Science.gov

Sample records for rad hard active

  1. Mongoose: Creation of a Rad-Hard MIPS R3000

    NASA Technical Reports Server (NTRS)

    Lincoln, Dan; Smith, Brian

    1993-01-01

    This paper describes the development of a 32 Bit, full MIPS R3000 code-compatible Rad-Hard CPU, code named Mongoose. Mongoose progressed from contract award, through the design cycle, to operational silicon in 12 months to meet a space mission for NASA. The goal was the creation of a fully static device capable of operation to the maximum Mil-883 derated speed, worst-case post-rad exposure with full operational integrity. This included consideration of features for functional enhancements relating to mission compatibility and removal of commercial practices not supported by Rad-Hard technology. 'Mongoose' developed from an evolution of LSI Logic's MIPS-I embedded processor, LR33000, code named Cobra, to its Rad-Hard 'equivalent', Mongoose. The term 'equivalent' is used to infer that the core of the processor is functionally identical, allowing the same use and optimizations of the MIPS-I Instruction Set software tool suite for compilation, software program trace, etc. This activity was started in September of 1991 under a contract from NASA-Goddard Space Flight Center (GSFC)-Flight Data Systems. The approach affected a teaming of NASA-GSFC for program development, LSI Logic for system and ASIC design coupled with the Rad-Hard process technology, and Harris (GASD) for Rad-Hard microprocessor design expertise. The program culminated with the generation of Rad-Hard Mongoose prototypes one year later.

  2. Rad-Hard/HI-REL FPGA

    NASA Technical Reports Server (NTRS)

    Wang, Jih-Jong; Cronquist, Brian E.; McGowan, John E.; Katz, Richard B.

    1997-01-01

    The goals for a radiation hardened (RAD-HARD) and high reliability (HI-REL) field programmable gate array (FPGA) are described. The first qualified manufacturer list (QML) radiation hardened RH1280 and RH1020 were developed. The total radiation dose and single event effects observed on the antifuse FPGA RH1280 are reported on. Tradeoffs and the limitations in the single event upset hardening are discussed.

  3. The ESA RADGLASS activity: a radiation study of non rad-hard glasses

    NASA Astrophysics Data System (ADS)

    Manolis, Ilias; Bézy, Jean-Loup; Costantino, Alessandra; Vink, Ramon; Deep, Atul; Ahmad, Munadi; Amorim, Emmanuel; Miranda, Micael D.; Meynart, Roland

    2015-10-01

    Only a small set of radiation hardened optical glasses are currently offered in the market, thus drastically limiting the optical design choices available to the engineers at the early phases of an instrument development. Furthermore, availability of those glasses cannot be easily guaranteed for the long term horizon of future space instrument developments. Radiation tests on conventional glasses on the other hand have shown significant sensitivity to high radiation levels but such levels are not necessarily representative of typical low Earth (LEO) orbits. We have conducted irradiation campaigns on several different types of conventional, non-radiation hard glasses, selected from the wider pool of the Schott "new" arsenic and lead free series (N-*) and characterized their spectral transmission properties before and after ionizing dose deposition. We report our first findings here.

  4. Nonvolatile Rad-Hard Holographic Memory

    NASA Technical Reports Server (NTRS)

    Chao, Tien-Hsin; Zhou, Han-Ying; Reyes, George; Dragoi, Danut; Hanna, Jay

    2001-01-01

    We are investigating a nonvolatile radiation-hardened (rad-hard) holographic memory technology. Recently, a compact holographic data storage (CHDS) breadboard utilizing an innovative electro-optic scanner has been built and demonstrated for high-speed holographic data storage and retrieval. The successful integration of this holographic memory breadboard has paved the way for follow-on radiation resistance test of the photorefractive (PR) crystal, Fe:LiNbO3. We have also started the investigation of using two-photon PR crystals that are doubly doped with atoms of iron group (Ti, Cr, Mn, Cu) and of rare-earth group (Nd, Tb) for nonvolatile holographic recordings.

  5. Rad-Hard Microcontroller for Space Applications

    NASA Astrophysics Data System (ADS)

    Habinc, Sandi; Johansson, Fredrik; Sturesson, Fredrik; Simlastik, Martin; Hjorth, Magnus; Andersson, Jan; Redant, Steven; Sijbers, Wim; Thys, Geert; Monteleone, Claudio

    2015-09-01

    This paper describes a mixed-signal LEON3FT microcontroller ASIC (Application Specific Integrated Circuit) targeting embedded control applications with hard real-time requirements. The prototype device is currently in development at Cobham Gaisler, Sweden, and IMEC, Belgium, in the activity Microcontroller for embedded space applications, initiated and funded by the European Space Agency (ESA).

  6. JPIC-Rad-Hard JPEG2000 Image Compression ASIC

    NASA Astrophysics Data System (ADS)

    Zervas, Nikos; Ginosar, Ran; Broyde, Amitai; Alon, Dov

    2010-08-01

    JPIC is a rad-hard high-performance image compression ASIC for the aerospace market. JPIC implements tier 1 of the ISO/IEC 15444-1 JPEG2000 (a.k.a. J2K) image compression standard [1] as well as the post compression rate-distortion algorithm, which is part of tier 2 coding. A modular architecture enables employing a single JPIC or multiple coordinated JPIC units. JPIC is designed to support wide data sources of imager in optical, panchromatic and multi-spectral space and airborne sensors. JPIC has been developed as a collaboration of Alma Technologies S.A. (Greece), MBT/IAI Ltd (Israel) and Ramon Chips Ltd (Israel). MBT IAI defined the system architecture requirements and interfaces, The JPEG2K-E IP core from Alma implements the compression algorithm [2]. Ramon Chips adds SERDES interfaces and host interfaces and integrates the ASIC. MBT has demonstrated the full chip on an FPGA board and created system boards employing multiple JPIC units. The ASIC implementation, based on Ramon Chips' 180nm CMOS RadSafe[TM] RH cell library enables superior radiation hardness.

  7. The ESA Rad-Hard electron monitor (RADEM) for JUICE

    NASA Astrophysics Data System (ADS)

    Desorgher, Laurent; Hajdas, Wojtek; Goncalves, Patricia; Pinto, Costa; Marques, Arlindo; Chastellain, Frédéric; Gambarara, Fabio; Muff, Reto; Maehlum, Gunnar; Meier, Dirk

    2014-05-01

    The ESA Jupiter Icy moons explorer (JUICE) mission will encounter a harsh radiation environment that is known to be severe but that is not yet fully understood. The Rad-Hard electron monitor (RADEM), currently under development, is a compact instrument (1L, 1kg, 2.2W) that will be set on JUICE for measuring the radiation environment during the mission. Its design is adapted to the harsh Jovian radiation environment and optimized for the detection of high energetic electrons. RADEM will consist of three detector subunits. The magneto-spectrometer will measure the electron spectrum in the 0.3 to 40 MeV range. The directionality sensor will characterize the pitch angle distribution of the electron environment. The Silicon stack detector will be dedicated to measure the spectrum of solar and Jovian protons, as well as the LET spectrum of heavy ions. In this paper we present the status of the development of RADEM, as well as Geant4 Monte Carlo analysis of the capability of the instruments.

  8. Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

    NASA Technical Reports Server (NTRS)

    Lio, Yi-Ching; Mazin, Alexander V.; Kowalczykowski, Stephen C.; Chen, David J.

    2003-01-01

    The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.

  9. Rad-hard electronics study for SSC detectors

    SciTech Connect

    Ekenberg, T.; Dawson, J.; Stevens, A.; Haberichter, W.

    1991-01-01

    The radiation environment in a SSC detector operating at a luminosity of 10{sup 33} cm{sup {minus}2}s{sup {minus}1} will put stringent requirements on radiation hardness of the electronics. Over the expected 10 year life-time of a large detector, ionizing radiation doses of up to 20 MRad and neutron fluences of 10{sup 16} neutrons/cm{sup 2} are projected. At a luminosity of 10{sup 34} cm{sup {minus}2}s{sup {minus}1} even higher total doses are expected. the effect of this environment have been simulated by exposing CMOS/bulk and CMOS/SOS devices from monolithic processes to neutrons and ionizing radiation. leakage currents, noise variations, and DC characteristics have been measured before and after exposure in order to evaluate the effects of the irradiations. As expected the device characteristics remained virtually unchanged by neutron irradiation, while ionizing radiation caused moderate degradation of performance. 5 refs., 6 figs.

  10. Nanopatterned ferroelectrics for ultrahigh density rad-hard nonvolatile memories.

    SciTech Connect

    Brennecka, Geoffrey L.; Stevens, Jeffrey; Scrymgeour, David; Gin, Aaron V.; Tuttle, Bruce Andrew

    2010-09-01

    Radiation hard nonvolatile random access memory (NVRAM) is a crucial component for DOE and DOD surveillance and defense applications. NVRAMs based upon ferroelectric materials (also known as FERAMs) are proven to work in radiation-rich environments and inherently require less power than many other NVRAM technologies. However, fabrication and integration challenges have led to state-of-the-art FERAMs still being fabricated using a 130nm process while competing phase-change memory (PRAM) has been demonstrated with a 20nm process. Use of block copolymer lithography is a promising approach to patterning at the sub-32nm scale, but is currently limited to self-assembly directly on Si or SiO{sub 2} layers. Successful integration of ferroelectrics with discrete and addressable features of {approx}15-20nm would represent a 100-fold improvement in areal memory density and would enable more highly integrated electronic devices required for systems advances. Towards this end, we have developed a technique that allows us to carry out block copolymer self-assembly directly on a huge variety of different materials and have investigated the fabrication, integration, and characterization of electroceramic materials - primarily focused on solution-derived ferroelectrics - with discrete features of {approx}20nm and below. Significant challenges remain before such techniques will be capable of fabricating fully integrated NVRAM devices, but the tools developed for this effort are already finding broader use. This report introduces the nanopatterned NVRAM device concept as a mechanism for motivating the subsequent studies, but the bulk of the document will focus on the platform and technology development.

  11. Rad-Hard, Miniaturized, Scalable, High-Voltage Switching Module for Power Applications Rad-Hard, Miniaturized

    NASA Technical Reports Server (NTRS)

    Adell, Philippe C.; Mojarradi, Mohammad; DelCastillo, Linda Y.; Vo, Tuan A.

    2011-01-01

    A paper discusses the successful development of a miniaturized radiation hardened high-voltage switching module operating at 2.5 kV suitable for space application. The high-voltage architecture was designed, fabricated, and tested using a commercial process that uses a unique combination of 0.25 micrometer CMOS (complementary metal oxide semiconductor) transistors and high-voltage lateral DMOS (diffusion metal oxide semiconductor) device with high breakdown voltage (greater than 650 V). The high-voltage requirements are achieved by stacking a number of DMOS devices within one module, while two modules can be placed in series to achieve higher voltages. Besides the high-voltage requirements, a second generation prototype is currently being developed to provide improved switching capabilities (rise time and fall time for full range of target voltages and currents), the ability to scale the output voltage to a desired value with good accuracy (few percent) up to 10 kV, to cover a wide range of high-voltage applications. In addition, to ensure miniaturization, long life, and high reliability, the assemblies will require intensive high-voltage electrostatic modeling (optimized E-field distribution throughout the module) to complete the proposed packaging approach and test the applicability of using advanced materials in a space-like environment (temperature and pressure) to help prevent potential arcing and corona due to high field regions. Finally, a single-event effect evaluation would have to be performed and single-event mitigation methods implemented at the design and system level or developed to ensure complete radiation hardness of the module.

  12. RC64, a Rad-Hard Many-Core High- Performance DSP for Space Applications

    NASA Astrophysics Data System (ADS)

    Ginosar, Ran; Aviely, Peleg; Gellis, Hagay; Liran, Tuvia; Israeli, Tsvika; Nesher, Roy; Lange, Fredy; Dobkin, Reuven; Meirov, Henri; Reznik, Dror

    2015-09-01

    RC64, a novel rad-hard 64-core signal processing chip targets DSP performance of 75 GMACs (16bit), 150 GOPS and 38 single precision GFLOPS while dissipating less than 10 Watts. RC64 integrates advanced DSP cores with a multi-bank shared memory and a hardware scheduler, also supporting DDR2/3 memory and twelve 3.125 Gbps full duplex high speed serial links using SpaceFibre and other protocols. The programming model employs sequential fine-grain tasks and a separate task map to define task dependencies. RC64 is implemented as a 300 MHz integrated circuit on a 65nm CMOS technology, assembled in hermetically sealed ceramic CCGA624 package and qualified to the highest space standards.

  13. Low power hard-rad electronics for particle detection in space plasmas

    NASA Astrophysics Data System (ADS)

    Berthomier, Matthieu; Techer, Jean-Denis

    2014-05-01

    Particle detection in highly-radiative environment is one of the experimental challenges of planetary exploration. We present the design and performances of a compact electron detector that takes advantage of the development of a hard-rad and ultra low-power front-end electronics. The Applied Specific Integrated Circuit (ASIC) consists of charge sensitive amplifiers and discriminators allowing a 4.5MHz periodic counting rate. The 16-channels ASIC only takes 30mW of power which is the power budget of single channel hybrid components with similar performances. Each channel can be independently configured in order to adjust the detection threshold of the discriminator. An internal test circuitry is used to monitor the behavior of the electronics. This component, that has been tested at high ionizing doses, is immune to Single Event Latchups up to at least 80 MeV.cm^2/mg and it will fly on the Solar Orbiter ESA mission.

  14. Electron trapping in rad-hard RCA IC's irradiated with electrons and gamma rays

    NASA Technical Reports Server (NTRS)

    Danchenko, V.; Brashears, S. S.; Fang, P. H.

    1984-01-01

    Enhanced electron trapping has been observed in n-channels of rad-hard CMOS devices due to electron and gamma-ray irradiation. Room-temperature annealing results in a positive shift in the threshold potential far beyond its initial value. The slope of the annealing curve immediately after irradiation was found to depend strongly on the gate bias applied during irradiation. Some dependence was also observed on the electron dose rate. No clear dependence on energy and shielding over a delidded device was observed. The threshold shift is probably due to electron trapping at the radiation-induced interface states and tunneling of electrons through the oxide-silicon energy barrier to fill the radiation-induced electron traps. A mathematical analysis, based on two parallel annealing kinetics, hole annealing and electron trapping, is applied to the data for various electron dose rates.

  15. Type I interferon-dependent activation of NK cells by rAd28 or rAd35, but not rAd5, leads to loss of vector-insert expression.

    PubMed

    Johnson, Matthew J; Björkström, Niklas K; Petrovas, Constantinos; Liang, Frank; Gall, Jason G D; Loré, Karin; Koup, Richard A

    2014-02-01

    Vaccines constructed from rare-serotype recombinant adenovirus vectors (rAd) such as rAd serotype 28 (rAd28) and rAd35 are currently being explored as alternatives to rAd5-based vaccines because they circumvent the problems with pre-existing immunity that complicate the effectiveness of rAd5 vaccines. However, previous work has demonstrated that the immunogenicity of rAd28 and rAd35 is substantially lower than rAd5. Here we show that rAd28 and rAd35 increase apoptosis of antigen presenting cells (APCs), such as monocytes, relative to rAd5 and mock infected controls. APCs undergoing apoptosis showed an increased loss of vector-insert expression. Loss of vector-insert expression correlated with activation of NK cells, which resulted in apoptosis of co-cultured monocytes. Finally, we show that activation of NK cells is dependent on IFNα which is produced by exposure to rAd28 or rAd35, but not to rAd5. Taken together, these data demonstrate that IFNα-induced activation of NK cells leads to increased monocyte apoptosis and subsequent vector-insert loss. This may be a possible mechanism that results in reduced immunogenicity of rAd28 and rAd35-based vectors. PMID:24325826

  16. Atmel's New Rad-Hard Sparc V8 Processor 200Mhz & Low Power System on Chip

    NASA Astrophysics Data System (ADS)

    Ganry, Nicolas; Mantelet, Guy; Parkes, Steve; McClements, Chris

    2014-08-01

    The AT6981 is a new generation of processor designed for critical spaceflight applications, which combines a high-performance SPARC® V8 radiation hard processor, with enough on-chip memory for many aerospace applications and state-of-the-art SpaceWire networking technology from STARDundee. The AT6981 is implemented in Atmel 90nm rad-hard technology, enabling 200 MHz operating speed for the processor with power consumption levels around 1W. This advanced technology allows strong system integration in a SoC with embedded peripherals like CAN, 1553, Ethernet, DDR and embedded memory with 1Mbytes SRAM. The device is ITARfree and is developed in France by Atmel Aerospace having more than of 30years space experience. This paper describes this new SoC architecture and technical options considered to insure the best performances, the minimum power consumption and high reliability. This device will be available on the market in H2 2014 for evaluation with first flight models targeted end 2015.

  17. Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL*S⃞

    PubMed Central

    Takaku, Motoki; Machida, Shinichi; Hosoya, Noriko; Nakayama, Shugo; Takizawa, Yoshimasa; Sakane, Isao; Shibata, Takehiko; Miyagawa, Kiyoshi; Kurumizaka, Hitoshi

    2009-01-01

    The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone promotes single-stranded DNA annealing, and the recombination activities of the EVL protein are further enhanced by the RAD51B protein. The expression of the EVL protein is not ubiquitous, but it is significantly expressed in breast cancer-derived MCF7 cells. These results suggest that the EVL protein is a novel recombination factor that may be required for repairing specific DNA lesions, and that may cause tumor malignancy by its inappropriate expression. PMID:19329439

  18. Reconfigurable, Bi-Directional Flexfet Level Shifter for Low-Power, Rad-Hard Integration

    NASA Technical Reports Server (NTRS)

    DeGregorio, Kelly; Wilson, Dale G.

    2009-01-01

    Two prototype Reconfigurable, Bi-directional Flexfet Level Shifters (ReBiLS) have been developed, where one version is a stand-alone component designed to interface between external low voltage and high voltage, and the other version is an embedded integrated circuit (IC) for interface between internal low-voltage logic and external high-voltage components. Targeting stand-alone and embedded circuits separately allows optimization for these distinct applications. Both ReBiLS designs use the commercially available 180-nm Flex fet Independently Double-Gated (IDG) SOI CMOS (silicon on insulator, complementary metal oxide semiconductor) technology. Embedded ReBiLS circuits were integrated with a Reed-Solomon (RS) encoder using CMOS Ultra-Low-Power Radiation Tolerant (CULPRiT) double-gated digital logic circuits. The scope of the project includes: creation of a new high-voltage process, development of ReBiLS circuit designs, and adjustment of the designs to maximize performance through simulation, layout, and manufacture of prototypes. The primary technical objectives were to develop a high-voltage, thick oxide option for the 180-nm Flexfet process, and to develop a stand-alone ReBiLS IC with two 8-channel I/O busses, 1.8 2.5 I/O on the low-voltage pins, 5.0-V-tolerant input and 3.3-V output I/O on the high-voltage pins, and 100-MHz minimum operation with 10-pF external loads. Another objective was to develop an embedded, rad-hard ReBiLS I/O cell with 0.5-V low-voltage operation for interface with core logic, 5.0-V-tolerant input and 3.3-V output I/O pins, and 100-MHz minimum operation with 10- pF external loads. A third objective was to develop a 0.5- V Reed-Solomon Encoder with embedded ReBilS I/O: Transfer the existing CULPRiT RS encoder from a 0.35-micron bulk-CMOS process to the ASI 180-nm Flexfet, rad-hard SOI Process. 0.5-V low-voltage core logic. 5.0-V-tolerant input and 3.3-V output I/O pins. 100-MHz minimum operation with 10- pF external loads. The stand

  19. Silicon-on-insulator field effect transistor with improved body ties for rad-hard applications

    DOEpatents

    Schwank, James R.; Shaneyfelt, Marty R.; Draper, Bruce L.; Dodd, Paul E.

    2001-01-01

    A silicon-on-insulator (SOI) field-effect transistor (FET) and a method for making the same are disclosed. The SOI FET is characterized by a source which extends only partially (e.g. about half-way) through the active layer wherein the transistor is formed. Additionally, a minimal-area body tie contact is provided with a short-circuit electrical connection to the source for reducing floating body effects. The body tie contact improves the electrical characteristics of the transistor and also provides an improved single-event-upset (SEU) radiation hardness of the device for terrestrial and space applications. The SOI FET also provides an improvement in total-dose radiation hardness as compared to conventional SOI transistors fabricated without a specially prepared hardened buried oxide layer. Complementary n-channel and p-channel SOI FETs can be fabricated according to the present invention to form integrated circuits (ICs) for commercial and military applications.

  20. Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity

    PubMed Central

    Kumar, Pankaj; Magala, Pearl; Geiger-Schuller, Kathryn R.; Majumdar, Ananya; Tolman, Joel R.; Wolberger, Cynthia

    2015-01-01

    Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity. PMID:26286193

  1. Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

    PubMed

    Callender, Tracy L; Laureau, Raphaelle; Wan, Lihong; Chen, Xiangyu; Sandhu, Rima; Laljee, Saif; Zhou, Sai; Suhandynata, Ray T; Prugar, Evelyn; Gaines, William A; Kwon, YoungHo; Börner, G Valentin; Nicolas, Alain; Neiman, Aaron M; Hollingsworth, Nancy M

    2016-08-01

    During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1. PMID:27483004

  2. Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1

    PubMed Central

    Callender, Tracy L.; Laljee, Saif; Zhou, Sai; Suhandynata, Ray T.; Gaines, William A.; Kwon, YoungHo; Börner, G. Valentin; Nicolas, Alain; Neiman, Aaron M.

    2016-01-01

    During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1. PMID:27483004

  3. Recovery of damage in rad-hard MOS devices during and after irradiation by electrons, protons, alphas, and gamma rays

    NASA Technical Reports Server (NTRS)

    Brucker, G. J.; Van Gunten, O.; Stassinopoulos, E. G.; Shapiro, P.; August, L. S.; Jordan, T. M.

    1983-01-01

    This paper reports on the recovery properties of rad-hard MOS devices during and after irradiation by electrons, protons, alphas, and gamma rays. The results indicated that complex recovery properties controlled the damage sensitivities of the tested parts. The results also indicated that damage sensitivities depended on dose rate, total dose, supply bias, gate bias, transistor type, radiation source, and particle energy. The complex nature of these dependencies make interpretation of LSI device performance in space (exposure to entire electron and proton spectra) difficult, if not impossible, without respective ground tests and analyses. Complete recovery of n-channel shifts was observed, in some cases within hours after irradiation, with equilibrium values of threshold voltages greater than their pre-irradiation values. This effect depended on total dose, radiation source, and gate bias during exposure. In contrast, the p-channel shifts recovered only 20 percent within 30 days after irradiation.

  4. Development of Small Molecules that Specifically Inhibit the D-loop Activity of RAD51.

    PubMed

    Lv, Wei; Budke, Brian; Pawlowski, Michal; Connell, Philip P; Kozikowski, Alan P

    2016-05-26

    RAD51 is the central protein in homologous recombination (HR) DNA repair and represents a therapeutic target in oncology. Herein we report a novel class of RAD51 inhibitors that were identified by high throughput screening. In contrast to many previously reported RAD51 inhibitors, our lead compound 1 is capable of blocking RAD51-mediated D-loop formation (IC50 21.3 ± 7.8 μM) at concentrations that do not influence RAD51 binding to ssDNA. In human cells, 1 inhibits HR (IC50 13.1 ± 1.6 μM) without blocking RAD51's ability to assemble into subnuclear foci at sites of DNA damage. We determined that the active constituent of 1 is actually an oxidized derivative (termed RI(dl)-1 or 8) of the original screening compound. Our SAR campaign also yielded RI(dl)-2 (hereafter termed 9h), which effectively blocks RAD51's D-loop activity in biochemical systems (IC50 11.1 ± 1.3 μM) and inhibits HR activity in human cells (IC50 3.0 ± 1.8 μM). PMID:27049177

  5. GR740: Rad-Hard Quad-Core LEON4FT System-on-Chip

    NASA Astrophysics Data System (ADS)

    Hijorth, Magnus; Aberg, Martin; Wessman, Nils-Johan; Andersson, Jan; Chevallier, Remy; Forsyth, Russel; Weigand, Rolad; Fossati, Luca

    2015-09-01

    The GR740 microprocessor device is a SPARC V8(E) based multi-core architecture that provides a significant performance increase compared to earlier generations of European space processors. The GR740 is currently in development at Cobham Gaisler, Sweden, and STMicroelectronics, France, in activities to develop the Next Generation Microprocessor (NGMP) initiated and funded by the European Space Agency (ESA).

  6. The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae.

    PubMed Central

    Verhage, R; Zeeman, A M; de Groot, N; Gleig, F; Bang, D D; van de Putte, P; Brouwer, J

    1994-01-01

    The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D. D. Bang, R. A. Verhage, N. Goosen, J. Brouwer, and P. van de Putte, Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs. Images PMID:8065346

  7. Evaluation of the pharmacological activities of RAD1901, a selective estrogen receptor degrader.

    PubMed

    Wardell, Suzanne E; Nelson, Erik R; Chao, Christina A; Alley, Holly M; McDonnell, Donald P

    2015-10-01

    Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains a first-line treatment for estrogen receptor 1 (ESR1) positive breast cancer. However, tumor resistance limits the duration of response. The clinical efficacy of fulvestrant, a selective ER degrader (SERD) that triggers receptor degradation, has confirmed that ESR1 often remains engaged in endocrine therapy resistant cancers. Recently developed, selective ER modulators (SERMs)/SERD hybrids (SSHs) that facilitate ESR1 degradation in breast cancer cells and reproductive tissues have been advanced as an alternative treatment for advanced breast cancer, particularly in the metastatic setting. RAD1901 is one SSH currently being evaluated clinically that is unique among ESR1 modulators in that it readily enters the brain, a common site of breast cancer metastasis. In this study, RAD1901 inhibited estrogen activation of ESR1 in vitro and in vivo, inhibited estrogen-dependent breast cancer cell proliferation and xenograft tumor growth, and mediated dose-dependent downregulation of ESR1 protein. However, doses of RAD1901 insufficient to induce ESR1 degradation were shown to result in the activation of ESR1 target genes and in the stimulation of xenograft tumor growth. RAD1901 is an SSH that exhibits complex pharmacology in breast cancer models, having dose-dependent agonist/antagonist activity displayed in a tissue-selective manner. It remains unclear how this unique pharmacology will impact the utility of RAD1901 for breast cancer treatment. However, being the only SERD currently known to access the brain, RAD1901 merits evaluation as a targeted therapy for the treatment of breast cancer brain metastases. PMID:26162914

  8. Cdc18/CDC6 activates the Rad3-dependent checkpoint in the fission yeast.

    PubMed

    Fersht, Naomi; Hermand, Damien; Hayles, Jacqueline; Nurse, Paul

    2007-01-01

    A screen for genes that can ectopically activate a Rad3-dependent checkpoint block over mitosis in fission yeast has identified the DNA replication initiation factor cdc18 (known as CDC6 in other organisms). Either a stabilized form of Cdc18, the Cdc18-T6A phosphorylation mutant, or overexpression of wild type Cdc18, activate the Rad3-dependent S-M checkpoint in the apparent absence of detectable replication structures and gross DNA damage. This cell cycle block relies on the Rad checkpoint pathway and requires Chk1 phosphorylation and activation. Unexpectedly, Cdc18-T6A induces changes in the mobility of Chromosome III, affecting the size of a restriction fragment containing rDNA repeats and producing aberrant nucleolar structures. Recombination events within the rDNA appear to contribute at least in part to the cell cycle delay. We propose that an elevated level of Cdc18 activates the Rad3-dependent checkpoint either directly or indirectly, and additionally causes expansion of the rDNA repeats on Chromosome III. PMID:17690116

  9. Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain.

    PubMed

    Pike, Brietta L; Tenis, Nora; Heierhorst, Jörg

    2004-09-17

    Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways. PMID:15271990

  10. Sae2 Function at DNA Double-Strand Breaks Is Bypassed by Dampening Tel1 or Rad53 Activity

    PubMed Central

    Gnugnoli, Marco; Menin, Luca; Clerici, Michela; Longhese, Maria Pia

    2015-01-01

    The MRX complex together with Sae2 initiates resection of DNA double-strand breaks (DSBs) to generate single-stranded DNA (ssDNA) that triggers homologous recombination. The absence of Sae2 not only impairs DSB resection, but also causes prolonged MRX binding at the DSBs that leads to persistent Tel1- and Rad53-dependent DNA damage checkpoint activation and cell cycle arrest. Whether this enhanced checkpoint signaling contributes to the DNA damage sensitivity and/or the resection defect of sae2Δ cells is not known. By performing a genetic screen, we identify rad53 and tel1 mutant alleles that suppress both the DNA damage hypersensitivity and the resection defect of sae2Δ cells through an Sgs1-Dna2-dependent mechanism. These suppression events do not involve escaping the checkpoint-mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function at DSBs by decreasing the amount of Rad9 bound at DSBs. As a consequence, reduced Rad9 association to DNA ends relieves inhibition of Sgs1-Dna2 activity, which can then compensate for the lack of Sae2 in DSB resection and DNA damage resistance. We propose that persistent Tel1 and Rad53 checkpoint signaling in cells lacking Sae2 increases the association of Rad9 at DSBs, which in turn inhibits DSB resection by limiting the activity of the Sgs1-Dna2 resection machinery. PMID:26584331

  11. RAD51 and BRCA2 enhance oncolytic adenovirus type 5 activity in ovarian cancer

    PubMed Central

    Tookman, Laura A.; Browne, Ashley K.; Connell, Claire M.; Bridge, Gemma; Ingemarsdotter, Carin K.; Dowson, Suzanne; Shibata, Atsushi; Lockley, Michelle; Martin, Sarah A.; McNeish, Iain A.

    2015-01-01

    Homologous Recombination (HR) function is critically important in High Grade Serous Ovarian Cancer (HGSOC). HGSOC with intact HR has a worse prognosis and is less likely to respond to platinum chemotherapy and PARP inhibitors. Oncolytic adenovirus, a novel therapy for human malignancies, stimulates a potent DNA damage response that influences overall anti-tumor activity. Here, the importance of HR was investigated by determining the efficacy of adenovirus type 5 (Ad5) vectors in ovarian cancer. Using matched BRCA2 mutant and wild-type HGSOC cells, it was demonstrated that intact HR function promotes viral DNA replication and augments overall efficacy, without influencing viral DNA processing. These data were confirmed in a wider panel of HR competent and defective ovarian cancer lines. Mechanistically, both BRCA2 and RAD51 localize to viral replication centers within the infected cell nucleus and that RAD51 localization occurs independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Finally, using functional assays of HR competence, despite inducing degradation of MRE11, Ad5 infection does not alter cellular ability to repair DNA double strand break damage via HR. These data reveal that Ad5 redistributes critical HR components to viral replication centers and enhances cytotoxicity. Implications Oncolytic adenoviral therapy may be most clinically relevant in tumors with intact HR function. PMID:26452665

  12. Novel Attributes of Hed1 Affect Dynamics and Activity of the Rad51 Presynaptic Filament during Meiotic Recombination*

    PubMed Central

    Busygina, Valeria; Saro, Dorina; Williams, Gareth; Leung, Wing-Kit; Say, Amanda F.; Sehorn, Michael G.; Sung, Patrick; Tsubouchi, Hideo

    2012-01-01

    During meiosis, recombination events that occur between homologous chromosomes help prepare the chromosome pairs for proper disjunction in meiosis I. The concurrent action of the Rad51 and Dmc1 recombinases is necessary for an interhomolog bias. Notably, the activity of Rad51 is tightly controlled, so as to minimize the use of the sister chromatid as recombination partner. We demonstrated recently that Hed1, a meiosis-specific protein in Saccharomyces cerevisiae, restricts the access of the recombinase accessory factor Rad54 to presynaptic filaments of Rad51. We now show that Hed1 undergoes self-association in a Rad51-dependent manner and binds ssDNA. We also find a strong stabilizing effect of Hed1 on the Rad51 presynaptic filament. Biochemical and genetic analyses of mutants indicate that these Hed1 attributes are germane for its recombination regulatory and Rad51 presynaptic filament stabilization functions. Our results shed light on the mechanism of action of Hed1 in meiotic recombination control. PMID:22115747

  13. Enhanced Histone Deacetylase Activity in Malignant Melanoma Provokes RAD51 and FANCD2-Triggered Drug Resistance.

    PubMed

    Krumm, Andrea; Barckhausen, Christina; Kücük, Pelin; Tomaszowski, Karl-Heinz; Loquai, Carmen; Fahrer, Jörg; Krämer, Oliver Holger; Kaina, Bernd; Roos, Wynand Paul

    2016-05-15

    DNA-damaging anticancer drugs remain a part of metastatic melanoma therapy. Epigenetic reprogramming caused by increased histone deacetylase (HDAC) activity arising during tumor formation may contribute to resistance of melanomas to the alkylating drugs temozolomide, dacarbazine, and fotemustine. Here, we report on the impact of class I HDACs on the response of malignant melanoma cells treated with alkylating agents. The data show that malignant melanomas in situ contain a high level of HDAC1/2 and malignant melanoma cells overexpress HDAC1/2/3 compared with noncancer cells. Furthermore, pharmacologic inhibition of class I HDACs sensitizes malignant melanoma cells to apoptosis following exposure to alkylating agents, while not affecting primary melanocytes. Inhibition of HDAC1/2/3 caused sensitization of melanoma cells to temozolomide in vitro and in melanoma xenografts in vivo HDAC1/2/3 inhibition resulted in suppression of DNA double-strand break (DSB) repair by homologous recombination because of downregulation of RAD51 and FANCD2. This sensitized cells to the cytotoxic DNA lesion O(6)-methylguanine and caused a synthetic lethal interaction with the PARP-1 inhibitor olaparib. Furthermore, knockdown experiments identified HDAC2 as being responsible for the regulation of RAD51. The influence of class I HDACs on DSB repair by homologous recombination and the possible clinical implication on malignant melanoma therapy with temozolomide and other alkylating drugs suggests a combination approach where class I HDAC inhibitors such as valproic acid or MS-275 (entinostat) appear to counteract HDAC- and RAD51/FANCD2-mediated melanoma cell resistance. Cancer Res; 76(10); 3067-77. ©2016 AACR. PMID:26980768

  14. XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination

    SciTech Connect

    Yamada, N; Hinz, J; Kopf, V L; Segalle, K; Thompson, L

    2004-02-25

    Homologous recombinational repair is a major DNA repair pathway that preserves chromosomal integrity by removing double-strand breaks, crosslinks, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 appears to exist in a single complex with Rad51C. To begin to examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a non-conservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF CHO cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, while ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of the mutants' dysfunction, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon coexpression in bacteria, nickel affinity purification, and western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, while the K113R mutant did not and was predominantly insoluble. Addition of 5 mM ATP, but not ADP, also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 is likely to regulate the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis, with both processes being essential for the complex's ability to participate in HRR.

  15. A Structural Hinge in Eukaryotic MutY Homologues Mediates Catalytic Activity and Rad9-Rad1-Hus1 Checkpoint Complex Interactions

    SciTech Connect

    P Luncsford; D Chang; G Shi; J Bernstein; A Madabushi; D Patterson; A Lu; E Toth

    2011-12-31

    The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome MYH-associated polyposis. A key feature of MYH activity is its coordination with cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector (IDC, residues 295-350) between the catalytic domain and the 8-oxoguanine recognition domain of hMYH is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH as model system. In vitro studies of S. pombe MYH identified residues I261 and E262 of the IDC (equivalent to V315 and E316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes the repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.

  16. Design, realization and test of a rad-hard 2D-compressor and packing chip for high energy physics experiments

    NASA Astrophysics Data System (ADS)

    Antinori, Samuele; Falchieri, Davide; Gabrielli, Alessandro; Gandolfi, Enzo

    2004-09-01

    CARLOSv3 is a third version of a chip that plays a significant role in the data acquisition chain of the A Large Ion Collider Experiment Inner Tracking System experiment. It has been designed and realized with a 0.25 μm CMOS 3-metal rad-hard digital library. The chip elaborates and compresses, by means of a bi-dimensional compressor, data belonging to a so-called event. The compressor looks for cross-shaped clusters within the whole data set coming from the silicon detector. To test the chip a specific PCB has been designed; it contains the connectors for probing the ASIC with a pattern generator and a logic state analyzer. The chip is inserted on the PCB using a ZIF socket. This allows to test the 35 packaged samples out of the total amount of bare chips we have from the foundry. The test phase has shown that 32 out of 35 chips under test work well. It is planned to redesign a new version of the chip by adding extra features and to submit the final version of CARLOS upon the final DAQ chain will be totally tested both in Bologna and at CERN.

  17. Enhancement of the RAD51 Recombinase Activity by the Tumor Suppressor PALB2

    SciTech Connect

    Dray, Eloise; Etchin, Julia; Wiese, Claudia; Saro, Dorina; Williams, Gareth J.; Hammel, Michal; Yu, Xiong; Galkin, Vitold E.; Liu, Dongqing; Tsai, Miaw-Sheue; Sy, Shirley M-H.; Egelman, Edward; Chen, Junjie; Sung, Patrick; Schild, D.

    2010-08-24

    Homologous recombination mediated by the RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-stranded breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2 in the enhancement of RAD51's ability to form the D-loop. We show that PALB2 binds DNA and physically interacts with RAD51. Importantly, while PALB2 alone stimulates D-loop formation, a cooperative effect is seen with RAD51AP1, an enhancer of RAD51. This stimulation stems from PALB2's ability to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results help unveil a multi-faceted role of PALB2 in chromosome damage repair. Since PALB2 mutations can cause breast and other tumors or lead to Fanconi anemia, our findings are important for understanding the mechanism of tumor suppression in humans.

  18. Learning our ABCs: Rad50 directs MRN repair functions via adenylate kinase activity from the conserved ATP binding cassette.

    PubMed

    Williams, R Scott; Tainer, John A

    2007-03-23

    In groundbreaking work, Bhaskara et al. (2007) demonstrate in a recent issue of Molecular Cell that the Mre11/Rad50/Nbs1 (MRN) complex harbors distinct, yet chemically related, ATPase and adenylate kinase catalytic activities that together orchestrate multiple requisite MRN functional and conformational states in dsDNA break repair sensing and signaling with general implications for ABC ATPases. PMID:17386254

  19. Regulation of active genome integrity and expression by Rad26p

    PubMed Central

    Malik, Shivani; Bhaumik, Sukesh R

    2014-01-01

    Rad26p is a SWI/SNF-like ATPase in yeast, and is conserved among eukaryotes. Both Rad26p and its human homolog CSB (Cockayne syndrome group B) are involved in regulation of chromatin structure, transcription and DNA repair.  Thus, mutations or malfunctions of these proteins have significant effects on cellular functions. Mutations in CSB are associated with Cockayne syndrome (CS) that is characterized by heterogeneous pathologies such as mental and physical retardation, sun sensitivity, premature aging, muscular and skeletal abnormalities, and progressive decline in neurological and cognitive functions. Therefore, many research groups focused their studies to understand the mechanisms of Rad26p/CSB functions to illuminate the molecular bases of CS. These studies have provided significant functional and mechanistic insights of Rad26p/CSB in regulation of gene expression and genome integrity as described here. PMID:25484185

  20. RAD hard PROM design study

    NASA Technical Reports Server (NTRS)

    1981-01-01

    The results of a preliminary study on the design of a radiation hardened fusible link programmable read-only memory (PROM) are presented. Various fuse technologies and the effects of radiation on MOS integrated circuits are surveyed. A set of design rules allowing the fabrication of a radiation hardened PROM using a Si-gate CMOS process is defined. A preliminary cell layout was completed and the programming concept defined. A block diagram is used to describe the circuit components required for a 4 K design. A design goal data sheet giving target values for the AC, DC, and radiation parameters of the circuit is presented.

  1. Fission Yeast Pxd1 Promotes Proper DNA Repair by Activating Rad16XPF and Inhibiting Dna2

    PubMed Central

    Zhang, Jia-Min; Liu, Xiao-Man; Ding, Yue-He; Xiong, Liang-Yao; Ren, Jing-Yi; Zhou, Zhi-Xiong; Wang, Hai-Tao; Zhang, Mei-Jun; Yu, Yang; Dong, Meng-Qiu; Du, Li-Lin

    2014-01-01

    Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3′ endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5′ endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases. PMID:25203555

  2. RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells

    PubMed Central

    Mason, Jennifer M.; Dusad, Kritika; Wright, William Douglass; Grubb, Jennifer; Budke, Brian; Heyer, Wolf-Dietrich; Connell, Philip P.; Weichselbaum, Ralph R.; Bishop, Douglas K.

    2015-01-01

    The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors. PMID:25765654

  3. RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

    PubMed

    Mason, Jennifer M; Dusad, Kritika; Wright, William Douglass; Grubb, Jennifer; Budke, Brian; Heyer, Wolf-Dietrich; Connell, Philip P; Weichselbaum, Ralph R; Bishop, Douglas K

    2015-03-31

    The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors. PMID:25765654

  4. ComRAD Active/Passive Microwave Measurement of Tree Canopies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The NASA/GSFC and George Washington University network analyzer-based multifrequency truck-mounted radar system has recently been upgraded with the addition of a dual-polarized 1.4 GHz total power radiometer. The system, now called ComRAD for Combined Radar/Radiometer, can function as a ground-based...

  5. A Saccharomyces Cerevisiae Rad52 Allele Expressing a C-Terminal Truncation Protein: Activities and Intragenic Complementation of Missense Mutations

    PubMed Central

    Boundy-Mills, K. L.; Livingston, D. M.

