minimum salt medium. The dependence of output of single-strand and double-strand breaks depending on the radiatier doze of E coli cells in the salt medium and EDTA-borate buffer, is studied. The supposition is made on the presence of a regulative interaction between the accumulation of DNA single-breaks and their repair with the formation of double-strand breaks. The functionating of fast and superfast repair processes considerably affects the formation of double-strand breaks in DNA of a bacterium cell. A considerable amount of double-breaks registered immediately after irradiation forms due to a close position of single-strand breaks on the opposite DNA strands

Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.

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Zach Hensel

Full Text Available DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences O(R and O(L (separated by 2.3 kb, mediated by the λ repressor CI (accession number P03034, play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.

DNA sequence+shape kernel enables alignment-free modeling of transcription factor binding.

Science.gov (United States)

Ma, Wenxiu; Yang, Lin; Rohs, Remo; Noble, William Stafford

2017-10-01

Transcription factors (TFs) bind to specific DNA sequence motifs. Several lines of evidence suggest that TF-DNA binding is mediated in part by properties of the local DNA shape: the width of the minor groove, the relative orientations of adjacent base pairs, etc. Several methods have been developed to jointly account for DNA sequence and shape properties in predicting TF binding affinity. However, a limitation of these methods is that they typically require a training set of aligned TF binding sites. We describe a sequence + shape kernel that leverages DNA sequence and shape information to better understand protein-DNA binding preference and affinity. This kernel extends an existing class of k-mer based sequence kernels, based on the recently described di-mismatch kernel. Using three in vitro benchmark datasets, derived from universal protein binding microarrays (uPBMs), genomic context PBMs (gcPBMs) and SELEX-seq data, we demonstrate that incorporating DNA shape information improves our ability to predict protein-DNA binding affinity. In particular, we observe that (i) the k-spectrum + shape model performs better than the classical k-spectrum kernel, particularly for small k values; (ii) the di-mismatch kernel performs better than the k-mer kernel, for larger k; and (iii) the di-mismatch + shape kernel performs better than the di-mismatch kernel for intermediate k values. The software is available at https://bitbucket.org/wenxiu/sequence-shape.git. rohs@usc.edu or william-noble@uw.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

DNA damage in neurodegenerative diseases

Energy Technology Data Exchange (ETDEWEB)

Coppedè, Fabio, E-mail: fabio.coppede@med.unipi.it; Migliore, Lucia, E-mail: lucia.migliore@med.unipi.it

2015-06-15

Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

DNA damage in neurodegenerative diseases

International Nuclear Information System (INIS)

Coppedè, Fabio; Migliore, Lucia

2015-01-01

Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean.

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Yuguang Song

Full Text Available Epigenetic modification contributes to the regulation of gene expression and plant development under salinity stress. Here we describe the identification of 49 soybean transcription factors by microarray analysis as being inducible by salinity stress. A semi-quantitative RT-PCR-based expression assay confirmed the salinity stress inducibility of 45 of these 49 transcription factors, and showed that ten of them were up-regulated when seedlings were exposed to the demethylation agent 5-aza-2-deoxycytidine. Salinity stress was shown to affect the methylation status of four of these ten transcription factors (one MYB, one b-ZIP and two AP2/DREB family members using a combination of bisulfite sequencing and DNA methylation-sensitive DNA gel blot analysis. ChIP analysis indicated that the activation of three of the four DNA methylated transcription factors was correlated with an increased level of histone H3K4 trimethylation and H3K9 acetylation, and/or a reduced level of H3K9 demethylation in various parts of the promoter or coding regions. Our results suggest a critical role for some transcription factors' activation/repression by DNA methylation and/or histone modifications in soybean tolerance to salinity stress.

Top3 processes recombination intermediates and modulates checkpoint activity after DNA damage

DEFF Research Database (Denmark)

Mankouri, Hocine W; Hickson, Ian D

2006-01-01

Mutation of TOP3 in Saccharomyces cerevisiae causes poor growth, hyperrecombination, and a failure to fully activate DNA damage checkpoints in S phase. Here, we report that overexpression of a dominant-negative allele of TOP3, TOP3(Y356F), which lacks the catalytic (decatenation) activity of Top3......, the catalytic activity of Top3 is not required for DNA damage checkpoint activation, but it is required for normal S-phase progression after DNA damage. We also present evidence that the checkpoint-mediated cell cycle delay and persistence of X-shaped DNA molecules resulting from overexpression of TOP3(Y356F......) are downstream of Rad51 function. We propose that Top3 functions in S phase to both process homologous recombination intermediates and modulate checkpoint activity....

DNA repair and cancer

International Nuclear Information System (INIS)

Rathore, Shakuntla; Joshi, Pankaj Kumar; Gaur, Sudha

2012-01-01

DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecule that encode it's genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many one million individual molecular lesions per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions include potentially harmful mutation in cell's genome which affect the survival of it's daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. Inherited mutation that affect DNA repair genes are strongly associated with high cancer risks in humans. Hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutation in the DNA mismatch repair pathway. BRCA1, BRCA2 two famous mutation conferring a hugely increased risk of breast cancer on carrier, are both associated with a large number of DNA repair pathway, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side effect is that other non-cancerous but rapidly dividing cells such as stem cells in the bone marrow are also affected. Modern cancer treatment attempt to localize the DNA damage to cells and tissue only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). (author)

PRIMARILY RESULTS OF PHYTOPLANKTON DNA AND VARIATION TO ENVIRONMENTAL FACTORS IN DURRES`S BAY COASTAL WATERS (ALBANIA

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Laura Gjyli

2013-10-01

Full Text Available After isolation of phytoplankton DNA in coastal waters of Durres Bay, Albania, quantification and analysis of quality were investigated with spectrophotometric analysis. Analysis of UV absorption by the nucleotides provides a simple and accurate estimation of the concentration of nucleic acids in a sample. This method is however limited by the quantity of DNA and the purity of the preparation. Also biotic environment factors as Chlorophyll a and abiotic environment factors as temperature, salinity, pH, dissolved oxygen, turbidity, nitrate, phosphate were investigated to assess DNA quantities in different environment conditions. The Chlorophyll a was studied also to access the level of trophy. The sample stations were: Golem Beach (GB, Channel of Plepa (ChP, Hekurudha Beach (HB, Ex-Fuel Quay in Marine Durres Harbour (EFQ, Water Channel of Durres City (WChDC and Currila Beach (CB. Samples are taken in one meter depth from the water surface. Water samples were collected monthly from April to October 2011. The most abundant stations with phytoplankton DNA are Channel of Plepa and Water Channel of Durres City. This confirms that there are spills of fresh waters, sewage or agricultural water spills, often discharge in coastal waters. Referring Mutliple Regression Analysis and single regression analysis, the association between phytoplankton DNA and environment factors was strong (R2 = 0.75. Basing in single correlation and statistically significance (p-value ≤ 0.05, the enviroment factors that correlated to phytoplankton DNA were pH, salinity and phosphate; explaining thus the variation of total phytoplankton in Durres Bay coastal waters.

DPI-ELISA: a fast and versatile method to specify the binding of plant transcription factors to DNA in vitro

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Chaban Christina

2010-11-01

Full Text Available Abstract Background About 10% of all genes in eukaryote genomes are predicted to encode transcription factors. The specific binding of transcription factors to short DNA-motifs influences the expression of neighbouring genes. However, little is known about the DNA-protein interaction itself. To date there are only a few suitable methods to characterise DNA-protein-interactions, among which the EMSA is the method most frequently used in laboratories. Besides EMSA, several protocols describe the effective use of an ELISA-based transcription factor binding assay e.g. for the analysis of human NFκB binding to specific DNA sequences. Results We provide a unified protocol for this type of ELISA analysis, termed DNA-Protein-Interaction (DPI-ELISA. Qualitative analyses with His-epitope tagged plant transcription factors expressed in E. coli revealed that EMSA and DPI-ELISA result in comparable and reproducible data. The binding of AtbZIP63 to the C-box and AtWRKY11 to the W2-box could be reproduced and validated by both methods. We next examined the physical binding of the C-terminal DNA-binding domains of AtWRKY33, AtWRKY50 and AtWRKY75 to the W2-box. Although the DNA-binding domain is highly conserved among the WRKY proteins tested, the use of the DPI-ELISA discloses differences in W2-box binding properties between these proteins. In addition to these well-studied transcription factor families, we applied our protocol to AtBPC2, a member of the so far uncharacterised plant specific Basic Pentacysteine transcription factor family. We could demonstrate binding to GA/TC-dinucleotide repeat motifs by our DPI-ELISA protocol. Different buffers and reaction conditions were examined. Conclusions We successfully applied our DPI-ELISA protocol to investigate the DNA-binding specificities of three different classes of transcription factors from Arabidopsis thaliana. However, the analysis of the binding affinity of any DNA-binding protein to any given DNA

Isolation of a cDNA for a Growth Factor of Vascular Endothelial Cells from Human Lung Cancer Cells: Its Identity with Insulin‐like Growth Factor II

Science.gov (United States)

Hagiwara, Koichi; Kobayashi, Tatsuo; Tobita, Masato; Kikyo, Nobuaki; Yazaki, Yoshio

1995-01-01

We have found growth‐promoting activity for vascular endothelial cells in the conditioned medium of a human lung cancer cell line, T3M‐11. Purification and characterization of the growth‐promoting activity have been carried out using ammonium sulfate precipitation and gel‐exclusion chromatography. The activity migrated as a single peak just after ribonuclease. It did not bind to a heparin affinity column. These results suggest that the activity is not a heparin‐binding growth factor (including fibroblast growth factors) or a vascular endothelial growth factor. To identify the molecule exhibiting the growth‐promoting activity, a cDNA encoding the growth factor was isolated through functional expression cloning in COS‐1 cells from a cDNA library prepared from T3M‐11 cells. The nucleotide sequence encoded by the cDNA proved to be identical with that of insulin‐like growth factor II. PMID:7730145

Low-cost label-free electrical detection of artificial DNA nanostructures using solution-processed oxide thin-film transistors.

Science.gov (United States)

Kim, Si Joon; Jung, Joohye; Lee, Keun Woo; Yoon, Doo Hyun; Jung, Tae Soo; Dugasani, Sreekantha Reddy; Park, Sung Ha; Kim, Hyun Jae

2013-11-13

A high-sensitivity, label-free method for detecting deoxyribonucleic acid (DNA) using solution-processed oxide thin-film transistors (TFTs) was developed. Double-crossover (DX) DNA nanostructures with different concentrations of divalent Cu ion (Cu(2+)) were immobilized on an In-Ga-Zn-O (IGZO) back-channel surface, which changed the electrical performance of the IGZO TFTs. The detection mechanism of the IGZO TFT-based DNA biosensor is attributed to electron trapping and electrostatic interactions caused by negatively charged phosphate groups on the DNA backbone. Furthermore, Cu(2+) in DX DNA nanostructures generates a current path when a gate bias is applied. The direct effect on the electrical response implies that solution-processed IGZO TFTs could be used to realize low-cost and high-sensitivity DNA biosensors.

Age and metabolic risk factors associated with oxidatively damaged DNA in human peripheral blood mononuclear cells

DEFF Research Database (Denmark)

Løhr, Mille; Jensen, Annie; Eriksen, Louise

2015-01-01

Aging is associated with oxidative stress-generated damage to DNA and this could be related to metabolic disturbances. This study investigated the association between levels of oxidatively damaged DNA in peripheral blood mononuclear cells (PBMCs) and metabolic risk factors in 1,019 subjects, aged...... 18-93 years. DNA damage was analyzed as strand breaks by the comet assay and levels of formamidopyrimidine (FPG-) and human 8-oxoguanine DNA glycosylase 1 (hOGG1)-sensitive sites There was an association between age and levels of FPG-sensitive sites for women, but not for men. The same tendency......, cholesterol and glycosylated hemoglobin (HbA1c). In the group of men, there were significant positive associations between alcohol intake, HbA1c and FPG-sensitive sites in multivariate analysis. The levels of metabolic risk factors were positively associated with age, yet only few subjects fulfilled all...

The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

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Agarwal, Poonam; Miller, Kyle M

2016-10-01

DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

Compound Poisson Processes and Clustered Damage of Radiation Induced DNA Double Strand Breaks

International Nuclear Information System (INIS)

Gudowska-Nowak, E.; Ritter, S.; Taucher-Scholz, G.; Kraft, G.

2000-01-01

Recent experimental data have demonstrated that DNA damage induced by densely ionizing radiation in mammalian cells is distributed along the DNA molecule in the form of clusters. The principal constituent of DNA damage are double-strand breaks (DSB) which are formed when the breaks occur in both DNA strands and are directly opposite or separated by only a few base pairs. DSBs are believed to be most important lesions produced in chromosomes by radiation; interaction between DSBs can lead to cell killing, mutation or carcinogenesis. The paper discusses a model of clustered DSB formation viewed in terms of compound Poisson process along with the predictive essay of the formalism in application to experimental data. (author)

RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage.

Science.gov (United States)

Cristini, Agnese; Groh, Matthias; Kristiansen, Maiken S; Gromak, Natalia

2018-05-08

R-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

The effect of heat and radiation on the initiation and elongation processes of DNA synthesis

International Nuclear Information System (INIS)

Davies, R.C.; Bowden, G.T.; Cress, A.E.

1983-01-01

The pH step alkaline elution and alkaline sucrose gradient techniques were utilized to evaluate alterations in DNA replication (initiation and elongation) induced by heat and low dose X-irradiation in synchronized Chinese hamster ovary cells. The initiation and elongation processes of DNA synthesis were radioresistant at the G 1 /S boundary (4 hours after mitosis) while in mid S phase (9 hours after mitosis) DNA initiation and elongation were sensitive to X-irradiation. The initiation and elongation processes of DNA synthesis which were radiation resistant at the G 1 /S boundary could be inhibited by a hyperthermia treatment (43 0 C for 1 hour beginning at 4 hours after mitosis). The impairment of initiation in the heated cells was maintained through late S phase while that of elongation was reversible as judged by full recovery at 15 hours after mitosis. These data suggest that the known synergistic lethality of heat and radiation may be mediated by an impairment of initiation of DNA synthesis. (author)

Hypothetical physicochemical mechanisms of some intracellular processes: The hydrate hypothesis of mitosis and DNA replication

International Nuclear Information System (INIS)

Kadyshevich, E.A.; Ostrovskii, V.E.

2007-01-01

A DNA replication, mitosis, and binary fission hydrate hypothesis (MRH hypothesis) allowing non-trivial explanations for the physicochemical mechanisms of some intracellular processes is proposed. The hypothesis has a thermodynamic basis and is initiated by original experimental calorimetric and kinetic studies of the behavior of functional organic polymer and monomer substances in highly concentrated aqueous solutions. Experimental data demonstrating the occurrence of a short-range ordering in concentrated aqueous solutions of such substances are included. Hypothetical simple non-enzymatic unified mechanisms for the natural processes of DNA local unwinding preceding the start of duplication, DNA replication, formation and disappearance of the protein bonds between sister chromatids in the centromere region of eukaryotic DNA and in the centromere-like region of prokaryotic DNA, moving of daughter chromosomes apart to the opposite sides of cells in late anaphase, and formation of the nuclear envelopes in telophase and intracellular membranes between the newly formed nuclei in cytokinesis are formulated. The nature of a number of other intracellular phenomena is discussed

Cisplatin- and UV-damaged DNA lure the basal transcription factor TFIID/TBP.

NARCIS (Netherlands)

P. Vichi; F. Coin (Frédéric); J-P. Renaud (Jean-Paul); W. Vermeulen (Wim); J.H.J. Hoeijmakers (Jan); D. Moras; J-M. Egly (Jean-Marc)

1997-01-01

textabstractA connection between transcription and DNA repair was demonstrated previously through the characterization of TFIIH. Using filter binding as well as in vitro transcription challenge competition assays, we now show that the promoter recognition factor TATA box-binding protein (TBP)/TFIID

Epidermal growth factor stimulating reparation of γ-ray-induced single-strand breaks predominantly in untranscribed DNA of HeLa cells

International Nuclear Information System (INIS)

Igusheva, O.A.; Bil'din, V.N.; Zhestyanikov, V.D.

1994-01-01

Considerable evidence suggest that genomic DNA undergoes reparation unevenly because of different transcription activities of its particular sequence. It is highly probably that transcriptional factors are necessary for postion stages of excision reparation and for reparation of single-strand DNA breaks caused by ionizing radiation. There is evidence suggesting that DNA lesions inflicted by γ-radiation is preferentially initiated in transcribed rather than in untranscribed DNA species. This paper looks at the relationship between stimulatory effect of epidermal growth factor (EGF) on reparation of single-strand DNA breaks and reparation of the damage done to active and inert fragments of chromatin. The results show that EGF stimulates reparation of single-strand DNA breaks induced by γ-radiation more effectively in untranscribed than in transcribed DNA. 13 refs., 1 fig., 1 tab

Effect of DNA extraction in the Rosa canina L. identification under different processing temperature

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Jana Žiarovská

2017-01-01

Full Text Available Rosa canina, L. is widely used for medicinal purposes as well as in food industry where it is a valuable source, bioactive compounds and natural colorants. Actually, no specific method is recommended as suitable one for DNA extraction from rose hips. The aim of the study was to compare three commercial and three non-commercial methods to extract total genomic DNA from rose hips hyphanthium. Four methods are based on the precipitation in principle and two methods are based on resin-binding. Extracted DNA was proved for the effectivity in following PCR. In total, six different DNA isolations was performed for differently heat processes rose hips - fresh hyphanthium, 2-weeks frozen hyphanthium, dried hyphanthium (50 °C and boiled hyphanthium (100 °C. The amplification parameters of 500 bp chloroplast gene amplicon were evaluated. Obtained amounts of extracted DNA was very variable not only for every individual method used but for individual treatment of samples, too. In general, non-commercial method provided higher amount of extracted DNA, but the A260/280 ratio was lower. When regarding the processing treatment of the samples, high differences were found among the samples untreated by heat and those that were dried or boiled for three of the used extraction methods. All the samples were positive for amplification, but different amounts of amplified product were obtained. The comparison of data for concentrations of extracted DNA and concentrations of amplified product showed large differences when regarding the achieved purity of DNA in extraction.

Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles.

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Liu, Baoming; Panda, Debasis; Mendez-Rios, Jorge D; Ganesan, Sundar; Wyatt, Linda S; Moss, Bernard

2018-04-01

Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5. IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may

Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

Science.gov (United States)

Ballari, Rajashekhar V; Martin, Asha

2013-12-01

DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal. Copyright © 2013 Elsevier Ltd. All rights reserved.

Chromatin modifications and the DNA damage response to ionizing radiation

International Nuclear Information System (INIS)

Kumar, Rakesh; Horikoshi, Nobuo; Singh, Mayank; Gupta, Arun; Misra, Hari S.; Albuquerque, Kevin; Hunt, Clayton R.; Pandita, Tej K.

2013-01-01

In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response.

DNA damage mediated transcription arrest: Step back to go forward.

Science.gov (United States)

Mullenders, Leon

2015-12-01

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the central zinc-binding domain of the human Mcm10 DNA-replication factor

International Nuclear Information System (INIS)

Jung, Nam Young; Bae, Won Jin; Chang, Jeong Ho; Kim, Young Chang; Cho, Yunje

2008-01-01

Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2–7 complex and DNA polymerases. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. The initiation of eukaryotic DNA replication requires the tightly controlled assembly of a set of replication factors. Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2–7 complex and DNA polymerases. The central domain, which contains the CCCH zinc-binding motif, is most conserved within Mcm10 and binds to DNA and several proteins, including proliferative cell nuclear antigen. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. An X-ray diffraction data set was collected to a resolution of 2.6 Å on a synchrotron beamline. The crystals formed belonged to space group R3, with unit-cell parameters a = b = 99.5, c = 133.0 Å. According to Matthews coefficient calculations, the crystals were predicted to contain six MCM10 central domain molecules in the asymmetric unit

From structure to mechanism—understanding initiation of DNA replication

Science.gov (United States)

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L. Maximilian; Schneider, Sarah; Speck, Christian

2017-01-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2–7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. PMID:28717046

Repair of DNA DSB in higher eukaryotes

International Nuclear Information System (INIS)

Wang, H.; Perrault, A.R.; Takeda, Y.; Iliakis, G.

2003-01-01

Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a NHEJ apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4, and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK- dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. We studied the role of Ku and DNA-PKcs in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient error-free endjoining observed in such in-vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite that fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA endjoining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing endjoining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts sugggesting the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3' or 5' protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3' overhangs. We propose that the

Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

DEFF Research Database (Denmark)

Dufva, Martin; Petersen, Jesper; Poulsen, Lena

2009-01-01

DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to f...

Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC.

Directory of Open Access Journals (Sweden)

Elodie Caboux

Full Text Available The European Prospective Investigation into Cancer and nutrition (EPIC is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88% performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies.

Regulating DNA Self-assembly by DNA-Surface Interactions.

Science.gov (United States)

Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde

2017-12-14

DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends

DEFF Research Database (Denmark)

Weterings, Eric; Verkaik, Nicole S; Keijzers, Guido

2008-01-01

Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) an......Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA......-PK(CS)) and the XRCC4/ligase IV complex. Activation of the DNA-PK(CS) serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation...... was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PK(CS) autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis...

A DEMETER-like DNA demethylase governs tomato fruit ripening.

