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Nanoplasmonic molecular ruler for nuclease activity and DNAfootprinting

Description: We have constructed a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of DNA length changes and perform DNA footprinting. The ruler was created by tethering double-stranded DNA to single Au nanoparticles. The scattering spectra of Au-DNA nanoconjugates showed red-shifted peak plasmon resonance wavelength dependent on DNA length, which can be measured with sub-nanometer axial resolution, averaging {approx}1.24 nm peak wavelength shift per DNA base pair. The spectra of individual Au-DNA nanoconjugates in the presence of nuclease showed a time-resolved dependence on the reaction dynamics, allowing quantitative, kinetic and real-time measurement of nuclease activity. The ruler was further developed into a new DNA footprinting platform. We showed the specific binding of a protein to DNA and the accurate mapping of its footprint. This work promises a very fast and convenient platform for mapping DNA-protein interactions, for nuclease activity monitoring, and for other DNA size-based methods.
Date: August 15, 2006
Creator: Chen, Fanqing Frank; Liu, Gang L.; Yin, Yadong; Gerion, Daniele; Kunchakarra, Siri; Mukherjee, Bipasha et al.
Partner: UNT Libraries Government Documents Department

Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules

Description: The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.
Date: February 8, 2006
Creator: Dugan, L
Partner: UNT Libraries Government Documents Department

Coordinateendonucleolytic 5' and 3' trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase

Description: Previous work showed that, in the presence of DNA-PK, Artemis slowly trims 3'-phosphoglycolate-terminated blunt ends. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. In the absence of DNA-PK, Artemis catalyzed extensive 5' {yields} 3' exonucleolytic resection of double-stranded DNA. This resection required a 5'-phosphate but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3' overhang. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent, and did not require a 5'-phosphate. For a blunt end with either a 3'-phosphoglycolate or 3'-hydroxyl terminus, endonucleolytic trimming of 2-4 nucleotides from the 3'-terminal strand was accompanied by trimming of 6 nucleotides from the 5'-terminal strand. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5'-terminal strand, resulting in short 3' overhangs that are trimmed endonucleolytically. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase mediated patching and ligation, with minimal loss of terminal sequence. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.
Date: February 18, 2008
Creator: Povirk, Lawrence; Yannone, Steven M.; Khan, Imran S.; Zhou, Rui-Zhe; Zhou, Tong; Valerie, Kristoffer et al.
Partner: UNT Libraries Government Documents Department

Epigenetic reversion of breast carcinoma phenotype is accompanied by DNA sequestration

Description: The importance of microenvironment and context in regulation of tissue-specific genes is finally well established. DNA exposure to, or sequestration from, nucleases can be used to detect differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we utilized an established 3-D assay where normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like vs tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion where the malignant cells are resistant to digestion relative to non-malignant cells. Reversion of the T4-2 breast cancer cells by either cAMP analogs, or a phospatidylinositol 3-kinase (P13K) inhibitor not only reverted the phenotype, but also the chromatin sensitivity to AluI. By using different cAMP-analogs, we show that the cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependent-protein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin also reverted the malignant phenotype, polarized the acini, and changed chromatin sequestration. These experiments show not only that modifying the tumor microenvironment can alter the organization of tumor cells but also that architecture of the tissues and the global chromatin organization are coupled and yet highly plastic.
Date: July 19, 2006
Creator: Sandal, Tone; Valyi-Nagy, Klara; Spencer, Virginia A.; Folberg,Robert; Bissell, Mina J. & Maniotis, Andrew J.
Partner: UNT Libraries Government Documents Department

Processing of 3'-Phosphoglycolate-Terminated DNA Double-StrandBreaks by Artemis Nuclease