    1993-01-01

    A nonsense allele of the yeast RAD52 gene, rad52-327, which expresses the N-terminal 65% of the protein was compared to two missense alleles, rad52-1 and rad52-2, and to a deletion allele. While the rad52-1 and the deletion mutants have severe defects in DNA repair, recombination and sporulation, the rad52-327 and rad52-2 mutants retain either partial or complete capabilities in repair and recombination. These two mutants behave similarly in most tests of repair and recombination during mitotic growth. One difference between these two alleles is that a homozygous rad52-2 diploid fails to sporulate, whereas the homozygous rad52-327 diploid sporulates weakly. The low level of sporulation by the rad52-327 diploid is accompanied by a low percentage of spore viability. Among these viable spores the frequency of crossing over for markers along chromosome VII is the same as that found in wild-type spores. rad52-327 complements rad52-2 for repair and sporulation. Weaker intragenic complementation occurs between rad52-327 and rad52-1. PMID:8417987

  6. Regulation of Rad51 promoter.

    PubMed

    Hine, Christopher M; Li, Hongjie; Xie, Li; Mao, Zhiyong; Seluanov, Andrei; Gorbunova, Vera

    2014-01-01

    The DNA double-strand break repair and homologous recombination protein Rad51 is overexpressed in the majority of human cancers. This correlates with therapy resistance and decreased patient survival. We previously showed that constructs containing Rad51 promoter fused to a reporter gene are, on average, 850-fold more active in cancer cells than in normal cells. It is not well understood what factors and sequences regulate the Rad51 promoter and cause its high activity in cancerous cells. Here we characterized regulatory regions and examined genetic requirements for oncogenic stimulation of the Rad51 promoter. We identified specific regions responsible for up- and downregulation of the Rad51 promoter in cancerous cells. Furthermore, we show that Rad51 expression is positively regulated by EGR1 transcription factor. We then modeled the malignant transformation process by expressing a set of oncoproteins in normal human fibroblasts. Expression of different combinations of SV40 large T antigen, oncogenic Ras and SV40 small T antigen resulted in step-wise increase in Rad51 promoter activity, with all the 3 oncoproteins together leading to a 47-fold increase in expression. Cumulatively, these results suggest that Rad51 promoter is regulated by multiple factors, and that its expression is gradually activated as cells progress toward malignancy. PMID:24781030

  7. Decarbamoyl mitomycin C (DMC) activates p53-independent ataxia telangiectasia and rad3 related protein (ATR) chromatin eviction

    PubMed Central

    Xiao, Gu; Kue, Pao; Bhosle, Rahul; Bargonetti, Jill

    2015-01-01

    Interstrand crosslinks induce DNA replication fork stalling that in turn activates the ATR-dependent checkpoint and DNA repair on nuclear chromatin. Mitomycin C (MC) and Decarbamoyl Mitomycin C (DMC) induce different types of DNA crosslinks with DMC being a more cytotoxic agent. We previously reported that the novel DMC induced β–interstrand DNA crosslinks induce a p53-independent form of cell death. The p53-independent DMC cytotoxicity associates with the activation, and subsequent depletion, of Chk1. In this study we further dissect the novel DMC signal transduction pathway and asked how it influences chromatin-associated proteins. We found that treatment with DMC, but not MC, stimulated the disassociation of ATR from chromatin and re-localization of ATR to the cytoplasm. The chromatin eviction of ATR was coupled with the formation of nuclear Rad51 foci and the phosphorylation of Chk1. Furthermore, DMC but not MC, activated expression of gadd45α mRNA. Importantly, knocking down p53 via shRNA did not inhibit the DMC-induced disassociation of ATR from chromatin or reduce the activation of transcription of gadd45α. Our results suggest that DMC induces a p53-independent disassociation of ATR from chromatin that facilitates Chk1 checkpoint activation and Rad51 chromatin recruitment. Our findings provide evidence that ATR chromatin eviction in breast cancer cells is an area of study that should be focused on for inducing p53-independent cell death. PMID:25565400

  8. Suppression of RAD21 Induces Senescence of MDA-MB-231 Human Breast Cancer Cells Through RB1 Pathway Activation Via c-Myc Downregulation.

    PubMed

    Zhu, Shan; Zhao, Li; Li, Yueyang; Hou, Pingfu; Yao, Ruosi; Tan, Jiang; Liu, Dongxu; Han, Liping; Huang, Baiqu; Lu, Jun; Zhang, Yu

    2016-06-01

    Cellular senescence impedes cancer progression by limiting uncontrolled cell proliferation. To identify new genetic events controlling senescence, we performed a small interfering RNA screening human cancer cells and identified a number of targets potentially involved in senescence of MDA-MB-231 human breast cancer cells. Importantly, we showed that knockdown of RAD21 resulted in the appearance of several senescent markers, including enhanced senescence-associated β-galactosidase activity and heterochromatin focus formation, as well as elevated p21 protein levels and RB1 pathway activation. Further biochemical analyses revealed that RAD21 knockdown led to the downregulation of c-Myc and its targets, including CDK4, a negative regulator of RB1, and blockedRB1 phosphorylation (pRB1), and the RB1-mediated transcriptional repression of E2F. Moreover, c-Myc downregulation was partially mediated by proteasome-dependent degradation within promyelocytic leukemia (PML) nuclear bodies, which were found to be highly abundant during RAD21 knockdown-induced senescence. Exogenous c-Myc reconstitution rescued cells from RAD21 silencing-induced senescence. Altogether, data arising from this study implicate a novel function of RAD21 in cellular senescence in MDA-MB-231 cells that is mainly dependent onRB1 pathway activation via c-Myc downregulation. J. Cell. Biochem. 117: 1359-1369, 2016. © 2015 Wiley Periodicals, Inc. PMID:26529363

  9. RAD51AP2, a novel vertebrate- and meiotic-specific protein, sharesa conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    SciTech Connect

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-07-25

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1.

  10. Rare adipose disorders (RADs) masquerading as obesity

    PubMed Central

    Herbst, Karen L

    2012-01-01

    Rare adipose disorders (RADs) including multiple symmetric lipomatosis (MSL), lipedema and Dercum's disease (DD) may be misdiagnosed as obesity. Lifestyle changes, such as reduced caloric intake and increased physical activity are standard care for obesity. Although lifestyle changes and bariatric surgery work effectively for the obesity component of RADs, these treatments do not routinely reduce the abnormal subcutaneous adipose tissue (SAT) of RADs. RAD SAT likely results from the growth of a brown stem cell population with secondary lymphatic dysfunction in MSL, or by primary vascular and lymphatic dysfunction in lipedema and DD. People with RADs do not lose SAT from caloric limitation and increased energy expenditure alone. In order to improve recognition of RADs apart from obesity, the diagnostic criteria, histology and pathophysiology of RADs are presented and contrasted to familial partial lipodystrophies, acquired partial lipodystrophies and obesity with which they may be confused. Treatment recommendations focus on evidence-based data and include lymphatic decongestive therapy, medications and supplements that support loss of RAD SAT. Associated RAD conditions including depression, anxiety and pain will improve as healthcare providers learn to identify and adopt alternative treatment regimens for the abnormal SAT component of RADs. Effective dietary and exercise regimens are needed in RAD populations to improve quality of life and construct advanced treatment regimens for future generations. PMID:22301856

  11. The Schizosaccharomyces pombe rad3 checkpoint gene.

    PubMed Central

    Bentley, N J; Holtzman, D A; Flaggs, G; Keegan, K S; DeMaggio, A; Ford, J C; Hoekstra, M; Carr, A M

    1996-01-01

    The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei-41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the 'lipid kinase' subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei-41. ATR can functionally complement esr1-1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution. Images PMID:8978690

  12. Rare adipose disorders (RADs) masquerading as obesity.

    PubMed

    Herbst, Karen L

    2012-02-01

    Rare adipose disorders (RADs) including multiple symmetric lipomatosis (MSL), lipedema and Dercum's disease (DD) may be misdiagnosed as obesity. Lifestyle changes, such as reduced caloric intake and increased physical activity are standard care for obesity. Although lifestyle changes and bariatric surgery work effectively for the obesity component of RADs, these treatments do not routinely reduce the abnormal subcutaneous adipose tissue (SAT) of RADs. RAD SAT likely results from the growth of a brown stem cell population with secondary lymphatic dysfunction in MSL, or by primary vascular and lymphatic dysfunction in lipedema and DD. People with RADs do not lose SAT from caloric limitation and increased energy expenditure alone. In order to improve recognition of RADs apart from obesity, the diagnostic criteria, histology and pathophysiology of RADs are presented and contrasted to familial partial lipodystrophies, acquired partial lipodystrophies and obesity with which they may be confused. Treatment recommendations focus on evidence-based data and include lymphatic decongestive therapy, medications and supplements that support loss of RAD SAT. Associated RAD conditions including depression, anxiety and pain will improve as healthcare providers learn to identify and adopt alternative treatment regimens for the abnormal SAT component of RADs. Effective dietary and exercise regimens are needed in RAD populations to improve quality of life and construct advanced treatment regimens for future generations. PMID:22301856

  13. Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction.

    PubMed

    Subramanyam, Shyamal; Jones, William T; Spies, Maria; Spies, M Ashley

    2013-10-01

    RAD51 DNA strand exchange protein catalyzes the central step in homologous recombination, a cellular process fundamentally important for accurate repair of damaged chromosomes, preservation of the genetic integrity, restart of collapsed replication forks and telomere maintenance. BRCA2 protein, a product of the breast cancer susceptibility gene, is a key recombination mediator that interacts with RAD51 and facilitates RAD51 nucleoprotein filament formation on single-stranded DNA generated at the sites of DNA damage. An accurate atomistic level description of this interaction, however, is limited to a partial crystal structure of the RAD51 core fused to BRC4 peptide. Here, by integrating homology modeling and molecular dynamics, we generated a structure of the full-length RAD51 in complex with BRC4 peptide. Our model predicted previously unknown hydrogen bonding patterns involving the N-terminal domain (NTD) of RAD51. These interactions guide positioning of the BRC4 peptide within a cavity between the core and the NTDs; the peptide binding separates the two domains and restricts internal dynamics of RAD51 protomers. The model's depiction of the RAD51-BRC4 complex was validated by free energy calculations and in vitro functional analysis of rationally designed mutants. All generated mutants, RAD51(E42A), RAD51(E59A), RAD51(E237A), RAD51(E59A/E237A) and RAD51(E42A/E59A/E237A) maintained basic biochemical activities of the wild-type RAD51, but displayed reduced affinities for the BRC4 peptide. Strong correlation between the calculated and experimental binding energies confirmed the predicted structure of the RAD51-BRC4 complex and highlighted the importance of RAD51 NTD in RAD51-BRCA2 interaction. PMID:23935068

  14. RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

    PubMed Central

    Chen, Su; Wang, Chen; Sun, Luxi; Wang, Da-Liang; Chen, Lu; Huang, Zhuan; Yang, Qi; Gao, Jie; Yang, Xi-Bin; Chang, Jian-Feng; Chen, Ping; Lan, Li

    2014-01-01

    Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients. PMID:25384975

  15. Post-translational environmental switch of RadA activity by extein–intein interactions in protein splicing

    PubMed Central

    Topilina, Natalya I.; Novikova, Olga; Stanger, Matthew; Banavali, Nilesh K.; Belfort, Marlene

    2015-01-01

    Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control. PMID:26101259

  16. MSL-RAD Cruise Operations Concept

    NASA Technical Reports Server (NTRS)

    Brinza, David E.; Zeitlin, Cary; Hassler, Donald; Weigle, Gerald E.; Boettcher, Stephan; Martin, Cesar; Wimmer-Schweingrubber, Robert

    2012-01-01

    The Mars Science Laboratory (MSL) payload includes the Radiation Assessment Detector (RAD) instrument, intended to fully characterize the radiation environment for the MSL mission. The RAD instrument operations concept is intended to reduce impact to spacecraft resources and effort for the MSL operations team. By design, RAD autonomously performs regular science observations without the need for frequent commanding from the Rover Compute Element (RCE). RAD operates with pre-defined "sleep" and "observe" periods, with an adjustable duty cycle for meeting power and data volume constraints during the mission. At the start of a new science observation, RAD performs a pre-observation activity to assess count rates for selected RAD detector elements. Based on this assessment, RAD can enter "solar event" mode, in which instrument parameters (including observation duration) are selected to more effectively characterize the environment. At the end of each observation period, RAD stores a time-tagged, fixed length science data packet in its non-volatile mass memory storage. The operating cadence is defined by adjustable parameters, also stored in non-volatile memory within the instrument. Periodically, the RCE executes an on-board sequence to transfer RAD science data packets from the instrument mass storage to the MSL downlink buffer. Infrequently, the RAD instrument operating configuration is modified by updating internal parameter tables and configuration entries.

  17. The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing.

    PubMed Central

    Davis, A P; Symington, L S

    2001-01-01

    The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing. PMID:11606529

  18. Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1)

    PubMed Central

    Dray, Eloïse; Dunlop, Myun Hwa; Kauppi, Liisa; Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

    2011-01-01

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 associated protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex-DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional cooperation is dependent on complex formation between DMC1 and RAD51AP1 and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci colocalize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination. PMID:21307306

  19. Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing

    PubMed Central

    Khade, Nilesh V.; Sugiyama, Tomohiko

    2016-01-01

    Yeast Rad52 (yRad52) has two important functions at homologous DNA recombination (HR); annealing complementary single-strand DNA (ssDNA) molecules and recruiting Rad51 recombinase onto ssDNA (recombination mediator activity). Its human homolog (hRAD52) has a lesser role in HR, and apparently lacks mediator activity. Here we show that yRad52 can load human Rad51 (hRAD51) onto ssDNA complexed with yeast RPA in vitro. This is biochemically equivalent to mediator activity because it depends on the C-terminal Rad51-binding region of yRad52 and on functional Rad52-RPA interaction. It has been reported that the N-terminal two thirds of both yRad52 and hRAD52 is essential for binding to and annealing ssDNA. Although a second DNA binding region has been found in the C-terminal region of yRad52, its role in ssDNA annealing is not clear. In this paper, we also show that the C-terminal region of yRad52, but not of hRAD52, is involved in ssDNA annealing. This suggests that the second DNA binding site is required for the efficient ssDNA annealing by yRad52. We propose an updated model of Rad52-mediated ssDNA annealing. PMID:27362509

  20. Rad-Release

    SciTech Connect

    2011-01-01

    The R&D 100 Award winning Rad-Release Chemical Decontamination Technology is a highly effective (up to 99% removal rate), affordable, patented chemical-foam-clay decontamination process tailored to specific radiological and metal contaminants, which is applicable to a wide variety of substrates. For more information about this project, visit http://www.inl.gov/rd100/2011/rad-release/

  1. Rad-Release

    ScienceCinema

    None

    2013-05-28

    The R&D 100 Award winning Rad-Release Chemical Decontamination Technology is a highly effective (up to 99% removal rate), affordable, patented chemical-foam-clay decontamination process tailored to specific radiological and metal contaminants, which is applicable to a wide variety of substrates. For more information about this project, visit http://www.inl.gov/rd100/2011/rad-release/

  2. RAD51AP2, a novel vertebrate- and meiotic-specific protein, shares a conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    PubMed Central

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-01-01

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate-specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1. PMID:16990250

  3. Human RAD18 Interacts with Ubiquitylated Chromatin Components and Facilitates RAD9 Recruitment to DNA Double Strand Breaks

    PubMed Central

    Inagaki, Akiko; Sleddens-Linkels, Esther; van Cappellen, Wiggert A.; Hibbert, Richard G.; Sixma, Titia K.; Hoeijmakers, Jan H. J.; Grootegoed, J. Anton; Baarends, Willy M.

    2011-01-01

    RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2. PMID:21858012

  4. Regulation of the Saccharomyces cerevisiae DNA repair gene RAD16.

    PubMed Central

    Bang, D D; Timmermans, V; Verhage, R; Zeeman, A M; van de Putte, P; Brouwer, J

    1995-01-01

    The RAD16 gene product has been shown to be essential for the repair of the silenced mating type loci [Bang et al. (1992) Nucleic Acids Res. 20, 3925-3931]. More recently we demonstrated that the RAD16 and RAD7 proteins are also required for repair of non-transcribed strands of active genes in Saccharomyces cerevisiae [Waters et al. (1993) Mol. Gen. Genet. 239, 28-32]. We have studied the regulation of the RAD16 gene and found that the RAD16 transcript levels increased up to 7-fold upon UV irradiation. Heat shock at 42 degrees C also results in elevated levels of RAD16 mRNA. In sporulating MAT alpha/MATa diploid cells RAD16 mRNA is also induced. The basal level of the RAD16 transcript is constant during the mitotic cell cycle. G1-arrested cells show normal induction of RAD16 mRNA upon UV irradiation demonstrating that the induction is not a secondary consequence of G2 cell cycle arrest following UV irradiation. However, in cells arrested in G1 the induction of RAD16 mRNA after UV irradiation is not followed by a rapid decline as occurs in normal growing cells suggesting that the down regulation of RAD16 transcription is dependent on progression into the cell cycle. Images PMID:7784171

  5. Theory of activated dynamics and glass transition of hard colloids in two dimensions.

    PubMed

    Zhang, Bo-kai; Li, Hui-shu; Tian, Wen-de; Chen, Kang; Ma, Yu-qiang

    2014-03-01

    The microscopic nonlinear Langevin equation theory is applied to study the localization and activated hopping of two-dimensional hard disks in the deeply supercooled and glass states. Quantitative comparisons of dynamic characteristic length scales, barrier, and their dependence on the reduced packing fraction are presented between hard-disk and hard-sphere suspensions. The dynamic barrier of hard disks emerges at higher absolute and reduced packing fractions and correspondingly, the crossover size of the dynamic cage which correlates to the Lindemann length for melting is smaller. The localization lengths of both hard disks and spheres decrease exponentially with packing fraction. Larger localization length of hard disks than that of hard spheres is found at the same reduced packing fraction. The relaxation time of hard disks rises dramatically above the reduced packing fraction of 0.88, which leads to lower reduced packing fraction at the kinetic glass transition than that of hard spheres. The present work provides a foundation for the subsequent study of the glass transition of binary or polydisperse mixtures of hard disks, normally adopted in experiments and simulations to avoid crystallization, and further, the rheology and mechanical response of the two-dimensional glassy colloidal systems. PMID:24606367

  6. Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

    PubMed Central

    Das, Maitreyi; Nuñez, Illyce; Rodriguez, Marbelys; Wiley, David J.; Rodriguez, Juan; Sarkeshik, Ali; Yates, John R.; Buchwald, Peter; Verde, Fulvia

    2015-01-01

    Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24–Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence. PMID:26246599

  7. Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

    PubMed Central

    Dupaigne, Pauline; Maloisel, Laurent; Guerois, Raphaël; Le Cam, Eric; Veaute, Xavier; Coïc, Eric

    2013-01-01

    The budding yeast Srs2 is the archetype of helicases that regulate several aspects of homologous recombination (HR) to maintain genomic stability. Srs2 inhibits HR at replication forks and prevents high frequencies of crossing-over. Additionally, sensitivity to DNA damage and synthetic lethality with replication and recombination mutants are phenotypes that can only be attributed to another role of Srs2: the elimination of lethal intermediates formed by recombination proteins. To shed light on these intermediates, we searched for mutations that bypass the requirement of Srs2 in DNA repair without affecting HR. Remarkably, we isolated rad52-L264P, a novel allele of RAD52, a gene that encodes one of the most central recombination proteins in yeast. This mutation suppresses a broad spectrum of srs2Δ phenotypes in haploid cells, such as UV and γ-ray sensitivities as well as synthetic lethality with replication and recombination mutants, while it does not significantly affect Rad52 functions in HR and DNA repair. Extensive analysis of the genetic interactions between rad52-L264P and srs2Δ shows that rad52-L264P bypasses the requirement for Srs2 specifically for the prevention of toxic Rad51 filaments. Conversely, this Rad52 mutant cannot restore viability of srs2Δ cells that accumulate intertwined recombination intermediates which are normally processed by Srs2 post-synaptic functions. The avoidance of toxic Rad51 filaments by Rad52-L264P can be explained by a modification of its Rad51 filament mediator activity, as indicated by Chromatin immunoprecipitation and biochemical analysis. Remarkably, sensitivity to DNA damage of srs2Δ cells can also be overcome by stimulating Rad52 sumoylation through overexpression of the sumo-ligase SIZ2, or by replacing Rad52 by a Rad52-SUMO fusion protein. We propose that, like the rad52-L264P mutation, sumoylation modifies Rad52 activity thereby changing the properties of Rad51 filaments. This conclusion is strengthened by the

  8. Environmental Radiation Studies Utilizing RadNet

    NASA Astrophysics Data System (ADS)

    Jackson, Steven

    2010-03-01

    RadNet is an Environmental Protection Agency (EPA) managed environmental radiation and air quality monitoring station that has been recently installed on the Angelo State University campus. RadNet cycles air through a filter which collects particulate matter for an examination of alpha and beta activities of contained material. This project includes defining dominant alpha and beta emitters in the local environment, conducting a correlation study involving the effect of weather and wind direction on filter activity, physical interpretation of air quality index (AQI), and the use of X-ray fluorescence to verify radon decay progeny in the local environment.

  9. Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis

    SciTech Connect

    Cole, G.M.; Mortimer, R.K. ); Schild, D. )

    1989-07-01

    The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/{alpha} cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of {beta}-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

  10. Rad6 is a Potential Early Marker of Melanoma Development

    PubMed Central

    Rosner, Karli; Adsule, Shreelekha; Haynes, Brittany; Kirou, Evangelia; Kato, Ikuko; Mehregan, Darius R.; Shekhar, Malathy P.V.

    2014-01-01

    Melanoma is the leading cause of death from skin cancer in industrialized countries. Several melanoma-related biomarkers and signaling pathways have been identified; however, their relevance to melanoma development/progression or to clinical outcome remains to be established. Aberrant activation of Wnt/β-catenin pathway is implicated in various cancers including melanoma. We have previously demonstrated Rad6, an ubiquitin-conjugating enzyme, as an important mediator of β-catenin stability in breast cancer cells. Similar to breast cancer, β-catenin-activating mutations are rare in melanomas, and since β-catenin signaling is implicated in melanoma, we examined the relationship between β-catenin levels/activity and expression of β-catenin transcriptional targets Rad6 and microphthalmia-associated transcription factor-M (Mitf-M) in melanoma cell models, and expression of Rad6, β-catenin, and Melan-A in nevi and cutaneous melanoma tissue specimens. Our data show that Rad6 is only weakly expressed in normal human melanocytes but is overexpressed in melanoma lines. Unlike Mitf-M, Rad6 overexpression in melanoma lines is positively associated with high molecular weight β-catenin protein levels and β-catenin transcriptional activity. Double-immunofluorescence staining of Rad6 and Melan-A in melanoma tissue microarray showed that histological diagnosis of melanoma is significantly associated with Rad6/Melan-A dual positivity in the melanoma group compared to the nevi group (P = .0029). In contrast to strong β-catenin expression in normal and tumor areas of superficial spreading malignant melanoma (SSMM), Rad6 expression is undetectable in normal areas and Rad6 expression increases coincide with increased Melan-A in the transformed regions of SSMM. These data suggest a role for Rad6 in melanoma pathogenesis and that Rad6 expression status may serve as an early marker for melanoma development. PMID:24831578

  11. Rad6 is a Potential Early Marker of Melanoma Development.

    PubMed

    Rosner, Karli; Adsule, Shreelekha; Haynes, Brittany; Kirou, Evangelia; Kato, Ikuko; Mehregan, Darius R; Shekhar, Malathy P V

    2014-05-12

    Melanoma is the leading cause of death from skin cancer in industrialized countries. Several melanoma-related biomarkers and signaling pathways have been identified; however, their relevance to melanoma development/progression or to clinical outcome remains to be established. Aberrant activation of Wnt/β-catenin pathway is implicated in various cancers including melanoma. We have previously demonstrated Rad6, an ubiquitin-conjugating enzyme, as an important mediator of β-catenin stability in breast cancer cells. Similar to breast cancer, β-catenin-activating mutations are rare in melanomas, and since β-catenin signaling is implicated in melanoma, we examined the relationship between β-catenin levels/activity and expression of β-catenin transcriptional targets Rad6 and microphthalmia-associated transcription factor-M (Mitf-M) in melanoma cell models, and expression of Rad6, β-catenin, and Melan-A in nevi and cutaneous melanoma tissue specimens. Our data show that Rad6 is only weakly expressed in normal human melanocytes but is overexpressed in melanoma lines. Unlike Mitf-M, Rad6 overexpression in melanoma lines is positively associated with high molecular weight β-catenin protein levels and β-catenin transcriptional activity. Double-immunofluorescence staining of Rad6 and Melan-A in melanoma tissue microarray showed that histological diagnosis of melanoma is significantly associated with Rad6/Melan-A dual positivity in the melanoma group compared to the nevi group (P=.0029). In contrast to strong β-catenin expression in normal and tumor areas of superficial spreading malignant melanoma (SSMM), Rad6 expression is undetectable in normal areas and Rad6 expression increases coincide with increased Melan-A in the transformed regions of SSMM. These data suggest a role for Rad6 in melanoma pathogenesis and that Rad6 expression status may serve as an early marker for melanoma development. PMID:24831578

  12. Effects of phosphorylation on function of the Rad GTPase.

    PubMed Central

    Moyers, J S; Zhu, J; Kahn, C R

    1998-01-01

    Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases. PMID:9677319

  13. Effects of phosphorylation on function of the Rad GTPase.

    PubMed

    Moyers, J S; Zhu, J; Kahn, C R

    1998-08-01

    Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases. PMID:9677319

  14. Rad51 ATP binding but not hydrolysis is required to recruit Rad10 in synthesis-dependent strand annealing sites in S. cerevisiae

    PubMed Central

    Karlin, Justin; Fischhaber, Paula L.

    2013-01-01

    Several modes of eukaryotic of DNA double strand break repair (DSBR) depend on synapsis of complementary DNA. The Rad51 ATPase, the S. cerevisiae homolog of E. coli RecA, plays a key role in this process by catalyzing homology searching and strand exchange between an invading DNA strand and a repair template (e.g. sister chromatid or homologous chromosome). Synthesis dependent strand annealing (SDSA), a mode of DSBR, requires Rad51. Another repair enzyme, the Rad1-Rad10 endonuclease, acts in the final stages of SDSA, hydrolyzing 3′ overhanging single-stranded DNA. Here we show in vivo by fluorescence microscopy that the ATP binding function of yeast Rad51 is required to recruit Rad10 SDSA sites indicating that Rad51 pre-synaptic filament formation must occur prior to the recruitment of Rad1-Rad10. Our data also show that Rad51 ATPase activity, an important step in Rad51 filament disassembly, is not absolutely required in order to recruit Rad1-Rad10 to DSB sites. PMID:25346869

  15. Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

    SciTech Connect

    Dray, Eloise; Dunlop, Myun Hwa; Kauppi, Liisa; San Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

    2010-11-05

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 Associated Protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional co-operation is dependent on complex formation between DMC1 and RAD51AP1, and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci co-localize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.

  16. Activity of everolimus (RAD001) in relapsed and/or refractory multiple myeloma: a phase I study

    PubMed Central

    Günther, Andreas; Baumann, Philipp; Burger, Renate; Kellner, Christian; Klapper, Wolfram; Schmidmaier, Ralf; Gramatzki, Martin

    2015-01-01

    The mammalian target of rapamycin plays an important role in multiple myeloma. The allosteric mammalian target of rapamycin inhibitor everolimus has long been approved for immunosuppression and has shown activity in certain cancers. This investigator-initiated phase I trial explored the use of everolimus in relapsed and/or refractory multiple myeloma patients who had received two or more lines of prior treatment. Following a dose-escalation design, it called for a fixed dose of oral everolimus. Blood drug levels were monitored and the biological activity of everolimus was evaluated in bone marrow. Seventeen patients were enrolled (age range, 52 to 76 years). All had been previously treated with stem cell transplantation and proteasome inhibitors and almost all with immunomodulatory drugs. No dose-limiting toxicity was observed and the intended final daily dose of 10 mg was reached. Only one severe adverse event was assessed as possibly related to the study drug, namely atypical pneumonia. Remarkably few infections were observed. Although the trial was mainly designed to evaluate feasibility, anti-myeloma activity, defined as clinical benefit, was documented in ten of 15 evaluable patients at every dose level including eight patients with stable disease, one patient with minor remission and one with partial remission. However, the median time to progression was 90 days (range, 13 to 278 days). The biomarker study documented on-target activity of everolimus in malignant plasma cells as well as the microenvironment. The observed responses are promising and allow further studies to be considered, including those testing combination strategies addressing escape pathways. This trial is registered with EudraCT number 2006-002675-41. PMID:25682600

  17. Rad Pole Cam Development

    SciTech Connect

    Heckendorn, F. M.; Odell, D. M. C; Harpring, L. J.; Peterson, K. D.

    2005-10-05

    The RadPoleCam was developed to provide Department Of Energy (DOE) first responders the capability to assess the radiological and visual condition of remote or inaccessible locations. Real time gamma isotopic identification is provided to the first responder in the form of audio feedback (i.e. spoken through head phones) from a gamma detector mounted on a collapsible pole that can extend from 1 to 9 meters (6 to 29 feet). Simultaneously, selectable direct and side looking visual images are provided from the 5cm (2in) diameter, waterproof probe tip. The lightweight, self contained, ruggedized, system will provide a rapidly deployable field system for visual and radiological search and assessment of confined spaces and extended reach locations.

  18. Swi2/Snf2-related translocases prevent accumulation of toxic Rad51 complexes during mitotic growth

    PubMed Central

    Shah, Parisha P.; Zheng, Xiuzhong; Epshtein, Anastasiya; Carey, Jeffrey N.; Bishop, Douglas K.; Klein, Hannah L.

    2010-01-01

    Summary Purified DNA translocases Rdh54 and Rad54 can dissociate complexes formed by eukaryotic RecA-like recombinases on double-stranded DNA. Here we show Rad51 complexes are dissociated by these translocases in mitotic cells. Rad51 overexpression blocked growth of cells deficient in Rdh54 activity. This toxicity was associated with accumulation of Rad51 foci on undamaged chromatin. At normal Rad51 levels, rdh54 deficiency resulted in slight elevation of Rad51 foci. A triple mutant lacking Rdh54, Rad54, and a third Swi2/Snf2 homologue Uls1, accumulated Rad51 foci, grew slowly, and suffered chromosome loss. Thus, Uls1 and Rad54 can partially substitute for Rdh54 in the removal of toxic, non–damage-associated Rad51-DNA complexes. Additional data suggest that the function of Rdh54 and Rad54 in removal of Rad51 foci is significantly specialized; Rad54 predominates for removal of damage-associated foci and Rdh54 predominates for removal of non-damage-associated foci. PMID:20864034

  19. THE GLOBAL IMPLICATIONS OF THE HARD X-RAY EXCESS IN TYPE 1 ACTIVE GALACTIC NUCLEI

    SciTech Connect

    Tatum, M. M.; Turner, T. J.; Reeves, J. N.; Miller, L.

    2013-01-10

    Recent evidence for a strong 'hard excess' of flux at energies {approx}> 20 keV in some Suzaku observations of type 1 active galactic nuclei (AGNs) has motivated an exploratory study of the phenomenon in the local type 1 AGN population. We have selected all type 1 AGNs in the Swift Burst Alert Telescope 58 month catalog and cross-correlated them with the holdings of the Suzaku public archive. We find the hard excess phenomenon to be a ubiquitous property of type 1 AGNs. Taken together, the spectral hardness and equivalent width of Fe K{alpha} emission are consistent with reprocessing by an ensemble of Compton-thick clouds that partially cover the continuum source. In the context of such a model, {approx}80% of the sample has a hardness ratio consistent with >50% covering of the continuum by low-ionization, Compton-thick gas. A more detailed study of the three hardest X-ray spectra in our sample reveal a sharp Fe K absorption edge at {approx}7 keV in each of them, indicating that blurred reflection is not responsible for the very hard spectral forms. Simple considerations place the distribution of Compton-thick clouds at or within the optical broad-line region.

  20. RAD001 (everolimus) attenuates experimental autoimmune neuritis by inhibiting the mTOR pathway, elevating Akt activity and polarizing M2 macrophages.

    PubMed

    Han, Ranran; Gao, Juan; Zhai, Hui; Xiao, Jinting; Ding, Ya'nan; Hao, Junwei

    2016-06-01

    Guillain-Barre' syndrome (GBS) is an acute, postinfectious, immune-mediated, demyelinating disease of peripheral nerves and nerve roots. As a classical animal model of GBS, experimental autoimmune neuritis (EAN) has become well-accepted. Additionally, the potent immune modulation exerted by mammalian target of rapamycin (mTOR) inhibitors has been used to treat cancers and showed beneficial effects. Here we demonstrate that the mTOR inhibitor RAD001 (everolimus) protected rats from the symptoms of EAN, as shown by decreased paralysis, diminished inflammatory cell infiltration, reductions in demyelination of peripheral nerves and improved nerve conduction. Furthermore, RAD001 shifted macrophage polarization toward the protective M2 phenotype and modified the inflammatory milieu by downregulating the production of pro-inflammatory cytokines including IFN-γ and IL-17as well as upregulating the release of anti-inflammatory cytokines such as IL-4 and TGF-β. Amounts of the mTOR downstream targets p-P70S6K and p-4E-BP1 in sciatic nerves decreased, whereas the level of its upstream protein p-Akt was elevated. This demonstrated that RAD001 inhibited the mTOR pathway and encouraged the expression of p-Akt, which led to M2 macrophage polarization, thus improved the outcome of EAN in rats. Consequently, RAD001 exhibits strong potential as a therapeutic strategy for ameliorating peripheral poly-neuropathy. PMID:27063582

  1. The RadAssessor manual

    SciTech Connect

    Seitz, Sharon L.

    2007-02-01

    THIS manual will describe the functions and capabilities that are available from the RadAssessor database and will demonstrate how to retrieve and view its information. You’ll learn how to start the database application, how to log in, how to use the common commands, and how to use the online help if you have a question or need extra guidance. RadAssessor can be viewed from any standard web browser. Therefore, you will not need to install any special software before using RadAssessor.

  2. Yeast Rad17/Mec3/Ddc1: A sliding clamp for the DNA damage checkpoint

    PubMed Central

    Majka, Jerzy; Burgers, Peter M. J.

    2003-01-01

    The Saccharomyces cerevisiae Rad24 and Rad17 checkpoint proteins are part of an early response to DNA damage in a signal transduction pathway leading to cell cycle arrest. Rad24 interacts with the four small subunits of replication factor C (RFC) to form the RFC-Rad24 complex. Rad17 forms a complex with Mec3 and Ddc1 (Rad17/3/1) and shows structural similarities with the replication clamp PCNA. This parallelism with a clamp-clamp loader system that functions in DNA replication has led to the hypothesis that a similar clamp-clamp loader relationship exists for the DNA damage response system. We have purified the putative checkpoint clamp loader RFC-Rad24 and the putative clamp Rad17/3/1 from a yeast overexpression system. Here, we provide experimental evidence that, indeed, the RFC-Rad24 clamp loader loads the Rad17/3/1 clamp around partial duplex DNA in an ATP-dependent process. Furthermore, upon ATP hydrolysis, the Rad17/3/1 clamp is released from the clamp loader and can slide across more than 1 kb of duplex DNA, a process which may be well suited for a search for damage. Rad17/3/1 showed no detectable exonuclease activity. PMID:12604797

  3. Mechanical activation of a hard magnetic Fe-Cr-Co alloy powder charge

    NASA Astrophysics Data System (ADS)

    Alymov, M. I.; Milyaev, I. M.; Sychev, A. E.; Kovalev, D. Yu.; Korneev, V. P.; Morozov, Yu. G.; Yusupov, V. S.; Bompe, T. A.

    2014-07-01

    The mechanical activation (MA) of a charge of a hard magnetic 22Kh15KT alloy is studied by wet and dry milling in a planetary mill in a medium of argon and ethyl alcohol with addition of surface-active materials and without them. It is shown that, upon dry MA, powder alloy components are alloyed with formation of two bcc solid solutions and, upon wet MA, charge particles are only intensely dispersed. Dispersion is developed at the highest degree in the first five minutes of MA.

  4. Human Rad54 protein stimulates human Mus81–Eme1 endonuclease

    PubMed Central

    Mazina, Olga M.; Mazin, Alexander V.

    2008-01-01

    Rad54, a key protein of homologous recombination, physically interacts with a DNA structure-specific endonuclease, Mus81–Eme1. Genetic data indicate that Mus81–Eme1 and Rad54 might function together in the repair of damaged DNA. In vitro, Rad54 promotes branch migration of Holliday junctions, whereas the Mus81–Eme1 complex resolves DNA junctions by endonucleolytic cleavage. Here, we show that human Rad54 stimulates Mus81–Eme1 endonuclease activity on various Holliday junction-like intermediates. This stimulation is the product of specific interactions between the human Rad54 (hRad54) and Mus81 proteins, considering that Saccharomyces cerevisiae Rad54 protein does not stimulate human Mus81–Eme1 endonuclease activity. Stimulation of Mus81–Eme1 cleavage activity depends on formation of specific Rad54 complexes on DNA substrates occurring in the presence of ATP and, to a smaller extent, of other nucleotide cofactors. Thus, our results demonstrate a functional link between the branch migration activity of hRad54 and the structure-specific endonuclease activity of hMus81–Eme1, suggesting that the Rad54 and Mus81–Eme1 proteins may cooperate in the processing of Holliday junction-like intermediates during homologous recombination or DNA repair. PMID:19017809

  5. Rad61/Wpl1 (Wapl), a cohesin regulator, controls chromosome compaction during meiosis

    PubMed Central

    Challa, Kiran; Lee, Min-Su; Shinohara, Miki; Kim, Keun P.; Shinohara, Akira

    2016-01-01

    Meiosis-specific cohesin, required for the linking of the sister chromatids, plays a critical role in various chromosomal events during meiotic prophase I, such as chromosome morphogenesis and dynamics, as well as recombination. Rad61/Wpl1 (Wapl in other organisms) negatively regulates cohesin functions. In this study, we show that meiotic chromosome axes are shortened in the budding yeast rad61/wpl1 mutant, suggesting that Rad61/Wpl1 negatively regulates chromosome axis compaction. Rad61/Wpl1 is required for efficient resolution of telomere clustering during meiosis I, indicating a positive effect of Rad61/Wpl1 on the cohesin function required for telomere dynamics. Additionally, we demonstrate distinct activities of Rad61/Wpl1 during the meiotic recombination, including its effects on the efficient processing of intermediates. Thus, Rad61/Wpl1 both positively and negatively regulates various cohesin-mediated chromosomal processes during meiosis. PMID:26825462

  6. Rad54 oligomers translocate and cross-bridge double-stranded DNA to stimulate synapsis

    PubMed Central

    Bianco, Piero R.; Bradfield, Justin J.; Castanza, Lauren R.; Donnelly, Andrea N.

    2007-01-01

    Rad54 is a key component of the eukaryotic recombination machinery. Its presence in DNA strand exchange reactions in vitro results in a significant stimulation in the overall reaction rate. Using untagged Rad54, we show that this stimulation can be attributed to enhancement of the formation of a key reaction intermediate known as DNA networks. Using a novel, single DNA molecule, dual-optical tweezers approach we show how Rad54 stimulates DNA network formation. We discovered that Rad54 oligomers possess a unique ability to cross-bridge or bind dsDNA molecules positioned in close proximity. Further, Rad54 oligomers rapidly translocate dsDNA while simultaneously inducing topological loops in the DNA at the locus of the oligomer. The combination of the cross-bridging and dsDNA translocation activities of Rad54 stimulates the formation of DNA networks, leading to rapid and efficient DNA strand exchange by Rad51. PMID:17949748

  7. A new protein complex promoting the assembly of Rad51 filaments

    PubMed Central

    Sasanuma, Hiroyuki; Tawaramoto, Maki S.; Lao, Jessica P.; Hosaka, Harumi; Sanda, Eri; Suzuki, Mamoru; Yamashita, Eiki; Hunter, Neil; Shinohara, Miki; Nakagawa, Atsushi; Shinohara, Akira

    2015-01-01

    During homologous recombination, eukaryotic RecA homologue Rad51 assembles into a nucleoprotein filament on single-stranded DNA to catalyse homologous pairing and DNA-strand exchange with a homologous template. Rad51 nucleoprotein filaments are highly dynamic and regulated via the coordinated actions of various accessory proteins including Rad51 mediators. Here, we identify a new Rad51 mediator complex. The PCSS complex, comprising budding yeast Psy3, Csm2, Shu1 and Shu2 proteins, binds to recombination sites and is required for Rad51 assembly and function during meiosis. Within the heterotetramer, Psy3-Csm2 constitutes a core sub-complex with DNA-binding activity. In vitro, purified Psy3-Csm2 stabilizes the Rad51–single-stranded DNA complex independently of nucleotide cofactor. The mechanism of Rad51 stabilization is inferred by our high-resolution crystal structure, which reveals Psy3-Csm2 to be a structural mimic of the Rad51-dimer, a fundamental unit of the Rad51-filament. Together, these results reveal a novel molecular mechanism for this class of Rad51-mediators, which includes the human Rad51 paralogues. PMID:23575680

  8. Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells

    NASA Technical Reports Server (NTRS)

    Wiese, Claudia; Collins, David W.; Albala, Joanna S.; Thompson, Larry H.; Kronenberg, Amy; Schild, David; Chatterjee, A. (Principal Investigator)

    2002-01-01

    Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require Rad51 and the five Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3), as has been shown in chicken and rodent cells. Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems. To test for interactions involving XRCC3 and Rad51C, stable human cell lines have been isolated that express (His)6-tagged versions of XRCC3 or Rad51C. Ni2+-binding experiments demonstrate that XRCC3 and Rad51C interact in human cells. In addition, we find that Rad51C, but not XRCC3, interacts directly or indirectly with Rad51B, Rad51D and XRCC2. These results argue that there are at least two complexes of Rad51 paralogs in human cells (Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2), both containing Rad51C. Moreover, Rad51 is not found in these complexes. X-ray treatment did not alter either the level of any Rad51 paralog or the observed interactions between paralogs. However, the endogenous level of Rad51C is moderately elevated in the XRCC3-overexpressing cell line, suggesting that dimerization between these proteins might help stabilize Rad51C.

  9. Lymphoid irradiation in intractable rheumatoid arthritis. A double-blind, randomized study comparing 750-rad treatment with 2,000-rad treatment

    SciTech Connect

    Hanly, J.G.; Hassan, J.; Moriarty, M.; Barry, C.; Molony, J.; Casey, E.; Whelan, A.; Feighery, C.; Bresnihan, B.