Science.gov (United States)

Liu, Ruie; How-Kit, Alexandre; Stammitti, Linda; Teyssier, Emeline; Rolin, Dominique; Mortain-Bertrand, Anne; Halle, Stefanie; Liu, Mingchun; Kong, Junhua; Wu, Chaoqun; Degraeve-Guibault, Charlotte; Chapman, Natalie H; Maucourt, Mickael; Hodgman, T Charlie; Tost, Jörg; Bouzayen, Mondher; Hong, Yiguo; Seymour, Graham B; Giovannoni, James J; Gallusci, Philippe

2015-08-25

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening- an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.

Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA

Energy Technology Data Exchange (ETDEWEB)

Suwa, Yoshiaki; Nakamura, Teruya; Toma, Sachiko; Ikemizu, Shinji; Kai, Hirofumi; Yamagata, Yuriko, E-mail: yamagata@gpo.kumamoto-u.ac.jp [Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973 (Japan)

2008-03-01

The complex between the Ets domain of Ets2 and its target DNA has been crystallized. The crystals diffracted to 3.0 Å resolution. The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down’s syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 Å, β = 101.7° and a V{sub M} value of 3.56 Å{sup 3} Da{sup −1}. Diffraction data were collected to a resolution of 3.0 Å.

Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA

International Nuclear Information System (INIS)

Suwa, Yoshiaki; Nakamura, Teruya; Toma, Sachiko; Ikemizu, Shinji; Kai, Hirofumi; Yamagata, Yuriko

2008-01-01

The complex between the Ets domain of Ets2 and its target DNA has been crystallized. The crystals diffracted to 3.0 Å resolution. The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down’s syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 Å, β = 101.7° and a V M value of 3.56 Å 3 Da −1 . Diffraction data were collected to a resolution of 3.0 Å

A novel electrochemical approach for nuclear factor kappa B detection based on triplex DNA and gold nanoparticles

International Nuclear Information System (INIS)

Shen Min; Yang Mei; Li Hao; Liang Zhiqiang; Li Genxi

2012-01-01

Highlights: ► A simple, selective, and sensitive electrochemical NF-κB sensor was presented. ► NF-κB was precisely qualified by chronocoulometry with a detection limit of 0.13 nM. ► NF-κB was also successfully detected in contaminated samples by our approach. - Abstract: The transcription factor nuclear factor kappa B (NF-κB) is always a standard for inducible transcription factors, while nearly all NF-κB studies require the measurement of the level of activated NF-κB in cells. Herein we report a novel electrochemical approach for accurate detection of NF-κB with the help of triplex DNA and gold nanoparticles (AuNPs). Firstly, double-stranded DNA (dsDNA) molecules are self-assembled on the surface of a gold electrode. Then, AuNPs are functionalized with triplex-forming oligonucleotide (TFO). Since TFO may act with the dsDNA to form triplex DNA, the TFO functionalized on the AuNPs surfaces will bind with the dsDNA immobilized on the electrode surface, bringing large amounts of electrochemical compounds [Ru(NH 3 ) 6 ] 3+ close to the electrode to generate an intense electrochemical signal. However, in the presence of NF-κB, the protein will capture and bind with the dsDNA to replace TFO–AuNPs, resulting in significant decrease of electrochemical signal of [Ru(NH 3 ) 6 ] 3+ . By using this “on-off” strategy, NF-κB has been quantified in the range from 0.4 to 12.0 nM, with a detection limit of 0.13 nM. This approach has also been successfully used to detect NF-κB in contaminated samples with high specificity.

Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates

Directory of Open Access Journals (Sweden)

Lavrik O. I.

2012-06-01

Full Text Available Aim. To investigate the TLS-activity of human DNA polymerases β and λ (pols β and λ using 5-formyluridine (5-foU containing DNA duplexes which are imitating the intermediates during replication of the leading DNA strand, and to study the influence of replication factors hRPA and hPCNA on this activity. Methods. The EMSA and the methods of enzyme’s kinetics were used. Results. The capability of pols β and λ to catalyze DNA synthesis across 5-foU was investigated and the kinetic characteristics of this process in the presence and in the absence of protein factors hRPA and hPCNA were evaluated. Conclusions. It was shown that: (i both proteins are able to catalyze TLS on used DNA substrates regardless of the reaction conditions, however, pol λ was more accurate enzyme; (ii hRPA can stimulate the efficacy of the nonmutagenic TLS catalyzed by pol at the nucleotide incorporation directly opposite of 5-foU, at the same time it doesn’t influence the incorporation efficacy if the damage displaced into the duplex; (iii hPCNA doesn’t influence the efficacy of TLS catalyzed by both enzymes.

DNA Processing and Reassembly on General Purpose FPGA-based Development Boards

Directory of Open Access Journals (Sweden)

SZÁSZ Csaba

2017-05-01

Full Text Available The great majority of researchers involved in microelectronics generally agree that many scientific challenges in life sciences have associated with them a powerful computational requirement that must be solved before scientific progress can be made. The current trend in Deoxyribonucleic Acid (DNA computing technologies is to develop special hardware platforms capable to provide the needed processing performance at lower cost. In this endeavor the FPGA-based (Field Programmable Gate Arrays configurations aimed to accelerate genome sequencing and reassembly plays a leading role. This paper emphasizes benefits and advantages using general purpose FPGA-based development boards in DNA reassembly applications beside the special hardware architecture solutions. An original approach is unfolded which outlines the versatility of high performance ready-to-use manufacturer development platforms endowed with powerful hardware resources fully optimized for high speed processing applications. The theoretical arguments are supported via an intuitive implementation example where the designer it is discharged from any hardware development effort and completely assisted in exclusive concentration only on software design issues providing greatly reduced application development cycles. The experiments prove that such boards available on the market are suitable to fulfill in all a wide range of DNA sequencing and reassembly applications.

Characterization of Bombyx mori mitochondrial transcription factor A, a conserved regulator of mitochondrial DNA.

Science.gov (United States)

Sumitani, Megumi; Kondo, Mari; Kasashima, Katsumi; Endo, Hitoshi; Nakamura, Kaoru; Misawa, Toshihiko; Tanaka, Hiromitsu; Sezutsu, Hideki

2017-04-15

In the present study, we initially cloned and characterized a mitochondrial transcription factor A (Tfam) homologue in the silkworm, Bombyx mori. Bombyx mori TFAM (BmTFAM) localized to mitochondria in cultured silkworm and human cells, and co-localized with mtDNA nucleoids in human HeLa cells. In an immunoprecipitation analysis, BmTFAM was found to associate with human mtDNA in mitochondria, indicating its feature as a non-specific DNA-binding protein. In spite of the low identity between BmTFAM and human TFAM (26.5%), the expression of BmTFAM rescued mtDNA copy number reductions and enlarged mtDNA nucleoids in HeLa cells, which were induced by human Tfam knockdown. Thus, BmTFAM compensates for the function of human TFAM in HeLa cells, demonstrating that the mitochondrial function of TFAM is highly conserved between silkworms and humans. BmTfam mRNA was strongly expressed in early embryos. Through double-stranded RNA (dsRNA)-based RNA interference (RNAi) in silkworm embryos, we found that the knockdown of BmTFAM reduced the amount of mtDNA and induced growth retardation at the larval stage. Collectively, these results demonstrate that BmTFAM is a highly conserved mtDNA regulator and may be a good candidate for investigating and modulating mtDNA metabolism in this model organism. Copyright © 2017 Elsevier B.V. All rights reserved.

Choosing and Using a Plant DNA Barcode

Science.gov (United States)

Hollingsworth, Peter M.; Graham, Sean W.; Little, Damon P.

2011-01-01

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance. PMID:21637336

Choosing and using a plant DNA barcode.

Directory of Open Access Journals (Sweden)

Peter M Hollingsworth

Full Text Available The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1 mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance.

SIRT participates at DNA damage response

Energy Technology Data Exchange (ETDEWEB)

Yun, Mi Yong; Joeng, Jae Min; Lee, Kee Ho [Korea Cancer Center Hospital, Seoul (Korea, Republic of); Park, Gil Hong [College of Medicine, Korea University, Seoul (Korea, Republic of)

2009-05-15

Sir2 maintains genomic stability in multiple ways in yeast. As a NAD{sup +}-dependent histone deacetylase, Sir2 has been reported to control chromatin silencing. In both budding yeast and Drosophila, overexpression of Sir2 extends life span. Previous reports have also demonstrated that Sir2 participate at DNA damage repair. A protein complex containing Sir2 has been reported to translocate to DNA double-strand breaks. Following DNA damage response, SIRT1 deacetylates p53 protein and attenuates its ability as a transcription factor. Consequently, SIRT1 over-expression increases cell survival under DNA damage inducing conditions. These previous observations mean a possibility that signals generated during the process of DNA repair are delivered through SIRT1 to acetylated p53. We present herein functional evidence for the involvement of SIRT1 in DNA repair response to radiation. In addition, this modulation of DNA repair activity may be connected to deacetylation of MRN proteins.

From structure to mechanism-understanding initiation of DNA replication.

Science.gov (United States)

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L Maximilian; Schneider, Sarah; Speck, Christian

2017-06-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. © 2017 Riera et al.; Published by Cold Spring Harbor Laboratory Press.

Different domains of P21Cip1/waf1 regulate DNA replication and DNA repair-associated processes after UV

International Nuclear Information System (INIS)

Soria, Gaston; Speroni, Juliana; Podhajcer, Osvaldo L.; Gottifredi, Vanesa; Prives, Carol

2007-01-01

Full text: Many genotoxic insults result in p21 up-regulation and p21-dependent cell cycle arrest but UV irradiation triggers p21 proteolysis. The significance of the increased p21 turnover is unclear and might be associated to DNA repair. While the role of p21 in Nucleotide Excision Repair (NER) remains controversial, two recent reports explore its effect on Translesion DNA Synthesis (TLS), a process that avoids replication blockage during S phase. The first report shows that p21 degradation is required for efficient PCNA ubiquitination, a post transcriptional modification that is relevant for TLS. The second report demonstrates that p21 (-/-) cells have increased TLS-associated mutagenic rates. Herein we analyze the effect of p21 on different PCNA-driven processes including DNA replication, NER and TLS. Whereas only the CDK binding domain of p21 is required for cell cycle arrest in unstressed cells; neither the CDK- nor the PCNA-binding domains of p21 are able to block early and late steps of NER. Intriguingly, through its PCNA binding domain, p21 inhibited recruitment of the TLS-polymerase, polη to PCNA foci after UV. Moreover, this obstruction correlates with accumulation of γH2AX and increased apoptosis. Taking together, our data emphasizes the link between p21 turnover and efficient TLS. This might also suggest a potential effect of p21 on other activities of polζ, a DNA polymerase with central roles in other biological scenarios such as genetic conversion, homologous recombination and modulation of the cellular response to genotoxic agents [es

Gastric lymphomas in Turkey. Analysis of prognostic factors with special emphasis on flow cytometric DNA content.

Science.gov (United States)

Aydin, Z D; Barista, I; Canpinar, H; Sungur, A; Tekuzman, G

2000-07-01

In contrast to DNA ploidy, to the authors' knowledge the prognostic significance of S-phase fraction (SPF) in gastric lymphomas has not been determined. In the current study, the prognostic significance of various parameters including SPF and DNA aneuploidy were analyzed and some distinct epidemiologic and biologic features of gastric lymphomas in Turkey were found. A series of 78 gastric lymphoma patients followed at Hacettepe University is reported. DNA flow cytometry was performed for 34 patients. The influence of various parameters on survival was investigated with the log rank test. The Cox proportional hazards model was fitted to identify independent prognostic factors. The median age of the patients was 50 years. There was no correlation between patient age and tumor grade. DNA content analysis revealed 4 of the 34 cases to be aneuploid with DNA index values < 1.0. The mean SPF was 33.5%. In the univariate analysis, surgical resection of the tumor, modified Ann Arbor stage, performance status, response to first-line chemotherapy, lactate dehydrogenase (LDH) level, and SPF were important prognostic factors for disease free survival (DFS). The same parameters, excluding LDH level, were important for determining overall survival (OS). In the multivariate analysis, surgical resection of the tumor, disease stage, performance status, and age were found to be important prognostic factors for OS. To the authors' knowledge the current study is the first to demonstrate the prognostic significance of SPF in gastric lymphomas. The distinguishing features of Turkish gastric lymphoma patients are 1) DNA indices of aneuploid cases that all are < 1.0, which is a unique feature; 2) a lower percentage of aneuploid cases; 3) a higher SPF; 4) a younger age distribution; and 5) lack of an age-grade correlation. The authors conclude that gastric lymphomas in Turkey have distinct biologic and epidemiologic characteristics. Copyright 2000 American Cancer Society.

Resident enhanced repair: novel repair process action on plasmid DNA transformed into Escherichia coli K-12

International Nuclear Information System (INIS)

Strike, P.; Roberts, R.J.

1982-01-01

The survival of UV-irradiated DNA of plasmid NTP16 was monitored after its transformation into recipient cells containing an essentially homologous undamaged plasmid, pLV9. The presence of pLV9 resulted in a substantial increase in the fraction of damaged NTP16 molecules which survived in the recipient cells. This enhanced survival requires the host uvrA + and uvrB + gene products, but not the host recA + gene product. The requirement for both homologous DNA and the uvrA + gene products suggests that a novel repair process may act on plasmid DNA. Possible mechanisms for this process are considered

Fast parallel molecular algorithms for DNA-based computation: factoring integers.

Science.gov (United States)

Chang, Weng-Long; Guo, Minyi; Ho, Michael Shan-Hui

2005-06-01

The RSA public-key cryptosystem is an algorithm that converts input data to an unrecognizable encryption and converts the unrecognizable data back into its original decryption form. The security of the RSA public-key cryptosystem is based on the difficulty of factoring the product of two large prime numbers. This paper demonstrates to factor the product of two large prime numbers, and is a breakthrough in basic biological operations using a molecular computer. In order to achieve this, we propose three DNA-based algorithms for parallel subtractor, parallel comparator, and parallel modular arithmetic that formally verify our designed molecular solutions for factoring the product of two large prime numbers. Furthermore, this work indicates that the cryptosystems using public-key are perhaps insecure and also presents clear evidence of the ability of molecular computing to perform complicated mathematical operations.

Imaging the DNA damage response with PET and SPECT

Energy Technology Data Exchange (ETDEWEB)

Knight, James C.; Koustoulidou, Sofia; Cornelissen, Bart [University of Oxford, CR-UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, Oxford (United Kingdom)

2017-06-15

DNA integrity is constantly challenged by endogenous and exogenous factors that can alter the DNA sequence, leading to mutagenesis, aberrant transcriptional activity, and cytotoxicity. Left unrepaired, damaged DNA can ultimately lead to the development of cancer. To overcome this threat, a series of complex mechanisms collectively known as the DNA damage response (DDR) are able to detect the various types of DNA damage that can occur and stimulate the appropriate repair process. Each DNA damage repair pathway leads to the recruitment, upregulation, or activation of specific proteins within the nucleus, which, in some cases, can represent attractive targets for molecular imaging. Given the well-established involvement of DDR during tumorigenesis and cancer therapy, the ability to monitor these repair processes non-invasively using nuclear imaging techniques may facilitate the earlier detection of cancer and may also assist in monitoring response to DNA damaging treatment. This review article aims to provide an overview of recent efforts to develop PET and SPECT radiotracers for imaging of DNA damage repair proteins. (orig.)

The yield, processing, and biological consequences of clustered DNA damage induced by ionizing radiation

International Nuclear Information System (INIS)

Shikazono, Naoya; Noguchi, Miho; Fujii, Kentaro; Urushibara, Ayumi; Yokoya, Akinari

2009-01-01

After living cells are exposed to ionizing radiation, a variety of chemical modifications of DNA are induced either directly by ionization of DNA or indirectly through interactions with water-derived radicals. The DNA lesions include single strand breaks (SSB), base lesions, sugar damage, and apurinic/apyrimidinic sites (AP sites). Clustered DNA damage, which is defined as two or more of such lesions within one to two helical turns of DNA induced by a single radiation track, is considered to be a unique feature of ionizing radiation. A double strand break (DSB) is a type of clustered DNA damage, in which single strand breaks are formed on opposite strands in close proximity. Formation and repair of DSBs have been studied in great detail over the years as they have been linked to important biological endpoints, such as cell death, loss of genetic material, chromosome aberration. Although non-DSB clustered DNA damage has received less attention, there is growing evidence of its biological significance. This review focuses on the current understanding of (1) the yield of non-DSB clustered damage induced by ionizing radiation (2) the processing, and (3) biological consequences of non-DSB clustered DNA damage. (author)

Polycyclic aromatic hydrocarbons and PAH-related DNA adducts.

Science.gov (United States)

Ewa, Błaszczyk; Danuta, Mielżyńska-Švach

2017-08-01

Investigations on the impact of chemicals on the environment and human health have led to the development of an exposome concept. The exposome refers to the totality of exposures received by a person during life, including exposures to life-style factors, from the prenatal period to death. The exposure to genotoxic chemicals and their reactive metabolites can induce chemical modifications of DNA, such as, for example, DNA adducts, which have been extensively studied and which play a key role in chemically induced carcinogenesis. Development of different methods for the identification of DNA adducts has led to adopting DNA adductomic approaches. The ability to simultaneously detect multiple PAH-derived DNA adducts may allow for the improved assessment of exposure, and offer a mechanistic insight into the carcinogenic process following exposure to PAH mixtures. The major advantage of measuring chemical-specific DNA adducts is the assessment of a biologically effective dose. This review provides information about the occurrence of the polycyclic aromatic hydrocarbons (PAHs) and their influence on human exposure and biological effects, including PAH-derived DNA adduct formation and repair processes. Selected methods used for determination of DNA adducts have been presented.

The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends.

Science.gov (United States)

Weterings, Eric; Verkaik, Nicole S; Keijzers, Guido; Florea, Bogdan I; Wang, Shih-Ya; Ortega, Laura G; Uematsu, Naoya; Chen, David J; van Gent, Dik C

2009-03-01

Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) and the XRCC4/ligase IV complex. Activation of the DNA-PK(CS) serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PK(CS) at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PK(CS) autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PK(CS) autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.

The Ku80 Carboxy Terminus Stimulates Joining and Artemis-Mediated Processing of DNA Ends▿

Science.gov (United States)

Weterings, Eric; Verkaik, Nicole S.; Keijzers, Guido; Florea, Bogdan I.; Wang, Shih-Ya; Ortega, Laura G.; Uematsu, Naoya; Chen, David J.; van Gent, Dik C.

2009-01-01

Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and the XRCC4/ligase IV complex. Activation of the DNA-PKCS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PKCS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PKCS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PKCS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction. PMID:19103741

DNA-Compatible Nitro Reduction and Synthesis of Benzimidazoles.

Science.gov (United States)

Du, Huang-Chi; Huang, Hongbing

2017-10-18

DNA-encoded chemical libraries have emerged as a cost-effective alternative to high-throughput screening (HTS) for hit identification in drug discovery. A key factor for productive DNA-encoded libraries is the chemical diversity of the small molecule moiety attached to an encoding DNA oligomer. The library structure diversity is often limited to DNA-compatible chemical reactions in aqueous media. Herein, we describe a facile process for reducing aryl nitro groups to aryl amines. The new protocol offers simple operation and circumvents the pyrophoric potential of the conventional method (Raney nickel). The reaction is performed in aqueous solution and does not compromise DNA structural integrity. The utility of this method is demonstrated by the versatile synthesis of benzimidazoles on DNA.

Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

Science.gov (United States)

Kang, Jian; Kusnadi, Eric P.; Ogden, Allison J.; Hicks, Rodney J.; Bammert, Lukas; Kutay, Ulrike; Hung, Sandy; Sanij, Elaine; Hannan, Ross D.; Hannan, Katherine M.; Pearson, Richard B.

2016-01-01

Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer. PMID:27385002

Fibroblast growth factor 2 and DNA repair involvement in the keratinocyte stem cells response to ionizing radiation

International Nuclear Information System (INIS)

Harfouche, L'Emira Ghida

2010-02-01

Keratinocyte stem cells (KSCs) from the human inter follicular epidermis are regarded as the major target to radiation during radiotherapy. We found herein that KSCs are more resistant to ionizing radiation than their direct progeny, and presented more rapid DNA damage repair kinetics than the progenitors. Furthermore, we provided evidence describing the effect of fibroblast growth factor 2 (FGF2) signaling on the ability of KSCs and progenitors to repair damaged DNA. Despite our knowledge of the fact, that FGF is an anti-apoptotic factor in multiple cell types, the direct link between DNA repair and FGF2 signaling has rarely been shown. Existence of such link is an important issue with implications not only to stem cell field but also to cancer therapy. (author)

Impairment of the DNA synthesis in roots of γ-irradiated seedlings, and the restorative processes

International Nuclear Information System (INIS)

Golikova, O.P.; Mironyuk, T.J.