Description: The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double-strand breaks. To assess the possibility that Artemis functions on oxidatively modified double-strand break termini, its activity toward model DNA substrates, bearing either 3{prime}-hydroxyl or 3{prime}-phosphoglycolate moieties, was examined. A 3{prime}-phosphoglycolate had little effect on Artemis-mediated trimming of long 3{prime} overhangs (>9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3{prime}-phosphoglycolates on overhangs of 4-5 bases promoted selective Artemis-mediated trimming of a single 3{prime}-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3{prime} overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was dependent upon Ku, DNA-dependent protein kinase, and ATP. Together, these data suggest that Artemis-mediated cleavage of 3{prime} overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3{prime} to the cleavage site. Shorter 3{prime}-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis, but much less efficiently. Consistent with the in vitro substrate specificity of Artemis, human cells lacking Artemis exhibited hypersensitivity to X-rays, bleomycin and neocarzinostatin, which all induce 3{prime}-phosphoglycolate-terminated double-strand breaks. Collectively, these results suggest that 3{prime}-phosphoglycolate termini and/or specific classes of DNA ends that arise from such blocked termini are relevant Artemis substrates in vivo.
Date: October 1, 2005
Creator: Povrik, Lawrence F.; Zhou, Tong; Zhou, Ruizhe; Cowan, Morton J. & Yannone, Steven M.
Partner: UNT Libraries Government Documents Department

Final report: FASEB Summer Research Conference on ''Post-transcriptional control of gene expression: Effectors of mRNA decay'' [agenda and attendees list]

Description: The goal of this meeting was to provide an interactive forum for scientists working on prokaryotic and eukaryotic mRNA decay. A special seminar presented by a leader in the field of mRNA decay in S. cerevisiae focused on what is known and what needs to be determined, not only for yeast but for other organisms. The large attendance (110 participants) reflects the awareness that mRNA decay is a key player in gene regulation in a way that is affected by the many steps that precede mRNA formation. Sessions were held on the following topics: mRNA transport and mRNP; multicomponent eukaryotic nucleases; nonsense-mediated mRNA decay and nonsense-associated altered splicing; Cis-acting sequences/Trans-acting factors of mRNA decay; translational accuracy; multicomponent bacterial nucleases; interplay between mRNA polyadenylation, translation and decay in prokaryotes and prokaryotic organelles; and RNA interference and other RNA mediators of gene expression. In addition to the talks and two poster sessions, there were three round tables: (1) Does translation occur in the nucleus? (2) Differences and similarities in the mechanisms of mRNA decay in different eukaryotes, and (3) RNA surveillance in bacteria?
Date: December 1, 2002
Creator: Maquat, Lynne
Partner: UNT Libraries Government Documents Department

Catalytic activity of nuclease P1: Experiment and theory

Description: Nuclease P1 from Penicillium citrinum is a zinc dependent glyco-enzyme that recognizes single stranded DNA and RNA as substrates and hydrolyzes the phosphate ester bond. Nuclease Pl seems to recognize particular conformations of the phosphodiester backbone and shows significant variation in the rate of hydrolytic activity depending upon which nucleosides are coupled by the phosphodiester bond. The efficiency of nuclease Pl in hydrolyzing the phosphodiester bonds of a substrate can be altered by modifications to one of the substrate bases induced by ionizing radiation or oxidative stress. Measurements have been made of the effect of several radiation induced lesions on the catalytic rate of nuclease Pl. A model of the structure of the enzyme has been constructed in order to better understand the binding and activity of this enzyme on various ssDNA substrates.
Date: October 1, 1994
Creator: Miller, J.H.; Falcone, J.M.; Shibata, M. & Box, H.C.
Partner: UNT Libraries Government Documents Department

WRN Exonuclease Structure, Molecular Mechanism, and DNA EndProcessing Role

Description: 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.
Date: February 15, 2006
Creator: Perry, J. Jefferson P.; Yannone, Steven M.; Holden, Lauren G.; Hitomi, Chiharu; Asaithamby, Aroumougame; Han, Seungil et al.
Partner: UNT Libraries Government Documents Department

Final Technical Report: Genetic and Molecular Analysis of a new control pathway in assimilate partitioning.