    1986-01-01

    Twenty patients with intractable rheumatoid arthritis were treated with 750-rad or 2,000-rad lymphoid irradiation in a randomized double-blind comparative study. Over a 12-month followup period, there was a significant improvement in 4 of 7 and 6 of 7 standard parameters of disease activity following treatment with 750 rads and 2,000 rads, respectively. Transient, short-term toxicity was less frequent with the lower dose. In both groups, there was a sustained peripheral blood lymphopenia, a selective depletion of T helper (Leu-3a+) lymphocytes, and reduced in vitro mitogen responses. These changes did not occur, however, in synovial fluid. These results suggest that 750-rad lymphoid irradiation is as effective as, but less toxic than, that with 2,000 rads in the management of patients with intractable rheumatoid arthritis.

  10. Strand pairing by Rad54 and Rad51 is enhanced by chromatin.

    PubMed

    Alexiadis, Vassilios; Kadonaga, James T

    2002-11-01

    We investigated the role of chromatin in the catalysis of homologous strand pairing by Rad54 and Rad51. Rad54 is related to the ATPase subunits of chromatin-remodeling factors, whereas Rad51 is related to bacterial RecA. In the absence of superhelical tension, we found that the efficiency of strand pairing with chromatin is >100-fold higher than that with naked DNA. In addition, we observed that Rad54 and Rad51 function cooperatively in the ATP-dependent remodeling of chromatin. These findings indicate that Rad54 and Rad51 have evolved to function with chromatin, the natural substrate, rather than with naked DNA. PMID:12414729

  11. Rad18 confers hematopoietic progenitor cell DNA damage tolerance independently of the Fanconi Anemia pathway in vivo

    PubMed Central

    Yang, Yang; Poe, Jonathan C.; Yang, Lisong; Fedoriw, Andrew; Desai, Siddhi; Magnuson, Terry; Li, Zhiguo; Fedoriw, Yuri; Araki, Kimi; Gao, Yanzhe; Tateishi, Satoshi; Sarantopoulos, Stefanie; Vaziri, Cyrus

    2016-01-01

    In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18−/− mice. Moreover, primary Rad18−/− mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18−/− HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18−/− mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting. PMID:26883629

  12. Rad18 confers hematopoietic progenitor cell DNA damage tolerance independently of the Fanconi Anemia pathway in vivo.

    PubMed

    Yang, Yang; Poe, Jonathan C; Yang, Lisong; Fedoriw, Andrew; Desai, Siddhi; Magnuson, Terry; Li, Zhiguo; Fedoriw, Yuri; Araki, Kimi; Gao, Yanzhe; Tateishi, Satoshi; Sarantopoulos, Stefanie; Vaziri, Cyrus

    2016-05-19

    In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18(-/-) mice. Moreover, primary Rad18(-/-) mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18(-/-) HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18(-/-) mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting. PMID:26883629

  13. Radio observations of a hard X-ray selected sample of active galaxies

    NASA Technical Reports Server (NTRS)

    Unger, S. W.; Lawrence, A.; Wilson, A. S.; Elvis, M.; Wright, A. E.

    1987-01-01

    Radio observations of a hard X-ray selected sample of active galaxies obtained with the VLA and Parkes radio telescopes are discussed, and the ratio of the radio to X-ray flux density is used to determine the degree of radio-loudness of the galaxies. A continuous distribution of the degree of radio loudness is found amongst the sample galaxies, and no evidence for distinct radio-quiet and radio-loud populations is noted. The X-ray and radio luminosity is shown to be nonlinearly correlated, with the radio-loud objects all having high X-ray luminosity.

  14. Caffeine inhibits gene conversion by displacing Rad51 from ssDNA.

    PubMed

    Tsabar, Michael; Mason, Jennifer M; Chan, Yuen-Ling; Bishop, Douglas K; Haber, James E

    2015-08-18

    Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1(ATR)/Tel1(ATM)-dependent DNA damage response or caffeine's inhibition of 5' to 3' resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

  15. Hard magnetohydrodynamic limit in 1/3 sawtooth like activity in LHD

    SciTech Connect

    Varela, J.; Watanabe, K. Y.; Ohdachi, S.; Narushima, Y.

    2014-03-15

    The optimization of LHD discharges in inward-shifted configurations with 1/3 sawtooth like activity is an open issue. These relaxation events limit the LHD performance driving a periodic plasma deconfinement. The aim of this study is to analyze the 1/3 sawtooth like activity in plasmas with different stability properties to foreseen the best operation conditions and minimize its undesired effects. We summarize the results of several MHD simulations for plasmas with Lundquist numbers between 10{sup 5} and 10{sup 6} in the slow reconnection regime, studying the equilibria properties during the onset of a chain of 1/3 sawtooth like events. The research conclusions point out that the hard MHD limit can be reached in the inner plasma region after the onset of a strong 1/3 resonant sawtooth like event and trigger a plasma collapse. The collapse can be avoided if the system remains in the soft MHD limit, namely, in a regime with a pressure gradient and a magnetic turbulence below the critical values to drive the soft-hard MHD transition. In the soft MHD limit the system relaxations are the non resonant 1/3 sawtooth like events or a weak version of the 1/3 resonant sawtooth like events. A system relaxation in the soft MHD regime drives a minor plasma deconfinement that does not limit the LHD performance if the event periodicity is not very high.

  16. Dissection of Rad9 BRCT Domain Function In The Mitotic Checkpoint Response To Telomere Uncapping

    PubMed Central

    Nnakwe, Chinonye C.; Altaf, Mohammed; Côté, Jacques; Kron, Stephen J.

    2009-01-01

    In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition—much like the response to an unrepaired DNA double-strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5' strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle. PMID:19880356

  17. Rad-hard computer elements for space applications

    NASA Technical Reports Server (NTRS)

    Krishnan, G. S.; Longerot, Carl D.; Treece, R. Keith

    1993-01-01

    Space Hardened CMOS computer elements emulating a commercial microcontroller and microprocessor family have been designed, fabricated, qualified, and delivered for a variety of space programs including NASA's multiple launch International Solar-Terrestrial Physics (ISTP) program, Mars Observer, and government and commercial communication satellites. Design techniques and radiation performance of the 1.25 micron feature size products are described.

  18. Quantum Dots Based Rad-Hard Computing and Sensors

    NASA Technical Reports Server (NTRS)

    Fijany, A.; Klimeck, G.; Leon, R.; Qiu, Y.; Toomarian, N.

    2001-01-01

    Quantum Dots (QDs) are solid-state structures made of semiconductors or metals that confine a small number of electrons into a small space. The confinement of electrons is achieved by the placement of some insulating material(s) around a central, well-conducting region. Thus, they can be viewed as artificial atoms. They therefore represent the ultimate limit of the semiconductor device scaling. Additional information is contained in the original extended abstract.

  19. Rad-Hard Structured ASIC Body of Knowledge

    NASA Technical Reports Server (NTRS)

    Heidecker, Jason

    2013-01-01

    Structured Application-Specific Integrated Circuit (ASIC) technology is a platform between traditional ASICs and Field-Programmable Gate Arrays (FPGA). The motivation behind structured ASICs is to combine the low nonrecurring engineering costs (NRE) costs of FPGAs with the high performance of ASICs. This report provides an overview of the structured ASIC platforms that are radiation-hardened and intended for space application

  20. Energetic electrons, hard x-ray emission and MHD activity studies in the IR-T1 tokamak.

    PubMed

    Agah, K Mikaili; Ghoranneviss, M; Elahi, A Salar

    2015-01-01

    Determinations of plasma parameters as well as the Magnetohydrodynamics (MHD) activity, energetic electrons energy and energy confinement time are essential for future fusion reactors experiments and optimized operation. Also some of the plasma information can be deduced from these parameters, such as plasma equilibrium, stability, and MHD instabilities. In this contribution we investigated the relation between energetic electrons, hard x-ray emission and MHD activity in the IR-T1 Tokamak. For this purpose we used the magnetic diagnostics and a hard x-ray spectroscopy in IR-T1 tokamak. A hard x-ray emission is produced by collision of the runaway electrons with the plasma particles or limiters. The mean energy was calculated from the slope of the energy spectrum of hard x-ray photons. PMID:25882736

  1. A novel interation of nucleolin with Rad51

    SciTech Connect

    De, Ananya; Donahue, Sarah L.; Tabah, Azah; Castro, Nancy E.; Mraz, Naomi; Cruise, Jennifer L.; Campbell, Colin . E-mail: campb034@umn.edu

    2006-05-26

    Nucleolin associates with various DNA repair, recombination, and replication proteins, and possesses DNA helicase, strand annealing, and strand pairing activities. Examination of nuclear protein extracts from human somatic cells revealed that nucleolin and Rad51 co-immunoprecipitate. Furthermore, purified recombinant Rad51 associates with in vitro transcribed and translated nucleolin. Electroporation-mediated introduction of anti-nucleolin antibody resulted in a 10- to 20-fold reduction in intra-plasmid homologous recombination activity in human fibrosarcoma cells. Additionally, introduction of anti-nucleolin antibody sensitized cells to death induced by the topoisomerase II inhibitor, amsacrine. Introduction of anti-Rad51 antibody also reduced intra-plasmid homologous recombination activity and induced hypersensitivity to amsacrine-induced cell death. Co-introduction of anti-nucleolin and anti-Rad51 antibodies did not produce additive effects on homologous recombination or on cellular sensitivity to amsacrine. The association of the two proteins raises the intriguing possibility that nucleolin binding to Rad51 may function to regulate homologous recombinational repair of chromosomal DNA.

  2. Radiation-hard active CMOS pixel sensors for HL-LHC detector upgrades

    NASA Astrophysics Data System (ADS)

    Backhaus, Malte

    2015-02-01

    The luminosity of the Large Hadron Collider (LHC) will be increased during the Long Shutdown of 2022 and 2023 (LS3) in order to increase the sensitivity of its experiments. A completely new inner detector for the ATLAS experiment needs to be developed to withstand the extremely harsh environment of the upgraded, so-called High-Luminosity LHC (HL-LHC). High radiation hardness as well as granularity is mandatory to cope with the requirements in terms of radiation damage as well as particle occupancy. A new silicon detector concept that uses commercial high voltage and/or high resistivity full complementary metal-oxide-semiconductor (CMOS) processes as active sensor for pixel and/or strip layers has risen high attention, because it potentially provides high radiation hardness and granularity and at the same time reduced price due to the commercial processing and possibly relaxed requirements for the hybridization technique. Results on the first prototypes characterized in a variety of laboratory as well as test beam environments are presented.

  3. Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding.

    PubMed

    Takeishi, Yukimasa; Iwaya-Omi, Rie; Ohashi, Eiji; Tsurimoto, Toshiki

    2015-08-01

    The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA damage. The 120-amino acid (aa) stretch of the Rad9 C terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent studies showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9(ΔC)-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA binding activity that manifests in the absence of the C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino acid sequence in the C-tail necessary for CRS binding to a 15-aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing the variant C-tail deficient for CRS binding, and we demonstrated that the mutant form restored DNA binding as efficiently as 9(ΔC)-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15-aa stretch, demonstrating that TopBP1 and CRS share the same binding region in the C-tail. Indeed, we observed their competitive binding to the C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA damage signaling suggests that the competitive interactions of TopBP1 and CRS with the C-tail will be crucial for the activation mechanism. PMID:26088138

  4. Targeting BRCA1- and BRCA2-deficient cells with RAD52 small molecule inhibitors

    PubMed Central

    Huang, Fei; Goyal, Nadish; Sullivan, Katherine; Hanamshet, Kritika; Patel, Mikir; Mazina, Olga M.; Wang, Charles X.; An, W. Frank; Spoonamore, James; Metkar, Shailesh; Emmitte, Kyle A.; Cocklin, Simon; Skorski, Tomasz; Mazin, Alexander V.

    2016-01-01

    RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair. PMID:26873923

  5. Targeting BRCA1- and BRCA2-deficient cells with RAD52 small molecule inhibitors.

    PubMed

    Huang, Fei; Goyal, Nadish; Sullivan, Katherine; Hanamshet, Kritika; Patel, Mikir; Mazina, Olga M; Wang, Charles X; An, W Frank; Spoonamore, James; Metkar, Shailesh; Emmitte, Kyle A; Cocklin, Simon; Skorski, Tomasz; Mazin, Alexander V

    2016-05-19

    RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair. PMID:26873923

  6. Muscle hardness characteristics of the masseter muscle after repetitive muscle activation: comparison to the biceps brachii muscle.

    PubMed

    Kashima, Koji; Higashinaka, Shuichi; Watanabe, Naoshi; Maeda, Sho; Shiba, Ryosuke

    2004-10-01

    The purpose of this study was to compare hardness characteristics of the masseter muscle to those of the biceps brachii muscle during repetitive muscle movements. Seventeen asymptomatic female subjects participated in this study. Each subject, on separate days, undertook a 5-minute unilateral chewing gum task on the right side and a 5-minute flexion-extension exercise on the right hand with a 2kg dumbbell. Using a handheld hardness meter, muscle hardness was measured in the right masseter and in the biceps brachii muscle at eight time points (before the task, immediately after the task, and at 1, 3, 5, 10, 30, and 60 minutes after the task), and the data obtained before and after the task on each muscle were compared. Comparisons of the normalized data were also performed between the two muscles at each time point. As a result, a significant increase in muscle hardness was seen at 1 minute after the task in the biceps brachii muscle (p=0.0093). In contrast, the masseter muscle showed a tendency to lower hardness, with the lowest point of hardness occurring at 10 minutes after the task (p = 0.0160). Between the two muscles, there was a difference in the normalized data immediately after the task, and at 1, 5, and 10 minutes after the task (0.01 hardness characteristics of the masseter muscle completely differed from those of the biceps brachii muscle after repetitive muscle activation. PMID:15532311

  7. A Search for Hard X-ray Emission from Active Stars Using CGRO/BATSE

    NASA Astrophysics Data System (ADS)

    White, S. M.; Harmon, B. A.; Lim, J.; Kundu, M. R.

    We report the results of a search for > 20 keV photons from active stars using CGRO/BATSE Earth-occultation observations. Twelve of the "usual suspects" together with 12 "placebo" locations have been analyzed using the BATSE software for occultation analysis developed at NASA/MSFC. There are four detections at the nominal 5sigma level, and eight at the 3sigma level. However the strongest detection (that of AB Dor) shows clear evidence for contamination from the nearby strong source LMC X-4. 18 of the 24 fields yield positive fluxes, indicating a clear bias in the results, and possibly indicating the presence of weak background hard X-ray sources detectable by BATSE in long-term studies.

  8. Development of radiation hard CMOS active pixel sensors for HL-LHC

    NASA Astrophysics Data System (ADS)

    Pernegger, Heinz

    2016-07-01

    New pixel detectors, based on commercial high voltage and/or high resistivity full CMOS processes, hold promise as next-generation active pixel sensors for inner and intermediate layers of the upgraded ATLAS tracker. The use of commercial CMOS processes allow cost-effective detector construction and simpler hybridisation techniques. The paper gives an overview of the results obtained on AMS-produced CMOS sensors coupled to the ATLAS Pixel FE-I4 readout chips. The SOI (silicon-on-insulator) produced sensors by XFAB hold great promise as radiation hard SOI-CMOS sensors due to their combination of partially depleted SOI transistors reducing back-gate effects. The test results include pre-/post-irradiation comparison, measurements of charge collection regions as well as test beam results.

  9. CGP57380 enhances efficacy of RAD001 in non-small cell lung cancer through abrogating mTOR inhibition-induced phosphorylation of eIF4E and activating mitochondrial apoptotic pathway.

    PubMed

    Wen, Qiuyuan; Wang, Weiyuan; Luo, Jiadi; Chu, Shuzhou; Chen, Lingjiao; Xu, Lina; Zang, Hongjing; Alnemah, Mohannad Ma; Ma, Jian; Fan, Songqing

    2016-05-10

    The mammalian target of rapamycin (mTOR) is a potentially important therapeutic target in a broad range of cancer types. mTOR inhibitors such as rapamycin and its analogs (rapalogs) have been proven effective as anticancer agents in non-small cell lung cancer (NSCLC), whereas they strongly enhance phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and activation of Akt, which cause resistance to mTOR-targeted therapy after an initial response. Rapamycin induces eIF4E phosphorylation by activating MAPK-interacting kinases (Mnks), and therefore targeting Mnk/eIF4E pathway represents a potential therapeutic strategy for the treatment of NSCLC. Here, our results showed that over-expression of p-Mnk1 and p-eIF4E was significantly associated with poor overall survival of NSCLC patients and high expression of p-Mnk1 might act as an independent prognostic biomarker for these patients. Meanwhile, inhibiting Mnk1 expression by Mnk inhibitor (CGP57380) could abrogate rapalogs (RAD001)-induced eIF4E phosphorylation and Akt activation. Furthermore, combination of CGP57380 and RAD001 could induce NSCLC cells apoptosis via activating intrinsic mitochondrial pathway, and exert synergistic antitumor efficacy both in vitro and in vivo. In conclusion, combination of targeting both mTOR and Mnk/eIF4E signaling pathways to enhance effectiveness of mTOR-targeted cancer therapy might be significant innovation for the personalized treatment of NSCLC. PMID:27050281

  10. HOST GALAXY PROPERTIES OF THE SWIFT BAT ULTRA HARD X-RAY SELECTED ACTIVE GALACTIC NUCLEUS

    SciTech Connect

    Koss, Michael; Mushotzky, Richard; Veilleux, Sylvain; Winter, Lisa M.; Baumgartner, Wayne; Tueller, Jack; Gehrels, Neil; Valencic, Lynne

    2011-10-01

    We have assembled the largest sample of ultra hard X-ray selected (14-195 keV) active galactic nucleus (AGN) with host galaxy optical data to date, with 185 nearby (z < 0.05), moderate luminosity AGNs from the Swift BAT sample. The BAT AGN host galaxies have intermediate optical colors (u - r and g - r) that are bluer than a comparison sample of inactive galaxies and optically selected AGNs from the Sloan Digital Sky Survey (SDSS) which are chosen to have the same stellar mass. Based on morphological classifications from the RC3 and the Galaxy Zoo, the bluer colors of BAT AGNs are mainly due to a higher fraction of mergers and massive spirals than in the comparison samples. BAT AGNs in massive galaxies (log M{sub *} >10.5) have a 5-10 times higher rate of spiral morphologies than in SDSS AGNs or inactive galaxies. We also see enhanced far-infrared emission in BAT AGN suggestive of higher levels of star formation compared to the comparison samples. BAT AGNs are preferentially found in the most massive host galaxies with high concentration indexes indicative of large bulge-to-disk ratios and large supermassive black holes. The narrow-line (NL) BAT AGNs have similar intrinsic luminosities as the SDSS NL Seyferts based on measurements of [O III] {lambda}5007. There is also a correlation between the stellar mass and X-ray emission. The BAT AGNs in mergers have bluer colors and greater ultra hard X-ray emission compared to the BAT sample as a whole. In agreement with the unified model of AGNs, and the relatively unbiased nature of the BAT sources, the host galaxy colors and morphologies are independent of measures of obscuration such as X-ray column density or Seyfert type. The high fraction of massive spiral galaxies and galaxy mergers in BAT AGNs suggest that host galaxy morphology is related to the activation and fueling of local AGN.

  11. The rad50 signature motif: essential to ATP binding and biological function.

    PubMed

    Moncalian, Gabriel; Lengsfeld, Bettina; Bhaskara, Venugopal; Hopfner, Karl-Peter; Karcher, Annette; Alden, Erinn; Tainer, John A; Paull, Tanya T

    2004-01-23

    The repair of double-strand breaks in DNA is an essential process in all organisms, and requires the coordinated activities of evolutionarily conserved protein assemblies. One of the most critical of these is the Mre11/Rad50 (M/R) complex, which is present in all three biological kingdoms, but is not well-understood at the biochemical level. Previous structural analysis of a Rad50 homolog from archaebacteria illuminated the catalytic core of the enzyme, an ATP-binding domain related to the ABC transporter family of ATPases. Here, we present the crystallographic structure of the Rad50 mutant S793R. This missense signature motif mutation changes the key serine residue in the signature motif that is conserved among Rad50 homologs and ABC ATPases. The S793R mutation is analogous to the mutation S549R in the cystic fibrosis transmembrane conductance regulator (CFTR) that results in cystic fibrosis. We show here that the serine to arginine change in the Rad50 protein prevents ATP binding and disrupts the communication among the other ATP-binding loops. This structural change, in turn, alters the communication between Rad50 monomers and thus prevents Rad50 dimerization. The equivalent mutation was made in the human Rad50 gene, and the resulting mutant protein did form a complex with Mre11 and Nbs1, but was specifically deficient in all ATP-dependent enzymatic activities. This signature motif structure-function homology extends to yeast, because the same mutation introduced into the Saccharomyces cerevisiae RAD50 gene generated an allele that failed to complement a rad50 deletion strain in DNA repair assays in vivo. These structural and biochemical results extend our understanding of the Rad50 catalytic domain and validate the use of the signature motif mutant to test the role of Rad50 ATP binding in diverse organisms. PMID:14698290

  12. Interdependence of the Rad50 hook and globular domain functions

    PubMed Central

    Hohl, Marcel; Kochańczyk, Tomasz; Tous, Cristina; Aguilera, Andrés; Krężel, Artur; Petrini, John H J

    2015-01-01

    SUMMARY Rad50 contains a conserved Zn2+ coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here we focused on rad50 mutations flanking the Zn2+-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end-joining, and DNA double strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled coil and globular ATPase domain, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions. PMID:25601756

  13. Association of the Rad9-Rad1-Hus1 checkpoint clamp with MYH DNA glycosylase and DNA.

    PubMed

    Hwang, Bor-Jang; Jin, Jin; Gunther, Randall; Madabushi, Amrita; Shi, Guoli; Wilson, Gerald M; Lu, A-Lien

    2015-07-01

    Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9-1-1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9-1-1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134-155) is a key determinant of MYH binding. Both the N-(residues 1-146) and C-terminal (residues 147-280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1(K136A) mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9(1-266) (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9(1-266)-1-1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9(1-266)-1-1 to 5'-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9-1-1 subunits to MYH-directed BER based on subunit asymmetry in protein-protein interactions and DNA binding events. PMID:26021743

  14. DUAL AND OPPOSITE EFFECTS OF hRAD51 CHEMICAL MODULATION ON HIV-1 INTEGRATION

    PubMed Central

    Thierry, Sylvain; Benleulmi, Mohamed Salah; Sinzelle, Ludivine; Thierry, Eloise; Calmels, Christina; Chaignepain, Stephane; Waffo-Teguo, Pierre; Merillon, Jean-Michel; Budke, Brian; Pasquet, Jean-Max; Litvak, Simon; Ciuffi, Angela; Sung, Patrick; Connell, Philip; Hauber, Ilona; Hauber, Joachim; Andreola, Marie-Line; Delelis, Olivier; Parissi, Vincent

    2016-01-01

    SUMMARY The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication. PMID:26051216

  15. Conversion of pesticides to biologically active products on urban hard surfaces.

    PubMed

    Jiang, Weiying; Gan, Jay

    2016-06-15

    Impervious pavements such as concrete are a dominant feature of urban landscapes, but their role in the fate of environmental contaminants is largely ignored. This study considered the case of urban-use pesticides, and demonstrated for the first time that surfaces such as concrete were capable of converting pesticides to other biologically active intermediates. Rapid transformation of pesticides was observed in both bench and field scale setups. Under outdoor conditions, permethrin, a heavily used pyrethroid insecticide, quickly formed 3-phenoxybenzoic acid (3-PBA) that is a known endocrine disruptor, and the level of 3-PBA was >100μg/L in the runoff water even 3months after the treatment. Fipronil, a product used for termite and ant control, was quickly transformed to desulfinyl and sulfone derivatives, with the desulfinyl level exceeding that of parent in the runoff water only 1week after treatment. Fipronil derivatives have aquatic toxicity similar or even greater than the parent fipronil. Direct sampling of deposited particles from residential exterior pavements revealed widespread presence of fipronil sulfone and desulfinyl and demonstrated their in-situ formation and accumulation on concrete. The extensive transformations were likely caused by the alkalinity and metal oxides in concrete and conducive photolytic conditions at the hard surfaces. The study findings highlight the role of urban pavements and urbanization in the geochemical cycling of anthropogenic contaminants. PMID:26971210

  16. Hard-X-ray spectra of active galactic nuclei in the INTEGRAL complete sample

    NASA Astrophysics Data System (ADS)

    Molina, M.; Bassani, L.; Malizia, A.; Stephen, J. B.; Bird, A. J.; Bazzano, A.; Ubertini, P.

    2013-08-01

    In this paper, we present the hard-X-ray spectral analysis of a complete sample of active galactic nuclei (AGNs) detected by INTEGRAL/IBIS. In conjunction with IBIS spectra, we make use of Swift/BAT data, with the aim of cross-calibrating the two instruments, studying source variability and constraining some important spectral parameters. We find that flux variability is present in at least 14 per cent of the sample, while spectral variability is found only in one object. There is general good agreement between BAT and IBIS spectra, despite a systematic mismatch of about 22 per cent in normalization. When fitted with a simple power-law model, type 1 and type 2 sources appear to have very similar average photon indices, suggesting that they are powered by the same mechanism. As expected, we also find that a simple power law does not always describe the data sufficiently well, thus indicating a certain degree of spectral complexity, which can be ascribed to features like a high energy cut-off and/or a reflection component. Fixing the reflection to be 0, 1 or 2, we find that our sample covers quite a large range in photon indices as well as cut-off energies; however, the spread is due only to a small number of objects, while the majority of the AGNs lie within well-defined boundaries of photon index (1 ≤ Γ ≤ 2) and cut-off energy (30 ≤ Ecut ≤ 300 keV).

  17. THE FIRST HARD X-RAY POWER SPECTRAL DENSITY FUNCTIONS OF ACTIVE GALACTIC NUCLEUS

    SciTech Connect

    Shimizu, T. Taro; Mushotzky, Richard F.

    2013-06-10

    We present results of our power spectral density (PSD) analysis of 30 active galactic nuclei (AGNs) using the 58 month light curves from Swift's Burst Alert Telescope (BAT) in the 14-150 keV band. PSDs were fit using a Monte Carlo based algorithm to take into account windowing effects and measurement error. All but one source were found to be fit very well using an unbroken power law with a slope of {approx} - 1, consistent at low frequencies with previous studies in the 2-10 keV band, with no evidence of a break in the PSD. For five of the highest signal-to-noise ratio sources, we tested the energy dependence of the PSD and found no significant difference in the PSD at different energies. Unlike previous studies of X-ray variability in AGNs, we do not find any significant correlations between the hard X-ray variability and different properties of the AGN including luminosity and black hole mass. The lack of break frequencies and correlations seem to indicate that AGNs are similar to the high state of Galactic black holes.

  18. A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59.

    PubMed Central

    Bai, Y; Davis, A P; Symington, L S

    1999-01-01

    With the use of an intrachromosomal inverted repeat as a recombination reporter, we have shown that mitotic recombination is dependent on the RAD52 gene, but reduced only fivefold by mutation of RAD51. RAD59, a component of the RAD51-independent pathway, was identified previously by screening for mutations that reduced inverted-repeat recombination in a rad51 strain. Here we describe a rad52 mutation, rad52R70K, that also reduced recombination synergistically in a rad51 background. The phenotype of the rad52R70K strain, which includes weak gamma-ray sensitivity, a fourfold reduction in the rate of inverted-repeat recombination, elevated allelic recombination, sporulation proficiency, and a reduction in the efficiency of mating-type switching and single-strand annealing, was similar to that observed for deletion of the RAD59 gene. However, rad52R70K rad59 double mutants showed synergistic defects in ionizing radiation resistance, sporulation, and mating-type switching. These results suggest that Rad52 and Rad59 have partially overlapping functions and that Rad59 can substitute for this function of Rad52 in a RAD51 rad52R70K strain. PMID:10545446

  19. Yeast cell-free system that catalyses joint-molecule formation in a Rad51p- and Rad52p-dependent fashion.

    PubMed Central

    Nagaraj, V; Norris, D

    2000-01-01

    One of the central reactions of homologous recombination is the invasion of a single strand of DNA into a homologous duplex to form a joint molecule. Here we describe the isolation of a cell-free system from meiotic yeast cells that catalyses joint-molecule formation in vitro. The active components in the system required ATP and homologous DNA and operated in both 0.5 and 13 mM MgCl(2). When the cell-free system was prepared from rad51/rad51 and rad52/rad52 mutants and joint-molecule formation was assayed at 0.5 mM MgCl(2), the specific activity decreased to 6% and 13.8% respectively of the wild-type level. However, when the same mutant extracts were premixed, joint-molecule formation increased 4-8-fold, i.e. the mutant extracts exhibited complementation in vitro. These results demonstrated that Rad51p and Rad52p were required for optimal joint-molecule formation at 0.5 mM MgCl(2). Intriguingly, however, Rad51p and Rad52p seemed to be more dispensable at higher concentrations of MgCl(2) (13 mM). Further purification of the responsible activity has proven problematical, but it did flow through a sizing column as a single peak (molecular mass 1.2 MDa) that was co-eluted with Rad51p and RFA, the eukaryotic single-stranded DNA-binding protein. All of these characteristics are consistent with the known properties of the reaction in vivo and suggest that the new cell-free system will be suitable for purifying enzymes involved in homologous recombination. PMID:10749664

  20. The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype.

    PubMed

    Marsden, Carolyn G; Jensen, Ryan B; Zagelbaum, Jennifer; Rothenberg, Eli; Morrical, Scott W; Wallace, Susan S; Sweasy, Joann B

    2016-08-01

    The RAD51 protein plays a key role in the homology-directed repair of DNA double-strand breaks and is important for maintaining genome stability. Here we report on a novel human RAD51 variant found in an aggressive and therapy-refractive breast carcinoma. Expression of the RAD51 G151D variant in human breast epithelial cells increases the levels of homology-directed repair. Expression of RAD51 G151D in cells also promotes high levels of chromosomal aberrations and sister chromatid exchanges. In vitro, the purified RAD51 G151D protein directly and significantly enhances DNA strand exchange activity in the presence of RPA. In concordance with this result, co-incubation of G151D with BRCA2 resulted in a much higher level of strand-exchange activity compared to WT RAD51. Strikingly, the RAD51 G151D variant confers resistance to multiple DNA damaging agents, including ionizing radiation, mitomycin C, and doxorubicin. Our findings demonstrate that the RAD51 G151D somatic variant has a novel hyper-recombination phenotype and suggest that this property of the protein is important for the repair of DNA damage, leading to drug resistance. PMID:27513445

  1. Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

    PubMed

    Su, Guan-Chin; Yeh, Hsin-Yi; Lin, Sheng-Wei; Chung, Chan-I; Huang, Yu-Shan; Liu, Yi-Chung; Lyu, Ping-Chiang; Chi, Peter

    2016-07-27

    During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5-SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51-SWI5-SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells. PMID:27131790

  2. The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype

    PubMed Central

    Marsden, Carolyn G.; Jensen, Ryan B.; Zagelbaum, Jennifer; Rothenberg, Eli; Morrical, Scott W.; Wallace, Susan S.; Sweasy, Joann B.

    2016-01-01

    The RAD51 protein plays a key role in the homology-directed repair of DNA double-strand breaks and is important for maintaining genome stability. Here we report on a novel human RAD51 variant found in an aggressive and therapy-refractive breast carcinoma. Expression of the RAD51 G151D variant in human breast epithelial cells increases the levels of homology-directed repair. Expression of RAD51 G151D in cells also promotes high levels of chromosomal aberrations and sister chromatid exchanges. In vitro, the purified RAD51 G151D protein directly and significantly enhances DNA strand exchange activity in the presence of RPA. In concordance with this result, co-incubation of G151D with BRCA2 resulted in a much higher level of strand-exchange activity compared to WT RAD51. Strikingly, the RAD51 G151D variant confers resistance to multiple DNA damaging agents, including ionizing radiation, mitomycin C, and doxorubicin. Our findings demonstrate that the RAD51 G151D somatic variant has a novel hyper-recombination phenotype and suggest that this property of the protein is important for the repair of DNA damage, leading to drug resistance. PMID:27513445

  3. The effect of ionic strength and hardness of trichloroethylene contaminated groundwater in remediation using granular activated carbon

    NASA Astrophysics Data System (ADS)

    Heo, J.; Chang, H.

    2005-12-01

    The objective of this study is to evaluate the trichloroethylene (TCE) removal by granular activated carbon (GAC) based on influential factors (ionic strength, hardness) of various groundwaters. The experimental method used in this study was batch experiments. Synthetic groundwater for test was made according to ionic strength, hardness and then it was artificially contaminated by TCE 5ppm. The variation of ions in synthetic groundwater was measured by I.C. and I.C.P. Surface area of GAC was determined by the Brunauer, Emmett and Teller (BET) adsorption data. The results of tests showed that TCE removal using GAC is affected by the hardness of synthetic groundwaters. It was founded that surface area of GAC was decreased by increasing of ions. Due to pore blocking of ions by functional group, the surface area of GAC decreased and the difference of the remediation appeared. This result was affected by the ionic strength and hardness of water. Therefore, the ionic strength and hardness of contaminated groundwater must be considered in remediation using GAC.

  4. Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels.

    PubMed

    Fornander, Louise H; Frykholm, Karolin; Fritzsche, Joachim; Araya, Joshua; Nevin, Philip; Werner, Erik; Çakır, Ali; Persson, Fredrik; Garcin, Edwige B; Beuning, Penny J; Mehlig, Bernhard; Modesti, Mauro; Westerlund, Fredrik

    2016-08-23

    RAD51 is the key component of the homologous recombination pathway in eukaryotic cells and performs its task by forming filaments on DNA. In this study we investigate the physical properties of RAD51 filaments formed on DNA using nanofluidic channels and fluorescence microscopy. Contrary to the bacterial ortholog RecA, RAD51 forms inhomogeneous filaments on long DNA in vitro, consisting of several protein patches. We demonstrate that a permanent "kink" in the filament is formed where two patches meet if the stretch of naked DNA between the patches is short. The kinks are readily seen in the present microscopy approach but would be hard to identify using conventional single DNA molecule techniques where the DNA is more stretched. We also demonstrate that protein patches separated by longer stretches of bare DNA roll up on each other and this is visualized as transiently overlapping filaments. RAD51 filaments can be formed at several different conditions, varying the cation (Mg(2+) or Ca(2+)), the DNA substrate (single-stranded or double-stranded), and the RAD51 concentration during filament nucleation, and we compare the properties of the different filaments formed. The results provide important information regarding the physical properties of RAD51 filaments but also demonstrate that nanofluidic channels are perfectly suited to study protein-DNA complexes. PMID:27479732

  5. Multiple regulation of Rad51-mediated homologous recombination by fission yeast Fbh1.

    PubMed

    Tsutsui, Yasuhiro; Kurokawa, Yumiko; Ito, Kentaro; Siddique, Md Shahjahan P; Kawano, Yumiko; Yamao, Fumiaki; Iwasaki, Hiroshi

    2014-08-01

    Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated. PMID:25165823

  6. Molecular basis of the essential s phase function of the rad53 checkpoint kinase.

    PubMed

    Hoch, Nicolas C; Chen, Eric S-W; Buckland, Robert; Wang, Shun-Chung; Fazio, Alessandro; Hammet, Andrew; Pellicioli, Achille; Chabes, Andrei; Tsai, Ming-Daw; Heierhorst, Jörg

    2013-08-01

    The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed. PMID:23754745

  7. Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

    PubMed Central

    Hoch, Nicolas C.; Chen, Eric S.-W.; Buckland, Robert; Wang, Shun-Chung; Fazio, Alessandro; Hammet, Andrew; Pellicioli, Achille; Chabes, Andrei; Tsai, Ming-Daw

    2013-01-01

    The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed. PMID:23754745

  8. Identification of a Small Molecule Inhibitor of RAD52 by Structure-Based Selection

    PubMed Central

    McElroy, Daniel L.; Ostrov, David A.; Haas, Kimberly; Childers, Wayne; Hromas, Robert; Skorski, Tomasz

    2016-01-01

    It has been reported that inhibition of RAD52 either by specific shRNA or a small peptide aptamer induced synthetic lethality in tumor cell lines carrying BRCA1 and BRCA2 inactivating mutations. Molecular docking was used to screen two chemical libraries: 1) 1,217 FDA approved drugs, and 2) 139,735 drug-like compounds to identify candidates for interacting with DNA binding domain of human RAD52. Thirty six lead candidate compounds were identified that were predicted to interfere with RAD52 –DNA binding. Further biological testing confirmed that 9 of 36 candidate compounds were able to inhibit the binding of RAD52 to single-stranded DNA in vitro. Based on molecular binding combined with functional assays, we propose a model in which the active compounds bind to a critical “hotspot” in RAD52 DNA binding domain 1. In addition, one of the 9 active compounds, adenosine 5’-monophosphate (A5MP), and also its mimic 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) 5’ phosphate (ZMP) inhibited RAD52 activity in vivo and exerted synthetic lethality against BRCA1 and BRCA2–mutated carcinomas. These data suggest that active, inhibitory RAD52 binding compounds could be further refined for efficacy and safety to develop drugs inducing synthetic lethality in tumors displaying deficiencies in BRCA1/2-mediated homologous recombination. PMID:26784987

  9. MSL-RAD Radiation Environment Measurements

    NASA Astrophysics Data System (ADS)

    Zeitlin, Cary; Hassler, Donald; Wimmer-Schweingruber, Robert; Boehm, Eckart; Boettcher, Stephan; Brinza, David; Burmeister, Soenke; Cucinotta, Francis; Ehresmann, Bent; Guo, Jingnan; Koehler, Jan; Martin, Cesar; Posner, Arik; Rafkin, Scot; Reitz, Guenther; MSL Science Team

    2013-04-01

    We present results from the Radiation Assessment Detector (RAD) obtained during MSL's cruise to Mars and during the first 150 sols after Curiosity's successful landing. RAD is designed to measure the energetic particle environment, which consists of Galactic Cosmic Rays (GCRs), Solar Energetic Particles (SEPs), and the secondary particles created by nuclear interactions of primary GCRs in shielding. During cruise, RAD was asymmetrically shielded inside the spacecraft. On the surface, RAD is shielded by the atmosphere, and the radiation dose rate is seen to vary slightly as the column depth of the atmosphere varies on a diurnal cycle. RAD's cruise measurements are a unique data set that provide a reasonable simulation of what might be encountered by a human crew headed for Mars or for some other destination in deep space. RAD successfully operated for 220 days of the 253 day journey to Mars. RAD has also operated stably on the surface of Mars, returning the first detailed radiation data from the surface of another planet. The data from the surface are also highly relevant for planning future crewed missions. We will present results for radiation dose and dose equivalent (the quantity most directly related to human health risk) obtained with both cruise and surface data. Dose and dose equivalent are dominated by the continuous GCR radiation, but five significant SEP events were seen during cruise and will be discussed.