1976-01-01

Degradation of a prelabelled H 3 -DNA and post-irradiation incorporation of 2-C 14 -thymidine into root DNA of mung beans, peas, and horse beans, have been studied as a function of a radiation dose. A marked dose-dependent decrease in the activity of H 3 -DNA has been detected in γ-irradiated roots. As the radiation dose increases, the specific activity of 2-C 14 -DNA also increases in roots of beans and mung beans. A maximum increase is registered at a dose of 1500 rads. The effects observed are thought to be due to the restorative processes

Theory on the mechanism of distal action of transcription factors: looping of DNA versus tracking along DNA

International Nuclear Information System (INIS)

Murugan, R

2010-01-01

In this paper, we develop a theory on the mechanism of distal action of the transcription factors, which are bound at their respective cis-regulatory enhancer modules on the promoter-RNA polymerase II (PR) complexes to initiate the transcription event in eukaryotes. We consider both the looping and tracking modes of their distal communication and calculate the mean first passage time that is required for the distal interactions of the complex of enhancer and transcription factor with the PR via both these modes. We further investigate how this mean first passage time is dependent on the length of the DNA segment (L, base-pairs) that connects the cis-regulatory binding site and the respective promoter. When the radius of curvature of this connecting segment of DNA is R that was induced upon binding of the transcription factor at the cis-acting element and RNAPII at the promoter in cis-positions, our calculations indicate that the looping mode of distal action will dominate when L is such that L > 2πR and the tracking mode of distal action will be favored when L 2 bps. It seems that the free energy associated with the binding of the transcription factor with its cis-acting element and the distance of this cis-acting element from the corresponding promoter of the gene of interest is negatively correlated. Our results suggest that the looping and tracking modes of distal action are concurrently operating on the transcription activation and the physics that determines the timescales associated with the looping/tracking in the mechanism of action of these transcription factors on the initiation of the transcription event must put a selection pressure on the distribution of the distances of cis-regulatory modules from their respective promoters of the genes. The computational analysis of the upstream sequences of promoters of various genes in the human and mouse genomes for the presence of putative cis-regulatory elements for a set of known transcription factors using

Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

International Nuclear Information System (INIS)

Spicer, E.K.; Horton, R.; Bloem, L.

1987-01-01

Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. λ Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC)

Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process.

Science.gov (United States)

Jia, Yuying; Yao, Xingdong; Zhao, Mingzhe; Zhao, Qiang; Du, Yanli; Yu, Cuimei; Xie, Futi

2015-08-07

The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert) with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman), which showed a relatively weak susceptibility. Gibberellin (GA) levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA). Higher zeatin riboside (ZR) content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA) content, polyphenol oxidase (PPO) and peroxidase (POD) activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process

Directory of Open Access Journals (Sweden)

Yuying Jia

2015-08-01

Full Text Available The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman, which showed a relatively weak susceptibility. Gibberellin (GA levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA. Higher zeatin riboside (ZR content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA content, polyphenol oxidase (PPO and peroxidase (POD activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

DNA Damages and White Blood Cell Death Processes in Victims with Severe Injury

Directory of Open Access Journals (Sweden)

V. V. Moroz

2014-01-01

Full Text Available Objective. To study the mechanisms of posttraumatic changes in the blood cells, by investigating DNA damages associat ed with hypoxia caused by massive blood loss (BL in severe injury.Subjects and methods. Ninetyfive patients aged 40.6±16.5 years (from 20 to 79 years who had sustained severe mechanical injury with different BL volumes (BLV (from 100 to 4000 ml and hemodynamic disorders were examined to study DNA damages and white blood cell necrotic and apop totic processes. In terms of the victims' weight, the mean BL was 21.5±16.5 ml/kg (from 1.4 to 61.5 ml/kg. The victimswere divided into 4 groups according to BLV: 1 26 victims whose BLV was less than 750 ml (5.93±2.41 ml/kg (grade I BL; 2 23 victims whose BLV was 750—1500 ml (11.5±1.5 ml/kg (grade 2 BL; 3 23 victims whose BLV was 1500—2000 ml (23.8±4.0 ml/kg (grade 3 BL; 4 23 victims whose BLV was over 2000 ml (45.6±10.1 ml/kg (grade 4 BL, according to the type of injury: 1 severe skeletal injury (SSI (n=17; 2 brain injury (BI (n=43; 3 a concurrence of SSI and BI (SSI+BI (n=35; according to the development of infectious complications: 1 69 victims who developed infectious com plications on days 5—7 postinjury; 2 26 victims who did not. To evaluate the impact of hypoxia on DNA damages, white blood cell apoptotic and necrotic processes, the victims were divided into 2 groups: 1 hypoxia (18 of the 95 victims who had 4 altered indicators, such as capillary blood pO2, plasma lactate levels, pH, and BE; 2 no hypoxia (10 of the 95 victims whose indicators were within the normal range. DNA damages and necrotic and apoptotic changes in the white blood cells were assessed by the DNA comet assay. The plasma concentration of extracellular DNA was fluorometrically determined using a QuantiTTM HS DNA Assay Kit (Invitrogen, USA. That of 8hydroxy2deoxyguanosine was estimated by enzyme immunoassay employing an 8hydroxy2deoxyGuanosine EIA Kit (Cayman Chemical, USA. The levels of cas

The relationship of transcription and repair of radioinduced DNA damage

International Nuclear Information System (INIS)

Zhestyanikov, V.D.; Igusheva, O.A.

1997-01-01

The data are discussed which has become a basement of such important findings as involvement of transcription into repair or existence of transcription-coupling repair factors. Thymine glycols which are appear under ionizing radiation exposure, are repaired preferentially in transcribed DNA. In present review the preferential repair of ionizing radiation-induced singlestrand breaks (SSBa) in transcribed DNA of human cells. Discontinuous distribution of DNA repair along hole genome has a grate role in biological processes

Identification of processed Chinese medicinal materials using DNA mini-barcoding.

Science.gov (United States)

Song, Ming; Dong, Gang-Qiang; Zhang, Ya-Qin; Liu, Xia; Sun, Wei

2017-07-01

Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and identification of processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA mini-barcoding, based on the sequencing of a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In the present study, six DNA barcode loci including ITS2, psbA-trnH, rbcL, matK, trnL (UAA) intron and its P6 loop, were employed for the authentication of 45 processed samples belonging to 15 species. We evaluated the amplification efficiency of each locus. We also examined the identification accuracy of the potential mini-barcode locus, of trnL (UAA) intron P6 loop. Our results showed that the five primary barcode loci were successfully amplified in only 8.89%-20% of the processed samples, while the amplification rates of the trnL (UAA) intron P6 loop were higher, at 75.56% successful amplification. We compared the mini-barcode sequences with Genbank using the Blast program. The analysis showed that 45.23% samples could be identified to genus level, while only one sample could be identified to the species level. We conclude that trnL (UAA) p6 loop is a candidate mini-barcode that has shown its potential and may become a universal mini-barcode as complementary barcode for authenticity testing and will play an important role in medicinal materials control. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination.

Science.gov (United States)

Ignarski, Michael; Singh, Aditi; Swart, Estienne C; Arambasic, Miroslav; Sandoval, Pamela Y; Nowacki, Mariusz

2014-10-29

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: complex theoretical and experimental studies.

Science.gov (United States)

Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Špérová, Miroslava; Schneider, Bohdan; Páv, Ondřej; Šebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

2013-01-01

Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. © 2013. Published by Elsevier B.V. All rights reserved.

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)

DNA repair synthesis in human fibroblasts requires DNA polymerase delta

International Nuclear Information System (INIS)

Nishida, C.; Reinhard, P.; Linn, S.

1988-01-01

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

DNA Checkpoint and Repair Factors Are Nuclear Sensors for Intracellular Organelle Stresses—Inflammations and Cancers Can Have High Genomic Risks

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Huihong Zeng

2018-05-01

Full Text Available Under inflammatory conditions, inflammatory cells release reactive oxygen species (ROS and reactive nitrogen species (RNS which cause DNA damage. If not appropriately repaired, DNA damage leads to gene mutations and genomic instability. DNA damage checkpoint factors (DDCF and DNA damage repair factors (DDRF play a vital role in maintaining genomic integrity. However, how DDCFs and DDRFs are modulated under physiological and pathological conditions are not fully known. We took an experimental database analysis to determine the expression of 26 DNA DDCFs and 42 DNA DDRFs in 21 human and 20 mouse tissues in physiological/pathological conditions. We made the following significant findings: (1 Few DDCFs and DDRFs are ubiquitously expressed in tissues while many are differentially regulated.; (2 the expression of DDCFs and DDRFs are modulated not only in cancers but also in sterile inflammatory disorders and metabolic diseases; (3 tissue methylation status, pro-inflammatory cytokines, hypoxia regulating factors and tissue angiogenic potential can determine the expression of DDCFs and DDRFs; (4 intracellular organelles can transmit the stress signals to the nucleus, which may modulate the cell death by regulating the DDCF and DDRF expression. Our results shows that sterile inflammatory disorders and cancers increase genomic instability, therefore can be classified as pathologies with a high genomic risk. We also propose a new concept that as parts of cellular sensor cross-talking network, DNA checkpoint and repair factors serve as nuclear sensors for intracellular organelle stresses. Further, this work would lead to identification of novel therapeutic targets and new biomarkers for diagnosis and prognosis of metabolic diseases, inflammation, tissue damage and cancers.

Normal breast tissue DNA methylation differences at regulatory elements are associated with the cancer risk factor age.

Science.gov (United States)

Johnson, Kevin C; Houseman, E Andres; King, Jessica E; Christensen, Brock C

2017-07-10

The underlying biological mechanisms through which epidemiologically defined breast cancer risk factors contribute to disease risk remain poorly understood. Identification of the molecular changes associated with cancer risk factors in normal tissues may aid in determining the earliest events of carcinogenesis and informing cancer prevention strategies. Here we investigated the impact cancer risk factors have on the normal breast epigenome by analyzing DNA methylation genome-wide (Infinium 450 K array) in cancer-free women from the Susan G. Komen Tissue Bank (n = 100). We tested the relation of established breast cancer risk factors, age, body mass index, parity, and family history of disease, with DNA methylation adjusting for potential variation in cell-type proportions. We identified 787 cytosine-guanine dinucleotide (CpG) sites that demonstrated significant associations (Q value breast cancer risk factors. Age-related DNA methylation changes are primarily increases in methylation enriched at breast epithelial cell enhancer regions (P = 7.1E-20), and binding sites of chromatin remodelers (MYC and CTCF). We validated the age-related associations in two independent populations, using normal breast tissue samples (n = 18) and samples of normal tissue adjacent to tumor tissue (n = 97). The genomic regions classified as age-related were more likely to be regions altered in both pre-invasive (n = 40, P = 3.0E-03) and invasive breast tumors (n = 731, P = 1.1E-13). DNA methylation changes with age occur at regulatory regions, and are further exacerbated in cancer, suggesting that age influences breast cancer risk in part through its contribution to epigenetic dysregulation in normal breast tissue.

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

Science.gov (United States)

Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

2016-07-26

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

DNA hybrids suggesting a recombination process repairing radiation-induced DNA double-strand breaks in Ehrlich Ascites tumor cells

International Nuclear Information System (INIS)

Barthel, H.R.

1984-01-01

The results presented suggest the possibility of repair of DNA double-strand breaks by recombination, at least in the S and G 2 -phases of the cell cycle, in mammalian cells. Further experiments with synchronized cell cultures will have to show whether this process may also occur in the G 1 -phase of the cell cycle. (orig./AJ) [de

TU-H-CAMPUS-TeP2-04: Measurement of Stereotactic Output Factors with DNA Double-Strand Breaks

Energy Technology Data Exchange (ETDEWEB)

Cline, K; Obeidat, M; Stathakis, S; Kabat, C; Markovic, M; Papanikolaou, N; Rasmussen, K; Gutierrez, A; Ha, C; Lee, S; Shim, E; Kirby, N [University of Texas HSC SA, San Antonio, TX (United States)

2016-06-15

Purpose: Radiotherapy treatment is specified by radiation dose prescriptions, but biological DNA damage actually controls treatment effectiveness. It is impractical to directly measure dose in the clinic, so we measure quantities, such as collected charge, and calculate the relationship to dose. At small fields, such as those in stereotactic radiosurgery (SRS), charged-particle equilibrium (CPE) breaks down and the accuracy of the measurement for delivered dose decreases. By measuring DNA double-strand breaks (DSB) directly, we believe treatment accuracy could improve by providing a more meaningful measurement. Methods: A DNA dosimeter, consisting of magnetic streptavidin beads attached to 4 kilobase pair DNA strands labeled with biotin and fluorescein amidite (FAM) on opposing ends, was suspended in phosphate-buffered saline (PBS). Twenty µL samples were placed in plastic micro-capillary tubes inside a water tank setup and irradiated with 10 cm, 3 cm, 1.25 cm, 0.75 cm, and 0.5 cm radiation field sizes, where the three smallest sizes were cones. After irradiation, the dosimeters were mechanically separated into beads (intact DNA) and supernatant (broken DNA/FAM) using a magnet. The fluorescence was read and the probability of DSB was calculated. This was used to calculate the output factor for an SRS beam and compared to that measured using a diode detector. Results: The output factors relative to a 10 cm field were 0.89±0.07, 0.76±0.08, 0.59±0.04, and 0.78±0.12 for the field sizes of 3 cm, 1.25 cm, 0.75 cm, and 0.5 cm, respectively. Some of the diode measurements do not fall within these uncertainties. Conclusion: This was the first attempt to measure output factors in a water tank with the DNA dosimeter. Although differences compared to the diode were observed, the uncertainty analysis ignored systematic errors. For future work, we will repeat this experiment to quantify and correct systematic errors, such as those caused by positional alignment and sample

The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor.

Science.gov (United States)

Townsend, Philip D; Dixon, Christopher H; Slootweg, Erik J; Sukarta, Octavina C A; Yang, Ally W H; Hughes, Timothy R; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Goverse, Aska; Cann, Martin J

2018-03-02

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming, and some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato is further able to bind and distort double-stranded DNA. However, Rx1 host targets that support a role for Rx1 in transcriptional reprogramming at DNA are unknown. Here, we report a functional interaction between Rx1 and Nb Glk1, a Golden2-like transcription factor. Rx1 binds to Nb Glk1 in vitro and in planta. Nb Glk1 binds to known Golden2-like consensus DNA sequences. Rx1 reduces the binding affinity of Nb Glk1 for DNA in vitro. Nb Glk1 activates cellular responses to potato virus X, whereas Rx1 associates with Nb Glk1 and prevents its assembly on DNA in planta unless activated by PVX. This study provides new mechanistic insight into how an NLR can coordinate an immune signaling response at DNA following pathogen perceptions. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

RTEL1 contributes to DNA replication and repair and telomere maintenance

NARCIS (Netherlands)

Uringa, Evert-Jan; Lisaingo, Kathleen; Pickett, Hilda A.; Brind'Amour, Julie; Rohde, Jan-Hendrik; Zelensky, Alex; Essers, Jeroen; Lansdorp, Peter M.

2012-01-01

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance

RTEL1 contributes to DNA replication and repair and telomere maintenance

NARCIS (Netherlands)

E.J. Uringa; K. Lisaingo (Kathleen); H.A. Pickett (Hilda); J. Brind'Amour (Julie); J.-H. Rohde (Jan-Hendrik); A. Zelensky (Alexander); J. Essers (Jeroen); P.M. Lansdorp (Peter)

2012-01-01

textabstractTelomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere

DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

KAUST Repository

Liu, Qian; Wang, Junguo; Miki, Daisuke; Xia, Ran; Yu, Wenxiang; He, Junna; Zheng, Zhimin; Zhu, Jian-Kang; Gonga, Zhizhong

2010-01-01

Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

KAUST Repository

Liu, Qian

2010-07-01

Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

Energy Technology Data Exchange (ETDEWEB)

Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

2014-10-31

Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

International Nuclear Information System (INIS)

Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

2014-01-01

Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K d 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy

Histone modifications in response to DNA damage

International Nuclear Information System (INIS)

Altaf, Mohammed; Saksouk, Nehme; Cote, Jacques

2007-01-01

The packaging of the eukaryotic genome into highly condensed chromatin makes it inaccessible to the factors required for gene transcription, DNA replication, recombination and repair. Eukaryotes have developed intricate mechanisms to overcome this repressive barrier imposed by chromatin. Histone modifying enzymes and ATP-dependent chromatin remodeling complexes play key roles here as they regulate many nuclear processes by altering the chromatin structure. Significantly, these activities are integral to the process of DNA repair where histone modifications act as signals and landing platforms for various repair proteins. This review summarizes the recent developments in our understanding of histone modifications and their role in the maintenance of genome integrity

DNA-binding proteins essential for protein-primed bacteriophage ø29 DNA replication

Directory of Open Access Journals (Sweden)

Margarita Salas

2016-08-01

Full Text Available Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5’ ends of the DNA. This protein, called terminal protein (TP, is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3’-5’ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding

Advances in the understanding of mitochondrial DNA as a pathogenic factor in inflammatory diseases [version 1; referees: 3 approved

Directory of Open Access Journals (Sweden)

Ray K. Boyapati

2017-02-01

Full Text Available Mitochondrial DNA (mtDNA has many similarities with bacterial DNA because of their shared common ancestry. Increasing evidence demonstrates mtDNA to be a potent danger signal that is recognised by the innate immune system and can directly modulate the inflammatory response. In humans, elevated circulating mtDNA is found in conditions with significant tissue injury such as trauma and sepsis and increasingly in chronic organ-specific and systemic illnesses such as steatohepatitis and systemic lupus erythematosus. In this review, we examine our current understanding of mtDNA-mediated inflammation and how the mechanisms regulating mitochondrial homeostasis and mtDNA release represent exciting and previously under-recognised important factors in many human inflammatory diseases, offering many new translational opportunities.

Only one ATP-binding DnaX subunit is required for initiation complex formation by the Escherichia coli DNA polymerase III holoenzyme.

Science.gov (United States)

Wieczorek, Anna; Downey, Christopher D; Dallmann, H Garry; McHenry, Charles S

2010-09-17

The DnaX complex (DnaX(3)δδ'χ psi) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β(2), onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β(2) binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β(2). DNA synthesis activity can be restored by increased concentrations of β(2). In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β(2) loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.

A MapReduce Framework for DNA Sequencing Data Processing

Directory of Open Access Journals (Sweden)

Samy Ghoneimy

2016-12-01

Full Text Available Genomics and Next Generation Sequencers (NGS like Illumina Hiseq produce data in the order of ‎‎200 billion base pairs in a single one-week run for a 60x human genome coverage, which ‎requires modern high-throughput experimental technologies that can ‎only be tackled with high performance computing (HPC and specialized software algorithms called ‎‎“short read aligners”. This paper focuses on the implementation of the DNA sequencing as a set of MapReduce programs that will accept a DNA data set as a FASTQ file and finally generate a VCF (variant call format file, which has variants for a given DNA data set. In this paper MapReduce/Hadoop along with Burrows-Wheeler Aligner (BWA, Sequence Alignment/Map (SAM ‎tools, are fully utilized to provide various utilities for manipulating alignments, including sorting, merging, indexing, ‎and generating alignments. The Map-Sort-Reduce process is designed to be suited for a Hadoop framework in ‎which each cluster is a traditional N-node Hadoop cluster to utilize all of the Hadoop features like HDFS, program ‎management and fault tolerance. The Map step performs multiple instances of the short read alignment algorithm ‎‎(BoWTie that run in parallel in Hadoop. The ordered list of the sequence reads are used as input tuples and the ‎output tuples are the alignments of the short reads. In the Reduce step many parallel instances of the Short ‎Oligonucleotide Analysis Package for SNP (SOAPsnp algorithm run in the cluster. Input tuples are sorted ‎alignments for a partition and the output tuples are SNP calls. Results are stored via HDFS, and then archived in ‎SOAPsnp format. ‎ The proposed framework enables extremely fast discovering somatic mutations, inferring population genetical ‎parameters, and performing association tests directly based on sequencing data without explicit genotyping or ‎linkage-based imputation. It also demonstrate that this method achieves comparable

Molecular Processes Studied at a Single-Molecule Level Using DNA Origami Nanostructures and Atomic Force Microscopy

Directory of Open Access Journals (Sweden)

Ilko Bald

2014-09-01

Full Text Available DNA origami nanostructures allow for the arrangement of different functionalities such as proteins, specific DNA structures, nanoparticles, and various chemical modifications with unprecedented precision. The arranged functional entities can be visualized by atomic force microscopy (AFM which enables the study of molecular processes at a single-molecular level. Examples comprise the investigation of chemical reactions, electron-induced bond breaking, enzymatic binding and cleavage events, and conformational transitions in DNA. In this paper, we provide an overview of the advances achieved in the field of single-molecule investigations by applying atomic force microscopy to functionalized DNA origami substrates.

Damage to cellular DNA from particulate radiations, the efficacy of its processing and the radiosensitivity of mammalian cells. Emphasis on DNA double strand breaks and chromatin breaks

Science.gov (United States)

Lett, J. T.