Description: Assimilate partitioning refers to the systemic distribution of photoassimilate from sites of primary assimilation (source tissue) to import-dependent tissues and organs (sinks). One of the defining questions in this area is how plants balance source productivity with sink demand. We discovered a sucrose-sensing signal transduction pathway that controls the activity of BvSUT1, a proton-sucrose symporter in sugar beet leaf tissue. Sucrose symporters are responsible for sucrose accumulation in the phloem of many plants and, therefore, they mediate the pivotal step in the long-distance transport of photoassimilate to non-photosynthetic tissues, such as roots and seed. We previously showed that sucrose transport activity is directly proportional to the transcription rate of BvSUT1 and that symporter mRNA and protein have high rates of turnover with half-lives on the order of 2 h. We further demonstrated that symporter transcription is regulated by sucrose levels in the leaf and that sucrose-dependent regulation of BvSUT1 transcription is mediated, at least in part, by a protein phosphorylation relay pathway. The goal of the experiments during this current grant were to use genetic and molecular approaches to identify essential components of this vital regulatory system. The initial objectives were to: (1) to characterize Arabidopsis mutants we've isolated that are resistant to growth inhibition by sucrose analogues that are recognized by the sucrose-sensor, (2) to screen for loss of function mutants in BvSUT1-promoter:luciferase transgenic plants that no longer respond to sucrose accumulation in the leaf using non-destructive visualization of luciferase activity, (3) to use gel mobility-shift assays and nuclease protection experiments to identify cis elements in the symporter promoter and DNA-binding proteins that are involved in sucrose regulation of symporter expression.
Date: March 10, 2009
Creator: Bush, Daniel, R.
Partner: UNT Libraries Government Documents Department

Comprehensive progress report, 1977-1980

Description: Research accomplishments and activities during the past three year period are described. Specific projects were: (1) restriction endonuclease studies of tetrahymena mtDNA; (2) biosynthesis of mitochondrial tRNA; and (3) structure and synthesis of tRNA charging enzymes. (ACR)
Date: January 1, 1980
Creator: Suyama, Y
Partner: UNT Libraries Government Documents Department

Pyrimidine dimer excision in exonuclease deficient mutants of Escherichia coli

Description: The rate of pyrimidine dimer excision has been measured in Escherichia coli strains deficient in exonuclease V, exonuclease VII, and the 5' ..-->.. 3' exonuclease of DNA polymerase I. The results suggest that a reduced level of the 5' ..-->.. 3' exonuclease of DNA polymerase I diminishes the rate of dimer excision and that an additional deficiency in exonuclease VII causes a significantly greater reduction in the cell's ability to remove dimers.
Date: January 1, 1978
Creator: Masker, W. E. & Chase, J. W.
Partner: UNT Libraries Government Documents Department

Chimeric Proteins to Detect DNA Damage and Mismatches

Description: The goal of this project was to develop chimeric proteins composed of a DNA mismatch or damage binding protein and a nuclease, as well as methods to detect DNA mismatches and damage. We accomplished this through protein engineering based on using polymerase chain reactions (PCRs) to create chimeras with novel functions for damage and mismatch detection. This project addressed fundamental questions relating to disease susceptibility and radiation-induced damage in cells. It also supported and enhanced LLNL's competency in the emerging field of proteomics. In nature, DNA is constantly being subjected to damaging agents such as exposure to ultraviolet (UV) radiation and various environmental and dietary carcinogens. If DNA damage is not repaired however, mutations in DNA result that can eventually manifest in cancer and other diseases. In addition to damage-induced DNA mutations, single nucleotide polymorphisms (SNPs), which are variations in the genetic sequence between individuals, may predispose some to disease. As a result of the Human Genome Project, the integrity of a person's DNA can now be monitored. Therefore, methods to detect DNA damage, mutations, and SNPs are useful not only in basic research but also in the health and biotechnology industries. Current methods of detection often use radioactive labeling and rely on expensive instrumentation that is not readily available in many research settings. Our methods to detect DNA damage and mismatches employ simple gel electrophoresis and flow cytometry, thereby alleviating the need for radioactive labeling and expensive equipment. In FY2001, we explored SNP detection by developing methods based on the ability of the chimeric proteins to detect mismatches. Using multiplex assays with flow cytometry and fluorescent beads to which the DNA substrates where attached, we showed that several of the chimeras possess greater affinity for damaged and mismatched DNA than for native DNA. This affinity was demonstrated in assays ...
Date: January 14, 2002
Creator: McCutchen-Maloney, S; Malfatti, M & Robbins, K M
Partner: UNT Libraries Government Documents Department