  10. A molecular genetic dissection of the evolutionarily conserved N terminus of yeast Rad52.

    PubMed Central

    Mortensen, Uffe H; Erdeniz, Naz; Feng, Qi; Rothstein, Rodney

    2002-01-01

    Rad52 is a DNA-binding protein that stimulates the annealing of complementary single-stranded DNA. Only the N terminus of Rad52 is evolutionarily conserved; it contains the core activity of the protein, including its DNA-binding activity. To identify amino acid residues that are important for Rad52 function(s), we systematically replaced 76 of 165 amino acid residues in the N terminus with alanine. These substitutions were examined for their effects on the repair of gamma-ray-induced DNA damage and on both interchromosomal and direct repeat heteroallelic recombination. This analysis identified five regions that are required for efficient gamma-ray damage repair or mitotic recombination. Two regions, I and II, also contain the classic mutations, rad52-2 and rad52-1, respectively. Interestingly, four of the five regions contain mutations that impair the ability to repair gamma-ray-induced DNA damage yet still allow mitotic recombinants to be produced at rates that are similar to or higher than those obtained with wild-type strains. In addition, a new class of separation-of-function mutation that is only partially deficient in the repair of gamma-ray damage, but exhibits decreased mitotic recombination similar to rad52 null strains, was identified. These results suggest that Rad52 protein acts differently on lesions that occur spontaneously during the cell cycle than on those induced by gamma-irradiation. PMID:12072453

  11. Genetic analysis of Escherichia coli RadA: functional motifs and genetic interactions.

    PubMed

    Cooper, Deani L; Boyle, Daniel C; Lovett, Susan T

    2015-03-01

    The RadA/Sms protein is a RecA-related protein found universally in eubacteria and plants, implicated in processing of recombination intermediates. Here we show that the putative Zn finger, Walker A motif, KNRXG motif and Lon protease homology domain of the Escherichia coli RadA protein are required for DNA damage survival. RadA is unlikely to possess protease activity as the putative active site serine is not required. Mutants in RadA have strong synergistic phenotypes with those in the branch migration protein RecG. Sensitivity of radA recG mutants to azidothymidine (AZT) can be rescued by blocking recombination with recA or recF mutations or by overexpression of RuvAB, suggesting that lethal recombination intermediates accumulate in the absence of RadA and RecG. Synthetic genetic interactions for survival to AZT or ciprofloxacin exposure were observed between RadA and known or putative helicases including DinG, Lhr, PriA, Rep, RuvAB, UvrD, YejH and YoaA. These represent the first affected phenotypes reported for Lhr, YejH and YoaA. The specificity of these effects sheds new light on the role of these proteins in DNA damage avoidance and repair and implicates a role in replication gap processing for DinG and YoaA and a role in double-strand break repair for YejH. PMID:25484163

  12. Use of the Rad51 promoter for targeted anti-cancer therapy.

    PubMed

    Hine, Christopher M; Seluanov, Andrei; Gorbunova, Vera

    2008-12-30

    Rad51 protein, involved in homologous recombination, is overexpressed in a variety of tumors, and its expression is correlated with a poor prognosis. Here we propose to exploit the overexpression of Rad51 in cancer cells to design a Rad51 promoter-based anticancer therapy. On average, Rad51 mRNA and protein levels are increased in cancer cells four- and sixfold, respectively. Serendipitously, we discovered that when the Rad51 ORF is replaced with another ORF, the difference in promoter activity between normal and cancer cells increases to an average of 840-fold with a maximum difference of 12,500-fold. This dramatic difference in activity has high therapeutic potential. We demonstrate that the fusion of Rad51 promoter to diphtheria toxin A (DTA) gene kills a variety of cancer cell types, including breast cancer, fibrosarcoma, and cervical cancer cells, with minimal effect on normal breast epithelial cells and normal fibroblasts. Our results suggest that therapies based on the Rad51 promoter will be highly tumor specific and open new avenues for targeting a broad range of cancers. PMID:19106292

  13. Genetic analysis of Escherichia coli RadA: functional motifs and genetic interactions

    PubMed Central

    Cooper, Deani L; Boyle, Daniel C; Lovett, Susan T

    2015-01-01

    The RadA/Sms protein is a RecA-related protein found universally in eubacteria and plants, implicated in processing of recombination intermediates. Here we show that the putative Zn finger, Walker A motif, KNRXG motif and Lon protease homology domain of the Escherichia coli RadA protein are required for DNA damage survival. RadA is unlikely to possess protease activity as the putative active site serine is not required. Mutants in RadA have strong synergistic phenotypes with those in the branch migration protein RecG. Sensitivity of radA recG mutants to azidothymidine (AZT) can be rescued by blocking recombination with recA or recF mutations or by overexpression of RuvAB, suggesting that lethal recombination intermediates accumulate in the absence of RadA and RecG. Synthetic genetic interactions for survival to AZT or ciprofloxacin exposure were observed between RadA and known or putative helicases including DinG, Lhr, PriA, Rep, RuvAB, UvrD, YejH and YoaA. These represent the first affected phenotypes reported for Lhr, YejH and YoaA. The specificity of these effects sheds new light on the role of these proteins in DNA damage avoidance and repair and implicates a role in replication gap processing for DinG and YoaA and a role in double-strand break repair for YejH. PMID:25484163

  14. RAD51 variant proteins from human lung and kidney tumors exhibit DNA strand exchange defects.

    PubMed

    Silva, Michelle C; Morrical, Milagros D; Bryan, Katie E; Averill, April M; Dragon, Julie; Bond, Jeffrey P; Morrical, Scott W

    2016-06-01

    In human cells, error-free repair of DNA double-strand breaks requires the DNA pairing and strand exchange activities of RAD51 recombinase. Activation of RAD51 recombination activities requires the assembly of RAD51 presynaptic filaments on the single-stranded DNA that forms at resected DSB ends. Mutations in proteins that control presynaptic filament assembly, such as BRCA2, and in RAD51 itself, are associated with human breast cancer. Here we describe the properties of two mutations in RAD51 protein that derive from human lung and kidney tumors, respectively. Sequence variants Q268P and Q272L both map to the DNA binding loop 2 (L2) region of RAD51, a motif that is involved in DNA binding and in the allosteric activation of ATP hydrolysis and DNA strand exchange activities. Both mutations alter the thermal stability, DNA binding, and ATPase properties of RAD51, however both variants retain intrinsic DNA strand exchange activity towards oligonucleotide substrates under optimized conditions. In contrast, both Q268P and Q272L variants exhibit drastically reduced DNA strand exchange activity in reaction mixtures containing long homologous ssDNA and dsDNA substrates and human RPA protein. Mixtures of wild-type and variant proteins also exhibit reduced DNA strand exchange activity, suggesting that heterozygous mutations could negatively affect DNA recombination and repair processes in vivo. Together, the findings of this study suggest that hypomorphic missense mutations in RAD51 protein could be drivers of genomic instability in cancer cells, and thereby contribute to the etiology of metastatic disease. PMID:27153211

  15. Identification and characterization of human Rad51 inhibitors by screening of an existing drug library.

    PubMed

    Normand, Anaïs; Rivière, Emmanuelle; Renodon-Cornière, Axelle

    2014-10-01

    Homologous Recombination (HR) plays an essential role in cellular proliferation and in maintaining genomic stability by repairing DNA double-stranded breaks that appear during replication. Rad51, a key protein of HR in eukaryotes, can have an elevated expression level in tumor cells, which correlates with their resistance to anticancer therapies. Therefore, targeted inhibition of Rad51 through inhibitor may improve the tumor response to these therapies. In order to identify small molecules that inhibit Rad51 activity, we screened the Prestwick Library (1120 molecules) for their effect on the strand exchange reaction catalyzed by Rad51. We found that Chicago Sky Blue (CSB) is a potent inhibitor of Rad51, showing IC₅₀ values in the low nanomolar range (400 nM). Biochemical analysis demonstrated that the inhibitory mechanism probably occurs by disrupting the Rad51 association with the single-stranded DNA, which prevents the nucleoprotein filament formation, the first step of the protein activity. Structure Activity Relationship analysis with a number of compounds that shared structure homology with CSB was also performed. The sensitivity of Rad51 inhibition to CSB modifications suggests specific interactions between the molecule and Rad51 nucleofilament. CSB and some of its analogs open up new perspectives in the search for agents capable of potentiating chemo- and radio-therapy treatments for cancer. Moreover, these compounds may be excellent tools to analyze Rad51 cellular functions. Our study also highlights how CSB and its analogs, which are frequently used in colorants, stains and markers, could be responsible of unwanted side effects by perturbing the DNA repair process. PMID:25124703

  16. The PSO4 Protein Complex Associates with Replication Protein A (RPA) and Modulates the Activation of Ataxia Telangiectasia-mutated and Rad3-related (ATR)*

    PubMed Central

    Wan, Li; Huang, Jun

    2014-01-01

    The PSO4 core complex is composed of PSO4/PRP19/SNEV, CDC5L, PLRG1, and BCAS2/SPF27. Besides its well defined functions in pre-mRNA splicing, the PSO4 complex has been shown recently to participate in the DNA damage response. However, the specific role for the PSO4 complex in the DNA damage response pathways is still not clear. Here we show that both the BCAS2 and PSO4 subunits of the PSO4 complex directly interact and colocalize with replication protein A (RPA). Depletion of BCAS2 or PSO4 impairs the recruitment of ATR-interacting protein (ATRIP) to DNA damage sites and compromises CHK1 activation and RPA2 phosphorylation. Moreover, we demonstrate that both the RPA1-binding ability of BCAS2 and the E3 ligase activity of PSO4 are required for efficient accumulation of ATRIP at DNA damage sites and the subsequent CHK1 activation and RPA2 phosphorylation. Our results suggest that the PSO4 complex functionally interacts with RPA and plays an important role in the DNA damage response. PMID:24443570

  17. The Saccharomyces Cerevisiae Rad30 Gene, a Homologue of Escherichia Coli Dinb and Umuc, Is DNA Damage Inducible and Functions in a Novel Error-Free Postreplication Repair Mechanism

    PubMed Central

    McDonald, J. P.; Levine, A. S.; Woodgate, R.

    1997-01-01

    Damage-inducible mutagenesis in prokaryotes is largely dependent upon the activity of the UmuD'C-like proteins. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized Saccharomyces cerevisiae DNA repair gene related to the Escherichia coli dinB, umuC and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Similar to its prokaryotic homologues, RAD30 was found to be damage inducible. Like many S. cerevisiae genes involved in error-prone DNA repair, epistasis analysis clearly places RAD30 in the RAD6 group and rad30 mutants display moderate UV sensitivity reminiscent of rev mutants. However, unlike rev mutants, no defect in UV-induced reversion was seen in rad30 strains. While rad6 and rad18 are both epistatic to rad30, no epistasis was observed with rev1, rev3, rev7 or rad5, all of which are members of the RAD6 epistasis group. These findings suggest that RAD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5. PMID:9409821

  18. Negative superhelicity promotes ATP-dependent binding of yeast RAD3 protein to ultraviolet-damaged DNA.

    PubMed

    Sung, P; Watkins, J F; Prakash, L; Prakash, S

    1994-03-18

    The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of UV-damaged DNA and is essential for cell viability. Remarkable homology exists between RAD3 and the human excision repair gene XPD, whose mutational inactivation underlies the cancer-prone disorder in xeroderma pigmentosum group D patients. Our previous work demonstrated that RAD3-encoded protein contains a DNA helicase activity. Here, we show that RAD3 binds preferentially to UV-damaged DNA over nondamaged DNA. Removal of pyrimidine dimers from damaged DNA by enzymatic photoreactivation does not affect binding, suggesting an affinity of RAD3 for pyrimidine (6-4) pyrimidone photoproducts. Damage-specific binding by RAD3 is strongly dependent on ATP and on the degree of negative superhelicity in DNA. The requirement of superhelicity in damage binding may target RAD3 to regions of DNA undergoing transcription, resulting in the preferential repair of these regions. The rad3 Arg-48 mutant protein, which lacks the DNA helicase activity, also binds UV-damaged DNA preferentially, indicating that DNA helicase and damage binding are two distinct and separable functional entities in RAD3. PMID:8132553

  19. ATP half-sites in RadA and RAD51 recombinases bind nucleotides.

    PubMed

    Marsh, May E; Scott, Duncan E; Ehebauer, Matthias T; Abell, Chris; Blundell, Tom L; Hyvönen, Marko

    2016-05-01

    Homologous recombination is essential for repair of DNA double-strand breaks. Central to this process is a family of recombinases, including archeal RadA and human RAD51, which form nucleoprotein filaments on damaged single-stranded DNA ends and facilitate their ATP-dependent repair. ATP binding and hydrolysis are dependent on the formation of a nucleoprotein filament comprising RadA/RAD51 and single-stranded DNA, with ATP bound between adjacent protomers. We demonstrate that truncated, monomeric Pyrococcus furiosus RadA and monomerised human RAD51 retain the ability to bind ATP and other nucleotides with high affinity. We present crystal structures of both apo and nucleotide-bound forms of monomeric RadA. These structures reveal that while phosphate groups are tightly bound, RadA presents a shallow, poorly defined binding surface for the nitrogenous bases of nucleotides. We suggest that RadA monomers would be constitutively bound to nucleotides in the cell and that the bound nucleotide might play a structural role in filament assembly. PMID:27419043

  20. A Synthetic Interaction between CDC20 and RAD4 in Saccharomyces cerevisiae upon UV Irradiation

    PubMed Central

    Rochelle, Lauren; Roberts, Asela

    2014-01-01

    Regulation of DNA repair can be achieved through ubiquitin-mediated degradation of transiently induced proteins. In Saccharomyces cerevisiae, Rad4 is involved in damage recognition during nucleotide excision repair (NER) and, in conjunction with Rad23, recruits other proteins to the site of damage. We identified a synthetic interaction upon UV exposure between Rad4 and Cdc20, a protein that modulates the activity of the anaphase promoting complex (APC/C), a multisubunit E3 ubiquitin ligase complex. The moderately UV sensitive Δrad4 strain became highly sensitive when cdc20-1 was present, and was rescued by overexpression of CDC20. The double mutant is also deficient in elicting RNR3-lacZ transcription upon exposure to UV irradiation or 4-NQO compared with the Δrad4 single mutant. We demonstrate that the Δrad4/cdc20-1 double mutant is defective in double strand break repair by way of a plasmid end-joining assay, indicating that Rad4 acts to ensure that damaged DNA is repaired via a Cdc20-mediated mechanism. This study is the first to present evidence that Cdc20 may play a role in the degradation of proteins involved in nucleotide excision repair. PMID:24707403

  1. Down-Regulation of Rad51 and Decreased Homologous Recombination in Hypoxic Cancer Cells

    PubMed Central

    Bindra, Ranjit S.; Schaffer, Paul J.; Meng, Alice; Woo, Jennifer; Måseide, Kårstein; Roth, Matt E.; Lizardi, Paul; Hedley, David W.; Bristow, Robert G.; Glazer, Peter M.

    2004-01-01

    There is an emerging concept that acquired genetic instability in cancer cells can arise from the dysregulation of critical DNA repair pathways due to cell stresses such as inflammation and hypoxia. Here we report that hypoxia specifically down-regulates the expression of RAD51, a key mediator of homologous recombination in mammalian cells. Decreased levels of Rad51 were observed in multiple cancer cell types during hypoxic exposure and were not associated with the cell cycle profile or with expression of hypoxia-inducible factor. Analyses of RAD51 gene promoter activity, as well as mRNA and protein stability, indicate that the hypoxia-mediated regulation of this gene occurs via transcriptional repression. Decreased expression of Rad51 was also observed to persist in posthypoxic cells for as long as 48 h following reoxygenation. Correspondingly, we found reduced levels of homologous recombination in both hypoxic and posthypoxic cells, suggesting that the hypoxia-associated reduction in Rad51 expression has functional consequences for DNA repair. In addition, hypoxia-mediated down-regulation of Rad51 was confirmed in vivo via immunofluorescent image analysis of experimental tumors in mice. Based on these findings, we propose a novel mechanism of genetic instability in the tumor microenvironment mediated by hypoxia-induced suppression of the homologous recombination pathway in cancer cells. The aberrant regulation of Rad51 expression may also create heterogeneity in the DNA damage response among cells within tumors, with implications for the response to cancer therapies. PMID:15367671

  2. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.

    PubMed

    Hopfner, K P; Karcher, A; Shin, D S; Craig, L; Arthur, L M; Carney, J P; Tainer, J A

    2000-06-23

    To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins. PMID:10892749

  3. RadBall™ Technology Testing and MCNP Modeling of the Tungsten Collimator

    PubMed Central

    Farfán, Eduardo B.; Foley, Trevor Q.; Coleman, J. Rusty; Jannik, G. Timothy; Holmes, Christopher J.; Oldham, Mark; Adamovics, John; Stanley, Steven J.

    2010-01-01

    The United Kingdom’s National Nuclear Laboratory (NNL) has developed a remote, non-electrical, radiation-mapping device known as RadBall™, which can locate and quantify radioactive hazards within contaminated areas of the nuclear industry. RadBall™ consists of a colander-like outer shell that houses a radiation-sensitive polymer sphere. The outer shell works to collimate radiation sources and those areas of the polymer sphere that are exposed react, becoming increasingly more opaque, in proportion to the absorbed dose. The polymer sphere is imaged in an optical-CT scanner, which produces a high resolution 3D map of optical attenuation coefficients. Subsequent analysis of the optical attenuation matrix provides information on the spatial distribution of sources in a given area forming a 3D characterization of the area of interest. RadBall™ has no power requirements and can be positioned in tight or hard-to reach locations. The RadBall™ technology has been deployed in a number of technology trials in nuclear waste reprocessing plants at Sellafield in the United Kingdom and facilities of the Savannah River National Laboratory (SRNL). This study focuses on the RadBall™ testing and modeling accomplished at SRNL. PMID:21617740

  4. RadBall Technology Testing and MCNP Modeling of the Tungsten Collimator.

    PubMed

    Farfán, Eduardo B; Foley, Trevor Q; Coleman, J Rusty; Jannik, G Timothy; Holmes, Christopher J; Oldham, Mark; Adamovics, John; Stanley, Steven J

    2010-01-01

    The United Kingdom's National Nuclear Laboratory (NNL) has developed a remote, non-electrical, radiation-mapping device known as RadBall(™), which can locate and quantify radioactive hazards within contaminated areas of the nuclear industry. RadBall(™) consists of a colander-like outer shell that houses a radiation-sensitive polymer sphere. The outer shell works to collimate radiation sources and those areas of the polymer sphere that are exposed react, becoming increasingly more opaque, in proportion to the absorbed dose. The polymer sphere is imaged in an optical-CT scanner, which produces a high resolution 3D map of optical attenuation coefficients. Subsequent analysis of the optical attenuation matrix provides information on the spatial distribution of sources in a given area forming a 3D characterization of the area of interest. RadBall(™) has no power requirements and can be positioned in tight or hard-to reach locations. The RadBall(™) technology has been deployed in a number of technology trials in nuclear waste reprocessing plants at Sellafield in the United Kingdom and facilities of the Savannah River National Laboratory (SRNL). This study focuses on the RadBall(™) testing and modeling accomplished at SRNL. PMID:21617740

  5. RadBallTM Technology Testing and MCNP Modeling of the Tungsten Collimator

    NASA Astrophysics Data System (ADS)

    Farfán, Eduardo B.; Foley, Trevor Q.; Rusty Coleman, J.; Jannik, G. Timothy; Holmes, Christopher J.; Oldham, Mark; Adamovics, John; Stanley, Steven J.

    2010-11-01

    The UK's National Nuclear Laboratory (NNL) has developed a remote, non-electrical, radiation-mapping device known as RadBallTM, which can locate and quantify radioactive hazards within contaminated areas of the nuclear industry. RadBallTM consists of a colander-like outer shell that houses a radiation-sensitive polymer sphere. The outer shell works to collimate radiation sources and those areas of the polymer sphere that are exposed react, becoming increasingly more opaque, in proportion to the absorbed dose. The polymer sphere is imaged in an optical-CT scanner, which produces a high resolution 3D map of optical attenuation coefficients. Subsequent analysis of the optical attenuation matrix provides information on the spatial distribution of sources in a given area forming a 3D characterization of the area of interest. RadBallTM has no power requirements and can be positioned in tight or hard-to reach locations. The RadBallTM technology has been deployed in a number of technology trials in nuclear waste reprocessing plants at Sellafield in the UK and facilities of the Savannah River National Laboratory (SRNL). This study focuses on the RadBallTM testing and modeling accomplished at SRNL.

  6. Phosphorylation-dependent Assembly and Coordination of the DNA Damage Checkpoint Apparatus by Rad4TopBP1

    PubMed Central

    Garcia, Valerie; Ren, Jing-Yi; Day, Matthew; Carr, Antony M.; Oliver, Antony W.; Du, Li-Lin; Pearl, Laurence H.

    2016-01-01

    Summary The BRCT-domain protein, Rad4TopBP1, facilitates activation of the DNA damage checkpoint in S. pombe by physically coupling the Rad9-Rad1-Hus1 clamp, the Rad3ATR-Rad26ATRIP kinase complex and Crb253BP1 mediator. We have now determined crystal structures of the BRCT repeats of Rad4TopBP1, revealing a distinctive domain architecture, and have characterized their phosphorylation-dependent interactions with Rad9 and Crb253BP1. We identify a cluster of phosphorylation sites in the N-terminal region of Crb253BP1 that mediate interaction with Rad4TopBP1, and reveal a hierarchical phosphorylation mechanism in which phosphorylation of Thr215 and Thr235 promotes phosphorylation of the non-canonical Thr187 site by scaffolding CDK recruitment. Finally we show that simultaneous interaction of a single Rad4TopBP1 molecule with both Thr187 phosphorylation sites in a Crb253BP1 dimer, is essential for establishing the DNA damage checkpoint. PMID:24074952

  7. Role of repair protein Rad51 in regulating the response to gefitinib in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Hong, Jhao-Hao; Wang, Lyu-Han; Cheng, Chau-Ming; Ciou, Shih-Ci; Lin, Szu-Ting; Jheng, Ming-Yan; Lin, Yun-Wei

    2008-11-01

    Gefitinib (Iressa, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that can block growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) activation. High-level Rad51 expression has been reported in chemoresistant or radioresistant carcinomas. In this study, we examined the role of Rad51 in regulating the response to gefitinib among different human lung cancer cell lines. The H520 line (human squamous cell carcinoma) was less sensitive to gefitinib compared with the H1650 (human adenocarcinoma) or A549 (human bronchioloalveolar carcinoma) lines. In H1650 and A549 cells but not in H520 cells, gefitinib decreased cellular levels of phospho-ERK1/2 and Rad51 protein and message levels. Moreover, gefitinib decreased Rad51 protein levels by enhancing Rad51 protein instability through 26S proteasome-mediated degradation. Inhibition of endogenous Rad51 levels by si-Rad51 RNA transfection significantly enhanced gefitinib-induced cytotoxicity. In contrast, transfection with constitutively active MKK1 vector could restore both Rad51 protein levels and cell survival inhibited by gefitinib. The MKK1/2-ERK1/2 signaling pathway constitutes the upstream signaling for maintaining Rad51 message and protein levels. Rad51 protein can protect lung cancer cells from cytotoxic effects induced by gefitinib. Suppression of Rad51 may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib. PMID:19001445

  8. INSIGHT INTO ACTIVE GALACTIC NUCLEUS AND HOST GALAXY CO-EVOLUTION FROM HARD X-RAY EMISSION

    SciTech Connect

    Wang, J.; Zhou, X. L.; Wei, J. Y.

    2013-05-10

    We study the issue of active galactic nucleus (AGN) and host co-evolution by focusing on the correlation between the hard X-ray emission from central AGNs and the stellar populations of the host galaxies. Focusing on galaxies with strong H{alpha} line emission (EW(H{alpha}) > 5 A), both X-ray and optical spectral analyses are performed on 67 (partially) obscured AGNs that are selected from the XMM-Newton 2XMMi/SDSS-DR7 catalog originally cross-matched by Pineau et al. The sample allows us to study central AGN activity and host galaxy activity directly and simultaneously in individual objects. Combining the spectral analysis in both bands reveals that the older the stellar population of the host galaxy, the harder the X-ray emission will be, which was missed in our previous study where ROSAT hardness ratios were used. By excluding the contamination from host galaxies and from jet beaming emission, the correlation indicates that Compton cooling in the accretion disk corona decreases with the mean age of the stellar population. We argue that this correlation is related to the correlation of L/L{sub Edd} with the host stellar population. In addition, the [O I]/H{alpha} and [S II]/H{alpha} narrow-line ratios are identified to correlate with the spectral slope in hard X-rays, which can be inferred from the currently proposed evolution of the X-ray emission because of the confirmed tight correlations between the two line ratios and stellar population age.

  9. The internal disruption as hard Magnetohydrodynamic limit of 1/2 sawtooth like activity in large helical device

    SciTech Connect

    Varela, J.; Watanabe, K. Y.; Ohdachi, S.

    2012-08-15

    Large helical device (LHD) inward-shifted configurations are unstable to resistive MHD pressure-gradient-driven modes. Sawtooth like activity was observed during LHD operation. The main drivers are the unstable modes 1/2 and 1/3 in the middle and inner plasma region which limit the plasma confinement efficiency of LHD advanced operation scenarios. The aim of the present research is to study the hard MHD limit of 1/2 sawtooth like activity, not observed yet in LHD operation, and to predict its effects on the device performance. Previous investigations pointed out this system relaxation can be an internal disruption [J. Varela et al., 'Internal disruptions and sawtooth like activity in LHD,' 38th EPS Conference on Plasma Physics (2011), P5.077]. In the present work, we simulate an internal disruption; we study the equilibria properties before and after the disruptive process, its effects on the plasma confinement efficiency during each disruptive phase, the relation between the n/m = 1/2 hard MHD events and the soft MHD events, and how to avoid or reduce their adverse effects. The simulation conclusions point out that the large stochastic region in the middle plasma strongly deforms and tears the flux surfaces when the pressure gradient increases above the hard MHD limit. If the instability reaches the inner plasma, the iota profiles will be perturbed near the plasma core and three magnetic islands can appear near the magnetic axis. If the instability is strong enough to link the stochastic regions in the middle plasma (around the half minor radius {rho}) and the plasma core ({rho}<0.25), an internal disruption is driven.

  10. HARD X-RAY LAGS IN ACTIVE GALACTIC NUCLEI: TESTING THE DISTANT REVERBERATION HYPOTHESIS WITH NGC 6814

    SciTech Connect

    Walton, D. J.; Harrison, F. A.; Zoghbi, A.; Reynolds, C. S.; Cackett, E. M.; Uttley, P.; Fabian, A. C.; Kara, E.; Miller, J. M.; Reis, R. C.

    2013-11-10

    We present an X-ray spectral and temporal analysis of the variable active galaxy NGC 6814, observed with Suzaku during 2011 November. Remarkably, the X-ray spectrum shows no evidence for the soft excess commonly observed amongst other active galaxies, despite its relatively low level of obscuration, and is dominated across the whole Suzaku bandpass by the intrinsic powerlaw-like continuum. Despite this, we clearly detect the presence of a low-frequency hard lag of ∼1600 s between the 0.5-2.0 and 2.0-5.0 keV energy bands at greater than 6σ significance, similar to those reported in the literature for a variety of other active galactic nuclei (AGNs). At these energies, any additional emission from, e.g., a very weak, undetected soft excess, or from distant reflection must contribute less than 3% of the observed countrates (at 90% confidence). Given the lack of any significant continuum emission component other than the powerlaw, we can rule out models that invoke distant reprocessing for the observed lag behavior, which must instead be associated with this continuum emission. These results are fully consistent with a propagating fluctuation origin for the low-frequency hard lags, and with the interpretation of the high-frequency soft lags—a common feature seen in the highest quality AGN data with strong soft excesses—as reverberation from the inner accretion disk.

  11. SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination.

    PubMed

    Silva, Sonia; Altmannova, Veronika; Eckert-Boulet, Nadine; Kolesar, Peter; Gallina, Irene; Hang, Lisa; Chung, Inn; Arneric, Milica; Zhao, Xiaolan; Buron, Line Due; Mortensen, Uffe H; Krejci, Lumir; Lisby, Michael

    2016-06-01

    Homologous recombination (HR) is essential for maintenance of genome stability through double-strand break (DSB) repair, but at the same time HR can lead to loss of heterozygosity and uncontrolled recombination can be genotoxic. The post-translational modification by SUMO (small ubiquitin-like modifier) has been shown to modulate recombination, but the exact mechanism of this regulation remains unclear. Here we show that SUMOylation stabilizes the interaction between the recombination mediator Rad52 and its paralogue Rad59 in Saccharomyces cerevisiae. Although Rad59 SUMOylation is not required for survival after genotoxic stress, it affects the outcome of recombination to promote conservative DNA repair. In some genetic assays, Rad52 and Rad59 SUMOylation act synergistically. Collectively, our data indicate that the described SUMO modifications affect the balance between conservative and non-conservative mechanisms of HR. PMID:27130983

  12. MSL-RAD radiation environment measurements.

    PubMed

    Guo, Jingnan; Zeitlin, Cary; Wimmer-Schweingruber, Robert F; Hassler, Donald M; Ehresmann, Bent; Köhler, Jan; Böhm, Eckart; Böttcher, Stephan; Brinza, David; Burmeister, Sönke; Cucinotta, Francis; Martin, Cesar; Posner, Arik; Rafkin, Scot; Reitz, Guenther

    2015-09-01

    In this study, results are presented from the on-board radiation assessment detector (RAD) of Mars Science Laboratory (MSL). RAD is designed to measure the energetic particle radiation environment, which consists of galactic cosmic rays (GCRs) and solar energetic particles (SEPs) as well as secondary particles created by nuclear interactions of primary particles in the shielding (during cruise) or Martian soil and atmosphere (surface measurements). During the cruise, RAD collected data on space radiation from inside the craft, thus allowing for a reasonable estimation of what a human crew travelling to/from Mars might be exposed to. On the surface of Mars, RAD is shielded by the atmosphere (from above) and the planet itself (from below). RAD measures the first detailed radiation data from the surface of another planet, and they are highly relevant for planning future crewed missions. The results for radiation dose and dose equivalent (a quantity most directly related to human health risk) are presented during the cruise phase, as well as on the Martian surface. Dose and dose equivalent are dominated by the continuous GCR radiation, but several SEP events were also detected and are discussed here. PMID:25969529

  13. Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

    PubMed Central

    Hengel, Sarah R; Malacaria, Eva; Folly da Silva Constantino, Laura; Bain, Fletcher E; Diaz, Andrea; Koch, Brandon G; Yu, Liping; Wu, Meng; Pichierri, Pietro; Spies, M Ashley; Spies, Maria

    2016-01-01

    The DNA repair protein RAD52 is an emerging therapeutic target of high importance for BRCA-deficient tumors. Depletion of RAD52 is synthetically lethal with defects in tumor suppressors BRCA1, BRCA2 and PALB2. RAD52 also participates in the recovery of the stalled replication forks. Anticipating that ssDNA binding activity underlies the RAD52 cellular functions, we carried out a high throughput screening campaign to identify compounds that disrupt the RAD52-ssDNA interaction. Lead compounds were confirmed as RAD52 inhibitors in biochemical assays. Computational analysis predicted that these inhibitors bind within the ssDNA-binding groove of the RAD52 oligomeric ring. The nature of the inhibitor-RAD52 complex was validated through an in silico screening campaign, culminating in the discovery of an additional RAD52 inhibitor. Cellular studies with our inhibitors showed that the RAD52-ssDNA interaction enables its function at stalled replication forks, and that the inhibition of RAD52-ssDNA binding acts additively with BRCA2 or MUS81 depletion in cell killing. DOI: http://dx.doi.org/10.7554/eLife.14740.001 PMID:27434671

  14. Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells.

    PubMed

    Hengel, Sarah R; Malacaria, Eva; Folly da Silva Constantino, Laura; Bain, Fletcher E; Diaz, Andrea; Koch, Brandon G; Yu, Liping; Wu, Meng; Pichierri, Pietro; Spies, M Ashley; Spies, Maria

    2016-01-01

    The DNA repair protein RAD52 is an emerging therapeutic target of high importance for BRCA-deficient tumors. Depletion of RAD52 is synthetically lethal with defects in tumor suppressors BRCA1, BRCA2 and PALB2. RAD52 also participates in the recovery of the stalled replication forks. Anticipating that ssDNA binding activity underlies the RAD52 cellular functions, we carried out a high throughput screening campaign to identify compounds that disrupt the RAD52-ssDNA interaction. Lead compounds were confirmed as RAD52 inhibitors in biochemical assays. Computational analysis predicted that these inhibitors bind within the ssDNA-binding groove of the RAD52 oligomeric ring. The nature of the inhibitor-RAD52 complex was validated through an in silico screening campaign, culminating in the discovery of an additional RAD52 inhibitor. Cellular studies with our inhibitors showed that the RAD52-ssDNA interaction enables its function at stalled replication forks, and that the inhibition of RAD52-ssDNA binding acts additively with BRCA2 or MUS81 depletion in cell killing. PMID:27434671

  15. From Compass to Hard Drive--Integrated Activities for Studying Magnets

    ERIC Educational Resources Information Center

    Dean, J.; Allwood, D. A.

    2014-01-01

    We describe a range of practical activities that allows students to investigate the properties and applications of magnets. The activities can be used in isolation or used together to build a rounded understanding of the subject area. The activities include simple demonstrations using common or inexpensive equipment, hands-on experiments for small…

  16. Radiation hardness of a 180 nm SOI monolithic active pixel sensor

    NASA Astrophysics Data System (ADS)

    Fernandez-Perez, S.; Backhaus, M.; Pernegger, H.; Hemperek, T.; Kishishita, T.; Krüger, H.; Wermes, N.

    2015-10-01

    The use of Silicon-on-Insulator (SOI) technology as a particle detector in a high radiation environment is, at present, limited mostly by radiation effects on the transistor characteristics, back gate effect, and mutual coupling between the Buried Oxide (BOX) and the sensor. We have fabricated and tested a new 0.18 μm SOI CMOS monolithic pixel sensor using the XFAB process. In contrast to the most commonly used SOI technologies, this particular technology uses partially depleted SOI transistors, offering a double well structure, which shields the thin gate oxide transistors from the BOX. In addition, an increased distance between transistors and a thicker BOX than has been previously used offers promising solutions to the performance limitations mentioned above. The process further allows the use of high voltages (up to 200 V), which are used to partially deplete the substrate. Thus, the newly fabricated device in the XFAB process is especially interesting for applications in extremely high radiation environments, such as LHC experiments. A four stage validation programme of the technology and the fabricated monolithic pixel sensor has been performed and its results are shown in this paper. The first targets radiation hardness of the transistor characteristics up to 700 Mrad, the second investigates the existence of the back gate effect, the third one targets the coupling between the BOX and the sensor, and the fourth investigates the characterization of charge collection in the sensor diode below the BOX.

  17. Bactericidal activity of chlorine dioxide against Escherichia coli in water and on hard surfaces.

    PubMed

    Foschino, R; Nervegna, I; Motta, A; Galli, A

    1998-06-01

    The efficacy of chlorine dioxide as a disinfectant was evaluated against cells of Escherichia coli ATCC 11229 in aqueous suspension and adhering to the surfaces of stainless steel AISI 304 and PVC. The concentrations tested ranged from 0.7 to 14 mg/liter; the exposure times investigated were 30 s and 1, 2, 4, and 8 min. When the bacteria were suspended in water with 1.4 mg/liter of chlorine dioxide, a 10(5)-fold reduction of the initial viable count occurred within 30 s; when cells were attached to the steel surface, the same rate of inactivation took place only after 6 min with 7 mg/liter or 4 min with 14 mg/liter of chlorine dioxide. A 5-log reduction was not obtained when organisms were adhered to polyvinyl chloride (PVC). Scanning electron microscope micrographs of contaminated surfaces revealed that the PVC was very rough with pores much larger in diameter than the cells. Time values determining a 90% reduction of the E. coli population (90% killing time) were calculated for each concentration of disinfectant tested in suspension and on the steel surface. If the same experimental conditions were strictly adopted, linear functions of the log of bacterial inactivation could be plotted (log 90% killing time versus log concentration of disinfectant). This work showed that results obtained with suspension tests could not be used to estimate disinfection of hard surfaces. PMID:9709246

  18. Promotion of Homologous Recombination and Genomic Stability by RAD51AP1 via RAD51 Recombinase Enhancement

    PubMed Central

    Wiese, Claudia; Dray, Eloïse; Groesser, Torsten; Filippo, Joseph San; Shi, Idina; Collins, David W.; Tsai, Miaw-Sheue; Williams, Gareth; Rydberg, Bjorn; Sung, Patrick; Schild, David

    2007-01-01

    Summary Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds both dsDNA and a D-loop structure, and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement. PMID:17996711

  19. From compass to hard drive—integrated activities for studying magnets

    NASA Astrophysics Data System (ADS)

    Dean, J.; Allwood, D. A.

    2014-11-01

    We describe a range of practical activities that allows students to investigate the properties and applications of magnets. The activities can be used in isolation or used together to build a rounded understanding of the subject area. The activities include simple demonstrations using common or inexpensive equipment, hands-on experiments for small groups, and interactive problem solving suitable for whole classes. These can be tailored for students in either primary or secondary education.

  20. Synthesis, molecular modeling, and biological evaluation of novel RAD51 inhibitors.

    PubMed

    Zhu, Jiewen; Chen, Hongyuan; Guo, Xuning Emily; Qiu, Xiao-Long; Hu, Chun-Mei; Chamberlin, A Richard; Lee, Wen-Hwa

    2015-01-01

    RAD51 recombinase plays a critical role for cancer cell proliferation and survival. Targeting RAD51 is therefore an attractive strategy for treating difficult-to-treat cancers, e.g. triple negative breast cancers which are often resistant to existing therapeutics. To this end, we have designed, synthesized and evaluated a panel of new RAD51 inhibitors, denoted IBR compounds. Among these compounds, we have identified a novel small molecule RAD51 inhibitor, IBR120, which exhibited a 4.8-fold improved growth inhibition activity in triple negative human breast cancer cell line MBA-MD-468. IBR120 also inhibited the proliferation of a broad spectrum of other cancer cell types. Approximately 10-fold difference between the IC50 values in normal and cancer cells were observed. Moreover, IBR120 was capable of disrupting RAD51 multimerization, impairing homologous recombination repair, and inducing apoptotic cell death. Therefore, these novel RAD51 inhibitors may serve as potential candidates for the development of pharmaceutical strategies against difficult-to-treat cancers. PMID:25874343

  1. Elastically Cooperative Activated Hopping Theory of Relaxation in Viscous Liquids. I. General Formulation and Application to Hard Sphere Fluids.

    SciTech Connect

    Mirigian, Stephen; Schweizer, Kenneth

    2014-01-01

    We generalize the force-level nonlinear Langevin equation theory of single particle hopping to include collective effects associated with long range elastic distortion of the liquid. The activated alpha relaxation event is of a mixed spatial character, involving two distinct, but inter-related, local and collective barriers. There are no divergences at volume fractions below jamming or temperatures above zero Kelvin. The ideas are first developed and implemented analytically and numerically in the context of hard sphere fluids. In an intermediate volume fraction crossover regime, the local cage process is dominant in a manner consistent with an apparent Arrhenius behavior. The super-Arrhenius collective barrier is more strongly dependent on volume fraction, dominates the highly viscous regime, and is well described by a nonsingular law below jamming. The increase of the collective barrier is determined by the amplitude of thermal density fluctuations, dynamic shear modulus or transient localization length, and a growing microscopic jump length. Alpha relaxation time calculations are in good agreement with recent experiments and simulations on dense fluids and suspensions of hard spheres. Comparisons of the theory with elastic models and entropy crisis ideas are explored. The present work provides a foundation for constructing a quasi-universal, fit-parameter-free theory for relaxation in thermal molecular liquids over 14 orders of magnitude in time.

  2. Elastically cooperative activated barrier hopping theory of relaxation in viscous fluids. I. General formulation and application to hard sphere fluids

    NASA Astrophysics Data System (ADS)

    Mirigian, Stephen; Schweizer, Kenneth S.

    2014-05-01

    We generalize the force-level nonlinear Langevin equation theory of single particle hopping to include collective effects associated with long range elastic distortion of the liquid. The activated alpha relaxation event is of a mixed spatial character, involving two distinct, but inter-related, local and collective barriers. There are no divergences at volume fractions below jamming or temperatures above zero Kelvin. The ideas are first developed and implemented analytically and numerically in the context of hard sphere fluids. In an intermediate volume fraction crossover regime, the local cage process is dominant in a manner consistent with an apparent Arrhenius behavior. The super-Arrhenius collective barrier is more strongly dependent on volume fraction, dominates the highly viscous regime, and is well described by a nonsingular law below jamming. The increase of the collective barrier is determined by the amplitude of thermal density fluctuations, dynamic shear modulus or transient localization length, and a growing microscopic jump length. Alpha relaxation time calculations are in good agreement with recent experiments and simulations on dense fluids and suspensions of hard spheres. Comparisons of the theory with elastic models and entropy crisis ideas are explored. The present work provides a foundation for constructing a quasi-universal, fit-parameter-free theory for relaxation in thermal molecular liquids over 14 orders of magnitude in time.

  3. Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase

    PubMed Central

    Chen, Jianhong; Morrical, Milagros D.; Donigan, Katherine A.; Weidhaas, Joanne B.; Sweasy, Joann B.; Averill, April M.; Tomczak, Jennifer A.; Morrical, Scott W.

    2015-01-01

    Human RAD51 protein catalyzes DNA pairing and strand exchange reactions that are central to homologous recombination and homology-directed DNA repair. Successful recombination/repair requires the formation of a presynaptic filament of RAD51 on ssDNA. Mutations in BRCA2 and other proteins that control RAD51 activity are associated with human cancer. Here we describe a set of mutations associated with human breast tumors that occur in a common structural motif of RAD51. Tumor-associated D149N, R150Q and G151D mutations map to a Schellman loop motif located on the surface of the RecA homology domain of RAD51. All three variants are proficient in DNA strand exchange, but G151D is slightly more sensitive to salt than wild-type (WT). Both G151D and R150Q exhibit markedly lower catalytic efficiency for adenosine triphosphate hydrolysis compared to WT. All three mutations alter the physical properties of RAD51 nucleoprotein filaments, with G151D showing the most dramatic changes. G151D forms mixed nucleoprotein filaments with WT RAD51 that have intermediate properties compared to unmixed filaments. These findings raise the possibility that mutations in RAD51 itself may contribute to genome instability in tumor cells, either directly through changes in recombinase properties, or indirectly through changes in interactions with regulatory proteins. PMID:25539919

  4. RAD21 Mutations Cause a Human Cohesinopathy

    PubMed Central

    Deardorff, Matthew A.; Wilde, Jonathan J.; Albrecht, Melanie; Dickinson, Emma; Tennstedt, Stephanie; Braunholz, Diana; Mönnich, Maren; Yan, Yuqian; Xu, Weizhen; Gil-Rodríguez, María Concepcion; Clark, Dinah; Hakonarson, Hakon; Halbach, Sara; Michelis, Laura Daniela; Rampuria, Abhinav; Rossier, Eva; Spranger, Stephanie; Van Maldergem, Lionel; Lynch, Sally Ann; Gillessen-Kaesbach, Gabriele; Lüdecke, Hermann-Josef; Ramsay, Robert G.; McKay, Michael J.; Krantz, Ian D.; Xu, Huiling; Horsfield, Julia A.; Kaiser, Frank J.