1992-01-01

For several years, it has been evident that cellular radiation biology is in a necessary period of consolidation and transition (Lett 1987, 1990; Lett et al. 1986, 1987). Both changes are moving apace, and have been stimulated by studies with heavy charged particles. From the standpoint of radiation chemistry, there is now a consensus of opinion that the DNA hydration shell must be distinguished from bulk water in the cell nucleus and treated as an integral part of DNA (chromatin) (Lett 1987). Concomitantly, sentiment is strengthening for the abandonment of the classical notions of "direct" and "indirect" action (Fielden and O'Neill 1991; O'Neill 1991; O'Neill et al. 1991; Schulte-Frohlinde and Bothe 1991 and references therein). A layer of water molecules outside, or in the outer edge of, the DNA (chromatin) hydration shell influences cellular radiosensitivity in ways not fully understood. Charge and energy transfer processes facilitated by, or involving, DNA hydration must be considered in rigorous theories of radiation action on cells. The induction and processing of double stand breaks (DSBs) in DNA (chromatin) seem to be the predominant determinants of the radiotoxicity of normally radioresistant mammalian cells, the survival curves of which reflect the patterns of damage induced and the damage present after processing ceases, and can be modelled in formal terms by the use of reaction (enzyme) kinetics. Incongruities such as sublethal damage are neither scientifically sound nor relevant to cellular radiation biology (Calkins 1991; Lett 1990; Lett et al. 1987a). Increases in linear energy transfer (LET infinity) up to 100-200 keV micron-1 cause increases in the extents of neighboring chemical and physical damage in DNA denoted by the general term DSB. Those changes are accompanied by decreasing abilities of cells normally radioresistant to sparsely ionizing radiations to process DSBs in DNA and chromatin and to recover from radiation exposure, so they make

Increased sister chromatid cohesion and DNA damage response factor localization at an enzyme-induced DNA double-strand break in vertebrate cells.

LENUS (Irish Health Repository)

Dodson, Helen

2009-10-01

The response to DNA damage in vertebrate cells involves successive recruitment of DNA signalling and repair factors. We used light microscopy to monitor the genetic dependencies of such localization to a single, induced DNA double strand break (DSB) in vertebrate cells. We used an inducible version of the rare-cutting I-SceI endonuclease to cut a chromosomally integrated I-SceI site beside a Tet operator array that was visualized by binding a Tet repressor-GFP fusion. Formation of gamma-H2AX foci at a single DSB was independent of ATM or Ku70. ATM-deficient cells showed normal kinetics of 53Bp1 recruitment to DSBs, but Rad51 localization was retarded. 53Bp1 and Rad51 foci formation at a single DSB was greatly reduced in H2AX-null DT40 cells. We also observed decreased inter-sister chromatid distances after DSB induction, suggesting that cohesin loading at DSBs causes elevated sister chromatid cohesion. Loss of ATM reduced DSB-induced cohesion, consistent with cohesin being an ATM target in the DSB response. These data show that the same genetic pathways control how cells respond to single DSBs and to multiple lesions induced by whole-cell DNA damage.

Kinetics of DNA tile dimerization.

Science.gov (United States)

Jiang, Shuoxing; Yan, Hao; Liu, Yan

2014-06-24

Investigating how individual molecular components interact with one another within DNA nanoarchitectures, both in terms of their spatial and temporal interactions, is fundamentally important for a better understanding of their physical behaviors. This will provide researchers with valuable insight for designing more complex higher-order structures that can be assembled more efficiently. In this report, we examined several spatial factors that affect the kinetics of bivalent, double-helical (DH) tile dimerization, including the orientation and number of sticky ends (SEs), the flexibility of the double helical domains, and the size of the tiles. The rate constants we obtained confirm our hypothesis that increased nucleation opportunities and well-aligned SEs accelerate tile-tile dimerization. Increased flexibility in the tiles causes slower dimerization rates, an effect that can be reversed by introducing restrictions to the tile flexibility. The higher dimerization rates of more rigid tiles results from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. We believe that the results presented here will assist in improved implementation of DNA tile based algorithmic self-assembly, DNA based molecular robotics, and other specific nucleic acid systems, and will provide guidance to design and assembly processes to improve overall yield and efficiency.

ATP-dependent chromatin remodeling and histone binding by the Cockayne syndrome B DNA repair-transcription coupling factor.

NARCIS (Netherlands)

E. Citterio (Elisabetta); V. van den Boom (Vincent); G. Schnitzler; R. Kanaar (Roland); E. Bonte (Edgar); R.E. Kingston; J.H.J. Hoeijmakers (Jan); W. Vermeulen (Wim)

2000-01-01

textabstractThe Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the

DNA repair in cancer: emerging targets for personalized therapy

International Nuclear Information System (INIS)

Abbotts, Rachel; Thompson, Nicola; Madhusudan, Srinivasan

2014-01-01

Genomic deoxyribonucleic acid (DNA) is under constant threat from endogenous and exogenous DNA damaging agents. Mammalian cells have evolved highly conserved DNA repair machinery to process DNA damage and maintain genomic integrity. Impaired DNA repair is a major driver for carcinogenesis and could promote aggressive cancer biology. Interestingly, in established tumors, DNA repair activity is required to counteract oxidative DNA damage that is prevalent in the tumor microenvironment. Emerging clinical data provide compelling evidence that overexpression of DNA repair factors may have prognostic and predictive significance in patients. More recently, DNA repair inhibition has emerged as a promising target for anticancer therapy. Synthetic lethality exploits intergene relationships where the loss of function of either of two related genes is nonlethal, but loss of both causes cell death. Exploiting this approach by targeting DNA repair has emerged as a promising strategy for personalized cancer therapy. In the current review, we focus on recent advances with a particular focus on synthetic lethality targeting in cancer

TET2 functions as a resistance factor against DNA methylation acquisition during Epstein-Barr virus infection.

Science.gov (United States)

Namba-Fukuyo, Hiroe; Funata, Sayaka; Matsusaka, Keisuke; Fukuyo, Masaki; Rahmutulla, Bahityar; Mano, Yasunobu; Fukayama, Masashi; Aburatani, Hiroyuki; Kaneda, Atsushi

2016-12-06

Extensive DNA methylation is observed in gastric cancer with Epstein-Barr virus (EBV) infection, and EBV infection is the cause to induce this extensive hypermethylaton phenotype in gastric epithelial cells. However, some 5' regions of genes do not undergo de novo methylation, despite the induction of methylation in surrounding regions, suggesting the existence of a resistance factor against DNA methylation acquisition. We conducted an RNA-seq analysis of gastric epithelial cells with and without EBV infection and found that TET family genes, especially TET2, were repressed by EBV infection at both mRNA and protein levels. TET2 was found to be downregulated by EBV transcripts, e.g. BARF0 and LMP2A, and also by seven human miRNAs targeting TET2, e.g., miR-93 and miR-29a, which were upregulated by EBV infection, and transfection of which into gastric cells repressed TET2. Hydroxymethylation target genes by TET2 were detected by hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq) with and without TET2 overexpression, and overlapped significantly with methylation target genes in EBV-infected cells. When TET2 was knocked down by shRNA, EBV infection induced de novo methylation more severely, including even higher methylation in methylation-acquired promoters or de novo methylation acquisition in methylation-protected promoters, leading to gene repression. TET2 knockdown alone without EBV infection did not induce de novo DNA methylation. These data suggested that TET2 functions as a resistance factor against DNA methylation in gastric epithelial cells and repression of TET2 contributes to DNA methylation acquisition during EBV infection.

A conserved helicase processivity factor is needed for conjugation and replication of an integrative and conjugative element.

Directory of Open Access Journals (Sweden)

Jacob Thomas

Full Text Available Integrative and conjugative elements (ICEs are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT by the ICE-encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP, encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.

Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

Science.gov (United States)

Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

2016-12-02

Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

Theory on the mechanism of distal action of transcription factors: looping of DNA versus tracking along DNA

Energy Technology Data Exchange (ETDEWEB)

Murugan, R, E-mail: rmurugan@gmail.co [Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036 (India)

2010-10-15

In this paper, we develop a theory on the mechanism of distal action of the transcription factors, which are bound at their respective cis-regulatory enhancer modules on the promoter-RNA polymerase II (PR) complexes to initiate the transcription event in eukaryotes. We consider both the looping and tracking modes of their distal communication and calculate the mean first passage time that is required for the distal interactions of the complex of enhancer and transcription factor with the PR via both these modes. We further investigate how this mean first passage time is dependent on the length of the DNA segment (L, base-pairs) that connects the cis-regulatory binding site and the respective promoter. When the radius of curvature of this connecting segment of DNA is R that was induced upon binding of the transcription factor at the cis-acting element and RNAPII at the promoter in cis-positions, our calculations indicate that the looping mode of distal action will dominate when L is such that L > 2{pi}R and the tracking mode of distal action will be favored when L < 2{pi}R. The time required for the distal action will be minimum when L = 2{pi}R where the typical value of R for the binding of histones will be R {approx} 16 bps and L {approx} 10{sup 2} bps. It seems that the free energy associated with the binding of the transcription factor with its cis-acting element and the distance of this cis-acting element from the corresponding promoter of the gene of interest is negatively correlated. Our results suggest that the looping and tracking modes of distal action are concurrently operating on the transcription activation and the physics that determines the timescales associated with the looping/tracking in the mechanism of action of these transcription factors on the initiation of the transcription event must put a selection pressure on the distribution of the distances of cis-regulatory modules from their respective promoters of the genes. The computational analysis

Systems Biology Model of Interactions Between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFbeta and ATM Signaling

Energy Technology Data Exchange (ETDEWEB)

O' Neill, Peter [University of Oxford; Anderson, Jennifer [University of Oxford

2014-10-02

The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low dose responses and cross-talk between the ATM and TGFβ pathways initiated by low and high LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to cross- talk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental

Systems Biology Model of Interactions between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFβ and ATM Signaling

International Nuclear Information System (INIS)

Cucinotta, Francis A

2016-01-01

The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently, the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses, and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low-dose responses and cross-talk between the ATM and TGFβ pathways initiated by low- and high-LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to crosstalk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental

Systems Biology Model of Interactions between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFβ and ATM Signaling

Energy Technology Data Exchange (ETDEWEB)

Cucinotta, Francis A [Univ. of Nevada, Las Vegas, NV (United States)

2016-09-01

The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently, the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses, and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low-dose responses and cross-talk between the ATM and TGFβ pathways initiated by low- and high-LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to crosstalk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental

Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

International Nuclear Information System (INIS)

Hirose, Tetsuro; Terajima, Hiroaki; Yamauchi, Akira

1997-01-01

Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125 I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3 H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125 I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125 I-insulin specific binding was not affected. The decrease in 125 I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/10 5 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125 I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125 I-epidermal growth factor binding

Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

Science.gov (United States)

Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

2018-03-01

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly

Cruciform structures are a common DNA feature important for regulating biological processes

Czech Academy of Sciences Publication Activity Database

Brázda, Václav; Laister, R.C.; Jagelská, Eva; Arrowsmith, Ch.

2011-01-01

Roč. 12, č. 33 (2011), s. 1-16 ISSN 1471-2199 R&D Projects: GA ČR(CZ) GAP301/10/1211; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : cruciform structure * inverted repeat * protein- DNA binding Subject RIV: BO - Biophysics Impact factor: 2.857, year: 2011

Conformational and mechanical changes of DNA upon transcription factor binding detected by a QCM and transmission line model.

Science.gov (United States)

de-Carvalho, Jorge; Rodrigues, Rogério M M; Tomé, Brigitte; Henriques, Sílvia F; Mira, Nuno P; Sá-Correia, Isabel; Ferreira, Guilherme N M

2014-04-21

A novel quartz crystal microbalance (QCM) analytical method is developed based on the transmission line model (TLM) algorithm to analyze the binding of transcription factors (TFs) to immobilized DNA oligoduplexes. The method is used to characterize the mechanical properties of biological films through the estimation of the film dynamic shear moduli, G and G, and the film thickness. Using the Saccharomyces cerevisiae transcription factor Haa1 (Haa1DBD) as a biological model two sensors were prepared by immobilizing DNA oligoduplexes, one containing the Haa1 recognition element (HRE(wt)) and another with a random sequence (HRE(neg)) used as a negative control. The immobilization of DNA oligoduplexes was followed in real time and we show that DNA strands initially adsorb with low or non-tilting, laying flat close to the surface, which then lift-off the surface leading to final film tilting angles of 62.9° and 46.7° for HRE(wt) and HRE(neg), respectively. Furthermore we show that the binding of Haa1DBD to HRE(wt) leads to a more ordered and compact film, and forces a 31.7° bending of the immobilized HRE(wt) oligoduplex. This work demonstrates the suitability of the QCM to monitor the specific binding of TFs to immobilized DNA sequences and provides an analytical methodology to study protein-DNA biophysics and kinetics.

Effects of Environmental Factors and Metallic Electrodes on AC Electrical Conduction Through DNA Molecule.

Science.gov (United States)

Abdalla, S; Obaid, A; Al-Marzouki, F M

2017-12-01

Deoxyribonucleic acid (DNA) is one of the best candidate materials for various device applications such as in electrodes for rechargeable batteries, biosensors, molecular electronics, medical- and biomedical-applications etc. Hence, it is worthwhile to examine the mechanism of charge transport in the DNA molecule, however, still a question without a clear answer is DNA a molecular conducting material (wire), semiconductor, or insulator? The answer, after the published data, is still ambiguous without any confirmed and clear scientific answer. DNA is found to be always surrounded with different electric charges, ions, and dipoles. These surrounding charges and electric barrier(s) due to metallic electrodes (as environmental factors (EFs)) play a substantial role when measuring the electrical conductivity through λ-double helix (DNA) molecule suspended between metallic electrodes. We found that strong frequency dependence of AC-complex conductivity comes from the electrical conduction of EFs. This leads to superimposing serious incorrect experimental data to measured ones. At 1 MHz, we carried out a first control experiment on electrical conductivity with and without the presence of DNA molecule. If there are possible electrical conduction due to stray ions and contribution of substrate, we will detected them. This control experiment revealed that there is an important role played by the environmental-charges around DNA molecule and any experiment should consider this role. We have succeeded to measure both electrical conductivity due to EFs (σ ENV ) and electrical conductivity due to DNA molecule (σ DNA ) independently by carrying the measurements at different DNA-lengths and subtracting the data. We carried out measurements as a function of frequency (f) and temperature (T) in the ranges 0.1 Hz molecule from all EFs effects that surround the molecule, but also to present accurate values of σ DNA and the dielectric constant of the molecule ε' DNA as a

The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques

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Burnside Kellie L

2009-11-01

Full Text Available Abstract Background ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV, is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication. Results We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1 and three species of macaques (RFHVMm, RFHVMn and RFHVMf, and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively. We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus and MneRV2 (pig-tail, with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses

UvrD in Deinococcus radiodurans is optimized for processing G-quadruplex DNA

International Nuclear Information System (INIS)

Das, Anubrata; Misra, H.S.

2015-01-01

Deinococcus radiodurans R1 is a radiation resistant Gram-positive bacterium capable of tolerating very high doses of DNA-damaging agents such as gamma radiation (D10 ∼ 12kGy) desiccation (∼ 5% relative humidity), UVC radiation (D10 ∼ 800J/m 2 ) and hydrogen peroxide (40 mM). It achieves this by using a complex regulatory mechanism and novel proteins. Recently bioinformatic analysis showed several stretches of guanine runs in D.radiodurans genome, which could form G-quartets. The role of G-quartets in regulatory processes is well documented in various organisms. The presence of G -quartets in D. radiodurans means that there are regulatory or structural proteins which would bind to these elements. Several proteins are known to bind G-quartets. Finding the proteins which would bind to G4 DNA is difficult as no specific motifs are available for binding these elements. Also most of the known proteins that are shown to bind to G-quadruplex DNA are of eukaryotic nature. To overcome these challenges we defined a set of known G-quadruplex binding proteins and used a smith-waterman algorithm with our own scoring matrix to homologs of G-quadruplex binding proteins in D.radiodurans. Using bioinformatics analysis, we showed that UvrD (DR 1775) of D. radiodurans has ability to bind/translocate along G-quadruplex DNA, a novel feature in prokaryotes. The translocase activity of DR1775 is ATP specific and this ATPase activity is attenuated by ssDNA. Data supporting UvrD of D. radiodurans as a G-quadruplex DNA metabolizing proteins would be presented. (author)

Crystal structure and DNA binding of the homeodomain of the stem cell transcription factor Nanog.

Science.gov (United States)

Jauch, Ralf; Ng, Calista Keow Leng; Saikatendu, Kumar Singh; Stevens, Raymond C; Kolatkar, Prasanna R

2008-02-22

The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level.

Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

Energy Technology Data Exchange (ETDEWEB)

Jauch, Ralf; Ng, Calista Keow Leng; Saikatendu, Kumar Singh; Stevens, Raymond C.; Kolatkar, Prasanna R. (GI-Singapore); (Scripps)

2010-02-08

The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level.

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

DEFF Research Database (Denmark)

Larsen, Dorthe Helena; Poinsignon, Catherine; Gudjonsson, Thorkell

2010-01-01

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate...

Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability.

Science.gov (United States)

Schalbetter, Stephanie A; Mansoubi, Sahar; Chambers, Anna L; Downs, Jessica A; Baxter, Jonathan

2015-08-18

Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.

Concentration and length dependence of DNA looping in transcriptional regulation.

Directory of Open Access Journals (Sweden)

Lin Han

2009-05-01

Full Text Available In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage, to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the for looping.

Isolation, nucleotide sequence and expression of a cDNA encoding feline granulocyte colony-stimulating factor.

Science.gov (United States)

Dunham, S P; Onions, D E

2001-06-21

A cDNA encoding feline granulocyte colony stimulating factor (fG-CSF) was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction. The cDNA is 949 bp in length and encodes a predicted mature protein of 174 amino acids. Recombinant fG-CSF was expressed as a glutathione S-transferase fusion and purified by affinity chromatography. Biological activity of the recombinant protein was demonstrated using the murine myeloblastic cell line GNFS-60, which showed an ED50 for fG-CSF of approximately 2 ng/ml. Copyright 2001 Academic Press.

Preferential recognition of auto-antibodies against 4-hydroxynonenal modified DNA in the cancer patients.

Science.gov (United States)

Faisal, Mohammad; Shahab, Uzma; Alatar, Abdulrahman A; Ahmad, Saheem

2017-11-01

The structural perturbations in DNA molecule may be caused by a break in a strand, a missing base from the backbone, or a chemically changed base. These alterations in DNA that occurs naturally can result from metabolic or hydrolytic processes. DNA damage plays a major role in the mutagenesis, carcinogenesis, aging and various other patho-physiological conditions. DNA damage can be induced through hydrolysis, exposure to reactive oxygen species (ROS) and other reactive carbonyl metabolites including 4-hydroxynonenal (HNE). 4-HNE is an important lipid peroxidation product which has been implicated in the mutagenesis and carcinogenesis processes. The present study examines to probe the presence of auto-antibodies against 4-hydroxynonenal damaged DNA (HNE-DNA) in various cancer subjects. In this study, the purified calf thymus DNA was damaged by the action of 4-HNE. The DNA was incubated with 4-HNE for 24 h at 37°C temperature. The binding characteristics of cancer auto-antibodies were assessed by direct binding and competitive inhibition ELISA. DNA modifications produced hyperchromicity in UV spectrum and decreased fluorescence intensity. Cancer sera exhibited enhanced binding with the 4-HNE modified calf thymus DNA as compared to its native conformer. The 4-HNE modified DNA presents unique epitopes which may be one of the factors for the auto-antibody induction in cancer patients. The HNE modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients. © 2017 Wiley Periodicals, Inc.

ssDNA degradation along capillary electrophoresis process using a Tris buffer.

Science.gov (United States)

Ric, Audrey; Ong-Meang, Varravaddheay; Poinsot, Verena; Martins-Froment, Nathalie; Chauvet, Fabien; Boutonnet, Audrey; Ginot, Frédéric; Ecochard, Vincent; Paquereau, Laurent; Couderc, François

2017-06-01

Tris-Acetate buffer is currently used in the selection and the characterization of ssDNA by capillary electrophoresis (CE). By applying high voltage, the migration of ionic species into the capillary generates a current that induces water electrolysis. This phenomenon is followed by the modification of the pH and the production of Tris derivatives. By injecting ten times by capillary electrophoresis ssDNA (50 nM), the whole oligonucleotide was degraded. In this paper, we will show that the Tris buffer in the running vials is modified along the electrophoretic process by electrochemical reactions. We also observed that the composition of the metal ions changes in the running buffer vials. This phenomenon, never described in CE, is important for fluorescent ssDNA analysis using Tris buffer. The oligonucleotides are degraded by electrochemically synthesized species (present in the running Tris vials) until it disappears, even if the separation buffer in the capillary is clean. To address these issues, we propose to use a sodium phosphate buffer that we demonstrate to be electrochemically inactive. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

Science.gov (United States)

Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

2013-01-01

WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

Repair of DNA-polypeptide crosslinks by human excision nuclease

Science.gov (United States)

Reardon, Joyce T.; Sancar, Aziz

2006-03-01

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

DNA end resection by CtIP and exonuclease 1 prevents genomic instability

DEFF Research Database (Denmark)

Eid, Wassim; Steger, Martin; El-Shemerly, Mahmoud

2010-01-01

End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombination-relies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and exonucleases such as exonuclease 1 (EXO1). In this s......End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombination-relies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and exonucleases such as exonuclease 1 (EXO1...... of DNA-PK-dependent radial chromosome formation. Thus, our study identifies new functions of CtIP and EXO1 in DNA end resection and provides new information on the regulation of DSB repair pathways, which is a key factor in the maintenance of genome integrity....

Predicting Variation of DNA Shape Preferences in Protein-DNA Interaction in Cancer Cells with a New Biophysical Model.