Biologically important radiation damage in DNA. Annual progress report, May 1, 1993--January 31, 1994

Description: Most DNA damage by the hydroxyl radical is confined to the bases, and this base damage represents an important component of locally multiply demanded sites (LMOS). The yields of the major damaged bases have been determined by gas chromatography mass spectrometry. For our propose, it was necessary to convert a known fraction of these damaged bases to strand breaks and then assay these labile sites as the increase in strand break yield over the normally observed level. Three potential agents by which this strategy of conversion of base damage to strand break could be implemented were identified in the original application: 1, Sl nuclease; 2, piperidine; and 3, base damage specific enzymes.
Date: March 1, 1994
Creator: Ward, J. F.
Partner: UNT Libraries Government Documents Department

Enzymatic studies of radiation damage. Progress report, July 1, 1976-September 30, 1977

Description: The major accomplishments of the year were a new method and a totally unexpected discovery that venom phosphodiesterase belongs to the group of single-strand specific nucleases. It acts on viral duplex DNA by producing a series of shorter fragments of duplex DNA. It was also found that the relaxed circular DNA of PM2 virus, that was previously enzymatically ligated, may be opened by very high amounts of phosphodiesterase. The new method deals with a simplified way of preparing polynucleotide kinase and with fast removal of the excess of hot ATP used to label a nick in DNA. This is done by imbedding DNA within an agarose pellet, which retains labeled DNA, but allows the excess of ATP to diffuse out.
Date: June 1, 1977
Creator: Laskowski, M. Sr.
Partner: UNT Libraries Government Documents Department

Steps in the process of DNA binding and entry in transformation. [Pneumococcus]

Description: The DNA uptake phase of genetic transformation in S. pheumoniae is reviewed with emphasis on molecular interactions at each step. An initial, reversible binding appears to be dependent on the molecular concentration of donor DNA. Subsequent irreversible binding, limited to the numbr of molecules corresponding to a fixed number of receptor sites, requires potassium ions and energy. Competition of different DNAs for uptake occurs at the initial step, but depends on the size of the DNA as well as its molecular concentration. Single-strand breakage accompanies irreversible binding of DNA. The frequency of breaks does not appear to depend on DNA concentration. Entry of DNA follows irreversible binding. The entry step, in which donor DNA is converted to single strands, requires action of a membrane nuclease. In the membrane this nuclease is part ofa specific multiprotein structure, which may function as a unit in DNA binding and entry.
Date: January 1, 1978
Creator: Lacks, S
Partner: UNT Libraries Government Documents Department

Differential regulation of plastid mRNA stability. Progress report

Description: Our goal is to identify cis-acting sequences and transacting factors that function in plastid mRNA maturation, stabilization, and/or decay through an in vitro and in vivo analysis of mRNA:protein interactions. Our previous results emphasized the study of 3{prime}end inverted repeat sequences (IRs) that serve both as mRNA processing elements and stability determinants, and associate with plastid proteins that potentially play enzymatic, structural and/or regulatory roles. We seek to define, by single base and internal deletion mutagenesis, the sequence and structural requirements for protein binding to the 3{prime} IRs of petD and psbA mRNAs; to purify RNA-binding proteins that demonstrate gene- or sequence-specific binding, or that are implicated in RNA stabilization or decay; and to investigate the native form of mRNA in the plastid, by attempting to purify ribonucleoprotein (RNP) particles from organelles. Our view of mRNA decay is that it is regulated by three interactive components: RNA structure, ribonucleases and RNA-binding proteins. We have used mutagenesis to study the role of RNA structure in regulating RNA decay rates, and to identify protein binding and endonuclease recognition sites. We have identified at least three endonuclease activities; one that cleaves psbA RNA; and two whose cleavage patterns with petD 3{prime} IR-RNA has been studied (endoC1 and endoC2). Additionally, we have continued to analyze the properties of the major RNA processing exoribonuclease. We have concentrated our efforts on three RNA-binding proteins. A 100 kd protein with properties suggestive of a mammalian RNP component has been purified. A protein of 55 kd that may also be an endonuclease has been partially purified. We have studied the interaction of a 29 kd protein with the petD stem/loop, and its role in RNA processing. Recently, we have used a novel gel shift/SDS-PAGE technique to identify new RNA-binding proteins.
Date: September 1, 1993
Creator: Stern, D. B.
Partner: UNT Libraries Government Documents Department