    2012-01-01

    The evolutionarily conserved cohesin complex was originally described for its role in regulating sister-chromatid cohesion during mitosis and meiosis. Cohesin and its regulatory proteins have been implicated in several human developmental disorders, including Cornelia de Lange (CdLS) and Roberts syndromes. Here we show that human mutations in the integral cohesin structural protein RAD21 result in a congenital phenotype consistent with a “cohesinopathy.” Children with RAD21 mutations display growth retardation, minor skeletal anomalies, and facial features that overlap findings in individuals with CdLS. Notably, unlike children with mutations in NIPBL, SMC1A, or SMC3, these individuals have much milder cognitive impairment than those with classical CdLS. Mechanistically, these mutations act at the RAD21 interface with the other cohesin proteins STAG2 and SMC1A, impair cellular DNA damage response, and disrupt transcription in a zebrafish model. Our data suggest that, compared to loss-of-function mutations, dominant missense mutations result in more severe functional defects and cause worse structural and cognitive clinical findings. These results underscore the essential role of RAD21 in eukaryotes and emphasize the need for further understanding of the role of cohesin in human development. PMID:22633399

  5. Promotion of Homologous Recombination and Genomic Stability byRAD51AP1 via RAD51 Recombinase Enhancement

    SciTech Connect

    Wiese, Claudia; Dray, Eloise; Groesser, Torsten; San Filippo,Joseph; Shi, Idina; Collins, David W.; Tsai, Miaw-Sheue; Williams,Gareth; Rydberg, Bjorn; Sung, Patrick; Schild, David

    2007-04-11

    Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds dsDNA and RAD51, and it greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide the first evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

  6. RAD51B in Familial Breast Cancer

    PubMed Central

    Pelttari, Liisa M.; Khan, Sofia; Vuorela, Mikko; Kiiski, Johanna I.; Vilske, Sara; Nevanlinna, Viivi; Ranta, Salla; Schleutker, Johanna; Winqvist, Robert; Kallioniemi, Anne; Dörk, Thilo; Bogdanova, Natalia V.; Figueroa, Jonine; Pharoah, Paul D. P.; Schmidt, Marjanka K.; Dunning, Alison M.; García-Closas, Montserrat; Bolla, Manjeet K.; Dennis, Joe; Michailidou, Kyriaki; Wang, Qin; Hopper, John L.; Southey, Melissa C.; Rosenberg, Efraim H.; Fasching, Peter A.; Beckmann, Matthias W.; Peto, Julian; dos-Santos-Silva, Isabel; Sawyer, Elinor J.; Tomlinson, Ian; Burwinkel, Barbara; Surowy, Harald; Guénel, Pascal; Truong, Thérèse; Bojesen, Stig E.; Nordestgaard, Børge G.; Benitez, Javier; González-Neira, Anna; Neuhausen, Susan L.; Anton-Culver, Hoda; Brenner, Hermann; Arndt, Volker; Meindl, Alfons; Schmutzler, Rita K.; Brauch, Hiltrud; Brüning, Thomas; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Hartikainen, Jaana M.; Chenevix-Trench, Georgia; Van Dyck, Laurien; Janssen, Hilde; Chang-Claude, Jenny; Rudolph, Anja; Radice, Paolo; Peterlongo, Paolo; Hallberg, Emily; Olson, Janet E.; Giles, Graham G.; Milne, Roger L.; Haiman, Christopher A.; Schumacher, Fredrick; Simard, Jacques; Dumont, Martine; Kristensen, Vessela; Borresen-Dale, Anne-Lise; Zheng, Wei; Beeghly-Fadiel, Alicia; Grip, Mervi; Andrulis, Irene L.; Glendon, Gord; Devilee, Peter; Seynaeve, Caroline; Hooning, Maartje J.; Collée, Margriet; Cox, Angela; Cross, Simon S.; Shah, Mitul; Luben, Robert N.; Hamann, Ute; Torres, Diana; Jakubowska, Anna; Lubinski, Jan; Couch, Fergus J.; Yannoukakos, Drakoulis; Orr, Nick; Swerdlow, Anthony; Darabi, Hatef; Li, Jingmei; Czene, Kamila; Hall, Per; Easton, Douglas F.; Mattson, Johanna; Blomqvist, Carl; Aittomäki, Kristiina; Nevanlinna, Heli

    2016-01-01

    Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11–1.19, P = 8.88 x 10−16) and among familial cases (OR: 1.24, 95% CI: 1.16–1.32, P = 6.19 x 10−11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk. PMID:27149063

  7. RAD51B in Familial Breast Cancer.

    PubMed

    Pelttari, Liisa M; Khan, Sofia; Vuorela, Mikko; Kiiski, Johanna I; Vilske, Sara; Nevanlinna, Viivi; Ranta, Salla; Schleutker, Johanna; Winqvist, Robert; Kallioniemi, Anne; Dörk, Thilo; Bogdanova, Natalia V; Figueroa, Jonine; Pharoah, Paul D P; Schmidt, Marjanka K; Dunning, Alison M; García-Closas, Montserrat; Bolla, Manjeet K; Dennis, Joe; Michailidou, Kyriaki; Wang, Qin; Hopper, John L; Southey, Melissa C; Rosenberg, Efraim H; Fasching, Peter A; Beckmann, Matthias W; Peto, Julian; Dos-Santos-Silva, Isabel; Sawyer, Elinor J; Tomlinson, Ian; Burwinkel, Barbara; Surowy, Harald; Guénel, Pascal; Truong, Thérèse; Bojesen, Stig E; Nordestgaard, Børge G; Benitez, Javier; González-Neira, Anna; Neuhausen, Susan L; Anton-Culver, Hoda; Brenner, Hermann; Arndt, Volker; Meindl, Alfons; Schmutzler, Rita K; Brauch, Hiltrud; Brüning, Thomas; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Hartikainen, Jaana M; Chenevix-Trench, Georgia; Van Dyck, Laurien; Janssen, Hilde; Chang-Claude, Jenny; Rudolph, Anja; Radice, Paolo; Peterlongo, Paolo; Hallberg, Emily; Olson, Janet E; Giles, Graham G; Milne, Roger L; Haiman, Christopher A; Schumacher, Fredrick; Simard, Jacques; Dumont, Martine; Kristensen, Vessela; Borresen-Dale, Anne-Lise; Zheng, Wei; Beeghly-Fadiel, Alicia; Grip, Mervi; Andrulis, Irene L; Glendon, Gord; Devilee, Peter; Seynaeve, Caroline; Hooning, Maartje J; Collée, Margriet; Cox, Angela; Cross, Simon S; Shah, Mitul; Luben, Robert N; Hamann, Ute; Torres, Diana; Jakubowska, Anna; Lubinski, Jan; Couch, Fergus J; Yannoukakos, Drakoulis; Orr, Nick; Swerdlow, Anthony; Darabi, Hatef; Li, Jingmei; Czene, Kamila; Hall, Per; Easton, Douglas F; Mattson, Johanna; Blomqvist, Carl; Aittomäki, Kristiina; Nevanlinna, Heli

    2016-01-01

    Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11-1.19, P = 8.88 x 10-16) and among familial cases (OR: 1.24, 95% CI: 1.16-1.32, P = 6.19 x 10-11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk. PMID:27149063

  8. Constraining hot plasma in a non-flaring solar active region with FOXSI hard X-ray observations

    NASA Astrophysics Data System (ADS)

    Ishikawa, Shin-nosuke; Glesener, Lindsay; Christe, Steven; Ishibashi, Kazunori; Brooks, David H.; Williams, David R.; Shimojo, Masumi; Sako, Nobuharu; Krucker, Säm

    2014-12-01

    We present new constraints on the high-temperature emission measure of a non-flaring solar active region using observations from the recently flown Focusing Optics X-ray Solar Imager (FOXSI) sounding rocket payload. FOXSI has performed the first focused hard X-ray (HXR) observation of the Sun in its first successful flight on 2012 November 2. Focusing optics, combined with small strip detectors, enable high-sensitivity observations with respect to previous indirect imagers. This capability, along with the sensitivity of the HXR regime to high-temperature emission, offers the potential to better characterize high-temperature plasma in the corona as predicted by nanoflare heating models. We present a joint analysis of the differential emission measure (DEM) of active region 11602 using coordinated observations by FOXSI, Hinode/XRT, and Hinode/EIS. The Hinode-derived DEM predicts significant emission measure between 1 MK and 3 MK, with a peak in the DEM predicted at 2.0-2.5 MK. The combined XRT and EIS DEM also shows emission from a smaller population of plasma above 8 MK. This is contradicted by FOXSI observations that significantly constrain emission above 8 MK. This suggests that the Hinode DEM analysis has larger uncertainties at higher temperatures and that > 8 MK plasma above an emission measure of 3 × 1044 cm-3 is excluded in this active region.

  9. Characterization of the role of the RadS/RadR two-component system in the radiation resistance of Deinococcus radiodurans.

    PubMed

    Desai, Shruti S; Rajpurohit, Yogendra S; Misra, Hari S; Deobagkar, Dileep N

    2011-10-01

    Deinococcus radiodurans shows extraordinary tolerance to DNA damage, and exhibits differential gene expression and protein recycling. A putative response regulator, the DRB0091 (RadR) ORF, was identified from a pool of DNA-binding proteins induced in response to gamma radiation in this bacterium. radR is located upstream of drB0090, which encodes a putative sensor histidine kinase (RadS) on the megaplasmid. Deletion of these genes both individually and together resulted in hypersensitivity to DNA-damaging agents and a delayed or altered double-strand break repair. A ΔradRradS double mutant and a ΔradR single mutant showed nearly identical responses to gamma radiation and UVC. Wild-type RadR and RadS complemented the corresponding mutant strains, but also exhibited significant cross-complementation, albeit at lower doses of gamma radiation. The radS transcript was not detected in the ΔradR mutant, suggesting the existence of a radRS operon. Recombinant RadS was autophosphorylated and could catalyse the transfer of γ phosphate from ATP to RadR in vitro. These results indicated the functional interaction of RadS and RadR, and suggested a role for the RadS/RadR two-component system in the radiation resistance of this bacterium. PMID:21737498

  10. Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase).

    PubMed Central

    Lewis, L Kevin; Karthikeyan, G; Westmoreland, James W; Resnick, Michael A

    2002-01-01

    Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs). A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1. Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4). Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes. Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains. exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination. These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ. We suggest that the 5'-3' exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures. Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways. PMID:11805044

  11. Small-Molecule Disruption of RAD52 Rings as a Mechanism for Precision Medicine in BRCA-Deficient Cancers.

    PubMed

    Chandramouly, Gurushankar; McDevitt, Shane; Sullivan, Katherine; Kent, Tatiana; Luz, Antonio; Glickman, J Fraser; Andrake, Mark; Skorski, Tomasz; Pomerantz, Richard T

    2015-11-19

    Suppression of RAD52 causes synthetic lethality in BRCA-deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-DL-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-Dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA-deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small-molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA-deficient cancers. PMID:26548611

  12. 1500 Gate standard cell compatible radiation hard gate array

    SciTech Connect

    Mills, B.D.; Shafer, B.D.; Melancon, E.P.

    1984-11-01

    The G1500 gate array combines Sandia Labs' 4/3..mu.. CMOS silicon gate radiation hard process with a novel gate isolated standard cell compatible design for quick turnaround time, low cost, and radiation hardness. This device is hard to 5 x 10/sup 5/ rads, utilizes a configuration that provides high packing density, and is supported on both the Daisy and Mentor workstations. This paper describes Sandia Labs' radiation hard 4/3..mu.. process, the G1500's unique design, and the complete design capabilities offered by the workstations.

  13. Tel1 and Rad51 are involved in the maintenance of telomeres with capping deficiency

    PubMed Central

    Di Domenico, Enea Gino; Mattarocci, Stefano; Cimino-Reale, Graziella; Parisi, Paola; Cifani, Noemi; D’Ambrosio, Ettore; Zakian, Virginia A.; Ascenzioni, Fiorentina

    2013-01-01

    Vertebrate-like T2AG3 telomeres in tlc1-h yeast consist of short double-stranded regions and long single-stranded overhang (G-tails) and, although based on Tbf1-capping activity, they are capping deficient. Consistent with this idea, we observe Y’ amplification because of homologous recombination, even in the presence of an active telomerase. In these cells, Y’ amplification occurs by different pathways: in Tel1+ tlc1h cells, it is Rad51-dependent, whereas in the absence of Tel1, it depends on Rad50. Generation of telomeric G-tail, which is cell cycle regulated, depends on the MRX (Mre11-Rad50-Xrs2) complex in tlc1h cells or is MRX-independent in tlc1h tel1Δ mutants. Unexpectedly, we observe telomere elongation in tlc1h lacking Rad51 that seems to act as a telomerase competitor for binding to telomeric G-tails. Overall, our results show that Tel1 and Rad51 have multiple roles in the maintenance of vertebrate-like telomeres in yeast, supporting the idea that they may participate to evolutionary conserved telomere protection mechanism/s acting at uncapped telomeres. PMID:23677619

  14. Overexpression of Rad inhibits glucose uptake in cultured muscle and fat cells.

    PubMed

    Moyers, J S; Bilan, P J; Reynet, C; Kahn, C R

    1996-09-20

    Rad is a Ras-like GTPase that was isolated by subtraction cloning of human muscle and shown to have increased expression in some individuals with Type II diabetes. To ascertain the potential role of Rad in insulin-mediated signaling, we have overexpressed Rad in myocyte and adipocyte cell lines. Expression of Rad resulted in a 50-90% reduction in insulin-stimulated 2-deoxyglucose glucose uptake in C2C12 murine myotubes, L6 rat myotubes, and 3T3-L1 adipocytes and a 25% reduction in 3-O-methylglucose uptake in 3T3-L1 adipocytes. This occurred despite unaltered levels of glucose transporter expression, with no detectable change in Glut4 translocation and with no alteration in insulin receptor or substrate phosphorylation or phosphatidylinositol 3-kinase activity. These data indicate that Rad is a negative regulator of glucose uptake and that this effect may be due to a decrease in the intrinsic activity of the transporter molecules, rather than an effect on the translocation of Glut4. PMID:8798502

  15. INFRARED AND HARD X-RAY DIAGNOSTICS OF ACTIVE GALACTIC NUCLEUS IDENTIFICATION FROM THE SWIFT/BAT AND AKARI ALL-SKY SURVEYS

    SciTech Connect

    Matsuta, K.; Dotani, T.; Yamamura, I.; Gandhi, P.; Nakagawa, T.; Isobe, N.; Stawarz, L.; Ueda, Y.; Ichikawa, K.; Terashima, Y.; Oyabu, S.

    2012-07-10

    We combine data from two all-sky surveys in order to study the connection between the infrared and hard X-ray (>10 keV) properties for local active galactic nuclei (AGNs). The Swift Burst Alert Telescope all-sky survey provides an unbiased, flux-limited selection of hard X-ray-detected AGNs. Cross-correlating the 22 month hard X-ray survey with the AKARI all-sky survey, we studied 158 AGNs detected by the AKARI instruments. We find a strong correlation for most AGNs between the infrared (9, 18, and 90 {mu}m) and hard X-ray (14-195 keV) luminosities, and quantify the correlation for various subsamples of AGNs. Partial correlation analysis confirms the intrinsic correlation after removing the redshift contribution. The correlation for radio galaxies has a slope and normalization identical to that for Seyfert 1 galaxies, implying similar hard X-ray/infrared emission processes in both. In contrast, Compton-thick (CT) sources show a large deficit in the hard X-ray band, because high gas column densities diminish even their hard X-ray luminosities. We propose two photometric diagnostics for source classification: one is an X-ray luminosity versus infrared color diagram, in which type 1 radio-loud AGNs are well isolated from the others in the sample. The other uses the X-ray versus infrared color as a useful redshift-independent indicator for identifying CT AGNs. Importantly, CT AGNs and starburst galaxies in composite systems can also be differentiated in this plane based upon their hard X-ray fluxes and dust temperatures. This diagram may be useful as a new indicator to classify objects in new and upcoming surveys such as WISE and NuSTAR.

  16. 3D active edge silicon sensors with different electrode configurations: Radiation hardness and noise performance

    NASA Astrophysics Data System (ADS)

    Da Viá, C.; Bolle, E.; Einsweiler, K.; Garcia-Sciveres, M.; Hasi, J.; Kenney, C.; Linhart, V.; Parker, Sherwood; Pospisil, S.; Rohne, O.; Slavicek, T.; Watts, S.; Wermes, N.

    2009-06-01

    3D detectors, with electrodes penetrating the entire silicon wafer and active edges, were fabricated at the Stanford Nano Fabrication Facility (SNF), California, USA, with different electrode configurations. After irradiation with neutrons up to a fluence of 8.8×10 15 n eq cm -2, they were characterised using an infrared laser tuned to inject ˜2 minimum ionising particles showing signal efficiencies as high as 66% for the configuration with the shortest (56 μm) inter-electrode spacing. Sensors from the same wafer were also bump-bonded to the ATLAS FE-I3 pixel readout chip and their noise characterised. Most probable signal-to-noise ratios were calculated before and after irradiation to be as good as 38:1 after the highest irradiation level with a substrate thickness of 210 μm. These devices are promising candidates for application at the LHC such as the very forward detectors at ATLAS and CMS, the ATLAS B-Layer replacement and the general pixel upgrade. Moreover, 3D sensors could play a role in applications where high speed, high-resolution detectors are required, such as the vertex locators at the proposed Compact Linear Collider (CLIC) at CERN.

  17. Fault-Tolerant, Radiation-Hard DSP

    NASA Technical Reports Server (NTRS)

    Czajkowski, David

    2011-01-01

    Commercial digital signal processors (DSPs) for use in high-speed satellite computers are challenged by the damaging effects of space radiation, mainly single event upsets (SEUs) and single event functional interrupts (SEFIs). Innovations have been developed for mitigating the effects of SEUs and SEFIs, enabling the use of very-highspeed commercial DSPs with improved SEU tolerances. Time-triple modular redundancy (TTMR) is a method of applying traditional triple modular redundancy on a single processor, exploiting the VLIW (very long instruction word) class of parallel processors. TTMR improves SEU rates substantially. SEFIs are solved by a SEFI-hardened core circuit, external to the microprocessor. It monitors the health of the processor, and if a SEFI occurs, forces the processor to return to performance through a series of escalating events. TTMR and hardened-core solutions were developed for both DSPs and reconfigurable field-programmable gate arrays (FPGAs). This includes advancement of TTMR algorithms for DSPs and reconfigurable FPGAs, plus a rad-hard, hardened-core integrated circuit that services both the DSP and FPGA. Additionally, a combined DSP and FPGA board architecture was fully developed into a rad-hard engineering product. This technology enables use of commercial off-the-shelf (COTS) DSPs in computers for satellite and other space applications, allowing rapid deployment at a much lower cost. Traditional rad-hard space computers are very expensive and typically have long lead times. These computers are either based on traditional rad-hard processors, which have extremely low computational performance, or triple modular redundant (TMR) FPGA arrays, which suffer from power and complexity issues. Even more frustrating is that the TMR arrays of FPGAs require a fixed, external rad-hard voting element, thereby causing them to lose much of their reconfiguration capability and in some cases significant speed reduction. The benefits of COTS high

  18. Photodynamic Inactivation of Root Canal Bacteria by Light Activation through Human Dental Hard and Simulated Surrounding Tissue

    PubMed Central

    Cieplik, Fabian; Pummer, Andreas; Leibl, Christoph; Regensburger, Johannes; Schmalz, Gottfried; Buchalla, Wolfgang; Hiller, Karl-Anton; Maisch, Tim

    2016-01-01

    Introduction: Photodynamic inactivation of bacteria (PIB) may be a supportive antimicrobial approach for use in endodontics, but sufficient activation of photosensitizers (PS) in root canals is a critical point. Therefore, aim of this study was to evaluate the ability of PS absorbing blue (TMPyP) or red light (Methylene Blue; MB) for light activation through human dental hard and simulated surrounding tissue to inactivate root canal bacteria. Methods: A tooth model was fabricated with a human premolar and two molars in an acrylic resin bloc simulating the optical properties of a porcine jaw. The distal root canal of the first molar was enlarged to insert a glass tube (external diameter 2 mm) containing PS and stationary-phase Enterococcus faecalis. Both PS (10 μM) were irradiated for 120 s with BlueV (20 mW/cm2; λem = 400–460 nm) or PDT 1200L (37.8 mW/cm2; λem = 570–680 nm; both: Waldmann Medizintechnik), respectively. Irradiation parameters ensured identical numbers of photons absorbed by each PS. Three setups were chosen: irradiating the glass pipette only (G), the glass pipette inside the single tooth without (GT) and with (GTM) simulated surrounding tissues. Colony forming units (CFU) were evaluated. Transmission measurements of the buccal halves of hemisected mandibular first molars were performed by means of a photospectrometer. Results: PIB with both PS led to reduction by ≥ 5 log10 of E. faecalis CFU for each setup. From transmission measurements, a threshold wavelength λth for allowing an amount of light transmission for sufficient activation of PS was determined to be 430 nm. Conclusion: This study can be seen as proof of principle that light activation of given intra-canal PS from outside a tooth may be possible at wavelengths ≥ 430 nm, facilitating clinical application of PIB in endodontics. PMID:27379059

  19. Both the Charged Linker Region and ATPase Domain of Hsp90 Are Essential for Rad51-Dependent DNA Repair

    PubMed Central

    Suhane, Tanvi; Laskar, Shyamasree; Advani, Siddheshwari; Roy, Nabamita; Varunan, Shalu; Bhattacharyya, Dibyendu

    2014-01-01

    The inhibition of Hsp90 in cancerous cells has been correlated with the reduction in double-strand break (DSB repair) activity. However, the precise effect of Hsp90 on the DSB repair pathway in normal cells has remained enigmatic. Our results show that the Hsp82 chaperone, the ortholog of mammalian Hsp90, is indispensable for homologous-recombination (HR)-mediated DNA repair in the budding yeast Saccharomyces cerevisiae. A considerable reduction in cell viability is observed in an Hsp82-inactivated mutant upon methyl methanesulfonate (MMS) treatment as well as upon UV treatment. The loss of Hsp82 function results in a dramatic decrease in gene-targeting efficiency and a marked decrease in the endogenous levels of the key recombination proteins Rad51 and Rad52 without any notable change in the levels of RAD51 or RAD52 transcripts. Our results establish Rad51 as a client of Hsp82, since they interact physically in vivo, and also show that when Hsp82 is inhibited by 17-AAG, Rad51 undergoes proteasomal degradation. By analyzing a number of point mutants with mutations in different domains of Hsp82, we observe a strong association between the sensitivity of an ATPase mutant of Hsp82 to DNA damage and the decreases in the amounts of Rad51 and Rad52 proteins. The most significant observations include the dramatic abrogation of HR activity and the marked decrease in Rad51 focus formation in the charged linker deletion mutant of Hsp82 upon MMS treatment. The charged linker region of Hsp82 is evolutionarily conserved in all eukaryotes, but until now, no biological significance has been assigned to it. Our findings elucidate the importance of this region in DNA repair for the first time. PMID:25380755

  20. Hsp90 induces increased genomic instability toward DNA-damaging agents by tuning down RAD53 transcription.

    PubMed

    Khurana, Nidhi; Laskar, Shyamasree; Bhattacharyya, Mrinal K; Bhattacharyya, Sunanda

    2016-08-01

    It is well documented that elevated body temperature causes tumors to regress upon radiotherapy. However, how hyperthermia induces DNA damage sensitivity is not clear. We show that a transient heat shock and particularly the concomitant induction of Hsp90 lead to increased genomic instability under DNA-damaging conditions. Using Saccharomyces cerevisiae as a model eukaryote, we demonstrate that elevated levels of Hsp90 attenuate efficient DNA damage signaling and dictate preferential use of the potentially mutagenic double-strand break repair pathway. We show that under normal physiological conditions, Hsp90 negatively regulates RAD53 transcription to suppress DNA damage checkpoint activation. However, under DNA damaging conditions, RAD53 is derepressed, and the increased level of Rad53p triggers an efficient DNA damage response. A higher abundance of Hsp90 causes increased transcriptional repression on RAD53 in a dose-dependent manner, which could not be fully derepressed even in the presence of DNA damage. Accordingly, cells behave like a rad53 loss-of-function mutant and show reduced NHEJ efficiency, with a drastic failure to up-regulate RAD51 expression and manifestly faster accumulation of CLN1 and CLN2 in DNA-damaged G1, cells leading to premature release from checkpoint arrest. We further demonstrate that Rad53 overexpression is able to rescue all of the aforementioned deleterious effects caused by Hsp90 overproduction. PMID:27307581

  1. DNA strand annealing is promoted by the yeast Rad52 protein.

    PubMed Central

    Mortensen, U H; Bendixen, C; Sunjevaric, I; Rothstein, R

    1996-01-01

    The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855248

  2. Radiation-hard Active Pixel Sensors for HL-LHC Detector Upgrades based on HV-CMOS Technology

    NASA Astrophysics Data System (ADS)

    Miucci, A.; Gonella, L.; Hemperek, T.; Hügging, F.; Krüger, H.; Obermann, T.; Wermes, N.; Garcia-Sciveres, M.; Backhaus, M.; Capeans, M.; Feigl, S.; Nessi, M.; Pernegger, H.; Ristic, B.; Gonzalez-Sevilla, S.; Ferrere, D.; Iacobucci, G.; La Rosa, A.; Muenstermann, D.; George, M.; Große-Knetter, J.; Quadt, A.; Rieger, J.; Weingarten, J.; Bates, R.; Blue, A.; Buttar, C.; Hynds, D.; Kreidl, C.; Peric, I.; Breugnon, P.; Pangaud, P.; Godiot-Basolo, S.; Fougeron, D.; Bompard, F.; Clemens, J. C.; Liu, J.; Barbero, M.; Rozanov, A.; HV-CMOS Collaboration

    2014-05-01

    Luminosity upgrades are discussed for the LHC (HL-LHC) which would make updates to the detectors necessary, requiring in particular new, even more radiation-hard and granular, sensors for the inner detector region. A proposal for the next generation of inner detectors is based on HV-CMOS: a new family of silicon sensors based on commercial high-voltage CMOS technology, which enables the fabrication of part of the pixel electronics inside the silicon substrate itself. The main advantages of this technology with respect to the standard silicon sensor technology are: low material budget, fast charge collection time, high radiation tolerance, low cost and operation at room temperature. A traditional readout chip is still needed to receive and organize the data from the active sensor and to handle high-level functionality such as trigger management. HV-CMOS has been designed to be compatible with both pixel and strip readout. In this paper an overview of HV2FEI4, a HV-CMOS prototype in 180 nm AMS technology, will be given. Preliminary results after neutron and X-ray irradiation are shown.

  3. The hard X-ray luminosity function of high-redshift (3 < z ≲ 5) active galactic nuclei

    NASA Astrophysics Data System (ADS)

    Vito, F.; Gilli, R.; Vignali, C.; Comastri, A.; Brusa, M.; Cappelluti, N.; Iwasawa, K.

    2014-12-01

    We present the hard-band (2-10 keV) X-ray luminosity function (HXLF) of 0.5-2 keV band selected active galactic nuclei (AGN) at high redshift. We have assembled a sample of 141 AGN at 3 < z ≲ 5 from X-ray surveys of different size and depth, in order to sample different regions in the LX - z plane. The HXLF is fitted in the range log LX ˜ 43-45 with standard analytical evolutionary models through a maximum likelihood procedure. The evolution of the HXLF is well described by a pure density evolution, with the AGN space density declining by a factor of ˜10 from z = 3 to 5. A luminosity-dependent density evolution model, which, normally, best represents the HXLF evolution at lower redshift, is also consistent with the data, but a larger sample of low-luminosity (log LX < 44), high-redshift AGN is necessary to constrain this model. We also estimated the intrinsic fraction of AGN obscured by a column density log NH ≥ 23 to be 0.54 ± 0.05, with no strong dependence on luminosity. This fraction is higher than the value in the Local Universe, suggesting an evolution of the luminous (LX > 1044 erg s-1) obscured AGN fraction from z = 0 to z > 3.

  4. Must "Hard Problems" Be Hard?

    ERIC Educational Resources Information Center

    Kolata, Gina

    1985-01-01

    To determine how hard it is for computers to solve problems, researchers have classified groups of problems (polynomial hierarchy) according to how much time they seem to require for their solutions. A difficult and complex proof is offered which shows that a combinatorial approach (using Boolean circuits) may resolve the problem. (JN)

  5. Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae.

    PubMed Central

    Morrison, A; Miller, E J; Prakash, L

    1988-01-01

    The RAD6 gene of Saccharomyces cerevisiae, which is required for normal tolerance of DNA damage and for sporulation, encodes a 172-residue protein whose 23 carboxyl-terminal residues are almost all acidic. We show that this polyacidic sequence appends to RAD6 protein as a polyanionic tail and that its function in vivo does not require stoichiometry of length. RAD6 protein was purified to near homogeneity from a yeast strain carrying a RAD6 overproducing plasmid. Approximately the first 150 residues of RAD6 protein composed a structural domain that was resistant to proteinase K and had a Stokes radius typical of a globular protein of its calculated mass. The carboxyl-terminal polyacidic sequence was sensitive to proteinase K, and it endowed RAD6 protein with an aberrantly large Stokes radius that indicates an asymmetric shape. We deduce that RAD6 protein is monomeric and comprises a globular domain with a freely extending polyacidic tail. We tested the phenotypic effects of partial or complete deletion of the polyacidic sequence, demonstrating the presence of the shortened proteins in the cell by using antibody to RAD6 protein. Removal of the entire polyacidic sequence severely reduced sporulation but only slightly affected survival after UV irradiation or UV-induced mutagenesis. Strains with deletions of all but the first 4 or 15 residues of the polyacidic sequence were phenotypically almost wild type or wild type, respectively. We conclude that the intrinsic activity of RAD6 protein resides in the globular domain, that the polyacidic sequence has a stimulatory or modifying role evident primarily in sporulation, and that only a short section apparently functions as effectively as the entire polyacidic sequence. Images PMID:3285176

  6. The KYxxL motif in Rad17 protein is essential for the interaction with the 9-1-1 complex.

    PubMed

    Fukumoto, Yasunori; Ikeuchi, Masayoshi; Nakayama, Yuji; Yamaguchi, Naoto

    2016-09-01

    ATR-dependent DNA damage checkpoint is the major DNA damage checkpoint against UV irradiation and DNA replication stress. The Rad17-RFC and Rad9-Rad1-Hus1 (9-1-1) complexes interact with each other to contribute to ATR signaling, however, the precise regulatory mechanism of the interaction has not been established. Here, we identified a conserved sequence motif, KYxxL, in the AAA+ domain of Rad17 protein, and demonstrated that this motif is essential for the interaction with the 9-1-1 complex. We also show that UV-induced Rad17 phosphorylation is increased in the Rad17 KYxxL mutants. These data indicate that the interaction with the 9-1-1 complex is not required for Rad17 protein to be an efficient substrate for the UV-induced phosphorylation. Our data also raise the possibility that the 9-1-1 complex plays a negative regulatory role in the Rad17 phosphorylation. We also show that the nucleotide-binding activity of Rad17 is required for its nuclear localization. PMID:27387238

  7. Genetic re-engineering of Saccharomyces cerevisiae RAD51 leads to a significant increase in the frequency of gene repair in vivo

    PubMed Central

    Liu, Li; Maguire, Katie K.; Kmiec, Eric B.

    2004-01-01

    Oligonucleotides can be used to direct the alteration of single nucleotides in chromosomal genes in yeast. Rad51 protein appears to play a central role in catalyzing the reaction, most likely through its DNA pairing function. Here, we re-engineer the RAD51 gene in order to produce proteins bearing altered levels of known activities. Overexpression of wild-type ScRAD51 elevates the correction of an integrated, mutant hygromycin resistance gene ∼3-fold. Overexpression of an altered RAD51 gene, which encodes a protein that has a higher affinity for ScRad54, enhances the targeting frequency nearly 100-fold. Another mutation which increases the affinity of Rad51 for DNA was also found to increase gene repair when overexpressed in the cell. Other mutations in the Rad51 protein, such as one that reduces interaction with Rad52, has little or no effect on the frequency of gene repair. These data provide the first evidence that the Rad51 protein can be modified so as to increase the frequency of gene repair in yeast. PMID:15087488

  8. Active and passive shielding design optimization and technical solutions for deep sensitivity hard x-ray focusing telescopes

    NASA Astrophysics Data System (ADS)

    Malaguti, G.; Pareschi, G.; Ferrando, P.; Caroli, E.; Di Cocco, G.; Foschini, L.; Basso, S.; Del Sordo, S.; Fiore, F.; Bonati, A.; Lesci, G.; Poulsen, J. M.; Monzani, F.; Stevoli, A.; Negri, B.

    2005-08-01

    The 10-100 keV region of the electromagnetic spectrum contains the potential for a dramatic improvement in our understanding of a number of key problems in high energy astrophysics. A deep inspection of the universe in this band is on the other hand still lacking because of the demanding sensitivity (fraction of μCrab in the 20-40 keV for 1 Ms integration time) and imaging (≈ 15" angular resolution) requirements. The mission ideas currently being proposed are based on long focal length, grazing incidence, multi-layer optics, coupled with focal plane detectors with few hundreds μm spatial resolution capability. The required large focal lengths, ranging between 8 and 50 m, can be realized by means of extendable optical benches (as foreseen e.g. for the HEXITSAT, NEXT and NuSTAR missions) or formation flight scenarios (e.g. Simbol-X and XEUS). While the final telescope design will require a detailed trade-off analysis between all the relevant parameters (focal length, plate scale value, angular resolution, field of view, detector size, and sensitivity degradation due to detector dead area and telescope vignetting), extreme attention must be dedicated to the background minimization. In this respect, key issues are represented by the passive baffling system, which in case of large focal lengths requires particular design assessments, and by the active/passive shielding geometries and materials. In this work, the result of a study of the expected background for a hard X-ray telescope is presented, and its implication on the required sensitivity, together with the possible implementation design concepts for active and passive shielding in the framework of future satellite missions, are discussed.

  9. Rad18 is required for functional interactions between FANCD2, BRCA2, and Rad51 to repair DNA topoisomerase 1-poisons induced lesions and promote fork recovery

    PubMed Central

    Tripathi, Kaushlendra; Mani, Chinnadurai; Clark, David W; Palle, Komaraiah

    2016-01-01

    Camptothecin (CPT) and its analogues are chemotherapeutic agents that covalently and reversibly link DNA Topoisomerase I to its nicked DNA intermediate eliciting the formation of DNA double strand breaks (DSB) during replication. The repair of these DSB involves multiple DNA damage response and repair proteins. Here we demonstrate that CPT-induced DNA damage promotes functional interactions between BRCA2, FANCD2, Rad18, and Rad51 to repair the replication-associated DSB through homologous recombination (HR). Loss of any of these proteins leads to equal disruption of HR repair, causes chromosomal aberrations and sensitizes cells to CPT. Rad18 appears to function upstream in this repair pathway as its downregulation prevents activation of FANCD2, diminishes BRCA2 and Rad51 protein levels, formation of nuclear foci of all three proteins and recovery of stalled or collapsed replication forks in response to CPT. Taken together this work further elucidates the complex interplay of DNA repair proteins in the repair of replication-associated DSB. PMID:26871286

  10. Nuclear localization of Rad51B is independent of BRCA2

    SciTech Connect

    Miller, K A; Hinz, J M; Yamada, A; Thompson, L H; Albala, J S

    2005-06-28

    Human Rad51 is critical for the maintenance of genome stability through its role in the repair of DNA double-strand breaks. Rad51B (Rad51L1/hRec2) is one of the five known paralogs of human Rad51 found in a multi-protein complex with three other Rad51 paralogs, Rad51C, Rad51D and Xrcc2. Examination of EGFP-Rad51B fusion protein in HeLa S3 cells and immunofluorescence in several human cell lines confirms the nuclear localization of Rad51B. This is the first report to detail putative interactions of a Rad51 paralog protein with BRCA2. Utilization of a BRCA2 mutant cell line, CAPAN-1 suggests that Rad51B localizes to the nucleus independent of BRCA2. Although both Rad51B and BRCA2 are clearly involved in the homologous recombinational repair pathway, Rad51B and BRCA2 do not appear to associate directly. Furthermore, mutations in the KKLK motif of Rad51B, amino acid residues 4-7, mislocalizes Rad51B to the cytoplasm suggesting that this is the nuclear localization signal for the Rad51B protein. Examination of wild-type EGFP-Rad51B fusion protein in mammalian cells deficient in Rad51C showed that Rad51B localizes to the nucleus independent of Rad51C; further suggesting that Rad51B, like Rad51C, contains its own nuclear localization signal.

  11. Promotion of RAD51-Mediated Homologous DNA Pairing by the RAD51AP1-UAF1 Complex.

    PubMed

    Liang, Fengshan; Longerich, Simonne; Miller, Adam S; Tang, Caroline; Buzovetsky, Olga; Xiong, Yong; Maranon, David G; Wiese, Claudia; Kupfer, Gary M; Sung, Patrick

    2016-06-01

    The UAF1-USP1 complex deubiquitinates FANCD2 during execution of the Fanconi anemia DNA damage response pathway. As such, UAF1 depletion results in persistent FANCD2 ubiquitination and DNA damage hypersensitivity. UAF1-deficient cells are also impaired for DNA repair by homologous recombination. Herein, we show that UAF1 binds DNA and forms a dimeric complex with RAD51AP1, an accessory factor of the RAD51 recombinase, and a trimeric complex with RAD51 through RAD51AP1. Two small ubiquitin-like modifier (SUMO)-like domains in UAF1 and a SUMO-interacting motif in RAD51AP1 mediate complex formation. Importantly, UAF1 enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1 but independent of USP1. Mechanistically, RAD51AP1-UAF1 co-operates with RAD51 to assemble the synaptic complex, a critical nucleoprotein intermediate in homologous recombination, and cellular studies reveal the biological significance of the RAD51AP1-UAF1 protein complex. Our findings provide insights into an apparently USP1-independent role of UAF1 in genome maintenance. PMID:27239033

  12. Lessons learned from the Radiation measurements of the Mars Science Lab Radiation Assessment Detector (MSL-RAD)

    NASA Astrophysics Data System (ADS)

    Reitz, Guenther; Ottolenghi, Andrea

    2016-07-01

    The Radiation Assessment Detector (RAD) was designed to characterize the radiation environment on the Mars surface and to contribute to an improved assessment of radiation risk for a future human mission to Mars. The flight was chosen to cover a period of solar maximum activity to allow besides the measurement of the galactic cosmic rays an intense study of exposures by solar particle events. The Mars Science Laboratory spacecraft (MSL), containing the Curiosity rover, in which RAD was integrated, was launched to Mars on November 26, 2011. Although not part of the mission planning, RAD was operated already during the 253 day and 560 million km cruise to Mars and made the first time detailed measurements of a radiation environment comparable to that inside a future spacecraft carrying humans to Mars and in other deep space missions. Exactly 100 years after the discovery of cosmic rays on August 7, 1912 RAD makes the first observation of the radiation environment on the surface of another planet and is still gathering data until today. Meanwhile the maximum activity of the current solar cycle has been passed and the solar activity is decreasing. Unfortunately the present solar cycle was an unexpected weak cycle. As a matter of fact only very small solar particle events could be observed during the still ongoing RAD measurements. The paper highlights the achievements of RAD by presenting selected data measured during the cruise and on the Mars surface and describes its impact on predictive models for health risks of astronauts during space missions.

  13. Special features of RAD Sequencing data: implications for genotyping

    PubMed Central

    Davey, John W; Cezard, Timothée; Fuentes-Utrilla, Pablo; Eland, Cathlene; Gharbi, Karim; Blaxter, Mark L

    2013-01-01

    Restriction site-associated DNA Sequencing (RAD-Seq) is an economical and efficient method for SNP discovery and genotyping. As with other sequencing-by-synthesis methods, RAD-Seq produces stochastic count data and requires sensitive analysis to develop or genotype markers accurately. We show that there are several sources of bias specific to RAD-Seq that are not explicitly addressed by current genotyping tools, namely restriction fragment bias, restriction site heterozygosity and PCR GC content bias. We explore the performance of existing analysis tools given these biases and discuss approaches to limiting or handling biases in RAD-Seq data. While these biases need to be taken seriously, we believe RAD loci affected by them can be excluded or processed with relative ease in most cases and that most RAD loci will be accurately genotyped by existing tools. PMID:23110438

  14. Special features of RAD Sequencing data: implications for genotyping.

    PubMed

    Davey, John W; Cezard, Timothée; Fuentes-Utrilla, Pablo; Eland, Cathlene; Gharbi, Karim; Blaxter, Mark L

    2013-06-01

    Restriction site-associated DNA Sequencing (RAD-Seq) is an economical and efficient method for SNP discovery and genotyping. As with other sequencing-by-synthesis methods, RAD-Seq produces stochastic count data and requires sensitive analysis to develop or genotype markers accurately. We show that there are several sources of bias specific to RAD-Seq that are not explicitly addressed by current genotyping tools, namely restriction fragment bias, restriction site heterozygosity and PCR GC content bias. We explore the performance of existing analysis tools given these biases and discuss approaches to limiting or handling biases in RAD-Seq data. While these biases need to be taken seriously, we believe RAD loci affected by them can be excluded or processed with relative ease in most cases and that most RAD loci will be accurately genotyped by existing tools. PMID:23110438

  15. Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation.

    PubMed

    Vallerga, María Belén; Mansilla, Sabrina F; Federico, María Belén; Bertolin, Agustina P; Gottifredi, Vanesa

    2015-12-01

    After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates. PMID:26627254

  16. RadCat 2.0 User Guide.

    SciTech Connect

    Osborn, Douglas.; Weiner, Ruth F.; Mills, George Scott; Hamp, Steve C.; O'Donnell, Brandon, M.; Orcutt, David J.; Heames, Terence J.; Hinojosa, Daniel

    2005-01-01

    This document provides a detailed discussion and a guide for the use of the RadCat 2.0 Graphical User Interface input file generator for the RADTRAN 5.5 code. The differences between RadCat 2.0 and RadCat 1.0 can be attributed to the differences between RADTRAN 5 and RADTRAN 5.5 as well as clarification for some of the input parameters. 3

  17. OPTICAL SPECTRAL PROPERTIES OF SWIFT BURST ALERT TELESCOPE HARD X-RAY-SELECTED ACTIVE GALACTIC NUCLEI SOURCES

    SciTech Connect

    Winter, Lisa M.; Keeney, Brian; Lewis, Karen T.; Koss, Michael; Veilleux, Sylvain; Mushotzky, Richard F.

    2010-02-10

    The Swift Burst Alert Telescope survey of active galactic nuclei (AGNs) is providing an unprecedented view of local AGNs ((z) {approx} 0.03) and their host galaxy properties. In this paper, we present an analysis of the optical spectra of a sample of 64 AGNs from the nine month survey, detected solely based on their 14-195 keV flux. Our analysis includes both archived spectra from the Sloan Digital Sky Survey and our own observations from the 2.1 m Kitt Peak National Observatory telescope. Among our results, we include line ratio classifications utilizing standard emission line diagnostic plots, [O III] 5007 A luminosities, and Hbeta-derived black hole masses. As in our X-ray study, we find the type 2 sources to be less luminous (in [O III] 5007 A and 14-195 keV luminosities) with lower accretion rates than the type 1 sources. We find that the optically classified low-ionization narrow emission line regions, H II/composite galaxies, and ambiguous sources have the lowest luminosities, while both broad-line and narrow-line Seyferts have similar luminosities. From a comparison of the hard X-ray (14-195 keV) and [O III] luminosities, we find that both the observed and extinction-corrected [O III] luminosities are weakly correlated with X-ray luminosity. In a study of the host galaxy properties from both continuum fits and measurements of the stellar absorption indices, we find that the hosts of the narrow-line sources have properties consistent with late-type galaxies.