Science.gov (United States)

Batmanov, Kirill; Wang, Junbai

2017-09-18

DNA shape readout is an important mechanism of transcription factor target site recognition, in addition to the sequence readout. Several machine learning-based models of transcription factor-DNA interactions, considering DNA shape features, have been developed in recent years. Here, we present a new biophysical model of protein-DNA interactions by integrating the DNA shape properties. It is based on the neighbor dinucleotide dependency model BayesPI2, where new parameters are restricted to a subspace spanned by the dinucleotide form of DNA shape features. This allows a biophysical interpretation of the new parameters as a position-dependent preference towards specific DNA shape features. Using the new model, we explore the variation of DNA shape preferences in several transcription factors across various cancer cell lines and cellular conditions. The results reveal that there are DNA shape variations at FOXA1 (Forkhead Box Protein A1) binding sites in steroid-treated MCF7 cells. The new biophysical model is useful for elucidating the finer details of transcription factor-DNA interaction, as well as for predicting cancer mutation effects in the future.

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

Science.gov (United States)

Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

2014-10-31

Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Relationship between DNA repair and cell recovery: Importance of competing biochemical and metabolic processes

International Nuclear Information System (INIS)

Van Ankeren, S.C.; Wheeler, K.T.; Kansas Univ., Lawrence

1985-01-01

The relationship between the inhibition of repair of radiation-induced DNA damage and the inhibition of recovery from radiation-induced potentially lethal damage (PLD) by hypertonic treatment was compared in 9L/Ro rat brain tumor cells. Fed plateau phase cultures were γ-irradiated with 1500 rad and then immediately treated for 20 min with a 37 0 C isotonic (0.15 M) or hypertonic (0.50 M) salt solution. The kinetics of repair of radiation-induced DNA damage as assayed using alkaline filter elution were compared to those of recovery from radiation-induced PLD as assayed by colony formation. hypertonic treatment of unirradiated cells produced neither DNA damage nor cell kill. Post-irradiation hypertonic treatment inhibited both DNA repair and PLD recovery, while post-irradiation istonic treatment inhibited neither phenomenon. However, by 2 h after irradiation, the amount of DNA damage remaining after a 20 min hypertonic treatment was equivalent to that remaining after a 20 min isotonic treatment. In contrast, cell survival after hypertonic treatment remained 2 logs lower than after isotonic treatment even at times up to 24 h. These results suggest that the repair of radiation-induced DNA damage per per se is not causally related to recovery from radiation-induced PLD. However, the data are consistent with the time of DNA repair as an important parameter in determining cell survival and, therefore, tend to support the hypothesis that imbalances in sets of competing biochemical or metabolic processes determine survival rather than the presence of a single class of unrepaired DNA lesions. (orig.)

Investigation of arc repressor DNA-binding specificity by comparative molecular dynamics simulations.

Science.gov (United States)

Song, Wei; Guo, Jun-Tao

2015-01-01

Transcription factors regulate gene expression through binding to specific DNA sequences. How transcription factors achieve high binding specificity is still not well understood. In this paper, we investigated the role of protein flexibility in protein-DNA-binding specificity by comparative molecular dynamics (MD) simulations. Protein flexibility has been considered as a key factor in molecular recognition, which is intrinsically a dynamic process involving fine structural fitting between binding components. In this study, we performed comparative MD simulations on wild-type and F10V mutant P22 Arc repressor in both free and complex conformations. The F10V mutant has lower DNA-binding specificity though both the bound and unbound main-chain structures between the wild-type and F10V mutant Arc are highly similar. We found that the DNA-binding motif of wild-type Arc is structurally more flexible than the F10V mutant in the unbound state, especially for the six DNA base-contacting residues in each dimer. We demonstrated that the flexible side chains of wild-type Arc lead to a higher DNA-binding specificity through forming more hydrogen bonds with DNA bases upon binding. Our simulations also showed a possible conformational selection mechanism for Arc-DNA binding. These results indicate the important roles of protein flexibility and dynamic properties in protein-DNA-binding specificity.

DNA dynamics is likely to be a factor in the genomic nucleotide repeats expansions related to diseases.

Directory of Open Access Journals (Sweden)

Boian S Alexandrov

Full Text Available Trinucleotide repeats sequences (TRS represent a common type of genomic DNA motif whose expansion is associated with a large number of human diseases. The driving molecular mechanisms of the TRS ongoing dynamic expansion across generations and within tissues and its influence on genomic DNA functions are not well understood. Here we report results for a novel and notable collective breathing behavior of genomic DNA of tandem TRS, leading to propensity for large local DNA transient openings at physiological temperature. Our Langevin molecular dynamics (LMD and Markov Chain Monte Carlo (MCMC simulations demonstrate that the patterns of openings of various TRSs depend specifically on their length. The collective propensity for DNA strand separation of repeated sequences serves as a precursor for outsized intermediate bubble states independently of the G/C-content. We report that repeats have the potential to interfere with the binding of transcription factors to their consensus sequence by altered DNA breathing dynamics in proximity of the binding sites. These observations might influence ongoing attempts to use LMD and MCMC simulations for TRS-related modeling of genomic DNA functionality in elucidating the common denominators of the dynamic TRS expansion mutation with potential therapeutic applications.

Direct PCR amplification of forensic touch and other challenging DNA samples: A review.

Science.gov (United States)

Cavanaugh, Sarah E; Bathrick, Abigail S

2018-01-01

DNA evidence sample processing typically involves DNA extraction, quantification, and STR amplification; however, DNA loss can occur at both the DNA extraction and quantification steps, which is not ideal for forensic evidence containing low levels of DNA. Direct PCR amplification of forensic unknown samples has been suggested as a means to circumvent extraction and quantification, thereby retaining the DNA typically lost during those procedures. Direct PCR amplification is a method in which a sample is added directly to an amplification reaction without being subjected to prior DNA extraction, purification, or quantification. It allows for maximum quantities of DNA to be targeted, minimizes opportunities for error and contamination, and reduces the time and monetary resources required to process samples, although data analysis may take longer as the increased DNA detection sensitivity of direct PCR may lead to more instances of complex mixtures. ISO 17025 accredited laboratories have successfully implemented direct PCR for limited purposes (e.g., high-throughput databanking analysis), and recent studies indicate that direct PCR can be an effective method for processing low-yield evidence samples. Despite its benefits, direct PCR has yet to be widely implemented across laboratories for the processing of evidentiary items. While forensic DNA laboratories are always interested in new methods that will maximize the quantity and quality of genetic information obtained from evidentiary items, there is often a lag between the advent of useful methodologies and their integration into laboratories. Delayed implementation of direct PCR of evidentiary items can be attributed to a variety of factors, including regulatory guidelines that prevent laboratories from omitting the quantification step when processing forensic unknown samples, as is the case in the United States, and, more broadly, a reluctance to validate a technique that is not widely used for evidence samples. The

High Efficiency Hydrodynamic DNA Fragmentation in a Bubbling System.

Science.gov (United States)

Li, Lanhui; Jin, Mingliang; Sun, Chenglong; Wang, Xiaoxue; Xie, Shuting; Zhou, Guofu; van den Berg, Albert; Eijkel, Jan C T; Shui, Lingling

2017-01-18

DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling system. We expect that hydrodynamic forces generated during the bubbling process shear the DNA molecules, extending and breaking them at the points where shearing forces are larger than the strength of the phosphate backbone. Factors of applied pressure, bubbling time and temperature have been investigated. Genomic DNA could be fragmented down to controllable 1-10 Kbp fragment lengths with a yield of 75.30-91.60%. We demonstrate that the ends of the genomic DNAs generated from hydrodynamic shearing can be ligated by T4 ligase and the fragmented DNAs can be used as templates for polymerase chain reaction. Therefore, in the bubbling system, DNAs could be hydrodynamically sheared to achieve smaller pieces in dsDNAs available for further processes. It could potentially serve as a DNA sample pretreatment technique in the future.

Archaeal RNA polymerase arrests transcription at DNA lesions.

Science.gov (United States)

Gehring, Alexandra M; Santangelo, Thomas J

2017-01-01

Transcription elongation is not uniform and transcription is often hindered by protein-bound factors or DNA lesions that limit translocation and impair catalysis. Despite the high degree of sequence and structural homology of the multi-subunit RNA polymerases (RNAP), substantial differences in response to DNA lesions have been reported. Archaea encode only a single RNAP with striking structural conservation with eukaryotic RNAP II (Pol II). Here, we demonstrate that the archaeal RNAP from Thermococcus kodakarensis is sensitive to a variety of DNA lesions that pause and arrest RNAP at or adjacent to the site of DNA damage. DNA damage only halts elongation when present in the template strand, and the damage often results in RNAP arresting such that the lesion would be encapsulated with the transcription elongation complex. The strand-specific halt to archaeal transcription elongation on modified templates is supportive of RNAP recognizing DNA damage and potentially initiating DNA repair through a process akin to the well-described transcription-coupled DNA repair (TCR) pathways in Bacteria and Eukarya.

Assembly and melting of DNA nanotubes from single-sequence tiles

International Nuclear Information System (INIS)

Sobey, T L; Renner, S; Simmel, F C

2009-01-01

DNA melting and renaturation studies are an extremely valuable tool to study the kinetics and thermodynamics of duplex dissociation and reassociation reactions. These are important not only in a biological or biotechnological context, but also for DNA nanotechnology which aims at the construction of molecular materials by DNA self-assembly. We here study experimentally the formation and melting of a DNA nanotube structure, which is composed of many copies of an oligonucleotide containing several palindromic sequences. This is done using temperature-controlled UV absorption measurements correlated with atomic force microscopy, fluorescence microscopy and transmission electron microscopy techniques. In the melting studies, important factors such as DNA strand concentration, hierarchy of assembly and annealing protocol are investigated. Assembly and melting of the nanotubes are shown to proceed via different pathways. Whereas assembly occurs in several hierarchical steps related to the formation of tiles, lattices and tubes, melting of DNA nanotubes appears to occur in a single step. This is proposed to relate to fundamental differences between closed, three-dimensional tube-like structures and open, two-dimensional lattices. DNA melting studies can lead to a better understanding of the many factors that affect the assembly process which will be essential for the assembly of increasingly complex DNA nanostructures.

Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus

DEFF Research Database (Denmark)

Peng, Wenfang; Li, Huan; Hallstrøm, Søren

2013-01-01

-adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3' and Cas3......" are essential for PAM-dependent DNA targeting activity, whereas Csa5, along with all other Cas modules, is dispensable for the targeting in the crenarchaeon. Cas6 is implicated as the only enzyme for pre-crRNA processing and the crRNA maturation is independent of the DNA targeting activity. Importantly, we show...

RTEL1 contributes to DNA replication and repair and telomere maintenance.

OpenAIRE

Uringa, E.-J.; Lisaingo, K.; Pickett, H. A.; Brind'Amour, J.; Rohde, J.-H.; Zelensky, A.; Essers, J.; Lansdorp, P. M.

2012-01-01

textabstractTelomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and...

Generalized theory on the mechanism of site-specific DNA-protein interactions

Science.gov (United States)

Niranjani, G.; Murugan, R.

2016-05-01

We develop a generalized theoretical framework on the binding of transcription factor proteins (TFs) with specific sites on DNA that takes into account the interplay of various factors regarding overall electrostatic potential at the DNA-protein interface, occurrence of kinetic traps along the DNA sequence, presence of other roadblock protein molecules along DNA and crowded environment, conformational fluctuations in the DNA binding domains (DBDs) of TFs, and the conformational state of the DNA. Starting from a Smolochowski type theoretical framework on site-specific binding of TFs we logically build our model by adding the effects of these factors one by one. Our generalized two-step model suggests that the electrostatic attractive forces present inbetween the positively charged DBDs of TFs and the negatively charged phosphate backbone of DNA, along with the counteracting shielding effects of solvent ions, is the core factor that creates a fluidic type environment at the DNA-protein interface. This in turn facilitates various one-dimensional diffusion (1Dd) processes such as sliding, hopping and intersegmental transfers. These facilitating processes as well as flipping dynamics of conformational states of DBDs of TFs between stationary and mobile states can enhance the 1Dd coefficient on a par with three-dimensional diffusion (3Dd). The random coil conformation of DNA also plays critical roles in enhancing the site-specific association rate. The extent of enhancement over the 3Dd controlled rate seems to be directly proportional to the maximum possible 1Dd length. We show that the overall site-specific binding rate scales with the length of DNA in an asymptotic way. For relaxed DNA, the specific binding rate will be independent of the length of DNA as length increases towards infinity. For condensed DNA as in in vivo conditions, the specific binding rate depends on the length of DNA in a turnover way with a maximum. This maximum rate seems to scale with the

DNA damage assessment by visualization and quantification of DNA damage response

International Nuclear Information System (INIS)

Matsuda, Shun; Matsuda, Tomonari; Ikura, Tsuyoshi

2017-01-01

DNA damage response (DDR) carries out signal transduction for DNA repair, activation of cell cycle checkpoint, and apoptosis to maintain genome integrity, in response to DNA damage. Many proteins and their post-translational modifications participate in the process. Especially, S139-phosphorylated histone H2AX (γH2AX), which is formed by DNA double-strand breaks (DSBs), is an important factor to bring and retain other DDR proteins to DSB sites, Thus, γH2AX is used as a good indicator of DSBs in clinical study and pharmacology for efficacy evaluation of chemotherapy and radiotherapy, detection of precancerous regions, and others. In regulatory science, γH2AX is also a useful biomarker of genotoxicity of chemicals, since a wide range of genotoxic chemicals induce γH2AX. However, conventional detection methods of γH2AX absolutely require anti-γH2AX antibody whose staining is burdensome and time-consuming, and some of these methods are not so superior in quantitativity. In this review, we introduce two new methods to overcome these limitations, involving an easy-to-use genotoxicity assay using DDR-visualizing cells and an absolute quantification method of γH2AX using liquid chromatography-tandem mass spectrometry (LC/MS/MS). (author)

Analysis of the DNA-Binding Activities of the Arabidopsis R2R3-MYB Transcription Factor Family by One-Hybrid Experiments in Yeast.

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Zsolt Kelemen

Full Text Available The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs. Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences.

THE YIELD OF DNA IN THERMAL TERATED DEER MEAT

Directory of Open Access Journals (Sweden)

Jozef Golian

2011-07-01

Full Text Available Residuals of DNA are one of the most important factors for detection, traceability and reverse authentication of deer meat. In this project we isolated DNA from deer processed meat and analysed by electrophoresis. Goal of the study was compute ratio between raw meat and several heat processed deer meat. Samples were prepared by five heat treatment techniques (pan roasted with temperature 180-240°C, fried with 156°C, braised with temperature 100-150°C, boiled in 100.2°C water and autoclaved in different time intervals. The highest amount of residual DNA 1927ng was obtained with two hours boiled sample. The lowest value 89.89ng was obtained with one hour braised sample. In technological adjustments highest amount of DNA and 1927ng, so the total yield of 192.7ng.-l was observed in the sample we cooked for two hours at boiling temperature.   doi:10.5219/153 

CEBPG transcription factor correlates with antioxidant and DNA repair genes in normal bronchial epithelial cells but not in individuals with bronchogenic carcinoma

International Nuclear Information System (INIS)

Mullins, D'Anna N; Crawford, Erin L; Khuder, Sadik A; Hernandez, Dawn-Alita; Yoon, Youngsook; Willey, James C

2005-01-01

Cigarette smoking is the primary cause of bronchogenic carcinoma (BC), yet only 10–15% of heavy smokers develop BC and it is likely that this variation in risk is, in part, genetically determined. We previously reported a set of antioxidant genes for which transcript abundance was lower in normal bronchial epithelial cells (NBEC) of BC individuals compared to non-BC individuals. In unpublished studies of the same NBEC samples, transcript abundance values for several DNA repair genes were correlated with these antioxidant genes. From these data, we hypothesized that antioxidant and DNA repair genes are co-regulated by one or more transcription factors and that inter-individual variation in expression and/or function of one or more of these transcription factors is responsible for inter-individual variation in risk for BC. The putative transcription factor recognition sites common to six of the antioxidant genes were identified through in silico DNA sequence analysis. The transcript abundance values of these transcription factors (n = 6) and an expanded group of antioxidant and DNA repair genes (n = 16) were measured simultaneously by quantitative PCR in NBEC of 24 non-BC and 25 BC individuals. CEBPG transcription factor was significantly (p < 0.01) correlated with eight of the antioxidant or DNA repair genes in non-BC individuals but not in BC individuals. In BC individuals the correlation with CEBPG was significantly (p < 0.01) lower than that of non-BC individuals for four of the genes (XRCC1, ERCC5, GSTP1, and SOD1) and the difference was nearly significant for GPX1. The only other transcription factor correlated with any of these five target genes in non-BC individuals was E2F1. E2F1 was correlated with GSTP1 among non-BC individuals, but in contrast to CEBPG, there was no significant difference in this correlation in non-BC individuals compared to BC individuals. We conclude that CEBPG is the transcription factor primarily responsible for regulating

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.

SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair

DEFF Research Database (Denmark)

McCord, Ronald A; Michishita, Eriko; Hong, Tao

2009-01-01

-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor......-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA......, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA...

Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

Science.gov (United States)

Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta’ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

2016-01-01

The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology. PMID:27435636

Impact of Age and Insulin-Like Growth Factor-1 on DNA Damage Responses in UV-Irradiated Human Skin.

Science.gov (United States)

Kemp, Michael G; Spandau, Dan F; Travers, Jeffrey B

2017-02-26

The growing incidence of non-melanoma skin cancer (NMSC) necessitates a thorough understanding of its primary risk factors, which include exposure to ultraviolet (UV) wavelengths of sunlight and age. Whereas UV radiation (UVR) has long been known to generate photoproducts in genomic DNA that promote genetic mutations that drive skin carcinogenesis, the mechanism by which age contributes to disease pathogenesis is less understood and has not been sufficiently studied. In this review, we highlight studies that have considered age as a variable in examining DNA damage responses in UV-irradiated skin and then discuss emerging evidence that the reduced production of insulin-like growth factor-1 (IGF-1) by senescent fibroblasts in the dermis of geriatric skin creates an environment that negatively impacts how epidermal keratinocytes respond to UVR-induced DNA damage. In particular, recent data suggest that two principle components of the cellular response to DNA damage, including nucleotide excision repair and DNA damage checkpoint signaling, are both partially defective in keratinocytes with inactive IGF-1 receptors. Overcoming these tumor-promoting conditions in aged skin may therefore provide a way to lower aging-associated skin cancer risk, and thus we will consider how dermal wounding and related clinical interventions may work to rejuvenate the skin, re-activate IGF-1 signaling, and prevent the initiation of NMSC.

Impact of Age and Insulin-Like Growth Factor-1 on DNA Damage Responses in UV-Irradiated Human Skin

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Michael G. Kemp

2017-02-01

Full Text Available The growing incidence of non-melanoma skin cancer (NMSC necessitates a thorough understanding of its primary risk factors, which include exposure to ultraviolet (UV wavelengths of sunlight and age. Whereas UV radiation (UVR has long been known to generate photoproducts in genomic DNA that promote genetic mutations that drive skin carcinogenesis, the mechanism by which age contributes to disease pathogenesis is less understood and has not been sufficiently studied. In this review, we highlight studies that have considered age as a variable in examining DNA damage responses in UV-irradiated skin and then discuss emerging evidence that the reduced production of insulin-like growth factor-1 (IGF-1 by senescent fibroblasts in the dermis of geriatric skin creates an environment that negatively impacts how epidermal keratinocytes respond to UVR-induced DNA damage. In particular, recent data suggest that two principle components of the cellular response to DNA damage, including nucleotide excision repair and DNA damage checkpoint signaling, are both partially defective in keratinocytes with inactive IGF-1 receptors. Overcoming these tumor-promoting conditions in aged skin may therefore provide a way to lower aging-associated skin cancer risk, and thus we will consider how dermal wounding and related clinical interventions may work to rejuvenate the skin, re-activate IGF-1 signaling, and prevent the initiation of NMSC.

DNA-based machines.

Science.gov (United States)

Wang, Fuan; Willner, Bilha; Willner, Itamar

2014-01-01

The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications.

The radioprotector O-phospho-tyrosine stimulates DNA-repair via epidermal growth factor receptor- and DNA-dependent kinase phosphorylation

International Nuclear Information System (INIS)

Dittmann, Klaus; Mayer, Claus; Wanner, Gabriele; Kehlbach, Rainer; Rodemann, H. Peter

2007-01-01

Background and purpose: Purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector O-phospho-tyrosine (P-Tyr). Methods: Molecular effects of P-Tyr at the level of EGFR responses were investigated in vitro with bronchial carcinoma cell line A549. Nuclear EGFR transport and DNA-PK activation were quantified after Western blotting. Residual DNA-damages were quantified by help of γH 2 AX focus assay. Results: As determined by dose-response curves, treatment of cells with P-Tyr for 16 h before irradiation results in radioprotection. Simultaneous treatment with EGFR blocking antibody Cetuximab abolished P-Tyr associated radioprotection. At the molecular level P-Tyr mediated a general phosphorylation of EGFR and a pronounced phosphorylation of nuclear EGFR at residue Thr No. 654, also observed after treatment with ionizing radiation. This phosphorylation was associated with nuclear EGFR accumulation. Moreover, P-Tyr-triggered EGFR nuclear accumulation was associated with phosphorylation of DNA-PK at Thr 2609. This activated form of DNA-PK was not DNA associated, but after radiation, DNA binding increased, particularly after P-Tyr pre-treatment. These molecular effects of P-Tyr resulted in a reduction of residual DNA-damage after irradiation. Conclusions: Radioprotection by P-Tyr is mediated through its stimulation of nuclear EGFR transport and concurrent, but DNA-damage independent, activation of DNA-PK. Thus, subsequent irradiation results in increased binding of DNA-PK to DNA, improved DNA-repair and increased cell survival

Overexpression of insulin-like growth factor (IGF)-I receptor enhances inhibition of DNA replication in mouse cells exposed to x-rays

International Nuclear Information System (INIS)

Wang, Y.; Cheong, N.; Miura, M.; Iliakis, G.