Removal of uv-induced pyrimidine dimers from the replicated and unreplicated DNA of human fibroblasts

Description: Excision repair in uv irradiated human fibroblasts has been examined in portions of DNA replicating after irradiation versus those remaining unreplicated. Two approaches, one using a uv-endonuclease to estimate pyrimidine dimers remaining in DNA, the other using density labeling to measure excision resynthesis, indicate that the extent of repair is the same for both replicated and unreplicated DNA.
Date: January 1, 1978
Creator: Waters, R.
Partner: UNT Libraries Government Documents Department

Enzymatic studies of radiation damage. Final report, January 1, 1979-September 30, 1981

Description: The susceptibility of the DNA in chromatin to single-strand specific nucleases was examined using nuclease P/sub 1/, mung beam nuclease, and venom phosphodiesterase. These experiments showed that chromatin contains a limited number of DNA sites which are susceptible to single-strand specific nucleases. These sites occur at intervals of 8 to 80 nucleosomes and are distributed throughout the chromatin. An endogenous nuclease was found in chicken erythrocyte nuclei. This enzyme resembles the nuclease of mammalian nuclei in requirements for bivalent cations and in production of large chromatin fragments that gradually decrease in size, but differs in that the products do not go through the stage of discrete bands on gel electrophoresis. Experiments in which chromatin fragments from micrococcal nuclease digestion were further digested with venom phosphodiesterase indicated that phosphodiesterase perferentially hydrolyzes all linkers, although this preference is not marked enough to produce and preserve particulate structures of chromatin. Analysis of the ribose-containing components resulting from digestion of chromatin by micrococcal nuclease and P/sub 1/ nuclease led to the conclusion that a minimal amount of Poly (ADP-Rib) is necessary to form and preserve the structure of nucleosomes.
Date: September 1, 1981
Creator: Kress, L. F. & Kowalski, D. F.
Partner: UNT Libraries Government Documents Department

Molecular basis for the mutagenic and lethal effects of ultraviolet irradiation. Research accomplishments (1968 to present)

Description: Earlier work on the chemical basis of mutagenesis led to certain chemical generalities sufficient to explain how certain mutagens such as uv light and hydroxylamine functioned in information transfer systems (replicative, transcriptive and translational). When such modifications were applied to biologically active DNA in a controlled manner biological expression was non-stoichiometric because much of the damage was removed from the DNA by repair systems. Our efforts were then directed to these systems which led to: (1) the isolation, purification and characterization of endonucleases responsible for the first and controlling step in DNA repair - referred to as incision in both M. luteus and E. coli. The biological role of these enzymes was inferred in appropriate mutants; (2) the isolation, purification and characterization of exonucleases responsible for the removal or excision of damaged nucleotides in M. luteus and human placental trophoblasts; (3) the repair of uv damaged biologically active transforming and transfecting DNAs by purified endonucleases, exonucleases, DNA polymerase I and polynucleotide ligase from M. luteus and E. coli; (4) the characterization of the dual gene control for incision phenomenon in M. luteus and E. coli; and (5) isolation, purification and characterization of repair enzymes from human placenta (currently in progress).
Date: January 1, 1978
Creator: Grossman, L.
Partner: UNT Libraries Government Documents Department

Transposon-induced nuclear mutations that alter chloroplast gene expression. Annual report, September 1, 1991--August 31, 1992

Description: The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene expression. We have found that two of these mutations are allelic. Both alleles have global defects in chloroplast translation initiation, as revealed by the failure to assemble chloroplast mRNAs into polysomes. We have isolated and characterized three new mutants from Mu lines that have novel defects in chloroplast RNA metabolism. We are now ready to begin the task of cloning several of these genes, by using the Mu transposon tag.
Date: December 31, 1992
Creator: Barkan, A.
Partner: UNT Libraries Government Documents Department