  18. Ancient microbial activity recorded in fracture fillings from granitic rocks (Äspö Hard Rock Laboratory, Sweden).

    PubMed

    Heim, C; Lausmaa, J; Sjövall, P; Toporski, J; Dieing, T; Simon, K; Hansen, B T; Kronz, A; Arp, G; Reitner, J; Thiel, V

    2012-07-01

    Fracture minerals within the 1.8-Ga-old Äspö Diorite (Sweden) were investigated for fossil traces of subterranean microbial activity. To track the potential organic and inorganic biosignatures, an approach combining complementary analytical techniques of high lateral resolution was applied to drill core material obtained at -450 m depth in the Äspö Hard Rock Laboratory. This approach included polarization microscopy, time-of-flight secondary ion mass spectrometry (ToF-SIMS), confocal Raman microscopy, electron microprobe (EMP) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The fracture mineral succession, consisting of fluorite and low-temperature calcite, showed a thin (20-100 μm), dark amorphous layer lining the boundary between the two phases. Microscopic investigations of the amorphous layer revealed corrosion marks and, in places, branched tubular structures within the fluorite. Geochemical analysis showed significant accumulations of Si, Al, Mg, Fe and the light rare earth elements (REE) in the amorphous layer. In the same area, ToF-SIMS imaging revealed abundant, partly functionalized organic moieties, for example, C(x)H(y)⁺, C(x)H(y)N⁺, C(x)H(y)O⁺. The presence of such functionalized organic compounds was corroborated by Raman imaging showing bands characteristic of C-C, C-N and C-O bonds. According to its organic nature and the abundance of relatively unstable N- and O- heterocompounds, the organic-rich amorphous layer is interpreted to represent the remains of a microbial biofilm that established much later than the initial cooling of the Precambrian host rock. Indeed, δ¹³C, δ¹⁸O and ⁸⁷Sr/⁸⁶Sr isotope data of the fracture minerals and the host rock point to an association with a fracture reactivation event in the most recent geological past. PMID:22506979

  19. A dominant mutation in human RAD51 reveals its function in DNA interstrand crosslink repair independent of homologous recombination

    PubMed Central

    Wang, Anderson T.; Kim, Taeho; Wagner, John E.; Conti, Brooke A.; Lach, Francis P.; Huang, Athena L.; Molina, Henrik; Sanborn, Erica M.; Zierhut, Heather; Cornes, Belinda K.; Abhyankar, Avinash; Sougnez, Carrie; Gabriel, Stacey B.; Auerbach, Arleen D.; Kowalczykowski, Stephen C.; Smogorzewska, Agata

    2015-01-01

    Summary Repair of DNA interstrand crosslinks requires action of multiple DNA repair pathways, including homologous recombination. Here, we report a de novo heterozygous T131P mutation in RAD51/FANCR, the key recombinase essential for homologous recombination, in a patient with Fanconi anemia-like phenotype. In vitro, RAD51-T131P displays DNA-independent ATPase activity, no DNA pairing capacity and a co-dominant negative effect on RAD51 recombinase function. However, the patient cells are homologous recombination proficient due to the low ratio of mutant to wildtype RAD51 in cells. Instead, patient cells are sensitive to crosslinking agents and display hyperphosphorylation of Replication Protein A due to increased activity of DNA2 and WRN at the DNA interstrand crosslinks. Thus, proper RAD51 function is important during DNA interstrand crosslink repair outside of homologous recombination. Our study provides a molecular basis for how RAD51 and its associated factors may operate in a homologous recombination-independent manner to maintain genomic integrity. PMID:26253028

  20. MR (Mre11-Rad50) complex in Giardia duodenalis: In vitro characterization and its response upon DNA damage.

    PubMed

    Sandoval-Cabrera, A; Zarzosa-Álvarez, A L; Martínez-Miguel, R M; Bermúdez-Cruz, R M

    2015-04-01

    Giardia duodenalis is a well-known protozoan parasite of humans and other mammals. The repair of DNA double strand breaks (DSBs) is crucial for genomic stability and homologous recombination is one of the primary mechanisms used by cells to repair DNA. The Mre11 complex is comprised by Mre11, an endonuclease and 3'-5' exonuclease known to resect ends during homologous recombination, and Rad50, a member of the structural maintenance of chromosomes (SMC) family of ATPases. In this work we cloned, expressed and characterized the catalytic activities of the giardial Mre11 (GdMre11) and Rad50 (GdRad50) proteins. Our results show that while purified recombinant GdMre11 and GdRad50 proteins bind DNA, GdMre11 contains a 3'-5' exonuclease and purified recombinant GdRad50 has ATPase activity. The predicted structure for GdMre11 revealed a conserved Mn(2+) dependent binding pocket. We also explored the expression of giardial mre11 and rad50 genes after ionizing radiation, and our results indicate that both specific transcripts were increased after 1-2 h while their protein levels were found to be significantly increased 4 h after gamma radiation treatment. These proteins were localized in the nuclei before and after irradiation. The implication of these observations is discussed. PMID:25662961

  1. A Dominant Mutation in Human RAD51 Reveals Its Function in DNA Interstrand Crosslink Repair Independent of Homologous Recombination.

    PubMed

    Wang, Anderson T; Kim, Taeho; Wagner, John E; Conti, Brooke A; Lach, Francis P; Huang, Athena L; Molina, Henrik; Sanborn, Erica M; Zierhut, Heather; Cornes, Belinda K; Abhyankar, Avinash; Sougnez, Carrie; Gabriel, Stacey B; Auerbach, Arleen D; Kowalczykowski, Stephen C; Smogorzewska, Agata

    2015-08-01

    Repair of DNA interstrand crosslinks requires action of multiple DNA repair pathways, including homologous recombination. Here, we report a de novo heterozygous T131P mutation in RAD51/FANCR, the key recombinase essential for homologous recombination, in a patient with Fanconi anemia-like phenotype. In vitro, RAD51-T131P displays DNA-independent ATPase activity, no DNA pairing capacity, and a co-dominant-negative effect on RAD51 recombinase function. However, the patient cells are homologous recombination proficient due to the low ratio of mutant to wild-type RAD51 in cells. Instead, patient cells are sensitive to crosslinking agents and display hyperphosphorylation of Replication Protein A due to increased activity of DNA2 and WRN at the DNA interstrand crosslinks. Thus, proper RAD51 function is important during DNA interstrand crosslink repair outside of homologous recombination. Our study provides a molecular basis for how RAD51 and its associated factors may operate in a homologous recombination-independent manner to maintain genomic integrity. PMID:26253028

  2. CSI: Hard Drive

    ERIC Educational Resources Information Center

    Sturgeon, Julie

    2008-01-01

    Acting on information from students who reported seeing a classmate looking at inappropriate material on a school computer, school officials used forensics software to plunge the depths of the PC's hard drive, searching for evidence of improper activity. Images were found in a deleted Internet Explorer cache as well as deleted file space.…

  3. Budgeting in Hard Times.

    ERIC Educational Resources Information Center

    Parrino, Frank M.

    2003-01-01

    Interviews with school board members and administrators produced a list of suggestions for balancing a budget in hard times. Among these are changing calendars and schedules to reduce heating and cooling costs; sharing personnel; rescheduling some extracurricular activities; and forming cooperative agreements with other districts. (MLF)

  4. Characterization of RAD51-Independent Break-Induced Replication That Acts Preferentially with Short Homologous Sequences

    PubMed Central

    Ira, Grzegorz; Haber, James E.

    2002-01-01

    Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase. PMID:12192038

  5. The set1Delta mutation unveils a novel signaling pathway relayed by the Rad53-dependent hyperphosphorylation of replication protein A that leads to transcriptional activation of repair genes.

    PubMed

    Schramke, V; Neecke, H; Brevet, V; Corda, Y; Lucchini, G; Longhese, M P; Gilson, E; Géli, V

    2001-07-15

    SET domain proteins are present in chromosomal proteins involved in epigenetic control of transcription. The yeast SET domain protein Set1p regulates chromatin structure, DNA repair, and telomeric functions. We investigated the mechanism by which the absence of Set1p increases DNA repair capacities of checkpoint mutants. We show that deletion of SET1 induces a response relayed by the signaling kinase Rad53p that leads to the MEC1/TEL1-independent hyperphosphorylation of replication protein A middle subunit (Rfa2p). Consequently, the binding of Rfa2p to upstream repressing sequences (URS) of repair genes is decreased, thereby leading to their derepression. Our results correlate the set1Delta-dependent phosphorylation of Rfa2p with the transcriptional induction of repair genes. Moreover, we show that the deletion of the amino-terminal region of Rfa2p suppresses the sensitivity to ultraviolet radiation of a mec3Delta checkpoint mutant, abolishes the URS-mediated repression, and increases the expression of repair genes. This work provides an additional link for the role of Rfa2p in the regulation of the repair capacity of the cell and reveals a role for the phosphorylation of Rfa2p and unveils unsuspected connections between chromatin, signaling pathways, telomeres, and DNA repair. PMID:11459833

  6. RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6.

    PubMed Central

    van Gool, A J; Verhage, R; Swagemakers, S M; van de Putte, P; Brouwer, J; Troelstra, C; Bootsma, D; Hoeijmakers, J H

    1994-01-01

    Transcription-coupled repair (TCR) is a universal sub-pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes. It accomplishes the preferential elimination of transcription-blocking DNA lesions and permits rapid resumption of the vital process of transcription. A defect in TCR is responsible for the rare hereditary disorder Cockayne syndrome (CS). Recently we found that mutations in the ERCC6 repair gene, encoding a putative helicase, underly the repair defect of CS complementation group B. Here we report the cloning and characterization of the Saccharomyces cerevisiae homolog of CSB/ERCC6, which we designate RAD26. A rad26 disruption mutant appears viable and grows normally, indicating that the gene does not have an essential function. In analogy with CS, preferential repair of UV-induced cyclobutane pyrimidine dimers in the transcribed strand of the active RBP2 gene is severely impaired. Surprisingly, in contrast to the human CS mutant, yeast RAD26 disruption does not induce any UV-, cisPt- or X-ray sensitivity, explaining why it was not isolated as a mutant before. Recovery of growth after UV exposure was somewhat delayed in rad26. These findings suggest that TCR in lower eukaryotes is not very important for cell survival and that the global genome repair pathway of NER is the major determinant of cellular resistance to genotoxicity. Images PMID:7957102

  7. RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA

    PubMed Central

    Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M. Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

    2016-01-01

    Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA. PMID:27230542

  8. RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA.

    PubMed

    Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

    2016-01-01

    Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA. PMID:27230542

  9. Fermi-LAT detection of hard spectrum gamma-ray activity from the FSRQ PKS 1532+01

    NASA Astrophysics Data System (ADS)

    Ciprini, S.; Cheung, C. C.

    2015-03-01

    The Large Area Telescope (LAT), one of the two instruments on the Fermi Gamma-ray Space Telescope, has observed increasing gamma-ray flux and an unusually hard gamma-ray spectrum from a source positionally consistent with the flat spectrum radio quasar (FSRQ) PKS 1532+01 (also known as 3FGL J1534.5+0128, Acero et al.

  10. A physiological significance of the functional interaction between Mus81 and Rad27 in homologous recombination repair

    PubMed Central

    Thu, Huong Phung Thi; Nguyen, Tuan Anh; Munashingha, Palinda Ruvan; Kwon, Buki; Dao Van, Quy; Seo, Yeon-Soo

    2015-01-01

    Fen1 and Mus81–Mms4 are endonucleases involved in the processing of various DNA structural intermediates, and they were shown to have genetic and functional interactions with each other. Here, we show the in vivo significance of the interactions between Mus81 and Rad27 (yeast Fen1). The N-terminal 120 amino-acid (aa) region of Mus81, although entirely dispensable for its catalytic activity, was essential for the abilities of Mus81 to bind to and be stimulated by Rad27. In the absence of SGS1, the mus81Δ120N mutation lacking the N-terminal 120 aa region exhibited synthetic lethality, and the lethality was rescued by deletion of RAD52, a key homologous recombination mediator. These findings, together with the fact that Sgs1 constitutes a redundant pathway with Mus81–Mms4, indicate that the N-terminus-mediated interaction of Mus81 with Rad27 is physiologically important in resolving toxic recombination intermediates. Mutagenic analyses of the N-terminal region identified two distinct motifs, named N21–26 (aa from 21–26) and N108–114 (aa from 108–114) important for the in vitro and in vivo functions of Mus81. Our findings indicate that the N-terminal region of Mus81 acts as a landing pad to interact with Rad27 and that Mus81 and Rad27 work conjointly for efficient removal of various aberrant DNA structures. PMID:25628354

  11. Reactive airways dysfunction syndrome (RADS): fact or fantasy?

    PubMed

    Bardana, E J

    1999-01-01

    Most evidence suggests that RADS is a distinct syndrome and not simply an exacerbation of pre-existing hyperresponsiveness. There is no doubt that much remains to be explained regarding pathogenesis. On the other hand, I urge caution about the claims of "low-level RADS". Here the data are much more anecdotal and leave much to be desired. PMID:10735647

  12. Can Reactive Airways Dysfunction Syndrome (RADS) be iatrogenic?

    PubMed

    Hewitt, David J

    2011-08-01

    Reactive airways dysfunction syndrome (RADS) is an asthma-like illness that develops after a single high-level exposure to a pulmonary irritant. Two different cases are reviewed, in which the exposure circumstances were not sufficient to result in adverse health effects yet resulted in persistent respiratory symptoms and a clinical diagnosis of RADS. Potential explanations for an erroneous diagnosis of RADS included an incomplete exposure assessment, medication adverse effects that can contribute to respiratory symptoms, and alternative explanations for respiratory symptoms or test findings. In particular, the empirical use of bronchodilator medications without a clear indication appeared to contribute to continued respiratory symptoms. Without a clear understanding of the patient's exposure, a RADS diagnosis should be carefully considered. The possibility of an iatrogenic sequence of events in which medication adverse effects facilitate respiratory symptoms and a mistaken RADS diagnosis should be considered, particularly in patients who have a poorly defined exposure history. PMID:21457622

  13. Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Wang, Tai-Jing; Chang, Po-Yuan; Syu, Jhan-Jhang; Chen, Jyh-Cheng; Chen, Chien-Yu; Jian, Yun-Ting; Jian, Yi-Jun; Zheng, Hao-Yu; Chen, Wen-Ching; Lin, Yun-Wei

    2015-10-01

    Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC. PMID:26212550

  14. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

    2016-04-01

    Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. PMID:26921637

  15. Enhanced non-homologous end joining contributes toward synthetic lethality of pathological RAD51C mutants with poly (ADP-ribose) polymerase.

    PubMed

    Somyajit, Kumar; Mishra, Anup; Jameei, Aida; Nagaraju, Ganesh

    2015-01-01

    Poly (ADP-ribose) polymerase 1 (PARP1) inhibitors are actively under clinical trials for the treatment of breast and ovarian cancers that arise due to mutations in BRCA1 and BRCA2. The RAD51 paralog RAD51C has been identified as a breast and ovarian cancer susceptibility gene. The pathological RAD51C mutants that were identified in cancer patients are hypomorphic with partial repair function. However, targeting cancer cells that express hypomorphic mutants of RAD51C is highly challenging. Here, we report that RAD51C-deficient cells can be targeted by a 'synthetic lethal' approach using PARP inhibitor and this sensitivity was attributed to accumulation of cells in the G2/M and chromosomal aberrations. In addition, spontaneous hyperactivation of PARP1 was evident in RAD51C-deficient cells. Interestingly, RAD51C-negative cells exhibited enhanced recruitment of non-homologous end joining (NHEJ) proteins onto chromatin and this accumulation correlated with increased activity of error-prone NHEJ as well as genome instability leading to cell death. Notably, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV rescued this phenotype. Strikingly, stimulation of NHEJ by low dose of ionizing radiation (IR) in the PARP inhibitor-treated RAD51C-deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity 'synergistically'. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a 'synergistic approach' and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other homologous recombination pathway genes. PMID:25292178

  16. The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination.

    PubMed Central

    Kooistra, R; Vreeken, K; Zonneveld, J B; de Jong, A; Eeken, J C; Osgood, C J; Buerstedde, J M; Lohman, P H; Pastink, A

    1997-01-01

    The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRADS4-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution. PMID:9315669

  17. Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation

    PubMed Central

    Bakr, A.; Oing, C.; Köcher, S.; Borgmann, K.; Dornreiter, I.; Petersen, C.; Dikomey, E.; Mansour, W.Y.

    2015-01-01

    Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3′-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR. PMID:25753674

  18. Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation.

    PubMed

    Bakr, A; Oing, C; Köcher, S; Borgmann, K; Dornreiter, I; Petersen, C; Dikomey, E; Mansour, W Y

    2015-03-31

    Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3'-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR. PMID:25753674

  19. Knockouts of RecA-like proteins RadC1 and RadC2 have distinct responses to DNA damage agents in Sulfolobus islandicus.

    PubMed

    Liang, Peng-Juan; Han, Wen-Yuan; Huang, Qi-Hong; Li, Yan-Ze; Ni, Jin-Feng; She, Qun-Xin; Shen, Yu-Long

    2013-10-20

    RecA family recombinases play essential roles in maintaining genome integrity. A group of RecA-like proteins named RadC are present in all archaea, but their in vivo functions remain unclear. In this study, we performed phylogenetic and genetic analysis of two RadC proteins from Sulfolobus islandicus. RadC is closer to the KaiC lineage of cyanobacteria and proteobacteria than to the lineage of the recombinases (RecA, RadA, and Rad51) and the recombinase paralogs (e.g., RadB, Rad55, and Rad51B). Using the recently-established S. islandicus genetic system, we constructed deletion and over-expression strains of radC1 and radC2. Deletion of radC1 rendered the cells more sensitive to DNA damaging agents, methyl methanesulfonate (MMS), hydroxyurea (HU), and ultraviolet (UV) radiation, than the wild type, and a ΔradC1ΔradC2 double deletion strain was more sensitive to cisplatin and MMS than the ΔradC1 single deletion mutant. In addition, ectopic expression of His-tagged RadC1 revealed that RadC1 was co-purified with a putative structure-specific nuclease and ATPase, which is highly conserved in archaea. Our results indicate that both RadC1 and RadC2 are involved in DNA repair. RadC1 may play a general or primary role in DNA repair, while RadC2 plays a role in DNA repair in response to specific DNA damages. PMID:24156919

  20. The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway

    SciTech Connect

    Schiestl, R.H.; Prakash, S.; Prakash, L. )

    1990-04-01

    rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, the authors have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6{Delta}) mutations and show that they also suppress the {gamma}-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of {gamma}-ray sensitivity. The six suppressor mutations they isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. They show that suppression of rad6{Delta} is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6{Delta} SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.

  1. Diphosphothreonine-specific interaction between an SQ/TQ cluster and an FHA domain in the Rad53-Dun1 kinase cascade.

    PubMed

    Lee, Hyun; Yuan, Chunhua; Hammet, Andrew; Mahajan, Anjali; Chen, Eric S-W; Wu, Ming-Ru; Su, Mei-I; Heierhorst, Jörg; Tsai, Ming-Daw

    2008-06-20

    Forkhead-associated (FHA) domains recognize phosphothreonines, and SQ/TQ cluster domains (SCDs) contain concentrated phosphorylation sites for ATM/ATR-like DNA-damage-response kinases. The Rad53-SCD1 has dual functions in regulating the activation of the Rad53-Dun1 checkpoint kinase cascade but with unknown molecular mechanisms. Here we present structural, biochemical, and genetic evidence that Dun1-FHA possesses an unprecedented diphosphothreonine-binding specificity. The Dun1-FHA has >100-fold increased affinity for diphosphorylated relative to monophosphorylated Rad53-SCD1 due to the presence of two separate phosphothreonine-binding pockets. In vivo, any single threonine of Rad53-SCD1 is sufficient for Rad53 activation and RAD53-dependent survival of DNA damage, but two adjacent phosphothreonines in the Rad53-SCD1 and two phosphothreonine-binding sites in the Dun1-FHA are necessary for Dun1 activation and DUN1-dependent transcriptional responses to DNA damage. The results uncover a phospho-counting mechanism that regulates the specificity of SCD, and provide mechanistic insight into a role of multisite phosphorylation in DNA-damage signaling. PMID:18570878

  2. The RING finger ATPase Rad5p of Saccharomyces cerevisiae contributes to DNA double-strand break repair in a ubiquitin-independent manner

    PubMed Central

    Chen, Shuhua; Davies, Adelina A.; Sagan, Daniel; Ulrich, Helle D.

    2005-01-01

    Tolerance to replication-blocking DNA lesions is achieved by means of ubiquitylation of PCNA, the processivity clamp for replicative DNA polymerases, by components of the RAD6 pathway. In the yeast Saccharomyces cerevisiae the ubiquitin ligase (E3) responsible for polyubiquitylation of the clamp is the RING finger protein Rad5p. Interestingly, the RING finger, responsible for the protein's E3 activity, is embedded in a conserved DNA-dependent ATPase domain common to helicases and chromatin remodeling factors of the SWI/SNF family. Here, we demonstrate that the Rad5p ATPase domain provides the basis for a function of the protein in DNA double-strand break repair via a RAD52- and Ku-independent pathway mediated by the Mre11/Rad50/Xrs2 protein complex. This activity is distinct and separable from the contribution of the RING domain to ubiquitin conjugation to PCNA. Moreover, we show that the Rad5 protein physically associates with the single-stranded DNA regions at a processed double-strand break in vivo. Our observations suggest that Rad5p is a multifunctional protein that—by means of independent enzymatic activities inherent in its RING and ATPase domains—plays a modulating role in the coordination of repair events and replication fork progression in response to various different types of DNA lesions. PMID:16224103

  3. Integration of imaging signs into RadLex.

    PubMed

    Shore, Matthew W; Rubin, Daniel L; Kahn, Charles E

    2012-02-01

    Imaging signs form an important part of the language of radiology, but are not represented in established lexicons. We sought to incorporate imaging signs into RSNA's RadLex® ontology of radiology terms. Names of imaging signs and their definitions were culled from books, journal articles, dictionaries, and biomedical web sites. Imaging signs were added into RadLex as subclasses of the term "imaging sign," which was defined in RadLex as a subclass of "imaging observation." A total of 743 unique imaging signs were added to RadLex with their 392 synonyms to yield a total of 1,135 new terms. All included definitions and related RadLex terms, including imaging modality, anatomy, and disorder, when appropriate. The information will allow RadLex users to identify imaging signs by modality (e.g., ultrasound signs) and to find all signs related to specific pathophysiology. The addition of imaging signs to RadLex augments its use to index the radiology literature, create and interpret clinical radiology reports, and retrieve relevant cases and images. PMID:21494902

  4. RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping.

    PubMed

    Ali, Omar A; O'Rourke, Sean M; Amish, Stephen J; Meek, Mariah H; Luikart, Gordon; Jeffres, Carson; Miller, Michael R

    2016-02-01

    Massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of DNA in many individuals is cost-prohibitive and unnecessary for many studies. Genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. Despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. Here we develop a much improved restriction site-associated DNA (RAD) sequencing protocol and a new method called Rapture ( R: AD c APTURE: ). The new RAD protocol improves versatility by separating RAD tag isolation and sequencing library preparation into two distinct steps. This protocol also recovers more unique (nonclonal) RAD fragments, which improves both standard RAD and Rapture analysis. Rapture then uses an in-solution capture of chosen RAD tags to target sequencing reads to desired loci. Rapture combines the benefits of both RAD and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. Our results demonstrate that Rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. The methods presented here should improve the efficiency of genetic analysis for many aspects of agricultural, environmental, and biomedical science. PMID:26715661

  5. Rad51 supports triple negative breast cancer metastasis

    PubMed Central

    Wiegmans, Adrian P; Al-Ejeh, Fares; Chee, Nicole; Yap, Pei-Yi; Gorski, Julia J; Silva, Leonard Da; Bolderson, Emma; Chenevix-Trench, Georgia; Anderson, Robin; Simpson, Peter T; Lakhani, Sunil R; Khanna, Kum Kum

    2014-01-01

    In contrast to extensive studies on familial breast cancer, it is currently unclear whether defects in DNA double strand break (DSB) repair genes play a role in sporadic breast cancer development and progression. We performed analysis of immunohistochemistry in an independent cohort of 235 were sporadic breast tumours. This analysis suggested that RAD51 expression is increased during breast cancer progression and metastasis and an oncogenic role for RAD51 when deregulated. Subsequent knockdown of RAD51 repressed cancer cell migration in vitro and reduced primary tumor growth in a syngeneic mouse model in vivo. Loss of RAD51 also inhibited associated metastasis not only in syngeneic mice but human xenografts and changed the metastatic gene expression profile of cancer cells, consistent with inhibition of distant metastasis. This demonstrates for the first time a new function of RAD51 that may underlie the proclivity of patients with RAD51 overexpression to develop distant metastasis. RAD51 is a potential biomarker and attractive drug target for metastatic triple negative breast cancer, with the capability to extend the survival of patients, which is less than 6 months. PMID:24811120

  6. Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells

    PubMed Central

    Lin, Ji-Fan; Lin, Yi-Chia; Yang, Shan-Che; Tsai, Te-Fu; Chen, Hung-En; Chou, Kuang-Yu; Hwang, Thomas I-Sheng

    2016-01-01

    Background Mammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited antitumor efficacy. We thus hypothesized that the use of autophagy inhibitor in combination with mTOR inhibition improves the cytotoxicity of mTOR inhibitors in bladder cancer. Materials and methods The cytotoxicity of RT4, 5637, HT1376, and T24 human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (Baf A1), chloroquine, or hydroxychloroquine) was assessed using the WST-8 cell viability kit. The autophagy status in cells was analyzed by the detection of microtubule-associated light chain 3 form II (LC3-II), using immunofluorescent staining and Western blot. Acidic vesicular organelle (AVO) formation in treated cells was determined by acridine orange vital staining. Inhibition of mTOR pathway by RAD001 was monitored by using a homemade quantitative polymerase chain reaction gene array, while phospho-mTOR was detected using Western blot. Induced apoptosis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment. Results Advanced bladder cancer cells (5637, HT1376, and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001-induced autophagy. AVO formation was detected in cells treated with RAD001 and was inhibited by the addition of 3-MA or Baf A1. Cotreatment of RAD001 with autophagy inhibitors further reduced cell viability and induced apoptosis in bladder cancer cells. Conclusion Our results indicate that

  7. BI-RADS 3 for magnetic resonance imaging.

    PubMed

    Comstock, Christopher; Sung, Janice S

    2013-08-01

    A BI-RADS (Breast Imaging Reporting and Data System) 3, or probably benign, assessment is given in approximately 7% to 12% of breast magnetic resonance (MR) images. However, the imaging features of probably benign lesions on MR imaging have not been well defined. As with mammography and ultrasonography, a BI-RADS 3 assessment should be used only when there is a less than 2% likelihood of malignancy. The use of BI-RADS 3 for classically benign findings should be avoided. Certain masses, foci, and areas of nonmass enhancement may be categorized as probably benign on baseline MR imaging. PMID:23928245

  8. RAD/COMM ''Cricket'' Test Report

    SciTech Connect

    Chiaro, P.J.

    2002-05-20

    A series of tests were performed at Oak Ridge National Laboratory (ORNL) to evaluate and characterize the radiological response of a ''Cricket'' radiation detection system. The ''Cricket'' is manufactured by RAD/COMM Systems Corp., which is located in Ontario, Canada. The system is designed to detect radioactive material that may be contained in scrap metal. The Cricket's detection unit is mounted to the base of a grappler and monitors material, while the grappler's tines hold the material. It can also be used to scan material in an attempt to isolate radioactive material if an alarm occurs. Testing was performed at the Environmental Effects Laboratory located at ORNL and operated by the Engineering Science and Technology Division. Tests performed included the following: (1) Background stability, (2) Energy response using {sup 241}Am, {sup 137}Cs, and {sup 60}Co, (3) Surface uniformity, (4) Angular dependence, (5) Alarm actuation, (6) Alarm threshold vs. background, (7) Shielding, (8) Response to {sup 235}U, (9) Response to neutrons using unmoderated {sup 252}Cf, and (10) Response to transient radiation. This report presents a summary of the test results. Background measurements were obtained prior to the performance of each individual test.

  9. RAD/COMM Cricket Test Report

    SciTech Connect

    Chiaro, PJ

    2003-08-28

    The Environmental Effects Laboratory of the Engineering Science and Technology Division of Oak Ridge National Laboratory performed a series of tests to further evaluate and characterize the radiological response of a ''Cricket'' radiation detection system. The Cricket, manufactured by Rad/Comm Systems Corporation of Ontario, Canada, is designed to detect radioactive material that may be contained in scrap metal. The Cricket's detection unit is designed to be mounted to the base of a grappler, allowing it to monitor material while the material is being held by the grappler tines. The Cricket was tested for background stability, energy response, spherical response, surface uniformity, angular dependence, and alarm actuation. Some of these tests were repeated from a prior test of a Cricket at the Environmental Effects Laboratory as reported in ORNL/TM-2002/94. Routine environmental tests--normal temperature and relatively humidity--were also performed as part of this testing process. Overall, the Cricket performed well during the testing process. The design of the instrument and the inherent photon energy of the radionuclides had some affect on portions of the tests but do not detract from the value-added benefits of the Cricket's detection capabilities.

  10. Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

    SciTech Connect

    Imura, Yoshiyuki Molho, Melissa; Chuang, Chingkai; Nagy, Peter D.

    2015-10-15

    Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.

  11. An Overview of the Reliability and Availability Data System (RADS)

    SciTech Connect

    T. E. Wierman; K. J. Kvarfordt; S. A. Eide; D. M. Rasmuson

    2005-09-01

    The Reliability and Availability Data System (RADS) is a database and analysis code, developed by the Idaho National Engineering and Environmental Laboratory (INEEL) for the U.S. Nuclear Regulatory Commission (USNRC). The code is designed to estimate industry and plant-specific reliability and availability parameters for selected components in risk-important systems and initiating events for use in risk-informed applications. The RADS tool contains data and information based on actual operating experience from U.S. commercial nuclear power plants. The data contained in RADS is kept up-to-date by loading the most current quarter's Equipment Performance and Information Exchange (EPIX) data and by yearly lods of initiating event data from licensee event reports (LERS). The reliability parameters estimated by RADS are (1) probability of failure on demand, (2) failure rate during operation (used to calculate failure to run probability) and (3) time trends in reliability parameters.

  12. The recombination mediator RAD51D promotes geminiviral infection.

    PubMed

    Richter, Kathrin S; Serra, Heϊdi; White, Charles I; Jeske, Holger

    2016-06-01

    To study a possible role for homologous recombination in geminivirus replication, we challenged Arabidopsis recombination gene knockouts by Euphorbia yellow mosaic virus infection. Our results show that the RAD51 paralog RAD51D, rather than RAD51 itself, promotes viral replication at early stages of infection. Blot hybridization analyses of replicative intermediates using one- and two-dimensional gels and deep sequencing point to an unexpected facet of recombination-dependent replication, the repair by single-strand annealing (SSA) during complementary strand replication. A significant decrease of both intramolecular, yielding defective DNAs and intermolecular recombinant molecules between the two geminiviral DNA components (A, B) were observed in the absence of RAD51D. By contrast, DNA A and B reacted differentially with the generation of inversions. A model to implicate single-strand annealing recombination in geminiviral recombination-dependent replication is proposed. PMID:27018825

  13. RADTRAN 6/RadCat 6 user guide.

    SciTech Connect

    Weiner, Ruth F.; Hinojosa, Daniel; Heames, Terence John; Farnum, Cathy Ottinger; Kalinina, Elena Arkadievna

    2013-09-01

    This document provides a detailed discussion and a guide for the use of the RadCat 6.0 Graphical User Interface input file generator for the RADTRAN code, Version 6. RadCat 6.0 integrates the newest analysis capabilities of RADTRAN 6.0, including an economic model, updated loss-of-lead shielding model, a new ingestion dose model, and unit conversion. As of this writing, the RADTRAN version in use is RADTRAN 6.02.

  14. Extracting BI-RADS Features from Portuguese Clinical Texts

    PubMed Central

    Nassif, Houssam; Cunha, Filipe; Moreira, Inês C.; Cruz-Correia, Ricardo; Sousa, Eliana; Page, David; Burnside, Elizabeth; Dutra, Inês

    2013-01-01

    In this work we build the first BI-RADS parser for Portuguese free texts, modeled after existing approaches to extract BI-RADS features from English medical records. Our concept finder uses a semantic grammar based on the BIRADS lexicon and on iterative transferred expert knowledge. We compare the performance of our algorithm to manual annotation by a specialist in mammography. Our results show that our parser’s performance is comparable to the manual method. PMID:23797461

  15. Evaluation of COTS Rad Detection Apps

    SciTech Connect

    Wagner, Eric

    2014-02-01

    Mobile applications are currently under distribution to smart phones utilizing the built-in charge coupled-device (CCD) camera as a radiation detector. The CCD detector has a very low but measurable gamma interaction cross section so the mechanism is feasible, especially for higher dose rate environments. Given that in a large release of radioactive material these ‘crowd sourced’ measurements will be put forth for consideration, a testing and evaluation of the accuracy and uncertainty of the Apps is a critical endeavor. Not only is the accuracy of the reported measurement of concern to the immediate user’s safety, a quantitative uncertainty is required for a government response such as the Federal Radiological Monitoring and Assessment Center (FRMAC) to accept the values for consideration in the determination of regions exceeding protective action guidelines. Already, prompted by the Fukushima nuclear material releases, several repositories of this crowd-sourced data have been created (http://japan.failedrobot.com, http://www.stubbytour.com/nuc/index_en.asp, and http://www.rdtn.org) although the question remains as to the reliability of measurements incorporated into these repositories. In cases of conflict between the real-time published crowd-sourced data and governmental protective actions prepared literature should be on-hand documenting why the difference, if any, exists. Four applications for iOS devices were obtained along with hardware to benchmark their performance. Gamma/X-Ray Detector by Stephan Hotto, Geiger Camera by Senscare, and RadioactivityCounter App by Hotray LTD are all the applications available for distribution within the US that utilize the CCD camera sensor for detection of radiation levels. The CellRad app under development by Idaho National Laboratory for the Android platform was evaluated. In addition, iRad Geiger with the associated hardware accessory was also benchmarked. Radiation fields were generated in a Cs-137 JL Shepherd

  16. The yeast Shu complex utilizes homologous recombination machinery for error-free lesion bypass via physical interaction with a Rad51 paralogue.

    PubMed

    Xu, Xin; Ball, Lindsay; Chen, Wangyang; Tian, Xuelei; Lambrecht, Amanda; Hanna, Michelle; Xiao, Wei

    2013-01-01

    DNA-damage tolerance (DDT) is defined as a mechanism by which eukaryotic cells resume DNA synthesis to fill the single-stranded DNA gaps left by replication-blocking lesions. Eukaryotic cells employ two different means of DDT, namely translesion DNA synthesis (TLS) and template switching, both of which are coordinately regulated through sequential ubiquitination of PCNA at the K164 residue. In the budding yeast Saccharomyces cerevisiae, the same PCNA-K164 residue can also be sumoylated, which recruits the Srs2 helicase to prevent undesired homologous recombination (HR). While the mediation of TLS by PCNA monoubiquitination has been extensively characterized, the method by which K63-linked PCNA polyubiquitination leads to template switching remains unclear. We recently identified a yeast heterotetrameric Shu complex that couples error-free DDT to HR as a critical step of template switching. Here we report that the Csm2 subunit of Shu physically interacts with Rad55, an accessory protein involved in HR. Rad55 and Rad57 are Rad51 paralogues and form a heterodimer to promote Rad51-ssDNA filament formation by antagonizing Srs2 activity. Although Rad55-Rad57 and Shu function in the same pathway and both act to inhibit Srs2 activity, Shu appears to be dedicated to error-free DDT while the Rad55-Rad57 complex is also involved in double-strand break repair. This study reveals the detailed steps of error-free lesion bypass and also brings to light an intrinsic interplay between error-free DDT and Srs2-mediated inhibition of HR. PMID:24339919

  17. Twenty Years of Rad-Hard K14 SPAD in Space Projects.

    PubMed

    Michálek, Vojtěch; Procházka, Ivan; Blažej, Josef

    2015-01-01

    During last two decades, several photon counting detectors have been developed in our laboratory. One of the most promising detector coming from our group silicon K14 Single Photon Avalanche Diode (SPAD) is presented with its valuable features and space applications. Based on the control electronics, it can be operated in both gated and non-gated mode. Although it was designed for photon counting detection, it can be employed for multiphoton detection as well. With respect to control electronics employed, the timing jitter can be as low as 20 ps RMS. Detection efficiency is about 40%in range of 500 nm to 800 nm. The detector including gating and quenching circuitry has outstanding timing stability. Due to its radiation resistivity, the diode withstands 100 krad gamma ray dose without parameters degradation. Single photon detectors based on K14 SPAD were used for planetary altimeter and atmospheric lidar in MARS92/96 and Mars Surveyor '98 space projects, respectively. Recent space applications of K14 SPAD comprises LIDAR and mainly time transfer between ground stations and artificial satellites. These include Laser Time Transfer, Time Transfer by Laser Link, and European Laser Timing projects. PMID:26213945

  18. Twenty Years of Rad-Hard K14 SPAD in Space Projects

    PubMed Central

    Michálek, Vojtěch; Procházka, Ivan; Blažej, Josef

    2015-01-01

    During last two decades, several photon counting detectors have been developed in our laboratory. One of the most promising detector coming from our group silicon K14 Single Photon Avalanche Diode (SPAD) is presented with its valuable features and space applications. Based on the control electronics, it can be operated in both gated and non-gated mode. Although it was designed for photon counting detection, it can be employed for multiphoton detection as well. With respect to control electronics employed, the timing jitter can be as low as 20 ps RMS. Detection efficiency is about 40 % in range of 500 nm to 800 nm. The detector including gating and quenching circuitry has outstanding timing stability. Due to its radiation resistivity, the diode withstands 100 krad gamma ray dose without parameters degradation. Single photon detectors based on K14 SPAD were used for planetary altimeter and atmospheric lidar in MARS92/96 and Mars Surveyor ’98 space projects, respectively. Recent space applications of K14 SPAD comprises LIDAR and mainly time transfer between ground stations and artificial satellites. These include Laser Time Transfer, Time Transfer by Laser Link, and European Laser Timing projects. PMID:26213945

  19. Role of the N-terminal forkhead-associated domain in the cell cycle checkpoint function of the Rad53 kinase.

    PubMed

    Pike, B L; Hammet, A; Heierhorst, J

    2001-04-27

    Forkhead-associated (FHA) domains are multifunctional phosphopeptide-binding modules and are the hallmark of the conserved family of Rad53-like checkpoint protein kinases. Rad53-like kinases, including the human tumor suppressor protein Chk2, play crucial roles in cell cycle arrest and activation of repair processes following DNA damage and replication blocks. Here we show that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle in G(1). This phenotype was highly specific for the Rad53-FHA1 domain and not observed with the similar Rad53-FHA2, Dun1-FHA, and Chk2-FHA domains, and it was abrogated by mutations that abolished binding to a phosphothreonine-containing peptide in vitro. Furthermore, replacement of the RAD53 gene with alleles containing amino acid substitutions in the FHA1 domain resulted in an increased DNA damage sensitivity in vivo. Taken together, these data demonstrate that the FHA1 domain contributes to the checkpoint function of Rad53, possibly by associating with a phosphorylated target protein in response to DNA damage in G(1). PMID:11278522

  20. Significance of ligand interactions involving Hop2-Mnd1 and the RAD51 and DMC1 recombinases in homologous DNA repair and XX ovarian dysgenesis.

    PubMed

    Zhao, Weixing; Sung, Patrick

    2015-04-30

    The evolutionarily conserved Hop2-Mnd1 complex is a key cofactor for the meiosis-specific recombinase Dmc1. However, emerging evidence has revealed that Hop2-Mnd1 is expressed in somatic tissues, primary human fibroblasts and cell lines, and that it functions in conjunction with the Rad51 recombinase to repair damaged telomeres via the alternate lengthening of telomeres mechanism. Here, we reveal how distinct DNA-binding activities of Hop2-Mnd1 mediate the stabilization of the RAD51-ssDNA presynaptic filament or stimulate the homologous DNA pairing reaction. We have also endeavored to define the interface that governs the assembly of the higher order complex of Hop2-Mnd1 with RAD51. Unexpectedly, we find that ATP enhances the interaction between Hop2-Mnd1 and RAD51, and that both Hop2 and Mnd1 are involved in RAD51 interaction via their C-terminal regions. Importantly, mutations introduced into these Hop2 and Mnd1 domains, including the HOP2 p.del201Glu mutation present in a patient of XX ovarian dysgenesis, diminish the association and functional synergy of Hop2-Mnd1 with both RAD51 and DMC1. Our findings help delineate the intricate manner in which Hop2-Mnd1 engages and functions with RAD51 and DMC1 in mammalian cells and speak to the possible cause of XX ovarian dysgenesis. PMID:25820426

  1. RAD51C deficiency in mice results in early prophase I arrest in males and sister chromatid separation at metaphase II in females

    PubMed Central

    Kuznetsov, Sergey; Pellegrini, Manuela; Shuda, Kristy; Fernandez-Capetillo, Oscar; Liu, Yilun; Martin, Betty K.; Burkett, Sandra; Southon, Eileen; Pati, Debananda; Tessarollo, Lino; West, Stephen C.; Donovan, Peter J.; Nussenzweig, Andre; Sharan, Shyam K.

    2007-01-01

    RAD51C is a member of the RecA/RAD51 protein family, which is known to play an important role in DNA repair by homologous recombination. In mice, it is essential for viability. Therefore, we have generated a hypomorphic allele of Rad51c in addition to a null allele. A subset of mice expressing the hypomorphic allele is infertile. This infertility is caused by sexually dimorphic defects in meiotic recombination, revealing its two distinct functions. Spermatocytes undergo a developmental arrest during the early stages of meiotic prophase I, providing evidence for the role of RAD51C in early stages of RAD51-mediated recombination. In contrast, oocytes can progress normally to metaphase I after superovulation but display precocious separation of sister chromatids, aneuploidy, and broken chromosomes at metaphase II. These defects suggest a possible late role of RAD51C in meiotic recombination. Based on the marked reduction in Holliday junction (HJ) resolution activity in Rad51c-null mouse embryonic fibroblasts, we propose that this late function may be associated with HJ resolution. PMID:17312021

  2. RadICalc. A program for estimating radiation intensity of radionuclide mixtures

    SciTech Connect

    Robinson, John W.; Dion, Michael P.; Eiden, Gregory C.; Farmer, Orville T.; Liezers, Martin

    2014-09-24

    RadICalc is a cross-platform program designed to calculate the intensity of radiation released by the decay of arbitrary isotopic mixtures. It was developed to address the need for a program that could calculate the composition, activity, and measurable radiation of a sample over time without significant effort from end-users. RadICalc uses Bateman's solutions for radioactive decay to determine activity over time. Radiation intensities are subsequently calculated using a database containing information about alpha particles, beta electrons, gamma- rays, conversion and Auger electrons, and X-rays. The user interface accepts input for isotopic mixture, initial number of atoms, and time passed since sample composition was known. Results are plotted graphically, and there is a search interface provided to and isotopes of interest. RadICalc can determine activity and radiation expected at specific masses with user-defined molecules in addition to atomic species; the latter is useful in mass based isotope separations for radiometric counting applications, a novel method under development at PNNL.