1997-01-01

Previous studies from our laboratory provided evidence for the operation of signal transduction pathways involving ras, myc, and staurosporine-sensitive protein kinases in the regulation of DNA replication in irradiated cells. Because ras and myc are also involved in the signal transduction elicited in response to ligand activation of growth factor receptors, we wondered whether growth factor receptors are upstream elements in the regulation of DNA replication in irradiated cells. Here, we report on the role of insulin-like growth factor I receptor (IGF-IR) in the regulation of DNA replication in irradiated cells. We compare radiation-induced inhibition of DNA replication in BALB/c 3T3 cells with that in P6 cells. P6 cells are derived from BALB/c 3T3 cells by transfection with a vector expressing IGF-IR, leading to 30-fold overexpression. We observe a significantly stronger inhibition of DNA replication after irradiation in P6 as compared with BALB/c 3T3 cells at all doses examined. Sedimentation in alkaline sucrose gradients shows that the increased inhibition in P6 cells is due to an increased inhibition of replicon initiation, the main controlling event in DNA replication. Staurosporine at 20 nM reduces radiation-induced inhibition of DNA replication in BALB/c 3T3 cells, but has only a small effect in P6 cells. Caffeine at a concentration of 1 mM, on the other hand, removes over 60% of the inhibition in both cell lines. The results implicate IGF-IR in the regulation of DNA replication in irradiated cells, but also suggest differences between cells of different origins in the proteins involved in the regulating signal transduction pathway. (orig.). With 5 figs

Inherited DNA repair defects in H. sapiens: their relation to uv-associated processes in xeroderma pigmentosum

International Nuclear Information System (INIS)

Robbins, J.H.; Kraemer, K.H.; Andrews, A.D.

1976-01-01

Xeroderma pigmentosum (XP) is an autosomal recessive disease in which patients develop pigmentation abnormalities and numerous malignancies on areas of skin exposed to sunlight. Some XP patients have neurological abnormalities in addition to their cutaneous pathology. Genetic defects in DNA repair have now been found in all studied XP patients. Here, we shall review and present studies relating the different inherited DNA repair defects of XP to several uv-associated processes. Peripheral blood lymphocytes and skin fibroblasts obtained from patients were cultured and the uv-induced thymidine incorporation in DNA was measured by autoradiography or by scintillation spectroscopy

Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family.

Science.gov (United States)

Dai, Qi; Ren, Aiming; Westholm, Jakub O; Duan, Hong; Patel, Dinshaw J; Lai, Eric C

2015-01-01

Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain ("BEN-solo" factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional

Nanostructures via DNA scaffold metallization

OpenAIRE

Ning, C.; Zinchenko, A.; Baigl, D.; Pyshkina, O.; Sergeyev, V.; Endo, Kazunaka; Yoshikawa, K.

2005-01-01

The critical role of polymers in process of noble metals nanostructures formation is well known, however, the use of DNA chain template in this process is yet largely unknown. In this study we demonstrate different ways of silver deposition on DNA template and report the influence of silver nanostructures formation on DNA conformational state. Metallization of DNA chain proceeds by two different scenarios depending on DNA conformation. If DNA chain is unfolded (elongated) chain, silver reduct...

Retroviral DNA Integration

Science.gov (United States)

2016-01-01

The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

Science.gov (United States)

Rajabi, H; Mohseni-Kouchesfehani, H; Eslami-Arshaghi, T; Salehi, M

2018-02-01

To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation. © 2017 Blackwell Verlag GmbH.

Mitochondrial and Nuclear DNA Damage and Repair in Age-Related Macular Degeneration

Directory of Open Access Journals (Sweden)

Janusz Blasiak

2013-01-01

Full Text Available Aging and oxidative stress seem to be the most important factors in the pathogenesis of age-related macular degeneration (AMD, a condition affecting many elderly people in the developed world. However, aging is associated with the accumulation of oxidative damage in many biomolecules, including DNA. Furthermore, mitochondria may be especially important in this process because the reactive oxygen species produced in their electron transport chain can damage cellular components. Therefore, the cellular response to DNA damage, expressed mainly through DNA repair, may play an important role in AMD etiology. In several studies the increase in mitochondrial DNA (mtDNA damage and mutations, and the decrease in the efficacy of DNA repair have been correlated with the occurrence and the stage of AMD. It has also been shown that mitochondrial DNA accumulates more DNA lesions than nuclear DNA in AMD. However, the DNA damage response in mitochondria is executed by nucleus-encoded proteins, and thus mutagenesis in nuclear DNA (nDNA may affect the ability to respond to mutagenesis in its mitochondrial counterpart. We reported that lymphocytes from AMD patients displayed a higher amount of total endogenous basal and oxidative DNA damage, exhibited a higher sensitivity to hydrogen peroxide and UV radiation, and repaired the lesions induced by these factors less effectively than did cells from control individuals. We postulate that poor efficacy of DNA repair (i.e., is impaired above average for a particular age when combined with the enhanced sensitivity of retinal pigment epithelium cells to environmental stress factors, contributes to the pathogenesis of AMD. Collectively, these data suggest that the cellular response to both mitochondrial and nuclear DNA damage may play an important role in AMD pathogenesis.

Epigenomic maintenance through dietary intervention can facilitate DNA repair process to slow down the progress of premature aging.

Science.gov (United States)

Ghosh, Shampa; Sinha, Jitendra Kumar; Raghunath, Manchala

2016-09-01

DNA damage caused by various sources remains one of the most researched topics in the area of aging and neurodegeneration. Increased DNA damage causes premature aging. Aging is plastic and is characterised by the decline in the ability of a cell/organism to maintain genomic stability. Lifespan can be modulated by various interventions like calorie restriction, a balanced diet of macro and micronutrients or supplementation with nutrients/nutrient formulations such as Amalaki rasayana, docosahexaenoic acid, resveratrol, curcumin, etc. Increased levels of DNA damage in the form of double stranded and single stranded breaks are associated with decreased longevity in animal models like WNIN/Ob obese rats. Erroneous DNA repair can result in accumulation of DNA damage products, which in turn result in premature aging disorders such as Hutchinson-Gilford progeria syndrome. Epigenomic studies of the aging process have opened a completely new arena for research and development of drugs and therapeutic agents. We propose here that agents or interventions that can maintain epigenomic stability and facilitate the DNA repair process can slow down the progress of premature aging, if not completely prevent it. © 2016 IUBMB Life, 68(9):717-721, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

LEDGF/p75 Deficiency Increases Deletions at the HIV-1 cDNA Ends.

Science.gov (United States)

Bueno, Murilo T D; Reyes, Daniel; Llano, Manuel

2017-09-15

Processing of unintegrated linear HIV-1 cDNA by the host DNA repair system results in its degradation and/or circularization. As a consequence, deficient viral cDNA integration generally leads to an increase in the levels of HIV-1 cDNA circles containing one or two long terminal repeats (LTRs). Intriguingly, impaired HIV-1 integration in LEDGF/p75-deficient cells does not result in a correspondent increase in viral cDNA circles. We postulate that increased degradation of unintegrated linear viral cDNA in cells lacking the lens epithelium-derived growth factor (LEDGF/p75) account for this inconsistency. To evaluate this hypothesis, we characterized the nucleotide sequence spanning 2-LTR junctions isolated from LEDGF/p75-deficient and control cells. LEDGF/p75 deficiency resulted in a significant increase in the frequency of 2-LTRs harboring large deletions. Of note, these deletions were dependent on the 3' processing activity of integrase and were not originated by aberrant reverse transcription. Our findings suggest a novel role of LEDGF/p75 in protecting the unintegrated 3' processed linear HIV-1 cDNA from exonucleolytic degradation.

Diet-Induced Weight Loss Reduces DNA Damage and Cardiometabolic Risk Factors in Overweight/Obese Women with Polycystic Ovary Syndrome.

Science.gov (United States)

Soares, Nayara Pereira; Santos, Ana Celly Souza dos; Costa, Eduardo Caldas; Azevedo, George Dantas; Damasceno, Débora Cristina; Fayh, Ana Paula Trussardi; Lemos, Telma Maria Araújo Moura

2016-01-01

We aimed to investigate the impact of following a diet to induce weight loss (500 kcal deficit per day) over DNA damage and cardiometabolic risk factors in women with overweight/obesity diagnosed with polycystic ovary syndrome (PCOS). A study was conducted in Natal, RN, Brazil selecting overweight/obese (body mass index ≥25 and weight loss, decreased sexual hormone and cardiometabolic markers such as insulin, homeostasis model assessment of insulin resistance and low-density lipoprotein cholesterol were verified In the multivariate regression analysis, quantitative insulin sensitivity check index and progesterone were responsible for the variation markers in DNA damage before the diet, losing its influence upon diet. DNA damage and the impact of cardiometabolic risk factors decreased after the intervention in women with PCOS, indicating the relevance of a nutritional approach in this group of patients. © 2016 S. Karger AG, Basel.

Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

International Nuclear Information System (INIS)

Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

1990-01-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

Science.gov (United States)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

[Identification of an auxin response factor-like protein cDNA from mango cotyledon section].

Science.gov (United States)

Xiao, Jie-Ning; Huang, Xue-Lin; Huang, Xia; Li, Xiao-Ju

2004-01-01

Auxin-responsive elements (AuxRE) interact with a new class of plant-specific transcription factors, auxin response factors (ARFs). Some of ARFs have been shown to repress or activate expression of genes with an AuxRE promotor element. In Arabidopsis, ARFs play important roles in early embryo development and vascular strand formation (ARF5), floral patterning (ARF3) and photo- and gravitropic responses (ARF7). Two cut surfaces (distal and proximal) of mango (Mangifera indica L. var. Zi-Hua) cotyledon showed different patterns of adventitious root formation, with only the proximal cut surface, but not the distal one, could be induced to form the roots. Thus, the mango cotyledon is a good system for studying adventitious root formation. A cDNA fragment homologous to the Arabidopsis auxin response factor-like protein and relates to adventitious root formation from the cut sections were isolated using suppressive subtractive hybridization (SSH). Two cDNA clones, designated as MiARF1 (mango auxin response factor 1 gene, GenBank accession number AY255705) and MiARF2 (mango auxin response factor 2 gene, GenBank accession number is AY300808), were identified by 3'RACE. MiARF1, 3 272bp long, contains an open reading frame (ORF) of 2 523bp, 5'UTR of 285bp and 3'UTR of 464bp, MiARF2, 1 474bp long, contains an ORF of 981bp, 5' UTR of 285bp and 3'UTR of 208bp. The deduced MiARF1 and MiARF2 are homologues of auxin response factor (ARF) family of transcriptional regulators, and show high similarity to ARF of Arabidopsis in conserved domains. The motifs of MiARF1 EL-WHACAGPL in DBD (DNA binding domain) and GDDPW in IV domain are identical to that of ARF-like protein of Arabidopsis. MiARF2 is identical to MiARF1 in a large part of DBD, but lacks a carboxyl-terminal domain containing conserved motifs III and IV. Virtual Northern blot showed that the expression of MiARF2 was high in rooting tissue of cultured cotyledon sections but low in non-rooting tissue, and the MiARF1 was

Signalign: An Ontology of DNA as Signal for Comparative Gene Structure Prediction Using Information-Coding-and-Processing Techniques.

Science.gov (United States)

Yu, Ning; Guo, Xuan; Gu, Feng; Pan, Yi

2016-03-01

Conventional character-analysis-based techniques in genome analysis manifest three main shortcomings-inefficiency, inflexibility, and incompatibility. In our previous research, a general framework, called DNA As X was proposed for character-analysis-free techniques to overcome these shortcomings, where X is the intermediates, such as digit, code, signal, vector, tree, graph network, and so on. In this paper, we further implement an ontology of DNA As Signal, by designing a tool named Signalign for comparative gene structure analysis, in which DNA sequences are converted into signal series, processed by modified method of dynamic time warping and measured by signal-to-noise ratio (SNR). The ontology of DNA As Signal integrates the principles and concepts of other disciplines including information coding theory and signal processing into sequence analysis and processing. Comparing with conventional character-analysis-based methods, Signalign can not only have the equivalent or superior performance, but also enrich the tools and the knowledge library of computational biology by extending the domain from character/string to diverse areas. The evaluation results validate the success of the character-analysis-free technique for improved performances in comparative gene structure prediction.

Cell- and virus-mediated regulation of the barrier-to-autointegration factor's phosphorylation state controls its DNA binding, dimerization, subcellular localization, and antipoxviral activity.

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Jamin, Augusta; Wicklund, April; Wiebe, Matthew S

2014-05-01

Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAF's ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAF's mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAF's dimerization and its DNA binding activity. Furthermore, our studies of BAF's antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus, both DNA binding and localization are important determinants of BAF's antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well. The barrier-to-autointegration factor (BAF) contributes to cellular genomic integrity in multiple ways

Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

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Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

2016-08-01

The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. Copyright © 2016 Elsevier B.V. All rights reserved.

Deuterium isotope effects and fractionation factors of hydrogen-bonded A:T base pairs of DNA

International Nuclear Information System (INIS)

Vakonakis, Ioannis; Salazar, Miguel; Kang, Mijeong; Dunbar, Kim R.; Li Wang, Andy C.

2003-01-01

Deuterium isotope effects and fractionation factors of N1...H3-N3 hydrogen bonded Watson-Crick A:T base pairs of two DNA dodecamers are presented here. Specifically, two-bond deuterium isotope effects on the chemical shifts of 13 C2 and 13 C4, 2 Δ 13 C2 and 2 Δ 13 C4, and equilibrium deuterium/protium fractionation factors of H3, Φ, were measured and seen to correlate with the chemical shift of the corresponding imino proton, δ H3 . Downfield-shifted imino protons associated with larger values of 2 Δ 13 C2 and 2 Δ 13 C4 and smaller Φ values, which together suggested that the effective H3-N3 vibrational potentials were more anharmonic in the stronger hydrogen bonds of these DNA molecules. We anticipate that 2 Δ 13 C2, 2 Δ 13 C4 and Φ values can be useful gauges of hydrogen bond strength of A:T base pairs

A systematic review on sperm DNA fragmentation in male factor infertility: Laboratory assessment

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Manesh Kumar Panner Selvam

2018-03-01

Full Text Available Objective: To review sperm DNA fragmentation (SDF testing as an important sperm function test in addition to conventional semen analysis. High SDF is negatively associated with semen quality, the fertilisation process, embryo quality, and pregnancy outcome. Over recent decades, different SDF assays have been developed and reviewed extensively to assess their applicability and accuracy as advanced sperm function tests. Amongst them, the standardisation of the terminal deoxynucleotidyl transferased UTP nick-end labelling (TUNEL assay with a bench top flow cytometer in clinical practice deserves special mention with a threshold value of 16.8% to differentiate infertile men with DNA damage from fertile men. Materials and methods: A systematic literature search was performed through the PubMed, Medline, and ScienceDirect databases using the keywords ‘sperm DNA fragmentation’ and ‘laboratory assessment’. Non-English articles were excluded and studies related to humans were only included. Results: Of the 618 identified, 87 studies (original research and reviews and in addition eight book chapters meeting the selection criteria were included in this review. In all, 366 articles were rejected in the preliminary screening and a further 165 articles related to non-human subjects were excluded. Conclusion: There are pros and cons to all the available SDF assays. TUNEL is a reliable technique with greater accuracy and as an additional diagnostic test in Andrology laboratories along with basic semen analysis can predict fertility outcome, and thus direct the choice of an assisted reproductive technology procedure for infertile couples. Also, the TUNEL assay can be used as a prognostic test and results are beneficial in deciding personalised treatment for infertile men. Keywords: Sperm DNA fragmentation (SDF, Terminal deoxynucleotidyl transferased UTP nick-end labelling (TUNEL, DNA damage, Sperm DNA fragmentation (SDF assay

WRN Exonuclease Structure, Molecular Mechanism, and DNA EndProcessing Role

Energy Technology Data Exchange (ETDEWEB)

Perry, J. Jefferson P.; Yannone, Steven M.; Holden, Lauren G.; Hitomi, Chiharu; Asaithamby, Aroumougame; Han, Seungil; Cooper, PriscillaK.; Chen, David J.; Tainer, John A.

2006-02-15

WRN is unique among the five human RecQ DNA helicases by having a functional exonuclease domain (WRN-exo) and being defective in the premature aging and cancer-related disorder Werner syndrome. Here, we characterize WRN-exo crystal structures, biochemical activity and participation in DNA end-joining. Metal ion complex structures, active site mutations and activity assays reveal a two-metal-ion mediated nuclease mechanism. The DNA end-binding Ku70/80 complex specifically stimulates WRN-exo activity, and structure-based mutational inactivation of WRN-exo alters DNA end-joining in human cells. We furthermore establish structural and biochemical similarities of WRN-exo to DnaQ family replicative proofreading exonucleases, with WRN-specific adaptations consistent with dsDNA specificity and functionally important conformational changes. These results indicate WRN-exo is a human DnaQ family member and support analogous proof-reading activities that are stimulated by Ku70/80 with implications for WRN functions in age related pathologies and maintenance of genomic integrity.

Identification and positional distribution analysis of transcription factor binding sites for genes from the wheat fl-cDNA sequences.

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Chen, Zhen-Yong; Guo, Xiao-Jiang; Chen, Zhong-Xu; Chen, Wei-Ying; Wang, Ji-Rui

2017-06-01

The binding sites of transcription factors (TFs) in upstream DNA regions are called transcription factor binding sites (TFBSs). TFBSs are important elements for regulating gene expression. To date, there have been few studies on the profiles of TFBSs in plants. In total, 4,873 sequences with 5' upstream regions from 8530 wheat fl-cDNA sequences were used to predict TFBSs. We found 4572 TFBSs for the MADS TF family, which was twice as many as for bHLH (1951), B3 (1951), HB superfamily (1914), ERF (1820), and AP2/ERF (1725) TFs, and was approximately four times higher than the remaining TFBS types. The percentage of TFBSs and TF members showed a distinct distribution in different tissues. Overall, the distribution of TFBSs in the upstream regions of wheat fl-cDNA sequences had significant difference. Meanwhile, high frequencies of some types of TFBSs were found in specific regions in the upstream sequences. Both TFs and fl-cDNA with TFBSs predicted in the same tissues exhibited specific distribution preferences for regulating gene expression. The tissue-specific analysis of TFs and fl-cDNA with TFBSs provides useful information for functional research, and can be used to identify relationships between tissue-specific TFs and fl-cDNA with TFBSs. Moreover, the positional distribution of TFBSs indicates that some types of wheat TFBS have different positional distribution preferences in the upstream regions of genes.

TbPIF5 is a Trypanosoma brucei mitochondrial DNA helicase involved in processing of minicircle Okazaki fragments.

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Beiyu Liu

2009-09-01

Full Text Available Trypanosoma brucei's mitochondrial genome, kinetoplast DNA (kDNA, is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei's six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5' to 3' DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb, are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments.

Elg1 forms an alternative RFC complex important for DNA replication and genome integrity

NARCIS (Netherlands)

Bellaoui, Mohammed; Chang, Michael; Ou, Jiongwen; Xu, Hong; Boone, Charles; Brown, Grant W

2003-01-01

Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA

Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition.

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Woodcock, Clayton B; Yakubov, Aziz B; Reich, Norbert O

2017-08-01

Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K m , k cat , k methylation , and K d for single-stranded and hemimethylated substrates, revealing discrimination of 10 7 -fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

Fast parallel DNA-based algorithms for molecular computation: quadratic congruence and factoring integers.

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Chang, Weng-Long

2012-03-01

Assume that n is a positive integer. If there is an integer such that M (2) ≡ C (mod n), i.e., the congruence has a solution, then C is said to be a quadratic congruence (mod n). If the congruence does not have a solution, then C is said to be a quadratic noncongruence (mod n). The task of solving the problem is central to many important applications, the most obvious being cryptography. In this article, we describe a DNA-based algorithm for solving quadratic congruence and factoring integers. In additional to this novel contribution, we also show the utility of our encoding scheme, and of the algorithm's submodules. We demonstrate how a variety of arithmetic, shifted and comparative operations, namely bitwise and full addition, subtraction, left shifter and comparison perhaps are performed using strands of DNA.

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

Science.gov (United States)

NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

FOXO3 Transcription Factor Is Essential for Protecting Hematopoietic Stem and Progenitor Cells from Oxidative DNA Damage.

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Bigarella, Carolina L; Li, Jianfeng; Rimmelé, Pauline; Liang, Raymond; Sobol, Robert W; Ghaffari, Saghi

2017-02-17

Accumulation of damaged DNA in hematopoietic stem cells (HSC) is associated with chromosomal abnormalities, genomic instability, and HSC aging and might promote hematological malignancies with age. Despite this, the regulatory pathways implicated in the HSC DNA damage response have not been fully elucidated. One of the sources of DNA damage is reactive oxygen species (ROS) generated by both exogenous and endogenous insults. Balancing ROS levels in HSC requires FOXO3, which is an essential transcription factor for HSC maintenance implicated in HSC aging. Elevated ROS levels result in defective Foxo3 -/- HSC cycling, among many other deficiencies. Here, we show that loss of FOXO3 leads to the accumulation of DNA damage in primitive hematopoietic stem and progenitor cells (HSPC), associated specifically with reduced expression of genes implicated in the repair of oxidative DNA damage. We provide further evidence that Foxo3 -/- HSPC are defective in DNA damage repair. Specifically, we show that the base excision repair pathway, the main pathway utilized for the repair of oxidative DNA damage, is compromised in Foxo3 -/- primitive hematopoietic cells. Treating mice in vivo with N -acetylcysteine reduces ROS levels, rescues HSC cycling defects, and partially mitigates HSPC DNA damage. These results indicate that DNA damage accrued as a result of elevated ROS in Foxo3 -/- mutant HSPC is at least partially reversible. Collectively, our findings suggest that FOXO3 serves as a protector of HSC genomic stability and health. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Estrogen signalling and the DNA damage response in hormone dependent breast cancers

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C Elizabeth Caldon

2014-05-01

Full Text Available Estrogen is necessary for the normal growth and development of breast tissue, but high levels of estrogen are a major risk factor for breast cancer. One mechanism by which estrogen could contribute to breast cancer is via the induction of DNA damage. This perspective discusses the mechanisms by which estrogen alters the DNA damage response (DDR and DNA repair through the regulation of key effector proteins including ATM, ATR, CHK1, BRCA1 and p53 and the feedback on estrogen receptor signalling from these proteins. We put forward the hypothesis that estrogen receptor signalling converges to suppress effective DNA repair and apoptosis in favour of proliferation. This is important in hormone-dependent breast cancer as it will affect processing of estrogen-induced DNA damage, as well as other genotoxic insults. DDR and DNA repair proteins are frequently mutated or altered in estrogen responsive breast cancer which will further change the processing of DNA damage. Finally the action of estrogen signalling on DNA damage is also relevant to the therapeutic setting as the suppression of a DNA damage response by estrogen has the potential to alter the response of cancers to anti-hormone treatment or chemotherapy that induces DNA damage.

Differential Processing of Low and High LET Radiation Induced DNA Damage: Investigation of Switch from ATM to ATR Signaling

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Saha, Janapriya; Wang, Minli; Hada, Megumi; Cucinotta, Francis A.

2011-01-01

The members of the phosphatidylinositol kinase-like kinase family of proteins namely ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) are directly responsible for the maintenance of genomic integrity by mounting DDR through signaling and facilitating the recruitment of repair factors at the sites of DNA damage along with coordinating the deployment of cell cycle checkpoints to permit repair by phosphorylating Checkpoint kinase Chk1, Chk2 and p53. High LET radiation from GCR (Galactic Cosmic Rays) consisting mainly of protons and high energy and charged (HZE) particles from SPE (Solar Particle Event) pose a major health risk for astronauts on their space flight missions. The determination of these risks and the design of potential safeguards require sound knowledge of the biological consequences of lesion induction and the capability of the cells to counter them. We here strive to determine the coordination of ATM and ATR kinases at the break sites directly affecting checkpoint signaling and DNA repair and whether differential processing of breaks induced by low and high LET radiation leads to possible augmentation of swap of these damage sensors at the sites of DNA damage. Exposure of cells to IR triggers rapid autophosphorylation of serine-1981 that causes dimer dissociation and initiates monomer formation of ATM. ATM kinase activity depends on the disruption of the dimer, which allows access and phosphorylation of downstream ATM substrates like Chk2. Evidence suggests that ATM is activated by the alterations in higher-order chromatin structure although direct binding of ATM to DSB ends may be a crucial step in its activation. On the other hand, in case of ATR, RPA (replication protein A)-coated ssDNA (single-stranded DNA) generated as a result of stalled DNA replication or during processing of chromosomal lesions is crucial for the localization of ATR to sites of DNA damage in association with ATR-interacting protein (ATRIP). Although the

Conformation-dependent DNA attraction

Science.gov (United States)

Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

2014-05-01

Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg2+ ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg2+ or Na+, benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg2+ bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by

Effect of processed and red meat on endogenous nitrosation and DNA damage.

Science.gov (United States)

Joosen, Annemiek M C P; Kuhnle, Gunter G C; Aspinall, Sue M; Barrow, Timothy M; Lecommandeur, Emmanuelle; Azqueta, Amaya; Collins, Andrew R; Bingham, Sheila A

2009-08-01

Haem in red meat (RM) stimulates the endogenous production of mutagenic nitroso compounds (NOC). Processed (nitrite-preserved red) meat additionally contains high concentrations of preformed NOC. In two studies, of a fresh RM versus a vegetarian (VEG) diet (six males and six females) and of a nitrite-preserved red meat (PM) versus a VEG diet (5 males and 11 females), we investigated whether processing of meat might increase colorectal cancer risk by stimulating nitrosation and DNA damage. Meat diets contained 420 g (males) or 366 g (females) meat/per day. Faecal homogenates from day 10 onwards were analysed for haem and NOC and associated supernatants for genotoxicity. Means are adjusted for differences in male to female ratios between studies. Faecal NOC concentrations on VEG diets were low (2.6 and 3.5 mmol/g) but significantly higher on meat diets (PM 175 +/- 19 nmol/g versus RM 185 +/- 22 nmol/g; P = 0.75). The RM diet resulted in a larger proportion of nitrosyl iron (RM 78% versus PM 54%; P meat diets (P Meats cured with nitrite have the same effect as fresh RM on endogenous nitrosation but show increased FW-induced oxidative DNA damage.

Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

Science.gov (United States)

Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

2018-05-22

Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

DNA lesions induced by replication stress trigger mitotic aberration and tetraploidy development.

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Yosuke Ichijima

Full Text Available During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.

Cell sensitivity to irradiation and DNA repair processes. II

International Nuclear Information System (INIS)

Kozubek, S.; Krasavin, E.A.

1984-01-01

A new model of DNA single-strand break (SSB) and double-strand break (DSB) induction by radiations of different linear energy transfer (LET) has been developed. Utilizing quadratic dependence of the dose that delta-electrons depart in the track of heavy particles the fraction of heavy particle energy deposited in the target of DNA dimensions has been calculated. SSBs arise from energy depositions in one strand of DNA, direct DSBs arise from two SSBs on opposite strands of DNA in the track of one particle. It is concluded that DSB's induced by γ-radiation are mostly of enzymatic origin, meanwhile DSB's induced by high-LET radiation are direct DSB's. The dependence of radiosensitivity D 0 -1 on LET (L) for isogenic mutants of E. coli with different sensitivity to γ-radiation has been determined on the bases of the model and considering microscopic energy fluctuations. The shape of D 0 -1 (L) function is formed both by physical characteristics of radiation and by the ability of cells to repair some types of DNA damage. The model provides a basis for further investigation. (author)

Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients.

Science.gov (United States)

Lyko, Frank; Stach, Dirk; Brenner, Axel; Stilgenbauer, Stephan; Döhner, Hartmut; Wirtz, Michaela; Wiessler, Manfred; Schmitz, Oliver J

2004-06-01

Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples.

Towards a DNA Nanoprocessor: Reusable Tile-Integrated DNA Circuits.

Science.gov (United States)

Gerasimova, Yulia V; Kolpashchikov, Dmitry M

2016-08-22

Modern electronic microprocessors use semiconductor logic gates organized on a silicon chip to enable efficient inter-gate communication. Here, arrays of communicating DNA logic gates integrated on a single DNA tile were designed and used to process nucleic acid inputs in a reusable format. Our results lay the foundation for the development of a DNA nanoprocessor, a small and biocompatible device capable of performing complex analyses of DNA and RNA inputs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

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Caroline M Li

Full Text Available We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE. In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40 origin of DNA replication and the viral large tumor antigen (T-antigen protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA, DNA topoisomerase I (topo I, DNA polymerase δ (Pol δ, DNA polymerase ɛ (Pol ɛ, replication protein A (RPA and replication factor C (RFC. Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

Science.gov (United States)

Li, Caroline M; Miao, Yunan; Lingeman, Robert G; Hickey, Robert J; Malkas, Linda H

2016-01-01

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

DNA-binding determinants promoting NHEJ by human Polμ.

Science.gov (United States)

Martin, Maria Jose; Juarez, Raquel; Blanco, Luis

2012-12-01

Non-homologous end-joining (NHEJ), the preferred pathway to repair double-strand breaks (DSBs) in higher eukaryotes, relies on a collection of molecular tools to process the broken ends, including specific DNA polymerases. Among them, Polµ is unique as it can catalyze DNA synthesis upon connection of two non-complementary ends. Here, we demonstrate that this capacity is intrinsic to Polµ, not conferred by other NHEJ factors. To understand the molecular determinants of its specific function in NHEJ, the interaction of human Polµ with DNA has been directly visualized by electromobility shift assay and footprinting assays. Stable interaction with a DNA gap requires the presence of a recessive 5'-P, thus orienting the catalytic domain for primer and nucleotide binding. Accordingly, recognition of the 5'-P is crucial to align the two DNA substrates of the NHEJ reaction. Site-directed mutagenesis demonstrates the relevance of three specific residues (Lys(249), Arg(253) and Arg(416)) in stabilizing the primer strand during end synapsis, allowing a range of microhomology-induced distortions beneficial for NHEJ. Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5'-P, thus boosting Polµ-mediated NHEJ reactions.

Wide-scale screening of T-DNA lines for transcription factor genes affecting male gametophyte development in Arabidopsis

Czech Academy of Sciences Publication Activity Database

Reňák, David; Dupľáková, Nikoleta; Honys, David

2012-01-01

Roč. 25, č. 1 (2012), s. 39-60 ISSN 0934-0882 R&D Projects: GA AV ČR KJB600380701; GA ČR GA522/09/0858; GA MŠk(CZ) OC10054 Institutional research plan: CEZ:AV0Z50380511 Keywords : Male gametophyte * Transcription factor * T-DNA insertion line Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.070, year: 2012

DNA replication stress restricts ribosomal DNA copy number.

Science.gov (United States)

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

DNA replication stress restricts ribosomal DNA copy number

Science.gov (United States)

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

DNA replication stress restricts ribosomal DNA copy number.

Directory of Open Access Journals (Sweden)

Devika Salim

2017-09-01

Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

Role of isolated and clustered DNA damage and the post-irradiating repair process in the effects of heavy ion beam irradiation

International Nuclear Information System (INIS)

Tokuyama, Yuka; Terato, Hiroaki; Furusawa, Yoshiya; Ide, Hiroshi; Yasui, Akira

2015-01-01

Clustered DNA damage is a specific type of DNA damage induced by ionizing radiation. Any type of ionizing radiation traverses the target DNA molecule as a beam, inducing damage along its track. Our previous study showed that clustered DNA damage yields decreased with increased linear energy transfer (LET), leading us to investigate the importance of clustered DNA damage in the biological effects of heavy ion beam radiation. In this study, we analyzed the yield of clustered base damage (comprising multiple base lesions) in cultured cells irradiated with various heavy ion beams, and investigated isolated base damage and the repair process in post-irradiation cultured cells. Chinese hamster ovary (CHO) cells were irradiated by carbon, silicon, argon and iron ion beams with LETs of 13, 55, 90 and 200 keV µm -1 , respectively. Agarose gel electrophoresis of the cells with enzymatic treatments indicated that clustered base damage yields decreased as the LET increased. The aldehyde reactive probe procedure showed that isolated base damage yields in the irradiated cells followed the same pattern. To analyze the cellular base damage process, clustered DNA damage repair was investigated using DNA repair mutant cells. DNA double-strand breaks accumulated in CHO mutant cells lacking Xrcc1 after irradiation, and the cell viability decreased. On the other hand, mouse embryonic fibroblast (Mef) cells lacking both Nth1 and Ogg1 became more resistant than the wild type Mef. Thus, clustered base damage seems to be involved in the expression of heavy ion beam biological effects via the repair process. (author)

Using sequence-specific chemical and structural properties of DNA to predict transcription factor binding sites.

Directory of Open Access Journals (Sweden)

Amy L Bauer

2010-11-01

Full Text Available An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF. Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate.

ThrR, a DNA-binding transcription factor involved in controlling threonine biosynthesis in Bacillus subtilis.

Science.gov (United States)

Rosenberg, Jonathan; Müller, Peter; Lentes, Sabine; Thiele, Martin J; Zeigler, Daniel R; Tödter, Dominik; Paulus, Henry; Brantl, Sabine; Stülke, Jörg; Commichau, Fabian M

2016-09-01

The threonine dehydratase IlvA is part of the isoleucine biosynthesis pathway in the Gram-positive model bacterium Bacillus subtilis. Consequently, deletion of ilvA causes isoleucine auxotrophy. It has been reported that ilvA pseudo-revertants having a derepressed hom-thrCB operon appear in the presence of threonine. Here we have characterized two classes of ilvA pseudo-revertants. In the first class the hom-thrCB operon was derepressed unmasking the threonine dehydratase activity of the threonine synthase ThrC. In the second class of mutants, threonine biosynthesis was more broadly affected. The first class of ilvA pseudo-revertants had a mutation in the Phom promoter (P*hom ), resulting in constitutive expression of the hom-thrCB operon. In the second class of ilvA pseudo-revertants, the thrR gene encoding a putative DNA-binding protein was inactivated, also resulting in constitutive expression of the hom-thrCB operon. Here we demonstrate that ThrR is indeed a DNA-binding transcription factor that regulates the hom-thrCB operon and the thrD aspartokinase gene. DNA binding assays uncovered the DNA-binding site of ThrR and revealed that the repressor competes with the RNA polymerase for DNA binding. This study also revealed that ThrR orthologs are ubiquitous in genomes from the Gram-positive phylum Firmicutes and in some Gram-negative bacteria. © 2016 John Wiley & Sons Ltd.

Single gene retrieval from thermally degraded DNA

Indian Academy of Sciences (India)

To simulate single gene retrieval from ancient DNA, several related factors have been investigated. By monitoring a 889 bp polymerase chain reaction (PCR) product and genomic DNA degradation, we find that heat and oxygen (especially heat) are both crucial factors influencing DNA degradation. The heat influence ...

Salicylic acid and nitric oxide alleviate high temperature induced oxidative damage in Lablab purpureus L plants by regulating bio-physical processes and DNA methylation.

Science.gov (United States)

Rai, Krishna Kumar; Rai, Nagendra; Rai, Shashi Pandey

2018-07-01

Salicylic acid (SA) and sodium nitroprusside (SNP, NO donor) modulates plant growth and development processes and recent findings have also revealed their involvement in the regulation of epigenetic factors under stress condition. In the present study, some of these factors were comparatively studied in hyacinth bean plants subjected to high temperature (HT) environment (40-42 °C) with and without exogenous application of SA and SNP under field condition. Exogenous application of SA and SNP substantially modulated the growth and biophysical process of hyacinth bean plants under HT environment. Exogenous application of SA and SNP also remarkably regulated the activities of antioxidant enzymes, modulated mRNA level of certain enzymes, improves plant water relation, enhance photosynthesis and thereby increasing plant defence under HT. Coupled restriction enzyme digestion-random amplification (CRED-RA) technique revealed that many methylation changes were "dose dependent" and HT significantly increased DNA damages as evidenced by both increase and decrease in bands profiles, methylation and de-methylation pattern. Thus, the result of the present study clearly shows that exogenous SA and SNP regulates DNA methylation pattern, modulates stress-responsive genes and can impart transient HT tolerance by synchronizing growth and physiological acclimatization of plants, thus narrowing the gaps between physio-biochemical and molecular events in addressing HT tolerance. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

The Mismatch-Binding Factor MutSβ Can Mediate ATR Activation in Response to DNA Double-Strand Breaks

Czech Academy of Sciences Publication Activity Database

Burdová, Kamila; Mihaljevic, B.; Sturzenegger, A.; Chappidi, N.; Janščák, Pavel

2015-01-01

Roč. 59, č. 4 (2015), s. 603-614 ISSN 1097-2765 R&D Projects: GA ČR GAP305/10/0281; GA ČR(CZ) GA14-05743S Grant - others:Oncosuisse(CH) KLS-02344-02-2009; Swiss National Science Foundation(CH) 31003A_146206; Novartis Foundation for Medical and Biological Research(CH) 11A16 Institutional support: RVO:68378050 Keywords : Ataxia telangiectasia-mutated and Rad3-related (ATR) protein kinase * DNA-damage response * DNA Double-Strand Breaks Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 13.958, year: 2015

Metaphors of DNA: a review of the popularisation processes

Directory of Open Access Journals (Sweden)

Sergi Cortiñas Rovira

2008-03-01

Full Text Available This article offers a 1953-present day review of the models that have popularised DNA, one of the fundamental molecules of biochemistry. DNA has become an iconic concept over the 20th century, overcoming the boundaries of science and spreading into literature, painting, sculpture or religion. This work analyses the reasons why DNA has penetrated society so effectively and examines some of the main metaphors used by the scientists and scientific popularisers. Furthermore, this article, taken from the author's PhD thesis, describes some recent popularisation models for this molecule.

Quantification of Epstein-Barr virus DNA load, interleukin-6, interleukin-10, transforming growth factor-β1 and stem cell factor in plasma of patients with nasopharyngeal carcinoma

International Nuclear Information System (INIS)

Tan, Eng-lai; Selvaratnam, G; Kananathan, R; Sam, Choon-kook

2006-01-01

Nasopharyngeal carcinoma (NPC) is a common epithelial neoplasm among the Chinese populations in Southern China and South East Asia. Epstein-Barr virus (EBV) is known to be an important etiologic agent of NPC and the viral gene products are frequently detected in NPC tissues along with elevated antibody titres to the viral proteins (VCA and EA) in a majority of patients. Elevated plasma EBV DNA load is regarded as an important marker for the presence of the disease and for the monitoring of disease progression. However, other serum/plasma parameters such as the levels of certain interleukins and growth factors have also been implicated in NPC. The objectives of the present study are, 1) to investigate the correlations between plasma EBV DNA load and the levels of interleukin (IL)-6, IL-10, TGF-β1 and SCF (steel factor) and 2) to relate these parameters to the stages of NPC and the effect of treatment. A total of 78 untreated NPC patients were enrolled in this study. Of these, 51 were followed-up after treatment. The remaining patients had irregular or were lost to follow-up. Plasma EBV DNA was quantified using real-time quantitative PCR. The levels of plasma interleukins and growth factors were quantified using ELISA. A significant decrease in EBV DNA load was detected in plasma of untreated NPC patients (1669 ± 637 copies/mL; n = 51) following treatment (57 ± 37 copies/mL, p < 0.05); n = 51). Plasma EBV DNA load was shown to be a good prognosticator for disease progression and clinical outcome in five of the follow-up patients. A significant difference in IL-6 levels was noted between the untreated patients (164 ± 37 pg/mL; n = 51) and following treatment (58 ± 16 pg/mL, p < 0.05; n = 51). Positive correlations between EBV DNA load and IL-10 (r(49) = 0.535, p < 0.01), between IL6 and IL-10 (r(49) = 0.474, p < 0.01) and between TGF and SCF (r(49) = 0.464, p < 0.01) were observed in patients following treatment. None of the parameters tested including Ig

A dynamic balanced scorecard for identification internal process factor

Directory of Open Access Journals (Sweden)

Javad sofiyabadi

2012-08-01

Full Text Available We present a dynamic balanced score card (BSC to investigate the strategic internal process management factors. The proposed dynamic BSC emphasizes on internal processes aspect, and using VIKOR and Shannon Entropy, determinants the internal processes, process management and improvement and all important factors are ranked. The current study first introduces dynamic BSC and examines effective factors on the process. The proposed model focuses on internal processes perspective of BSC and determines importance degree of each factor is used using VIKOR decision-making techniques.

Andrographolide interferes with binding of nuclear factor-κB to DNA in HL-60-derived neutrophilic cells

Science.gov (United States)

Hidalgo, María A; Romero, Alex; Figueroa, Jaime; Cortés, Patricia; Concha, Ilona I; Hancke, Juan L; Burgos, Rafael A

2005-01-01

Andrographolide, the major active component from Andrographis paniculata, has shown to possess anti-inflammatory activity. Andrographolide inhibits the expression of several proinflammatory proteins that exhibit a nuclear factor kappa B (NF-κB) binding site in their gene. In the present study, we analyzed the effect of andrographolide on the activation of NF-κB induced by platelet-activating factor (PAF) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) in HL-60 cells differentiated to neutrophils. PAF (100 nM) and fMLP (100 nM) induced activation of NF-κB as determined by degradation of inhibitory factor B α (IκBα) using Western blotting in cytosolic extracts and by binding to DNA using electrophoretic mobility shift assay (EMSA) in nuclear extracts. Andrographolide (5 and 50 μM) inhibited the NF-κB-luciferase activity induced by PAF. However, andrographolide did not reduce phosphorylation of p38 MAPK or ERK1/2 and did not change IκBα degradation induced by PAF and fMLP. Andrographolide reduced the DNA binding of NF-κB in whole cells and in nuclear extracts induced by PAF and fMLP. Andrographolide reduced cyclooxygenase-2 (COX-2) expression induced by PAF and fMLP in HL-60/neutrophils. It is concluded that andrographolide exerts its anti-inflammatory effects by inhibiting NF-κB binding to DNA, and thus reducing the expression of proinflammatory proteins, such as COX-2. PMID:15678086

Eukaryotic DNA Replication Fork.