  3. Roles of Rad51 paralogs for promoting homologous recombination in Leishmania infantum

    PubMed Central

    Genois, Marie-Michelle; Plourde, Marie; Éthier, Chantal; Roy, Gaétan; Poirier, Guy G.; Ouellette, Marc; Masson, Jean-Yves

    2015-01-01

    To achieve drug resistance Leishmania parasite alters gene copy number by using its repeated sequences widely distributed through the genome. Even though homologous recombination (HR) is ascribed to maintain genome stability, this eukaryote exploits this potent mechanism driven by the Rad51 recombinase to form beneficial extrachromosomal circular amplicons. Here, we provide insights on the formation of these circular amplicons by analyzing the functions of the Rad51 paralogs. We purified three Leishmania infantum Rad51 paralogs homologs (LiRad51-3, LiRad51-4 and LiRad51-6) all of which directly interact with LiRad51. LiRad51-3, LiRad51-4 and LiRad51-6 show differences in DNA binding and annealing capacities. Moreover, it is also noteworthy that LiRad51-3 and LiRad51-4 are able to stimulate Rad51-mediated D-loop formation. In addition, we succeed to inactivate the LiRad51-4 gene and report a decrease of circular amplicons in this mutant. The LiRad51-3 gene was found to be essential for cell viability. Thus, we propose that the LiRad51 paralogs play crucial functions in extrachromosomal circular DNA amplification to circumvent drug actions and preserve survival. PMID:25712090

  4. Roles of Rad51 paralogs for promoting homologous recombination in Leishmania infantum.

    PubMed

    Genois, Marie-Michelle; Plourde, Marie; Éthier, Chantal; Roy, Gaétan; Poirier, Guy G; Ouellette, Marc; Masson, Jean-Yves

    2015-03-11

    To achieve drug resistance Leishmania parasite alters gene copy number by using its repeated sequences widely distributed through the genome. Even though homologous recombination (HR) is ascribed to maintain genome stability, this eukaryote exploits this potent mechanism driven by the Rad51 recombinase to form beneficial extrachromosomal circular amplicons. Here, we provide insights on the formation of these circular amplicons by analyzing the functions of the Rad51 paralogs. We purified three Leishmania infantum Rad51 paralogs homologs (LiRad51-3, LiRad51-4 and LiRad51-6) all of which directly interact with LiRad51. LiRad51-3, LiRad51-4 and LiRad51-6 show differences in DNA binding and annealing capacities. Moreover, it is also noteworthy that LiRad51-3 and LiRad51-4 are able to stimulate Rad51-mediated D-loop formation. In addition, we succeed to inactivate the LiRad51-4 gene and report a decrease of circular amplicons in this mutant. The LiRad51-3 gene was found to be essential for cell viability. Thus, we propose that the LiRad51 paralogs play crucial functions in extrachromosomal circular DNA amplification to circumvent drug actions and preserve survival. PMID:25712090

  5. Genetic polymorphism at codon 546 of the human RAD17 contributes to the risk for esophageal squamous cell carcinoma

    PubMed Central

    Yasuda, Yukiko; Sakai, Akiko; Ito, Sachio; Mita, Yuichiro; Sonoyama, Takayuki; Tanabe, Shunsuke; Shirakawa, Yasuhiro; Naomoto, Yoshio; Katayama, Hiroshi; Shimizu, Kenji

    2016-01-01

    Human RAD17, a human homolog of the Schizosaccharomyces pombe cell cycle checkpoint gene RAD17, plays a significant role in activating checkpoint signals in response to DNA damage. We evaluated the association of hRAD17 Leu546Arg (rs1045051), a missense single nucleotide polymorphism, with the risk of esophageal squamous cell carcinoma in relation to smoking and alcohol consumption history in 154 esophageal squamous cell carcinoma male patients and 695 cancer-free male controls by a case-control study conducted in Japan. The results showed that the hRAD17 Arg/Arg genotype compared to the Leu/Leu and Leu/Arg genotypes was significantly associated with the risk of the esophageal squamous cell carcinoma with an adjusted odds ratios of 2.22 (95% CI: 1.19-4.16 P=0.013). In stratified studies, the risk of esophageal squamous cell carcinoma was markedly higher in light drinkers (less than 23 g ethanol/day) with the Arg/Arg genotype than in heavy drinkers (excess of 23 g ethanol/day) with the Arg/Arg genotype (OR=2.83, 95% CI: 1.05-7.61, P=0.04). We concluded that the genetic variant of hRAD17 Leu546Arg polymorphism exerts a significant effect on esophageal squamous cell carcinoma risk among Japanese men. PMID:27186329

  6. Investigation of the stability of yeast rad52 mutant proteins uncovers post-translational and transcriptional regulation of Rad52p.

    PubMed Central

    Asleson, Erin N; Livingston, Dennis M

    2003-01-01

    We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations--one a deletion of amino acids 210-327 and the other a missense mutation of residue 235--increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair. PMID:12586699

  7. Expression of the denV gene of coliphage T4 in UV-sensitive rad mutants of Saccharomyces cerevisiae

    SciTech Connect

    Valerie, K.; Fronko, G.; Henderson, E.E.; de Riel, J.K.

    1986-10-01

    A plasmid containing the denV gene from bacteriophage T4, under the control of the yeast alcohol dehydrogenase I (ADC1) promoter, conferred a substantial increase in UV resistance in the UV-sensitive Saccharomyces cerevisiae mutants rad1-2 and rad3-2. The UV resistance of the denV+ yeast cells was cell cycle dependent and correlated well with the level of the denV gene product as measured by immunoblotting and by a photoreversal assay for pyrimidine dimer-DNA glycosylase activity.

  8. Preliminary On-Orbit Neutron Dose Equivalent and Energy Spectrum Results from the ISS-RAD Fast Neutron Detector (FND)

    NASA Technical Reports Server (NTRS)

    Semones, Edward; Leitgab, Martin

    2016-01-01

    The ISS-RAD instrument was activated on ISS on February 1st, 2016. Integrated in ISS-RAD, the Fast Neutron Detector (FND) performs, for the first time on ISS, routine and precise direct neutron measurements between 0.5 and 8 MeV. Preliminary results for neutron dose equivalent and neutron flux energy distributions from online/on-board algorithms and offline ground analyses will be shown, along with comparisons to simulated data and previously measured neutron spectral data. On-orbit data quality and pre-launch analysis validation results will be discussed as well.

  9. Associations of UBE2I with RAD52, UBL1, p53, and RAD51 proteins in a yeast two-hybrid system

    SciTech Connect

    Shen, Zhiyuan; Pardington-Purtymun, P.E.; Comeaux, J.C.

    1996-10-15

    The yeast RAD52-dependent pathway is involved in DNA recombination and double-strand break repair. Yeast ubiquitin-conjugating enzyme UBC9 participates in S- and M-phase cyclin degradation and mitotic control. Using the human RAD52 protein as the bait in a yeast two-hybrid system, we have identified a human homolog of yeast UBC9, designated UBE2I, that interacts with RAD52, RAD51, p53, and a ubiquitin-like protein UBL1. These interactions are UBE2I-specific, since another DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), does not interact with these proteins. The interaction of UBE2I with RAD52 is mediated by RAD52`s self-association region. These results suggest that the RAD52-dependent processes, cell cycle control, p53-mediated pathway(s), and ubiquitination interact through human UBE2I. 22 refs., 3 figs.

  10. Protein-DNA interactions in high speed AFM: single molecule diffusion analysis of human RAD54.

    PubMed

    Sanchez, Humberto; Suzuki, Yuki; Yokokawa, Masatoshi; Takeyasu, Kunio; Wyman, Claire

    2011-11-01

    High-speed AFM (atomic force microscopy also called scanning force microscopy) provides nanometre spatial resolution and sub-second temporal resolution images of individual molecules. We exploit these features to study diffusion and motor activity of the RAD54 DNA repair factor. Human RAD54 functions at critical steps in recombinational-DNA repair. It is a member of the Swi2/Snf2 family of chromatin remodelers that translocate on DNA using ATP hydrolysis. A detailed single molecular description of DNA-protein interactions shows intermediate states and distribution of variable states, usually hidden by ensemble averaging. We measured the motion of individual proteins using single-particle tracking and observed that random walks were affected by imaging-buffer composition. Non-Brownian diffusion events were characterized in the presence and in the absence of nucleotide cofactors. Double-stranded DNA immobilized on the surface functioned as a trap reducing Brownian motion. Distinct short range slides and hops on DNA were visualized by high-speed AFM. These short-range interactions were usually inaccessible by other methods based on optical resolution. RAD54 monomers displayed a diffusive behavior unrelated to the motor activity. PMID:21986699

  11. Role of Rad52 in fractionated irradiation induced signaling in A549 lung adenocarcinoma cells.

    PubMed

    Ghosh, Somnath; Krishna, Malini

    2012-01-01

    The effect of fractionated doses of γ-irradiation (2Gy per fraction over 5 days), as delivered in cancer radiotherapy, was compared with acute doses of 10 and 2Gy, in A549 cells. A549 cells were found to be relatively more radioresistant if the 10Gy dose was delivered as a fractionated regimen. Microarray analysis showed upregulation of DNA repair and cell cycle arrest genes in the cells exposed to fractionated irradiation. There was intense activation of DNA repair pathway-associated genes (DNA-PK, ATM, Rad52, MLH1 and BRCA1), efficient DNA repair and phospho-p53 was found to be translocated to the nucleus of A549 cells exposed to fractionated irradiation. MCF-7 cells responded differently in fractionated regimen. Silencing of the Rad52 gene in fractionated group of A549 cells made the cells radiosensitive. The above result indicated increased radioresistance in A549 cells due to the activation of Rad52 gene. PMID:22001234

  12. 65nm RadSafe™ Technology for RC64 and Advanced SOCs

    NASA Astrophysics Data System (ADS)

    Liran, Tuvia; Ginosar, Ran; Lange, Fredy; Mandler, Alberto; Aviely, Peleg; Meirov, Henri; Goldberg, Michael; Meister, Zeev; Oliel, Mickey

    2015-09-01

    The trend of scaling of microelectronic provides certain advantages for space components, as well as some challenges. It enables implementing highly integrated and high performance ASICs, reducing power, area and weight. Scaling also improves the immunity to TID and SEL in most cases, but increases soft error rate significantly. Ramon Chips adopted the 65nm technology for implementing RC64 [1,2], a 64 core DSP for space applications, and for making other future products. The 65nm process node is widely used, very mature, and supported by wide range of IP providers. Thus the need for full custom design of cores and IPs is minimized, and radiation hardening is achievable by mitigating the radiation effects on the available IPs, and developing proprietary IPs only for complementing the available IPs. The RadSafe_65TM technology includes hardened standard cells and I/O libraries, methods for mitigation of radiation effects in COTS IP cores (SRAM, PLL, SERDES, DDR2/3 interface) and adding unique cores for monitoring radiation effects and junction temperature. We had developed RADIC6, a technology development vehicle, for verification of all hard cores and verification of the methodologies and design flow required for RC64. RADIC6 includes the test structures for characterizing the IP cores for immunity to all radiation effects. This paper describes the main elements and IP cores of RadSafe_65TM, as well as the contents of RADIC6 test chip.

  13. Pharmacokinetics of SDZ RAD and cyclosporin including their metabolites in seven kidney graft patients after the first dose of SDZ RAD

    PubMed Central

    Kirchner, Gabriele I; Winkler, Michael; Mueller, Lueke; Vidal, Christian; Jacobsen, Wolfgang; Franzke, Anke; Wagner, Siegfried; Blick, Stefan; Manns, Michael P; Sewing, Karl-Friedrich

    2000-01-01

    Aims The aim of the study was to investigate the pharmacokinetics and metabolism of the new immunosuppressant SDZ RAD during concomitant therapy with cyclosporin in stable renal transplant patients. Furthermore, we studied the influence of SDZ RAD on the pharmacokinetics of cyclosporin at steady state levels. Methods SDZ RAD was administered orally in different doses (0.25–15 mg day−1) to seven patients, who were on standard cyclosporin-based immunosuppression. The blood concentrations of both drugs including their main groups of metabolites were measured simultaneously by LC/electrospray-mass spectrometry. Results The mean area under the blood concentration-time curve to 12 h (AUC(0,12 h)) was 4244 ± 1311 µg l−1 h for cyclosporin before SDZ RAD treatment and 4683 ± 1174 µg l−1 h (P = 0.106) on the day of SDZ RAD treatment (95% CI for difference -126, 1003). On both study days Cmax, and tmax of cyclosporin were not significantly different. The metabolite pattern of cyclosporin did not change. The pharmacokinetic data of SDZ RAD dose-normalized to 1 mg SDZ RAD were as follows: AUC(0,24 h): 35.4 ± 13.1 µg l−1 h, Cmax: 7.9 ± 2.7 µg l−1 and tmax: 1.5 ± 0.9 h. The metabolites of SDZ RAD found in blood were hydroxy-SDZ RAD, dihydroxy-SDZ RAD, demethyl-SDZ RAD, and a ring-opened form of SDZ RAD. Conclusions A single dose of SDZ RAD did not influence significantly the pharmacokinetics of cyclosporin. The most important metabolite of SDZ RAD was the hydroxy-SDZ RAD, its AUC(0,24 h) being nearly half that of the parent compound SDZ RAD. PMID:11069439

  14. Determination of radon concentration in water using RAD7 with RAD H2O accessories

    NASA Astrophysics Data System (ADS)

    Malik, M. F. I.; Rabaiee, N. A.; Jaafar, M. S.

    2015-04-01

    In the last decade, the radon issue has become one of the major problems of radiation protection. Radon exposure occurs when using water for showering, washing dishes, cooking and drinking water. RAD7 and Rad H20 accessories were used in order to measure radon concentration in water sample. In this study, four types of water were concerns which are reverse osmosis (drinking water), mineral water, tap water and well water. Reverse osmosis (drinking water) and mineral water were bought from the nearest supermarket while tap water and well water were taken from selected areas of Pulau Pinang and Kedah. Total 20 samples were taken with 5 samples for each type of water. The measured radon concentration ranged from 2.9±2.9 to 79.5±17 pCi/L, 2.9±2.9 to 67.8±16 pCi/L, 15.97±7 to 144.25±24 pCi/L and 374.89±37 to 6409.03±130 pCi/L in reverse osmosis (drinking water), mineral water, tap water and well water. Well water has the highest radon compared to others. It was due to their geological element such as granite. Results for all types of water are presented and compared with maximum contamination limit (MCL) recommended by United State Environmental Protection Agency (USEPA) which is 300pCi/L. Reverse osmosis water, mineral water and tap water were fall below MCL. However, well water was exceeded maximum level that was recommended. Thus, these findings were suggested that an action should be taken to reduce radon concentration level in well water as well as reduce a health risk towards the public.

  15. RAD in the realm of next-generation sequencing technologies.

    PubMed

    Rowe, H C; Renaut, S; Guggisberg, A

    2011-09-01

    The first North American RAD Sequencing and Genomics Symposium, sponsored by Floragenex (http://www.floragenex.com/radmeeting/), took place in Portland, Oregon (USA) on 19 April 2011. This symposium was convened to promote and discuss the use of restriction-site-associated DNA (RAD) sequencing technologies. RAD sequencing is one of several strategies recently developed to increase the power of data generated via short-read sequencing technologies by reducing their complexity (Baird et al. 2008; Huang et al. 2009; Andolfatto et al. 2011; Elshire et al. 2011). RAD sequencing, as a form of genotyping by sequencing, has been effectively applied in genetic mapping and quantitative trait loci (QTL) analyses in a range of organisms including nonmodel, genetically highly heterogeneous organisms (Table 1; Baird et al. 2008; Baxter et al. 2011; Chutimanitsakun et al. 2011; Pfender et al. 2011). RAD sequencing has recently found applications in phylogeography (Emerson et al. 2010) and population genomics (Hohenlohe et al. 2010). Considering the diversity of talks presented during this meeting, more developments are to be expected in the very near future. PMID:21991593

  16. Determining Charged Particle Flux Direction in MSL/RAD

    NASA Astrophysics Data System (ADS)

    Appel, J. K.; Kohler, J.; Guo, J.; Ehresmann, B.; Zeitlin, C. J.; Wimmer-Schweingruber, R. F.; Hassler, D.; Rafkin, S. C.; Boehm, E.; Böttcher, S. I.; Martin-Garcia, C.; Brinza, D. E.; Weigle, E.; Lohf, H.; Burmeister, S.; Reitz, G.; Matthiae, D.; Posner, A.; Martín-Torres, J.; Zorzano, M. P.

    2014-12-01

    The Radiation Assessment Detector (RAD) is an instrument onboard the Mars Science Laboratory (MSL) rover Curiosity, currently characterizing the radiation environment on the surface of Mars. The radiation entering the instrument from above consists mostly of Galactic Cosmic Rays (GCRs) modulated by the Martian atmosphere. From below, the instrument is exposed to secondary radiation produced by the interactions of the GCR with the soil. This secondary radiation gets further modulated going through the rover body before entering RAD. We developed a method of determining the direction of the charged particles measured by RAD. This method also extends the energy range possible for measurements with RAD beyond the intruments design limit. Using a combination of GEANT4 and Planetocosmics simulations, we reconstructed the expected charged particle spectra and intensities for upward and downward directed radiation which can be compared with observations. With the developed method, we are able to, for the first time, measure the upward charged particle flux with RAD both during the cruise phase and the surface science phase. Comparing the results of the simulations with the instrument data sets enables us to evaluate the simulation tools used to predict the Martian radiation envronment.

  17. The C-terminal region of Rad52 is essential for Rad52 nuclear and nucleolar localization, and accumulation at DNA damage sites immediately after irradiation

    SciTech Connect

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2013-05-31

    Highlights: •Rad52 might play a key role in the repair of DSB immediately after irradiation. •EYFP-Rad52 accumulates rapidly at DSB sites and colocalizes with Ku80. •Accumulation of Rad52 at DSB sites is independent of the core NHEJ factors. •Localization and recruitment of Rad52 to DSB sites are dependent on the Rad52 CTR. •Basic amino acids in Rad52 CTR are highly conserved among vertebrate species. -- Abstract: Rad52 plays essential roles in homologous recombination (HR) and repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae. However, in vertebrates, knockouts of the Rad52 gene show no hypersensitivity to agents that induce DSBs. Rad52 localizes in the nucleus and forms foci at a late stage following irradiation. Ku70 and Ku80, which play an essential role in nonhomologous DNA-end-joining (NHEJ), are essential for the accumulation of other core NHEJ factors, e.g., XRCC4, and a HR-related factor, e.g., BRCA1. Here, we show that the subcellular localization of EYFP-Rad52(1–418) changes dynamically during the cell cycle. In addition, EYFP-Rad52(1–418) accumulates rapidly at microirradiated sites and colocalizes with the DSB sensor protein Ku80. Moreover, the accumulation of EYFP-Rad52(1–418) at DSB sites is independent of the core NHEJ factors, i.e., Ku80 and XRCC4. Furthermore, we observed that EYFP-Rad52(1–418) localizes in nucleoli in CHO-K1 cells and XRCC4-deficient cells, but not in Ku80-deficient cells. We also found that Rad52 nuclear localization, nucleolar localization, and accumulation at DSB sites are dependent on eight amino acids (411–418) at the end of the C-terminal region of Rad52 (Rad52 CTR). Furthermore, basic amino acids on Rad52 CTR are highly conserved among mammalian, avian, and fish homologues, suggesting that Rad52 CTR is important for the regulation and function of Rad52 in vertebrates. These findings also suggest that the mechanism underlying the regulation of subcellular localization of Rad52 is

  18. Sorption of water alkalinity and hardness from high-strength wastewater on bifunctional activated carbon: process optimization, kinetics and equilibrium studies.

    PubMed

    Amosa, Mutiu K

    2016-08-01

    Sorption optimization and mechanism of hardness and alkalinity on bifunctional empty fruit bunch-based powdered activation carbon (PAC) were studied. The PAC possessed both high surface area and ion-exchange properties, and it was utilized in the treatment of biotreated palm oil mill effluent. Batch adsorption experiments designed with Design Expert(®) were conducted in correlating the singular and interactive effects of the three adsorption parameters: PAC dosage, agitation speed and contact time. The sorption trends of the two contaminants were sequentially assessed through a full factorial design with three factor interaction models and a central composite design with polynomial models of quadratic order. Analysis of variance revealed the significant factors on each design response with very high R(2) values indicating good agreement between model and experimental values. The optimum operating conditions of the two contaminants differed due to their different regions of operating interests, thus necessitating the utility of desirability factor to get consolidated optimum operation conditions. The equilibrium data for alkalinity and hardness sorption were better represented by the Langmuir isotherm, while the pseudo-second-order kinetic model described the adsorption rates and behavior better. It was concluded that chemisorption contributed majorly to the adsorption process. PMID:26752149

  19. Roles of MKK1/2-ERK1/2 and phosphoinositide 3-kinase-AKT signaling pathways in erlotinib-induced Rad51 suppression and cytotoxicity in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Ciou, Shih-Ci; Jhan, Jhih-Yuan; Cheng, Chao-Min; Su, Ying-Jhen; Chuang, Show-Mei; Lin, Szu-Ting; Chang, Chia-Che; Lin, Yun-Wei

    2009-08-01

    Erlotinib (Tarceva) is a selective epidermal growth factor receptor tyrosine kinase inhibitor in the treatment of human non-small cell lung cancer (NSCLC). In this study, we investigated the roles of ERK1/2 and AKT signaling pathways in regulating Rad51 expression and cytotoxic effects in different NSCLC cell lines treated with erlotinib. Erlotinib decreased cellular levels of phosphorylated ERK1/2, phosphorylated AKT, Rad51 protein, and mRNA in erlotinib-sensitive H1650, A549, and H1869 cells, leading to cell death via apoptosis, but these results were not seen in erlotinib-resistant H520 and H1703 cells. Erlotinib decreased Rad51 protein levels by enhancing Rad51 mRNA and protein instability. Enforced expression of constitutively active MKK1 or AKT vectors could restore Rad51 protein levels, which were inhibited by erlotinib, and decrease erlotinib-induced cytotoxicity. Knocking down endogenous Rad51 expression by si-Rad51 RNA transfection significantly enhanced erlotinib-induced cytotoxicity. In contrast, overexpression of Rad51 by transfection with Rad51 vector could protect the cells from cytotoxic effects induced by erlotinib. Blocking the activations of ERK1/2 and AKT by MKK1/2 inhibitor (U0126) and phosphoinositide 3-kinase inhibitor (wortmannin) suppressed the expression of Rad51 and enhanced the erlotinib-induced cell death in erlotinib-resistant cells. In conclusion, suppression of Rad51 may be a novel therapeutic modality in overcoming drug resistance of erlotinib in NSCLC. PMID:19671683

  20. RAD18 mediates resistance to ionizing radiation in human glioma cells

    SciTech Connect

    Xie, Chen; Wang, Hongwei; Cheng, Hongbin; Li, Jianhua; Wang, Zhi Yue, Wu

    2014-02-28

    Highlights: • RAD18 is an important mediator of the IR-induced resistance in glioma cell lines. • RAD18 overexpression confers resistance to IR-mediated apoptosis. • The elevated expression of RAD18 is associated with recurrent GBM who underwent IR therapy. - Abstract: Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). RAD18 a central regulator of translesion DNA synthesis (TLS), has been shown to play an important role in regulating genomic stability and DNA damage response. In the present study, we investigate the relationship between RAD18 and resistance to ionizing radiation (IR) and examined the expression levels of RAD18 in primary and recurrent GBM specimens. Our results showed that RAD18 is an important mediator of the IR-induced resistance in GBM. The expression level of RAD18 in glioma cells correlates with their resistance to IR. Ectopic expression of RAD18 in RAD18-low A172 glioma cells confers significant resistance to IR treatment. Conversely, depletion of endogenous RAD18 in RAD18-high glioma cells sensitized these cells to IR treatment. Moreover, RAD18 overexpression confers resistance to IR-mediated apoptosis in RAD18-low A172 glioma cells, whereas cells deficient in RAD18 exhibit increased apoptosis induced by IR. Furthermore, knockdown of RAD18 in RAD18-high glioma cells disrupts HR-mediated repair, resulting in increased accumulation of DSB. In addition, clinical data indicated that RAD18 was significantly higher in recurrent GBM samples that were exposed to IR compared with the corresponding primary GBM samples. Collectively, our findings reveal that RAD18 may serve as a key mediator of the IR response and may function as a potential target for circumventing IR resistance in human GBM.

  1. Hard-metal (WC-Co) particles trigger a signaling cascade involving p38 MAPK, HIF-1α, HMOX1, and p53 activation in human PBMC.

    PubMed

    Lombaert, Noömi; Castrucci, Eleonora; Decordier, Ilse; Van Hummelen, Paul; Kirsch-Volders, Micheline; Cundari, Enrico; Lison, Dominique

    2013-02-01

    Hard-metals are made of tungsten carbide (WC) and metallic cobalt (Co) particles and are important industrial materials produced for their extreme hardness and high wear resistance properties. While occupational exposure to metallic Co alone is apparently not associated with an increased risk of cancer, the WC-Co particle mixture was shown to increase the risk of lung cancer in exposed workers. We have previously shown that WC-Co specifically induces a burst of reactive oxygen species (ROS) and in vitro mutagenic/apoptogenic effects in human peripheral blood mononucleated cells (PBMC) used as a validated experimental model. In the present study, PBMCs were treated during a short period (15 min) to focus on the very rapid ROS burst induced by WC-Co. We investigated by microarray the response to WC-Co versus Co(2+) ions (CoCl(2)) after 15 min exposure and found that the oxidative stress response HMOX1 gene was highly expressed in WC-Co-treated samples. This result was confirmed by qRT-PCR, and western blotting was carried out to analyze translational and post-translational regulation of genes belonging to the HMOX1 pathway. We show here that WC-Co, and metallic Co particles although with slower kinetics, but not CoCl(2) or WC alone, induced a temporally ordered cascade of events. This cascade implies p38/MAP kinase activation, HIF-1α stabilization, HMOX1 transcriptional activation, and ATM-independent p53 stabilization. These events, and in particular HIF-1α stabilization, could contribute to the carcinogenic activity of WC-Co dusts. PMID:23052192

  2. Cosmic ray dose monitoring using RadFET sensors of the Rosetta instruments SESAME and COSIMA

    NASA Astrophysics Data System (ADS)

    Falke, Peter; Fischer, Hans-Herbert; Seidensticker, Klaus J.; Thiel, Klaus; Fischer, Henning; Hilchenbach, Martin; Henkel, Hartmut; Koch, Andreas

    2016-08-01

    On its more than 10 years journey to comet 67P/Churyumov-Gerasimenko, Rosetta carried RadFET ionising dose monitors in the central electronics of the orbiter instrument COSIMA and the lander instrument SESAME. The readings of the dosimeters were corrected for the temperature of the devices during measurements. Because the sensitivity of RadFETs depends on the energy of impinging charged particles, a mean efficiency factor for the prevalent proton radiation was determined by applying nine efficiency models to proton energy spectra of Rosetta's radiation environment. The resulting dose profiles show linear increases of the accumulated dose with time, mainly caused by galactic cosmic radiation, and the arrival of two solar particle events in 2005. The accumulated dose (in Silicon) during 3909 days in space from 2004-03-02 to 2014-11-14 was 3.2 ± 0.6 Gy in case of COSIMA and 1.9 ± 0.4 Gy for SESAME. The deviation of the two measurements is mainly due to the solar particle event in September 2005, which had a 5.3 ± 1.0 times stronger impact on the COSIMA RadFET. Measured dose levels are one order of magnitude lower than those expected before launch for not being exceeded on the 90% confidence level, which is mainly due to the low solar activity during the mission so far.

  3. Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1β production.

    PubMed

    Roth, Susanne; Rottach, Andrea; Lotz-Havla, Amelie S; Laux, Verena; Muschaweckh, Andreas; Gersting, Søren W; Muntau, Ania C; Hopfner, Karl-Peter; Jin, Lei; Vanness, Katelynd; Petrini, John H J; Drexler, Ingo; Leonhardt, Heinrich; Ruland, Jürgen

    2014-06-01

    Double-stranded DNA (dsDNA) in the cytoplasm triggers the production of interleukin 1β (IL-1β) as an antiviral host response, and deregulation of the pathways involved can promote inflammatory disease. Here we report a direct cytosolic interaction between the DNA-damage sensor Rad50 and the innate immune system adaptor CARD9. Transfection of dendritic cells with dsDNA or infection of dendritic cells with a DNA virus induced the formation of dsDNA-Rad50-CARD9 signaling complexes for activation of the transcription factor NF-κB and the generation of pro-IL-1β. Primary cells conditionally deficient in Rad50 or lacking CARD9 consequently exhibited defective DNA-induced production of IL-1β, and Card9(-/-) mice had impaired inflammatory responses after infection with a DNA virus in vivo. Our results define a cytosolic DNA-recognition pathway for inflammation and a physical and functional connection between a conserved DNA-damage sensor and the innate immune response to pathogens. PMID:24777530

  4. Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

    PubMed

    Tsabar, Michael; Waterman, David P; Aguilar, Fiona; Katsnelson, Lizabeth; Eapen, Vinay V; Memisoglu, Gonen; Haber, James E

    2016-05-15

    To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair. PMID:27222517

  5. Electronic radon monitoring with the CMOS System-on-Chip AlphaRad

    NASA Astrophysics Data System (ADS)

    Higueret, S.; Husson, D.; Le, T. D.; Nourreddine, A.; Michielsen, N.

    2008-01-01

    The development of the integrated circuit AlphaRad as a new System-on-Chip for detection of α-particles has already been reported. This paper deals with electronic monitoring of atmospheric radon, which is one of the promising applications of the chip. The future electronic radon monitor (ERM) is designed to be compact, inexpensive, operating at low voltage and fully stand-alone. We present here the complete electronic board of the future ERM: it is made of three independent AlphaRad chips running in parallel, mounted on a small printed-circuit board which includes a numeric block for data treatment based on a Xilinx programmable gate array. The maximal counting rate of the AlphaRad chip has been pushed to at least 3×10 6 α-particles cm -2. The complete system for detection of the solid aerosols will be published separately, and this paper will focus on the electronic board alone. Already 20 times faster than our first measurement with a CMOS pixel sensor, the system was tested at low and high activities, showing an excellent linearity for 222Rn levels up to 80 kBq m -3.

  6. ATM/ATR-mediated phosphorylation of PALB2 promotes RAD51 function.

    PubMed

    Ahlskog, Johanna K; Larsen, Brian D; Achanta, Kavya; Sørensen, Claus S

    2016-05-01

    DNA damage activates the ATM and ATR kinases that coordinate checkpoint and DNA repair pathways. An essential step in homology-directed repair (HDR) of DNA breaks is the formation of RAD51 nucleofilaments mediated by PALB2-BRCA2; however, roles of ATM and ATR in this critical step of HDR are poorly understood. Here, we show that PALB2 is markedly phosphorylated in response to genotoxic stresses such as ionizing radiation and hydroxyurea. This response is mediated by the ATM and ATR kinases through three N-terminal S/Q-sites in PALB2, the consensus target sites for ATM and ATR Importantly, a phospho-deficient PALB2 mutant is unable to support proper RAD51 foci formation, a key PALB2 regulated repair event, whereas a phospho-mimicking PALB2 version supports RAD51 foci formation. Moreover, phospho-deficient PALB2 is less potent in HDR than wild-type PALB2. Further, this mutation reveals a separation in PALB2 function, as the PALB2-dependent checkpoint response is normal in cells expressing the phospho-deficient PALB2 mutant. Collectively, our findings highlight a critical importance of PALB2 phosphorylation as a novel regulatory step in genome maintenance after genotoxic stress. PMID:27113759

  7. Application of RAD-BCG calculator to Hanford's 300 area shoreline characterization dataset

    SciTech Connect

    Antonio, Ernest J.; Poston, Ted M.; Tiller, Brett L.; Patton, Gene W.

    2003-07-01

    Abstract. In 2001, a multi-agency study was conducted to characterize potential environmental effects from radiological and chemical contaminants on the near-shore environment of the Columbia River at the 300 Area of the U.S. Department of Energy’s Hanford Site. Historically, the 300 Area was the location of nuclear fuel fabrication and was the main location for research and development activities from the 1940s until the late 1980s. During past waste handling practices uranium, copper, and other heavy metals were routed to liquid waste streams and ponds near the Columbia River shoreline. The Washington State Department of Health and the Pacific Northwest National Laboratory’s Surface Environmental Surveillance Project sampled various environmental components including river water, riverbank spring water, sediment, fishes, crustaceans, bivalve mollusks, aquatic insects, riparian vegetation, small mammals, and terrestrial invertebrates for analyses of radiological and chemical constituents. The radiological analysis results for water and sediment were used as initial input into the RAD-BCG Calculator. The RAD-BCG Calculator, a computer program that uses an Excel® spreadsheet and Visual Basic® software, showed that maximum radionuclide concentrations measured in water and sediment were lower than the initial screening criteria for concentrations to produce dose rates at existing or proposed limits. Radionuclide concentrations measured in biota samples were used to calculate site-specific bioaccumulation coefficients (Biv) to test the utility of the RAD-BCG-Calculator’s site-specific screening phase. To further evaluate site-specific effects, the default Relative Biological Effect (RBE) for internal alpha particle emissions was reduced by half and the program’s kinetic/allometric calculation approach was initiated. The subsequent calculations showed the initial RAD-BCG Calculator results to be conservative, which is appropriate for screening purposes.

  8. Radiation Measurements during the Cruise to and on the Surface of Mars with MSL/RAD

    NASA Astrophysics Data System (ADS)

    Ehresmann, Bent

    The Radiation Assessment Detector (RAD) on board the Mars Science Laboratory's (MSL) Curiosity rover is the first ever instrument to measure the energetic particle radiation on the surface of Mars. The main scientific goal of the RAD instrument is to characterize the radiation environment on the surface of Mars by making detailed measurements of the radiation dose, linear energy transfer (LET) spectra, and charged and neutral particle spectra. In addition to the surface measurements, RAD was also operating for large parts of the 253-day cruise to Mars. Combined, these measurements give unique insight to the expected radiation exposure for a potential manned mission to the red planet. The average absorbed tissue-equivalent dose rate during the cruise was found to be 0.48 ± 0.08 mGy/day, while for the first 300 days of surface operations the measured dose was 0.21 ± 0.04 mGy/day. By measuring the LET spectrum the absorbed dose can be converted into the biologically-relevant dose equivalent, resulting in values of 1.84 ± 0.30 mSv/day during cruise and 0.64 ± 0.12 mSv/day on the surface of Mars. RAD further encountered several solar energetic particle (SEP) events during cruise and surface operations. SEP events can create significant enhancements of the radiation exposure on short time scales, depending on the magnitude of such an event. For example, the five SEP events encountered during cruise contributed about five percent to the total measured dose equivalent in 253 days. The occurrence rate of SEP events strongly depends on the state of the solar activity, emphasizing the importance of continued radiation measurements throughout the solar cycle.

  9. The Saccharomyces cerevisiae Mre11-Rad50-Xrs2 complex promotes trinucleotide repeat expansions independently of homologous recombination.

    PubMed

    Ye, Yanfang; Kirkham-McCarthy, Lucy; Lahue, Robert S

    2016-07-01

    Trinucleotide repeats (TNRs) are tandem arrays of three nucleotides that can expand in length to cause at least 17 inherited human diseases. Somatic expansions in patients can occur in differentiated tissues where DNA replication is limited and cannot be a primary source of somatic mutation. Instead, mouse models of TNR diseases have shown that both inherited and somatic expansions can be suppressed by the loss of certain DNA repair factors. It is generally believed that these repair factors cause misprocessing of TNRs, leading to expansions. Here we extend this idea to show that the Mre11-Rad50-Xrs2 (MRX) complex of Saccharomyces cerevisiae is a causative factor in expansions of short TNRs. Mutations that eliminate MRX subunits led to significant suppression of expansions whereas mutations that inactivate Rad51 had only a minor effect. Coupled with previous evidence, this suggests that MRX drives expansions of short TNRs through a process distinct from homologous recombination. The nuclease function of Mre11 was dispensable for expansions, suggesting that expansions do not occur by Mre11-dependent nucleolytic processing of the TNR. Epistasis between MRX and post-replication repair (PRR) was tested. PRR protects against expansions, so a rad5 mutant gave a high expansion rate. In contrast, the mre11 rad5 double mutant gave a suppressed expansion rate, indistinguishable from the mre11 single mutant. This suggests that MRX creates a TNR substrate for PRR. Protein acetylation was also tested as a mechanism regulating MRX activity in expansions. Six acetylation sites were identified in Rad50. Mutation of all six lysine residues to arginine gave partial bypass of a sin3 HDAC mutant, suggesting that Rad50 acetylation is functionally important for Sin3-mediated expansions. Overall we conclude that yeast MRX helps drive expansions of short TNRs by a mechanism distinct from its role in homologous recombination and independent of the nuclease function of Mre11. PMID:27173583

  10. Rad54B targeting to DNA double-strand break repair sites requires complex formation with S100A11.

    PubMed

    Murzik, Ulrike; Hemmerich, Peter; Weidtkamp-Peters, Stefanie; Ulbricht, Tobias; Bussen, Wendy; Hentschel, Julia; von Eggeling, Ferdinand; Melle, Christian

    2008-07-01

    S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21(WAF1/CIP1) was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. PMID:18463164

  11. Rad54B Targeting to DNA Double-Strand Break Repair Sites Requires Complex Formation with S100A11

    PubMed Central

    Murzik, Ulrike; Hemmerich, Peter; Weidtkamp-Peters, Stefanie; Ulbricht, Tobias; Bussen, Wendy; Hentschel, Julia; von Eggeling, Ferdinand

    2008-01-01

    S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21WAF1/CIP1 was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. PMID:18463164

  12. RadCat 3.0 user guide.

    SciTech Connect

    Hinojosa, Daniel; Penisten, Janelle J.; Dennis, Matthew L.; Osborn, Douglas M.; Weiner, Ruth F.; Heames, Terence John; Marincel, Michelle K.

    2009-05-01

    RADTRAN is an internationally accepted program and code for calculating the risks of transporting radioactive materials. The first versions of the program, RADTRAN I and II, were developed for NUREG-0170 (USNRC, 1977), the first environmental statement on transportation of radioactive materials. RADTRAN and its associated software have undergone a number of improvements and advances consistent with improvements in both available data and computer technology. The version of RADTRAN currently bundled with RadCat is RADTRAN 6.0. This document provides a detailed discussion and a guide for the use of the RadCat 3.0 Graphical User Interface input file generator for the RADTRAN code. RadCat 3.0 integrates the newest analysis capabilities of RADTRAN 6.0 which includes an economic model, updated loss-of-lead shielding model, and unit conversion. As of this writing, the RADTRAN version in use is RADTRAN 6.0.

  13. UV induced ubiquitination of the yeast Rad4-Rad23 complex promotes survival by regulating cellular dNTP pools.

    PubMed

    Zhou, Zheng; Humphryes, Neil; van Eijk, Patrick; Waters, Raymond; Yu, Shirong; Kraehenbuehl, Rolf; Hartsuiker, Edgar; Reed, Simon H

    2015-09-01

    Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4-Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes including DUN1, inhibiting their expression. UV radiation promotes the loss of occupancy of the Rad4-Rad23 complex from the regulatory regions of these genes, enabling their induction and thereby controlling the production of dNTPs. We demonstrate that this regulatory mechanism, which is dependent on the ubiquitination of Rad4 by the GG-NER E3 ligase, promotes UV survival in yeast cells. These results support an unanticipated regulatory mechanism that integrates ubiquitination of NER DNA repair factors with the regulation of the transcriptional response controlling dNTP production and cellular survival after UV damage. PMID:26150418

  14. Influence of light guide tip used in the photo-activation on degree of conversion and hardness of one nanofilled dental composite

    NASA Astrophysics Data System (ADS)

    Galvão, M. R.; Costa, S. X. S.; Victorino, K. R.; Ribeiro, A. A.; Menezes, F. C. H.; Rastelli, A. N. S.; Bagnato, V. S.; Andrade, M. F.

    2010-12-01

    The aim of this study was to evaluate the degree of conversion and hardness of a dental composite resin Filtek™ Z-350 (3M ESPE, Dental Products St. Paul, MN) photo-activated for 20 s of irradiation time with two different light guide tips, metal and polymer, coupled on blue LED Ultraled LCU (Dabi Atlante, SP, Brazil). With the metal light tip, power density was of 352 and with the polymer was of 456 mW/cm2, respectively. Five samples (4 mm in diameter and 2mm in thickness—ISO 4049), were made for each Group evaluated. The measurements for DC (%) were made in a Nexus-470 FT-IR, Thermo Nicolet, E.U.A. Spectroscopy (FTIR). Spectra for both uncured and cured samples were analyzed using an accessory of reflectance diffuse. The measurements were recorded in absorbance operating under the following conditions: 32 scans, 4 cm-1 resolution, 300-4000 cm-1 wavelength. The percentage of unreacted carbon double bonds (% C=C) was determined from the ratio of absorbance intensities of aliphatic C=C (peak at 1637 cm-1) against internal standard before and after curing of the sample: aromatic C-C (peak at 1610 cm-1). The Vickers hardness measurements (top and bottom surfaces) were performed in a universal testing machine (Buehler MMT-3 digital microhardness tester Lake Bluff, Illinois USA). A 50 gf load was used and the indenter with a dwell time of 30 s. The data were submitted to the test t Student at significance level of 5%. The mean values of degree of conversion for the polymer and metal light guide tip no were statistically different ( p = 0.8389). The hardness mean values were no statistically significant different among the light guide tips ( p = 0.6244), however, there was difference between top and bottom surfaces ( p < 0.001). The results show that so much the polymer light tip as the metal light tip can be used for the photo-activation, probably for the low quality of the light guide tip metal.