Science.gov (United States)

Burgers, Peter M J; Kunkel, Thomas A

2017-06-20

This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

Gefitinib radiosensitizes stem-like glioma cells: inhibition of epidermal growth factor receptor-Akt-DNA-PK signaling, accompanied by inhibition of DNA double-strand break repair.

Science.gov (United States)

Kang, Khong Bee; Zhu, Congju; Wong, Yin Ling; Gao, Qiuhan; Ty, Albert; Wong, Meng Cheong

2012-05-01

We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H(2)AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H(2)AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G(2)/M arrest and increased γ-H(2)AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H(2)AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G(2)/M arrest, and DNA DSBs, compared with nonstem

Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

Energy Technology Data Exchange (ETDEWEB)

Kang, Khong Bee, E-mail: dmskkb@nccs.com.sg [Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore (Singapore); Zhu Congju; Wong Yinling; Gao Qiuhan; Ty, Albert; Wong, Meng Cheong [Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore (Singapore)

2012-05-01

Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, {gamma}-H{sub 2}AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, {gamma}-H{sub 2}AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G{sub 2}/M arrest and increased {gamma}-H{sub 2}AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased {gamma}-H{sub 2}AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are

Conformation-dependent DNA attraction.

Science.gov (United States)

Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

2014-06-21

Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg(2+) ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg(2+) or Na(+), benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg(2+) bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.

Ter-dependent stress response systems: novel pathways related to metal sensing, production of a nucleoside-like metabolite, and DNA-processing.

Science.gov (United States)

Anantharaman, Vivek; Iyer, Lakshminarayan M; Aravind, L

2012-10-30

The mode of action of the bacterial ter cluster and TelA genes, implicated in natural resistance to tellurite and other xenobiotic toxic compounds, pore-forming colicins and several bacteriophages, has remained enigmatic for almost two decades. Using comparative genomics, sequence-profile searches and structural analysis we present evidence that the ter gene products and their functional partners constitute previously underappreciated, chemical stress response and anti-viral defense systems of bacteria. Based on contextual information from conserved gene neighborhoods and domain architectures, we show that the ter gene products and TelA lie at the center of membrane-linked metal recognition complexes with regulatory ramifications encompassing phosphorylation-dependent signal transduction, RNA-dependent regulation, biosynthesis of nucleoside-like metabolites and DNA processing. Our analysis suggests that the multiple metal-binding and non-binding TerD paralogs and TerC are likely to constitute a membrane-associated complex, which might also include TerB and TerY, and feature several, distinct metal-binding sites. Versions of the TerB domain might also bind small molecule ligands and link the TerD paralog-TerC complex to biosynthetic modules comprising phosphoribosyltransferases (PRTases), ATP grasp amidoligases, TIM-barrel carbon-carbon lyases, and HAD phosphoesterases, which are predicted to synthesize novel nucleoside-like molecules. One of the PRTases is also likely to interact with RNA by means of its Pelota/Ribosomal protein L7AE-like domain. The von Willebrand factor A domain protein, TerY, is predicted to be part of a distinct phosphorylation switch, coupling a protein kinase and a PP2C phosphatase. We show, based on the evidence from numerous conserved gene neighborhoods and domain architectures, that both the TerB and TelA domains have been linked to diverse lipid-interaction domains, such as two novel PH-like and the Coq4 domains, in different bacteria

Epigenetic subgroups of esophageal and gastric adenocarcinoma with differential GATA5 DNA methylation associated with clinical and lifestyle factors.

Directory of Open Access Journals (Sweden)

Xinhui Wang

Full Text Available BACKGROUND: Adenocarcinomas located near the gastroesophageal junction have unclear etiology and are difficult to classify. We used DNA methylation analysis to identify subtype-specific markers and new subgroups of gastroesophageal adenocarcinomas, and studied their association with epidemiological risk factors and clinical outcomes. METHODOLOGY/PRINCIPAL FINDINGS: We used logistic regression models and unsupervised hierarchical cluster analysis of 74 DNA methylation markers on 45 tumor samples (44 patients of esophageal and gastric adenocarcinomas obtained from a population-based case-control study to uncover epigenetic markers and cluster groups of gastroesophageal adenocarcinomas. No distinct epigenetic differences were evident between subtypes of gastric and esophageal cancers. However, we identified two gastroesophageal adenocarcinoma subclusters based on DNA methylation profiles. Group membership was best predicted by GATA5 DNA methylation status. We analyzed the associations between these two epigenetic groups and exposure using logistic regression, and the associations with survival time using Cox regression in a larger set of 317 tumor samples (278 patients. There were more males with esophageal and gastric cardia cancers in Cluster Group 1 characterized by higher GATA5 DNA methylation values (all p<0.05. This group also showed associations of borderline statistical significance with having ever smoked (p-value = 0.07, high body mass index (p-value = 0.06, and symptoms of gastroesophageal reflux (p-value = 0.07. Subjects in cluster Group 1 showed better survival than those in Group 2 after adjusting for tumor differentiation grade, but this was not found to be independent of tumor stage. CONCLUSIONS/SIGNIFICANCE: DNA methylation profiling can be used in population-based studies to identify epigenetic subclasses of gastroesophageal adenocarcinomas and class-specific DNA methylation markers that can be linked to

Oxidative DNA base modifications as factors in carcinogenesis

International Nuclear Information System (INIS)

Olinski, R.; Jaruga, P.; Zastawny, T.H.

1998-01-01

Reactive oxygen species can cause extensive DNA modifications including modified bases. Some of the DNA base damage has been found to possess premutagenic properties. Therefore, if not repaired, it can contribute to carcinogenesis. We have found elevated amounts of modified bases in cancerous and precancerous tissues as compared with normal tissues. Most of the agents used in anticancer therapy are paradoxically responsible for induction of secondary malignancies and some of them may generate free radicals. The results of our experiments provide evidence that exposure of cancer patients to therapeutic doses of ionizing radiation and anticancer drugs cause base modifications in genomic DNA of lymphocytes. Some of these base damages could lead to mutagenesis in critical genes and ultimately to secondary cancers such as leukemias. This may point to an important role of oxidative base damage in cancer initiation. Alternatively, the increased level of the modified base products may contribute to genetic instability and metastatic potential of tumor cells. (author)

Challenges in Simulating Light-Induced Processes in DNA

Directory of Open Access Journals (Sweden)

Philipp Marquetand

2016-12-01

Full Text Available In this contribution, we give a perspective on the main challenges in performing theoretical simulations of photoinduced phenomena within DNA and its molecular building blocks. We distinguish the different tasks that should be involved in the simulation of a complete DNA strand subject to UV irradiation: (i stationary quantum chemical computations; (ii the explicit description of the initial excitation of DNA with light; (iii modeling the nonadiabatic excited state dynamics; (iv simulation of the detected experimental observable; and (v the subsequent analysis of the respective results. We succinctly describe the methods that are currently employed in each of these steps. While for each of them, there are different approaches with different degrees of accuracy, no feasible method exists to tackle all problems at once. Depending on the technique or combination of several ones, it can be problematic to describe the stacking of nucleobases, bond breaking and formation, quantum interferences and tunneling or even simply to characterize the involved wavefunctions. It is therefore argued that more method development and/or the combination of different techniques are urgently required. It is essential also to exercise these new developments in further studies on DNA and subsystems thereof, ideally comprising simulations of all of the different components that occur in the corresponding experiments.

Amplification of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 lytic origin of DNA replication is dependent upon a cis-acting AT-rich region and an ORF50 response element and the trans-acting factors ORF50 (K-Rta) and K8 (K-bZIP)

International Nuclear Information System (INIS)

AuCoin, David P.; Colletti, Kelly S.; Cei, Sylvia A.; Papouskova, Iva; Tarrant, Margaret; Pari, Gregory S.

2004-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), has significant sequence homology to Epstein-Barr virus (EBV). In cell culture, HHV8 is primarily latent, and viral genes associated with lytic replication are not expressed. Two lytic origins of DNA replication (oriLyt) are present within the HHV8 genome and are composed of an AT-rich region adjacent to GC-rich DNA sequences. We have now identified essential cis- and trans-acting elements required for oriLyt-dependent DNA replication. The transient replication assay was used to show that two AT-rich elements, three consensus AP1 transcription factor-binding sites, an ORF50 response element (RE), and a consensus TATA box motif are essential for efficient origin-dependent DNA replication. Transient transfection of luciferase reporter constructs indicated that the downstream region of the HHV8 oriLyt responds to ORF50 and suggests that part of the oriLyt may be an enhancer/promoter. In addition, a transient cotransfection-replication assay elucidated the set of trans-acting factors required for lytic DNA replication. These factors consist of homologues to the core replication proteins: ORF6 (ssDNA binding protein), ORF9 (DNA polymerase), ORF40-41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor) common to all herpesviruses along with ORF50 (K-Rta) and K8 (K-bZIP)

The role of DNA dependent protein kinase in synapsis of DNA ends.

Science.gov (United States)

Weterings, Eric; Verkaik, Nicole S; Brüggenwirth, Hennie T; Hoeijmakers, Jan H J; van Gent, Dik C

2003-12-15

DNA 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 the action of exonucleases and ligases. The DNA termini become accessible after autophosphorylation of DNA-PK(CS), which we demonstrate to require synapsis of DNA ends. Interestingly, the presence of DNA-PK prevents ligation of the two synapsed termini, but allows ligation to another DNA molecule. This alteration of the ligation route is independent of the type of ligase that we used, indicating that the intrinsic architecture of the DNA-PK complex itself is not able to support ligation of the synapsed DNA termini. We present a working model in which DNA-PK creates a stable molecular bridge between two DNA ends that is remodeled after DNA-PK autophosphorylation in such a way that the extreme termini become accessible without disrupting synapsis. We infer that joining of synapsed DNA termini would require an additional protein factor.

Mono- and Di-Alkylation Processes of DNA Bases by Nitrogen Mustard Mechlorethamine.

Science.gov (United States)

Larrañaga, Olatz; de Cózar, Abel; Cossío, Fernando P

2017-12-06

The reactivity of nitrogen mustard mechlorethamine (mec) with purine bases towards formation of mono- (G-mec and A-mec) and dialkylated (AA-mec, GG-mec and AG-mec) adducts has been studied using density functional theory (DFT). To gain a complete overview of DNA-alkylation processes, direct chloride substitution and formation through activated aziridinium species were considered as possible reaction paths for adduct formation. Our results confirm that DNA alkylation by mec occurs via aziridine intermediates instead of direct substitution. Consideration of explicit water molecules in conjunction with polarizable continuum model (PCM) was shown as an adequate computational method for a proper representation of the system. Moreover, Runge-Kutta numerical kinetic simulations including the possible bisadducts have been performed. These simulations predicted a product ratio of 83:17 of GG-mec and AG-mec diadducts, respectively. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition of DNA replication, DNA repair synthesis, and DNA polymerases α and δ by butylphenyl deoxyguanosine triphosphate

International Nuclear Information System (INIS)

Dreslor, S.L.; Frattini, M.G.

1987-01-01

Semiconservative DNA replication in growing mammalian cells and ultraviolet (UV)-induced DNA repair synthesis in nongrowing mammalian cells are mediated by one or both of the aphidicolin-sensitive DNA polymerases, α and/or δ. They have studied the inhibition of replication and repair synthesis in permeable human cells by N 2 (p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh dGTP), an agent which inhibits polymerase α strongly and polymerase δ weakly. Both processes are inhibited by BuPh-dGTP in competition with dGTP. The K/sub i/'s are, for replication, 2-3 μM and, for repair synthesis, 3-4 μM, consistent with the involvement of the same DNA polymerase in both processes. Inhibition of isolated human polymerase α by BuPh-dGTP is also competitive with dGTP, but the K/sub i/ is approximately 10 nM, several hundred-fold lower than the K/sub i/'s of replication and repair synthesis. Isolated polymerase δ is inhibited by BuPh-dGTP at doses similar to those which inhibit replication and repair synthesis, however, attempts to determine the K/sub i/ of polymerase δ were hampered by the finding that the dependence of δ activity on deoxyribunucleotide concentration is parabolic at low doses. This behavior differs from the behavior of polymerase α and of cellular DNA replication and repair synthesis, all of which show a simple, hyperbolic relationship between activity and deoxyribonucleotide concentration. Thus, inhibition of DNA replication and UV induced DNA repair synthesis by BuPh dGTP is quantitatively similar to DNA polymerase δ, but some other characteristics of the cellular processes are more similar to those of polymerase α

Supramolecular 1-D polymerization of DNA origami through a dynamic process at the 2-dimensionally confined air-water interface.

Science.gov (United States)

Yonamine, Yusuke; Cervantes-Salguero, Keitel; Minami, Kosuke; Kawamata, Ibuki; Nakanishi, Waka; Hill, Jonathan P; Murata, Satoshi; Ariga, Katsuhiko

2016-05-14

In this study, a Langmuir-Blodgett (LB) system has been utilized for the regulation of polymerization of a DNA origami structure at the air-water interface as a two-dimensionally confined medium, which enables dynamic condensation of DNA origami units through variation of the film area at the macroscopic level (ca. 10-100 cm(2)). DNA origami sheets were conjugated with a cationic lipid (dioctadecyldimethylammonium bromide, 2C18N(+)) by electrostatic interaction and the corresponding LB-film was prepared. By applying dynamic pressure variation through compression-expansion processes, the lipid-modified DNA origami sheets underwent anisotropic polymerization forming a one-dimensionally assembled belt-shaped structure of a high aspect ratio although the thickness of the polymerized DNA origami was maintained at the unimolecular level. This approach opens up a new field of mechanical induction of the self-assembly of DNA origami structures.

Impedimetric aptasensor for nuclear factor kappa B with peroxidase-like mimic coupled DNA nanoladders as enhancer.

Science.gov (United States)

Peng, Kanfu; Zhao, Hongwen; Xie, Pan; Hu, Shuang; Yuan, Yali; Yuan, Ruo; Wu, Xiongfei

2016-07-15

In this work, we developed a sensitive and universal aptasensor for nuclear factor kappa B (NF-κB) detection based on peroxidase-like mimic coupled DNA nanoladders for signal amplification. The dsDNA formed by capture DNA S1 and NF-κB binding aptamer (NBA) was firstly assembled on electrode surface. The presence of target NF-κB then led to the leave of NBA from electrode surface and thus provided the binding sites for immobilizing initiator to trigger in situ formation of DNA nanoladders on electrode surface. Since the peroxidase-like mimic manganese (III) meso-tetrakis (4-Nmethylpyridyl)-porphyrin (MnTMPyP) interacts with DNA nanoladders via groove binding, the insoluble benzo-4-chlorohexadienone (4-CD) precipitation derived from the oxidation of 4-chloro-1-naphthol (4-CN) could be formed on electrode surface in the presence of H2O2, resulting in a significantly amplified EIS signal output for quantitative target analysis. As a result, the developed aptasensor showed a low detection limit of 7pM and a wide linear range of 0.01-20nM. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective and stable detection of different types of target proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

Diversification of DnaA dependency for DNA replication in cyanobacterial evolution.

Science.gov (United States)

Ohbayashi, Ryudo; Watanabe, Satoru; Ehira, Shigeki; Kanesaki, Yu; Chibazakura, Taku; Yoshikawa, Hirofumi

2016-05-01

Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor.

Superimposed Code Theoretic Analysis of Deoxyribonucleic Acid (DNA) Codes and DNA Computing

Science.gov (United States)

2010-01-01

DNA strand and its Watson - Crick complement can be used to perform mathematical computation. This research addresses how the...Acid dsDNA double stranded DNA MOSAIC Mobile Stream Processing Cluster PCR Polymerase Chain Reaction RAM Random Access Memory ssDNA single stranded DNA WC Watson – Crick A Adenine C Cytosine G Guanine T Thymine ...are 5′→3′ and strands with strikethrough are 3′→5′. A dsDNA duplex formed between a strand and its reverse complement is called a

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

Science.gov (United States)

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-06-01

Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

Synthesis of titanium oxide nanoparticles using DNA-complex as template for solution-processable hybrid dielectric composites

Energy Technology Data Exchange (ETDEWEB)

Ramos, J.C. [Center for Sustainable Materials Chemistry, 153 Gilbert Hall, Oregon State University, Corvallis, OR (United States); Mejia, I.; Murphy, J.; Quevedo, M. [Department of Materials Science and Engineering, University of Texas at Dallas, Dallas, TX (United States); Garcia, P.; Martinez, C.A. [Engineering and Technology Institute, Autonomous University of Ciudad Juarez, Ciudad Juarez, Chihuahua (Mexico)

2015-09-15

Highlights: • We developed a synthesis method to produce TiO{sub 2} nanoparticles using a DNA complex. • The nanoparticles were anatase phase (~6 nm diameter), and stable in alcohols. • Composites showed a k of 13.4, 4.6 times larger than the k of polycarbonate. • Maximum processing temperature was 90 °C. • Low temperature enables their use in low-voltage, low-cost, flexible electronics. - Abstract: We report the synthesis of TiO{sub 2} nanoparticles prepared by the hydrolysis of titanium isopropoxide (TTIP) in the presence of a DNA complex for solution processable dielectric composites. The nanoparticles were incorporated as fillers in polycarbonate at low concentrations (1.5, 5 and 7 wt%) to produce hybrid dielectric films with dielectric constant higher than thermally grown silicon oxide. It was found that the DNA complex plays an important role as capping agent in the formation and suspension stability of nanocrystalline anatase phase TiO{sub 2} at room temperature with uniform size (∼6 nm) and narrow distribution. The effective dielectric constant of spin-cast polycarbonate thin-films increased from 2.84 to 13.43 with the incorporation of TiO{sub 2} nanoparticles into the polymer host. These composites can be solution processed with a maximum temperature of 90 °C and could be potential candidates for its application in low-cost macro-electronics.

DNA-Destabilizing Agents as an Alternative Approach for Targeting DNA: Mechanisms of Action and Cellular Consequences

Directory of Open Access Journals (Sweden)

Gaëlle Lenglet

2010-01-01

Full Text Available DNA targeting drugs represent a large proportion of the actual anticancer drug pharmacopeia, both in terms of drug brands and prescription volumes. Small DNA-interacting molecules share the ability of certain proteins to change the DNA helix's overall organization and geometrical orientation via tilt, roll, twist, slip, and flip effects. In this ocean of DNA-interacting compounds, most stabilize both DNA strands and very few display helix-destabilizing properties. These types of DNA-destabilizing effect are observed with certain mono- or bis-intercalators and DNA alkylating agents (some of which have been or are being developed as cancer drugs. The formation of locally destabilized DNA portions could interfere with protein/DNA recognition and potentially affect several crucial cellular processes, such as DNA repair, replication, and transcription. The present paper describes the molecular basis of DNA destabilization, the cellular impact on protein recognition, and DNA repair processes and the latter's relationships with antitumour efficacy.

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

International Nuclear Information System (INIS)

Arneodo, Alain; Vaillant, Cedric; Audit, Benjamin; Argoul, Francoise; D'Aubenton-Carafa, Yves; Thermes, Claude

2011-01-01

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.

mtSSB may sequester UNG1 at mitochondrial ssDNA and delay uracil processing until the dsDNA conformation is restored

DEFF Research Database (Denmark)

Wollen Steen, Kristian; Doseth, Berit; westbye, Marianne

2012-01-01

Single-strand DNA binding proteins protect DNA from nucleolytic damage, prevent formation of secondary structures and prevent premature reannealing of DNA in DNA metabolic transactions. In eukaryotes, the nuclear single-strand DNA binding protein RPA is essential for chromosomal DNA replication...

[Single-molecule detection and characterization of DNA replication based on DNA origami].

Science.gov (United States)

Wang, Qi; Fan, Youjie; Li, Bin

2014-08-01

To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

A Comparison of Factors that Influence the Lyophilization Process

OpenAIRE

Mnerie, Dumitru; Anghel, Gabriela Victoria; Mnerie, Alin Vasile; Cheveresan, Constantin

2007-01-01

The lyophilization (or freeze drying) process for agro-foods products depends on a series of technological factors that are in an inter-dependence with the process performance. This paper presents an expert method and its application. This method characterizes the influence factors of the lyophilization process, after the importance level of some factors in correlation with other factors, is defined. Only the most important factors were considered; influence considerations were made in relati...

DNA dosimetry assessment for sunscreen genotoxic photoprotection.

Directory of Open Access Journals (Sweden)

André Passaglia Schuch

Full Text Available Due to the increase of solar ultraviolet radiation (UV incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter.The Sun Protection Factor for DNA (DNA-SPF is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF. Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations.The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.

DNA dosimetry assessment for sunscreen genotoxic photoprotection.

Science.gov (United States)

Schuch, André Passaglia; Lago, Juliana Carvalhães; Yagura, Teiti; Menck, Carlos Frederico Martins

2012-01-01

Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.

Dual DNA binding property of ABA insensitive 3 like factors targeted to promoters responsive to ABA and auxin.