  15. RadMonitor: radiology operations data mining in real time.

    PubMed

    Chen, Richard; Mongkolwat, Pattanasak; Channin, David S

    2008-09-01

    This paper describes the web-based visualization interface of RadMonitor, a platform-independent web application designed to help manage the complexity of information flow within a health care enterprise. The system eavesdrops on Health Layer 7 traffic and parses statistical operational information into a database. The information is then presented to the user as a treemap--a graphical visualization scheme that simplifies the display of hierarchical information. While RadMonitor has been implemented for the purpose of analyzing radiology operations, its XML backend allows it to be reused for virtually any other hierarchical data set. PMID:17534683

  16. First Results from the ISS-RAD Charged Particle Detector

    NASA Technical Reports Server (NTRS)

    Semones, Edward; Zeitland, Cary

    2016-01-01

    The Charged Particle Detector (CPD) subsystem of the ISS-RAD detector has been making measurements of high-energy charged and neutral particles since the unit was deployed on Feb. 1, 2016. The CPD is nearly identical to the MSL-RAD instrument, but onboard data processing has been significantly modified to meet ISS requirements. We will present dose rates and LET spectra obtained over the first six months of operations, as well as preliminary results obtained from the limited sample of pulse-height analyzed raw data that has been telemetered to Earth.

  17. Remote Sensing and GIS Integrated Approach for Assessment of Hydrogeoenvironment of a Hard-rock Terrain associated with an Active Fault System

    NASA Astrophysics Data System (ADS)

    Singh, A.; Mukherjee, S.

    2013-12-01

    Though faulted zones exhibit a crucial role in regulation of flow and accumulation of water, studying the way hydrological environment of any landscape is shaped by them can be very difficult. While aquifer recharge can be influenced by geological as well as structural changes, surface flow may be influenced depending upon the topography. This study aimed at characterization of surface as well as subsurface hydrological conditions in a hard-rock terrain, primarily under the influence of tensional type of faulting, through a remote sensing approach. The area selected lies in vicinity of an active fault, with quartzitic rocks showing signs of multiple folding. For surface water bodies hydrological analysis on elevation data (DEM) was performed whereas for subsurface recharge, margins of geological units were targeted using satellite images (both hyperspectral and multispectral). In total 30 tiles of multi-sensor multi-temporal satellite data (LANDSAT, HYPERION, ASTER and CARTOSAT) was used in this study. A conglomeration of different techniques was used to study patterns of water storage and flow in order to effectively assess the influence of structural lineaments (extent and connectivity), recharge zones and eventually the overall availability of water resource in the study area. The information was verified and supplemented with field geophysical (resistivity) surveys at suitable strategic locations and laboratory sample analysis. Spatial analysis for microzonation was also performed using the GIS database which yielded 14 sites with highly pronounced role of faulting activity in regulating connectivity between multiple aquifers and surface water bodies. 10 different subsurface recharge conditions were eventually identified for the study area. The resulting GIS database is also to be used for modeling, water resource management and vulnerability studies in near future. Similar strategy may also be applied elsewhere to assess and manage water resources in any hard

  18. Evolution of the optical and hard X-ray activity of AM Her in a season dominated by the high states

    NASA Astrophysics Data System (ADS)

    Šimon, Vojtěch

    2016-07-01

    The long-term activity of AM Her, the prototype of polars, consists of interchanging high and low states. This analysis uses the data from the BAT/ Swift, MAXI/ ISS, and AAVSO data archives (including long CCD V-band night series) for investigating the relation of the time evolution of intensities in the hard X-ray, medium/hard X-ray, and the optical bands on super-orbital timescale in a season dominated by the long high-state episodes. The observations mapped the relation of the cyclotron and bremsstrahlung luminosities in such episodes. Although an increase of intensity of the cyclotron emission is generally accompanied by a brightening of the bremsstrahlung component, this relation differs for the individual high-state episodes. These variations were accompanied by the large changes of the optical modulation. In my view, all of these variations suggest variations of the dimensions and structure of the cyclotron emitting region(s), not only the changes of the mass accretion rate. The activity during a decline (much less steep than the state transition) from an initial very bright peak can be explained by a gradual evolution of the conditions in stratified shock regions in a single high state episode. The observed behavior in the neighboring high-state episodes is also important for explaining the mechanisms which cause the low state between them. In the interpretation, some low-state episodes cause that AM Her develops a different accretion mode than the one before entering the low state. Each high-state episode is a complex phenomenon in the history of accretion in this system.

  19. Binding of nickel and copper to fish gills predicts toxicity when water hardness varies, but free-ion activity does not

    SciTech Connect

    Meyer, J.S.; Bobbitt, J.P.; Debrey, L.D.; Boese, C.J.; Bergman, H.L.; Santore, R.C.; Paquin, P.R.; Ditoro, D.M.; Allen, H.E.

    1999-03-15

    Based on a biotic-ligand model (BLM), the authors hypothesized that the concentration of a transition metal bound to fish gills ([M{sub gill}]) will be a constant predictor of mortality, whereas a free-ion activity model is generally interpreted to imply that the chemical activity of the aquo (free) ion of the metal will be a constant predictor of mortality. In laboratory tests, measured [Ni{sub gill}] and calculated [Cu{sub gill}] were constant predictors of acute toxicity of Ni and Cu to fathead minnows (Pimephales promelas) when water hardness varied up to 10-fold, whereas total aqueous concentrations and free-ion activities of Ni and Cu were not. Thus, the BLM, which simultaneously accounts for (a) metal speciation in the exposure water and (b) competitive binding of transition-metal ions and other cations to biotic ligands predicts acute toxicity better than does free-ion activity of Ni or Cu. Adopting a biotic-ligand modeling approach could help establish a more defensible, mechanistic basis for regulating aqueous discharges of metals.

  20. Digital radiology using active matrix readout of amorphous selenium: radiation hardness of cadmium selenide thin film transistors.

    PubMed

    Zhao, W; Waechter, D; Rowlands, J A

    1998-04-01

    A flat-panel x-ray imaging detector using active matrix readout of amorphous selenium (a-Se) is being investigated for digital radiography and fluoroscopy. The active matrix consists of a two-dimensional array of thin film transistors (TFTs). Radiation penetrating through the a-Se layer will interact with the TFTs and it is important to ensure that radiation induced changes will not affect the operation of the x-ray imaging detector. The methodology of the present work is to investigate the effects of radiation on the characteristic curves of the TFTs using individual TFT samples made with cadmium selenide (CdSe) semiconductor. Four characteristic parameters, i.e., threshold voltage, subthreshold swing, field effect mobility, and leakage current, were examined. This choice of parameters was based on the well established radiation damage mechanisms for crystalline silicon metal-oxide-semiconductor field-effect transistors (MOSFETs), which have a similar principle of operation as CdSe TFTs. It was found that radiation had no measurable effect on the leakage current and the field effect mobility. However, radiation shifted the threshold voltage and increased the subthreshold swing. But even the estimated lifetime dose (50 Gy) of a diagnostic radiation detector will not affect the normal operation of an active matrix x-ray detector made with CdSe TFTs. The mechanisms of the effects of radiation will be discussed and compared with those for MOSFETs and hydrogenated amorphous silicon (a-Si:H) TFTs. PMID:9571621

  1. Wear of hard materials by hard particles

    SciTech Connect

    Hawk, Jeffrey A.

    2003-10-01

    Hard materials, such as WC-Co, boron carbide, titanium diboride and composite carbide made up of Mo2C and WC, have been tested in abrasion and erosion conditions. These hard materials showed negligible wear in abrasion against SiC particles and erosion using Al2O3 particles. The WC-Co materials have the highest wear rate of these hard materials and a very different material removal mechanism. Wear mechanisms for these materials were different for each material with the overall wear rate controlled by binder composition and content and material grain size.

  2. Genetic and physical interaction of Ssp1 CaMKK and Rad24 14-3-3 during low pH and osmotic stress in fission yeast

    PubMed Central

    Freitag, Silja I.; Wong, Jimson; Young, Paul G.

    2014-01-01

    The Ssp1 calmodulin kinase kinase (CaMKK) is necessary for stress-induced re-organization of the actin cytoskeleton and initiation of growth at the new cell end following division in Schizosaccharomyces pombe. In addition, it regulates AMP-activated kinase and functions in low glucose tolerance. ssp1− cells undergo mitotic delay at elevated temperatures and G2 arrest in the presence of additional stressors. Following hyperosmotic stress, Ssp1-GFP forms transient foci which accumulate at the cell membrane and form a band around the cell circumference, but not co-localizing with actin patches. Hyperosmolarity-induced localization to the cell membrane occurs concomitantly with a reduction of its interaction with the 14-3-3 protein Rad24, but not Rad25 which remains bound to Ssp1. The loss of rad24 in ssp1− cells reduces the severity of hyperosmotic stress response and relieves mitotic delay. Conversely, overexpression of rad24 exacerbates stress response and concomitant cell elongation. rad24− does not impair stress-induced localization of Ssp1 to the cell membrane, however this response is almost completely absent in cells overexpressing rad24. PMID:24451546

  3. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens

    PubMed Central

    Suchan, Tomasz; Pitteloud, Camille; Gerasimova, Nadezhda S.; Kostikova, Anna; Schmid, Sarah; Arrigo, Nils; Pajkovic, Mila; Ronikier, Michał; Alvarez, Nadir

    2016-01-01

    In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes

  4. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens.

    PubMed

    Suchan, Tomasz; Pitteloud, Camille; Gerasimova, Nadezhda S; Kostikova, Anna; Schmid, Sarah; Arrigo, Nils; Pajkovic, Mila; Ronikier, Michał; Alvarez, Nadir

    2016-01-01

    In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes

  5. Curcumin enhances the mitomycin C-induced cytotoxicity via downregulation of MKK1/2-ERK1/2-mediated Rad51 expression in non-small cell lung cancer cells

    SciTech Connect

    Ko, Jen-Chung; Tsai, Min-Shao; Weng, Shao-Hsing; Kuo, Ya-Hsun; Chiu, Yu-Fan; Lin, Yun-Wei

    2011-09-15

    Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC) cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2-ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC. - Highlights: > Curcumin downregulates MKK-ERK-mediated Rad51 expression. > Curcumin enhances mitomycin C-induced cytotoxicity. > Rad51 protects cells from cytotoxic effects induced by curcumin and mitomycin C. > Rad51 inhibition enhances the chemosensitization of mitomycin C by

  6. Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

    PubMed Central

    Symington, Lorraine S.

    2002-01-01

    The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination. PMID:12456786

  7. Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant

    SciTech Connect

    Chen, Jianhong; Villanueva, Nicolas; Rould, Mark A.; Morrical, Scott W.

    2010-09-03

    Rad51 protein promotes homologous recombination in eukaryotes. Recombination activities are activated by Rad51 filament assembly on ssDNA. Previous studies of yeast Rad51 showed that His352 occupies an important position at the filament interface, where it could relay signals between subunits and active sites. To investigate, we characterized yeast Rad51 H352A and H352Y mutants, and solved the structure of H352Y. H352A forms catalytically competent but salt-labile complexes on ssDNA. In contrast, H352Y forms salt-resistant complexes on ssDNA, but is defective in nucleotide exchange, RPA displacement and strand exchange with full-length DNA substrates. The 2.5 {angstrom} crystal structure of H352Y reveals a right-handed helical filament in a high-pitch (130 {angstrom}) conformation with P61 symmetry. The catalytic core and dimer interface regions of H352Y closely resemble those of DNA-bound Escherichia coli RecA protein. The H352Y mutation stabilizes Phe187 from the adjacent subunit in a position that interferes with the {gamma}-phosphate-binding site of the Walker A motif/P-loop, potentially explaining the limited catalysis observed. Comparison of Rad51 H352Y, RecA-DNA and related structures reveals that the presence of bound DNA correlates with the isomerization of a conserved cis peptide near Walker B to the trans configuration, which appears to prime the catalytic glutamate residue for ATP hydrolysis.

  8. Appliation of rad-sequencing to linkage mapping in citrus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High density linkage maps can be developed for modest cost using high-throughput DNA sequencing to genotype a defined fraction (representation) of the genome. We developed linkage maps in two citrus populations using the RAD (Restriction site Associated DNA) genotyping method which involves restrict...

  9. Structural and functional evidence that Rad4 competes with Rad2 for binding to the Tfb1 subunit of TFIIH in NER

    PubMed Central

    Lafrance-Vanasse, Julien; Arseneault, Geneviève; Cappadocia, Laurent; Legault, Pascale; Omichinski, James G.

    2013-01-01

    XPC/Rad4 (human/yeast) recruits transcription faction IIH (TFIIH) to the nucleotide excision repair (NER) complex through interactions with its p62/Tfb1 and XPB/Ssl2 subunits. TFIIH then recruits XPG/Rad2 through interactions with similar subunits and the two repair factors appear to be mutually exclusive within the NER complex. Here, we show that Rad4 binds the PH domain of the Tfb1 (Tfb1PH) with high affinity. Structural characterization of a Rad4–Tfb1PH complex demonstrates that the Rad4-binding interface is formed using a motif similar to one used by Rad2 to bind Tfb1PH. In vivo studies in yeast demonstrate that the N-terminal Tfb1-binding motif and C-terminal TFIIH-binding motif of Rad4 are both crucial for survival following exposure to UV irradiation. Together, these results support the hypothesis that XPG/Rad2 displaces XPC/Rad4 from the repair complex in part through interactions with the Tfb1/p62 subunit of TFIIH. The Rad4–Tfb1PH structure also provides detailed information regarding, not only the interplay of TFIIH recruitment to the NER, but also links the role of TFIIH in NER and transcription. PMID:23295669

  10. Structural and functional evidence that Rad4 competes with Rad2 for binding to the Tfb1 subunit of TFIIH in NER.

    PubMed

    Lafrance-Vanasse, Julien; Arseneault, Geneviève; Cappadocia, Laurent; Legault, Pascale; Omichinski, James G

    2013-02-01

    XPC/Rad4 (human/yeast) recruits transcription faction IIH (TFIIH) to the nucleotide excision repair (NER) complex through interactions with its p62/Tfb1 and XPB/Ssl2 subunits. TFIIH then recruits XPG/Rad2 through interactions with similar subunits and the two repair factors appear to be mutually exclusive within the NER complex. Here, we show that Rad4 binds the PH domain of the Tfb1 (Tfb1PH) with high affinity. Structural characterization of a Rad4-Tfb1PH complex demonstrates that the Rad4-binding interface is formed using a motif similar to one used by Rad2 to bind Tfb1PH. In vivo studies in yeast demonstrate that the N-terminal Tfb1-binding motif and C-terminal TFIIH-binding motif of Rad4 are both crucial for survival following exposure to UV irradiation. Together, these results support the hypothesis that XPG/Rad2 displaces XPC/Rad4 from the repair complex in part through interactions with the Tfb1/p62 subunit of TFIIH. The Rad4-Tfb1PH structure also provides detailed information regarding, not only the interplay of TFIIH recruitment to the NER, but also links the role of TFIIH in NER and transcription. PMID:23295669

  11. A novel mode of nuclease action is revealed by the bacterial Mre11/Rad50 complex

    PubMed Central

    Lim, Chew Theng; Lai, Pey Jiun; Leach, David R. F.; Maki, Hisaji; Furukohri, Asako

    2015-01-01

    The Mre11/Rad50 complex is a central player in various genome maintenance pathways. Here, we report a novel mode of nuclease action found for the Escherichia coli Mre11/Rad50 complex, SbcC2/D2 complex (SbcCD). SbcCD cuts off the top of a cruciform DNA by making incisions on both strands and continues cleaving the dsDNA stem at ∼10-bp intervals. Using linear-shaped DNA substrates, we observed that SbcCD cleaved dsDNA using this activity when the substrate was 110 bp long, but that on shorter substrates the cutting pattern was changed to that predicted for the activity of a 3′-5′ exonuclease. Our results suggest that SbcCD processes hairpin and linear dsDNA ends with this novel DNA end-dependent binary endonuclease activity in response to substrate length rather than using previously reported activities. We propose a model for this mode of nuclease action, which provides new insight into SbcCD activity at a dsDNA end. PMID:26319016

  12. Nuclear localization of Rad52 is pre-requisite for its sumoylation

    SciTech Connect

    Ohuchi, Takashi; Seki, Masayuki Enomoto, Takemi

    2008-07-18

    In Saccharomyces cerevisiae, Rad52 plays major roles in several types of homologous recombination. Here, we found that rad52-K200R mutation greatly reduced sumoylation of Rad52. The rad52-K200R mutant exhibited defects in various types of recombination, such as intrachromosomal recombination and mating-type switching. The K200 residue of Rad52 is part of the nuclear localization signal (NLS), which is important for transport into the nucleus. Indeed, the addition of a SV40 NLS to Rad52-K200R suppressed the sumoylation defect of Rad52-K200R. These findings indicate that nuclear localization of Rad52 is pre-requisite for its sumoylation.

  13. Dysfunction of human Rad18 results in defective postreplication repair and hypersensitivity to multiple mutagens.

    PubMed

    Tateishi, S; Sakuraba, Y; Masuyama, S; Inoue, H; Yamaizumi, M

    2000-07-01

    Postreplication repair functions in gap-filling of a daughter strand on replication of damaged DNA. The yeast Saccharomyces cerevisiae Rad18 protein plays a pivotal role in the process together with the Rad6 protein. Here, we have cloned a human homologue of RAD18, hRAD18. It maps on chromosome 3p24-25, where deletions are often found in lung, breast, ovary, and testis cancers. In vivo, hRad18 protein binds to hHR6 protein through a conserved ring-finger motif. Stable transformants with hRad18 mutated in this motif become sensitive to UV, methyl methanesulfonate, and mitomycin C, and are defective in the replication of UV-damaged DNA. Thus, hRAD18 is a functional homologue of RAD18. PMID:10884424

  14. p53 modulates homologous recombination by transcriptional regulation of the RAD51 gene

    PubMed Central

    Arias-Lopez, Carmen; Lazaro-Trueba, Iciar; Kerr, Peter; Lord, Christopher J; Dexter, Tim; Iravani, Marjan; Ashworth, Alan; Silva, Augusto

    2006-01-01

    DNA repair by homologous recombination is involved in maintaining genome stability. Previous data report that wild-type p53 suppresses homologous recombination and physically interacts with Rad51. Here, we show the in vivo binding of wild-type p53 to a p53 response element in the promoter of Rad51 and the downregulation of Rad51 messenger RNA and protein by wild-type p53, favoured by DNA damage. Moreover, wild-type p53 inhibits Rad51 foci formation in response to double-strand breaks, whereas p53 contact mutant R280K fails to repress Rad51 mRNA and protein expression and Rad51 foci formation. We propose that transcriptional repression of Rad51 by p53 participates in regulating homologous recombination, and impaired Rad51 repression by p53 mutants may contribute to malignant transformation. PMID:16322760

  15. Enhanced cellular activities of polycaprolactone/alginate-based cell-laden hierarchical scaffolds for hard tissue engineering applications.

    PubMed

    Lee, HyeongJin; Kim, GeunHyung

    2014-09-15

    Biomedical scaffolds have been widely investigated because they are essential for support and promotion of cell adhesion, proliferation and differentiation in three-dimensional (3D) structures. An ideal scaffold should be highly porous to enable efficient nutrient and oxygen transfer and have a 3D structure that provides optimal micro-environmental conditions for the seeded cells to obtain homogeneous growth after a long culture period. In this study, new hierarchical osteoblast-like cell (MG-63)-laden scaffolds consisting of micro-sized struts/inter-layered micro-nanofibres and cell-laden hydrogel struts with mechanically stable and biologically superior properties were introduced. Poly(ethylene oxide) (PEO) was used as a sacrificial component to generate pores within the cell-laden hydrogel struts to attain a homogeneous cell distribution and rapid cell growth in the scaffold interior. The alginate-based cell-laden struts with PEO induced fast/homogeneous cell release, in contrast to nonporous cell-laden struts. Various weight fractions (0.5, 1, 2, 3 and 3.5 wt%) of PEO were used, of which 2 wt% PEO in the cell-laden strut resulted in the most appropriate cell release and enhanced biological activities (cell proliferation and calcium deposition), compared to nonporous cell-laden struts. PMID:24974244

  16. Top3-Rmi1 dissolve Rad51-mediated D-loops by a topoisomerase-based mechanism

    PubMed Central

    Fasching, Clare L.; Cejka, Petr; Kowalczykowski, Stephen C.; Heyer, Wolf-Dietrich

    2015-01-01

    Summary The displacement loop (D-loop) is the DNA strand invasion product formed during homologous recombination. Disruption of nascent D-loops represents a mechanism of anti-recombination. During Synthesis-Dependent Strand Annealing D-loop disruption after extension of the invading strand is an integral step of the pathway and ensures a non-crossover outcome. The proteins implicated in D-loop disruption are DNA motor proteins/helicases acting by migrating DNA junctions. Here we report an unanticipated mechanism of D-loop dissolution mediated by DNA topoisomerase 3 (Top3) and dependent on its catalytic activity. D-loop dissolution catalyzed by yeast Top3 is highly specific for yeast Rad51/Rad54-mediated D-loops, whereas protein-free D-loops or D-loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also the human Topoisomerase IIIα-RMI1–RMI2 complex is capable of dissolving D-loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is in part a result of unprocessed D-loops. PMID:25699708

  17. Sds22 participates in Glc7 mediated Rad53 dephosphorylation in MMS-induced DNA damage in Candida albicans.

    PubMed

    Yao, Guangyin; Wan, Junhua; Mu, Chunhua; Liu, Qizheng; Wang, Yue; Sang, Jianli

    2016-08-01

    The protein kinase Rad53 and its orthologs play a fundamental role in regulating the DNA damage checkpoint in eukaryotes. Rad53 is activated by phosphorylation in response to DNA damage and deactivated by dephosphorylation after the damage is repaired. However, the phosphatases involved in Rad53 deactivation are not entirely understood. In this study, by investigating the consequences of overexpressing SDS22, a gene encoding a regulatory subunit of the PP1 phosphatase Glc7, in the human fungal pathogen Candida albicans, we discovered that Sds22 plays an important role in Rad53 dephosphorylation and thus the deactivation of the DNA damage checkpoint. Sds22 cellular levels increase when cells are exposed to DNA damaging agents and decrease after removing the genotoxins. Depletion of Glc7 has similar phenotypes. We provide evidence that Sds2 acts through inhibitory physical association with Glc7. Our findings provide novel insights into the mechanisms for the control of DNA damage checkpoint. Furthermore, SDS22 overexpression reduces C. albicans virulence in a mouse model of systemic infection, suggesting potential targets for developing antifungal drugs. PMID:27328280

  18. Rad Chem data acquisition chassis users manual

    SciTech Connect

    Jones, B.A.

    1980-01-24

    The Shiva Laser at LLL requires many forms of diagnostics to measure and analyze fusion experiments. This manual describes the operation of a Micro-Processor controlled data acquisition system designed at LLL to measure Neutron Activation during fusion experiments on the Shiva Laser.

  19. Rad18 and Rnf8 facilitate homologous recombination by two distinct mechanisms, promoting Rad51 focus formation and suppressing the toxic effect of nonhomologous end joining.

    PubMed

    Kobayashi, S; Kasaishi, Y; Nakada, S; Takagi, T; Era, S; Motegi, A; Chiu, R K; Takeda, S; Hirota, K

    2015-08-13

    The E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligases Rad18 and Rnf8 promote homologous recombination (HR)-mediated double-strand break (DSB) repair by enhancing polymerization of the Rad51 recombinase at γ-ray-induced DSB sites. To analyze functional interactions between the three enzymes, we created RAD18(-/-), RNF8(-/-), RAD18(-/-)/RNF8(-/-) and UBC13(-/-)clones in chicken DT40 cells. To assess the capability of HR, we measured the cellular sensitivity to camptothecin (topoisomerase I poison) and olaparib (poly(ADP ribose)polymerase inhibitor) because these chemotherapeutic agents induce DSBs during DNA replication, which are repaired exclusively by HR. RAD18(-/-), RNF8(-/-) and RAD18(-/-)/RNF8(-/-) clones showed very similar levels of hypersensitivity, indicating that Rad18 and Rnf8 operate in the same pathway in the promotion of HR. Although these three mutants show less prominent defects in the formation of Rad51 foci than UBC13(-/-)cells, they are more sensitive to camptothecin and olaparib than UBC13(-/-)cells. Thus, Rad18 and Rnf8 promote HR-dependent repair in a manner distinct from Ubc13. Remarkably, deletion of Ku70, a protein essential for nonhomologous end joining (NHEJ) significantly restored tolerance of RAD18(-/-) and RNF8(-/-) cells to camptothecin and olaparib without affecting Rad51 focus formation. Thus, in cellular tolerance to the chemotherapeutic agents, the two enzymes collaboratively promote DSB repair by HR by suppressing the toxic effect of NHEJ on HR rather than enhancing Rad51 focus formation. In contrast, following exposure to γ-rays, RAD18(-/-), RNF8(-/-), RAD18(-/-)/RNF8(-/-) and UBC13(-/-)cells showed close correlation between cellular survival and Rad51 focus formation at DSB sites. In summary, the current study reveals that Rad18 and Rnf8 facilitate HR by two distinct mechanisms: suppression of the toxic effect of NHEJ on HR during DNA replication and the promotion of Rad51 focus formation at radiotherapy

  20. The QSAR study of flavonoid-metal complexes scavenging rad OH free radical

    NASA Astrophysics Data System (ADS)

    Wang, Bo-chu; Qian, Jun-zhen; Fan, Ying; Tan, Jun

    2014-10-01

    Flavonoid-metal complexes have antioxidant activities. However, quantitative structure-activity relationships (QSAR) of flavonoid-metal complexes and their antioxidant activities has still not been tackled. On the basis of 21 structures of flavonoid-metal complexes and their antioxidant activities for scavenging rad OH free radical, we optimised their structures using Gaussian 03 software package and we subsequently calculated and chose 18 quantum chemistry descriptors such as dipole, charge and energy. Then we chose several quantum chemistry descriptors that are very important to the IC50 of flavonoid-metal complexes for scavenging rad OH free radical through method of stepwise linear regression, Meanwhile we obtained 4 new variables through the principal component analysis. Finally, we built the QSAR models based on those important quantum chemistry descriptors and the 4 new variables as the independent variables and the IC50 as the dependent variable using an Artificial Neural Network (ANN), and we validated the two models using experimental data. These results show that the two models in this paper are reliable and predictable.

  1. Mutation Analysis of the RAD51C and RAD51D Genes in High-Risk Ovarian Cancer Patients and Families from the Czech Republic

    PubMed Central

    Janatova, Marketa; Soukupova, Jana; Stribrna, Jana; Kleiblova, Petra; Vocka, Michal; Boudova, Petra; Kleibl, Zdenek

    2015-01-01

    Recent studies have conferred that the RAD51C and RAD51D genes, which code for the essential proteins involved in homologous recombination, are ovarian cancer (OC) susceptibility genes that may explain genetic risks in high-risk patients. We performed a mutation analysis in 171 high-risk BRCA1 and BRCA2 negative OC patients, to evaluate the frequency of hereditary RAD51C and RAD51D variants in Czech population. The analysis involved direct sequencing, high resolution melting and multiple ligation-dependent probe analysis. We identified two (1.2%) and three (1.8%) inactivating germline mutations in both respective genes, two of which (c.379_380insG, p.P127Rfs*28 in RAD51C and c.879delG, p.C294Vfs*16 in RAD51D) were novel. Interestingly, an indicative family cancer history was not present in four carriers. Moreover, the ages at the OC diagnoses in identified mutation carriers were substantially lower than those reported in previous studies (four carriers were younger than 45 years). Further, we also described rare missense variants, two in RAD51C and one in RAD51D whose clinical significance needs to be verified. Truncating mutations and rare missense variants ascertained in OC patients were not detected in 1226 control samples. Although the cumulative frequency of RAD51C and RAD51D truncating mutations in our patients was lower than that of the BRCA1 and BRCA2 genes, it may explain OC susceptibility in approximately 3% of high-risk OC patients. Therefore, an RAD51C and RAD51D analysis should be implemented into the comprehensive multi-gene testing for high-risk OC patients, including early-onset OC patients without a family cancer history. PMID:26057125

  2. PTEN Loss Does Not Predict for Response to RAD001 (Everolimus) in a Glioblastoma Orthotopic Xenograft Test Panel

    PubMed Central

    Yang, Lin; Clarke, Michelle J.; Carlson, Brett L.; Mladek, Ann C.; Schroeder, Mark A.; Decker, Paul; Wu, Wenting; Kitange, Gaspar J.; Grogan, Patrick T.; Goble, Jennie M.; Uhm, Joon; Galanis, Evanthia; Giannini, Caterina; Lane, Heidi A.; James, C. David; Sarkaria, Jann N.

    2014-01-01

    Purpose Hyperactivation of the phosphatidylinositol 3-kinase/Akt signaling through disruption of PTEN function is common in glioblastoma multiforme, and these genetic changes are predicted to enhance sensitivity to mammalian target of rapamycin (mTOR) inhibitors such as RAD001 (everolimus). Experimental Design To test whether PTEN loss could be used as a predictive marker for mTOR inhibitor sensitivity, the response of 17 serially transplantable glioblastoma multiforme xenografts was evaluated in an orthotopic therapy evaluation model. Of these 17 xenograft lines, 7 have either genomic deletion or mutation of PTEN. Results Consistent with activation of Akt signaling, there was a good correlation between loss of PTEN function and elevated levels of Akt phosphorylation. However, of the 7 lines with disrupted PTEN function, only 1 tumor line (GBM10) was significantly sensitive to RAD001 therapy (25% prolongation in median survival), whereas1 of 10 xenograft lines with wild-type PTEN was significantly sensitive to RAD001 (GS22; 34% prolongation in survival). Relative to placebo, 5 days of RAD001 treatment was associated with a marked 66% reduction in the MIB1 proliferation index in the sensitive GBM10 line (deleted PTEN) compared with a 25% and 7% reduction in MIB1 labeling index in the insensitive GBM14 (mutant PTEN) and GBM15 (wild-type PTEN) lines, respectively. Consistent with a cytostatic antitumor effect, bioluminescent imaging of luciferase-transduced intracranial GBM10 xenografts showed slowed tumor growth without significant tumor regression during RAD001 therapy. Conclusion These data suggest that loss of PTEN function is insufficient to adequately predict responsiveness to mTOR inhibitors in glioblastoma multiforme. PMID:18559622

  3. Selective androgen receptor modulator RAD140 is neuroprotective in cultured neurons and kainate-lesioned male rats.

    PubMed

    Jayaraman, Anusha; Christensen, Amy; Moser, V Alexandra; Vest, Rebekah S; Miller, Chris P; Hattersley, Gary; Pike, Christian J

    2014-04-01

    The decline in testosterone levels in men during normal aging increases risks of dysfunction and disease in androgen-responsive tissues, including brain. The use of testosterone therapy has the potential to increase the risks for developing prostate cancer and or accelerating its progression. To overcome this limitation, novel compounds termed "selective androgen receptor modulators" (SARMs) have been developed that lack significant androgen action in prostate but exert agonist effects in select androgen-responsive tissues. The efficacy of SARMs in brain is largely unknown. In this study, we investigate the SARM RAD140 in cultured rat neurons and male rat brain for its ability to provide neuroprotection, an important neural action of endogenous androgens that is relevant to neural health and resilience to neurodegenerative diseases. In cultured hippocampal neurons, RAD140 was as effective as testosterone in reducing cell death induced by apoptotic insults. Mechanistically, RAD140 neuroprotection was dependent upon MAPK signaling, as evidenced by elevation of ERK phosphorylation and inhibition of protection by the MAPK kinase inhibitor U0126. Importantly, RAD140 was also neuroprotective in vivo using the rat kainate lesion model. In experiments with gonadectomized, adult male rats, RAD140 was shown to exhibit peripheral tissue-specific androgen action that largely spared prostate, neural efficacy as demonstrated by activation of androgenic gene regulation effects, and neuroprotection of hippocampal neurons against cell death caused by systemic administration of the excitotoxin kainate. These novel findings demonstrate initial preclinical efficacy of a SARM in neuroprotective actions relevant to Alzheimer's disease and related neurodegenerative diseases. PMID:24428527

  4. RAD51 potentiates synergistic effects of chemotherapy with PCI-24781 and cis-diamminedichloroplatinum on gastric cancer

    PubMed Central

    He, Wei-Ling; Li, Yu-Huang; Hou, Wei-Jian; Ke, Zun-Fu; Chen, Xin-Lin; Lu, Li-Ya; Cai, Shi-Rong; Song, Wu; Zhang, Chang-Hua; He, Yu-Long

    2014-01-01

    AIM: To explore the efficacy of PCI-24781, a broad-spectrum, hydroxamic acid-derived histone deacetylase inhibitor, in the treatment of gastric cancer (GC). METHODS: With or without treatment of PCI-24781 and/or cis-diamminedichloroplatinum (CDDP), GC cell lines were subjected to functional analysis, including cell growth, apoptosis and clonogenic assays. Chromatin immunoprecipitation and luciferase reporter assays were used to determine the interacting molecules and the activity of the enzyme. An in vivo study was carried out in GC xenograft mice. Cell culture-based assays were represented as mean ± SD. ANOVA tests were used to assess differences across groups. All pairwise comparisons between tumor weights among treatment groups were made using the Tukey-Kramer method for multiple comparison adjustment to control experimental-wise type I error rates. Significance was set at P < 0.05. RESULTS: PCI-24781 significantly reduced the growth of the GC cells, enhanced cell apoptosis and suppressed clonogenicity, and these effects synergized with the effects of CDDP. PCI-24781 modulated the cell cycle and significantly reduced the expression of RAD51, which is related to homologous recombination. Depletion of RAD51 augmented the biological functions of PCI-24781, CDDP and the combination treatment, whereas overexpressing RAD51 had the opposite effects. Increased binding of the transcription suppressor E2F4 on the RAD51 promoter appeared to play a major role in these processes. Furthermore, significant suppression of tumor growth and weight in vivo was obtained following PCI-24781 treatment, which synergized with the anticancer effect of CDDP. CONCLUSION: These data suggest that RAD51 potentiates the synergistic effects of chemotherapy with PCI-24781 and CDDP on GC. PMID:25110436

  5. Selective Androgen Receptor Modulator RAD140 Is Neuroprotective in Cultured Neurons and Kainate-Lesioned Male Rats

    PubMed Central

    Jayaraman, Anusha; Christensen, Amy; Moser, V. Alexandra; Vest, Rebekah S.; Miller, Chris P.; Hattersley, Gary

    2014-01-01

    The decline in testosterone levels in men during normal aging increases risks of dysfunction and disease in androgen-responsive tissues, including brain. The use of testosterone therapy has the potential to increase the risks for developing prostate cancer and or accelerating its progression. To overcome this limitation, novel compounds termed “selective androgen receptor modulators” (SARMs) have been developed that lack significant androgen action in prostate but exert agonist effects in select androgen-responsive tissues. The efficacy of SARMs in brain is largely unknown. In this study, we investigate the SARM RAD140 in cultured rat neurons and male rat brain for its ability to provide neuroprotection, an important neural action of endogenous androgens that is relevant to neural health and resilience to neurodegenerative diseases. In cultured hippocampal neurons, RAD140 was as effective as testosterone in reducing cell death induced by apoptotic insults. Mechanistically, RAD140 neuroprotection was dependent upon MAPK signaling, as evidenced by elevation of ERK phosphorylation and inhibition of protection by the MAPK kinase inhibitor U0126. Importantly, RAD140 was also neuroprotective in vivo using the rat kainate lesion model. In experiments with gonadectomized, adult male rats, RAD140 was shown to exhibit peripheral tissue-specific androgen action that largely spared prostate, neural efficacy as demonstrated by activation of androgenic gene regulation effects, and neuroprotection of hippocampal neurons against cell death caused by systemic administration of the excitotoxin kainate. These novel findings demonstrate initial preclinical efficacy of a SARM in neuroprotective actions relevant to Alzheimer's disease and related neurodegenerative diseases. PMID:24428527

  6. Ordering of hard particles between hard walls

    NASA Astrophysics Data System (ADS)

    Chrzanowska, A.; Teixeira, P. I. C.; Ehrentraut, H.; Cleaver, D. J.

    2001-05-01

    The structure of a fluid of hard Gaussian overlap particles of elongation κ = 5, confined between two hard walls, has been calculated from density-functional theory and Monte Carlo simulations. By using the exact expression for the excluded volume kernel (Velasco E and Mederos L 1998 J. Chem. Phys. 109 2361) and solving the appropriate Euler-Lagrange equation entirely numerically, we have been able to extend our theoretical predictions into the nematic phase, which had up till now remained relatively unexplored due to the high computational cost. Simulation reveals a rich adsorption behaviour with increasing bulk density, which is described semi-quantitatively by the theory without any adjustable parameters.

  7. RAD51 plays a crucial role in halting cell death program induced by ionizing radiation in bovine oocytes.

    PubMed

    Kujjo, Loro L; Ronningen, Reg; Ross, Pablo; Pereira, Ricardo J G; Rodriguez, Ramon; Beyhan, Zeki; Goissis, Marcelo D; Baumann, Thomas; Kagawa, Wataru; Camsari, Cagri; Smith, George W; Kurumizaka, Hitoshi; Yokoyama, Shigeyuki; Cibelli, Jose B; Perez, Gloria I

    2012-03-01

    Reproductive health of humans and animals exposed to daily irradiants from solar/cosmic particles remains largely understudied. We evaluated the sensitivities of bovine and mouse oocytes to bombardment by krypton-78 (1 Gy) or ultraviolet B (UV-B; 100 microjoules). Mouse oocytes responded to irradiation by undergoing massive activation of caspases, rapid loss of energy without cytochrome-c release, and subsequent necrotic death. In contrast, bovine oocytes became positive for annexin-V, exhibited cytochrome-c release, and displayed mild activation of caspases and downstream DNAses but with the absence of a complete cell death program; therefore, cytoplasmic fragmentation was never observed. However, massive cytoplasmic fragmentation and increased DNA damage were induced experimentally by both inhibiting RAD51 and increasing caspase 3 activity before irradiation. Microinjection of recombinant human RAD51 prior to irradiation markedly decreased both cytoplasmic fragmentation and DNA damage in both bovine and mouse oocytes. RAD51 response to damaged DNA occurred faster in bovine oocytes than in mouse oocytes. Therefore, we conclude that upon exposure to irradiation, bovine oocytes create a physiologically indeterminate state of partial cell death, attributed to rapid induction of DNA repair and low activation of caspases. The persistence of these damaged cells may represent an adaptive mechanism with potential implications for livestock productivity and long-term health risks associated with human activity in space. PMID:22190703

  8. Gefitinib Synergizes with Irinotecan to Suppress Hepatocellular Carcinoma via Antagonizing Rad51-Mediated DNA-Repair

    PubMed Central

    Peng, Xueming; Chen, Min; Zhu, Yuanrun; Xu, Li; Zhu, Hong; Yang, Bo; Luo, Peihua; He, Qiaojun

    2016-01-01

    Chemotherapy is the only choice for most of the advanced hepatocellular carcinoma (HCC) patients, while few agents were available, making it an urgent need to develop new chemotherapy strategies. A phase II clinical trial suggested that the efficacy of irinotecan in HCC was limited due to dose-dependent toxicities. Here, we found that gefitinib exhibited synergistic activity in combination with SN-38, an active metabolite of irinotecan, in HCC cell lines. And the enhanced apoptosis induced by gefitinib plus SN-38 was a result from caspase pathway activation. Mechanistically, gefitinib dramatically promoted the ubiquitin–proteasome-dependent degradation of Rad51 protein, suppressed the DNA repair, gave rise to more DNA damages, and ultimately resulted in the synergism of these two agents. In addition, the increased antitumor efficacy of gefitinib combined with irinotecan was further validated in a HepG2 xenograft mice model. Taken together, our data demonstrated for the first time that the combination of irinotecan and gefitinib showed potential benefit in HCC, which suggests that Rad51 is a promising target and provides a rationale for clinical trials investigating the efficacy of the combination of topoisomerase I inhibitors and gefitinib in HCC. PMID:26752698

  9. Gefitinib Synergizes with Irinotecan to Suppress Hepatocellular Carcinoma via Antagonizing Rad51-Mediated DNA-Repair.

    PubMed

    Shao, Jinjin; Xu, Zhifei; Peng, Xueming; Chen, Min; Zhu, Yuanrun; Xu, Li; Zhu, Hong; Yang, Bo; Luo, Peihua; He, Qiaojun

    2016-01-01

    Chemotherapy is the only choice for most of the advanced hepatocellular carcinoma (HCC) patients, while few agents were available, making it an urgent need to develop new chemotherapy strategies. A phase II clinical trial suggested that the efficacy of irinotecan in HCC was limited due to dose-dependent toxicities. Here, we found that gefitinib exhibited synergistic activity in combination with SN-38, an active metabolite of irinotecan, in HCC cell lines. And the enhanced apoptosis induced by gefitinib plus SN-38 was a result from caspase pathway activation. Mechanistically, gefitinib dramatically promoted the ubiquitin-proteasome-dependent degradation of Rad51 protein, suppressed the DNA repair, gave rise to more DNA damages, and ultimately resulted in the synergism of these two agents. In addition, the increased antitumor efficacy of gefitinib combined with irinotecan was further validated in a HepG2 xenograft mice model. Taken together, our data demonstrated for the first time that the combination of irinotecan and gefitinib showed potential benefit in HCC, which suggests that Rad51 is a promising target and provides a rationale for clinical trials investigating the efficacy of the combination of topoisomerase I inhibitors and gefitinib in HCC. PMID:26752698

  10. Understanding Active and Passive Users: The Effects of an Active User Using Normal, Hard and Unreliable Technologies on User Assessment of Trust in Technology and Co-User

    PubMed Central

    Montague, Enid; JieXu

    2011-01-01

    The aim of this study was to understand how passive users perceive the trustworthiness of active users and technologies under varying technological conditions. An experimental study was designed to vary the functioning of technologies that active users interacted with, while passive users observed these interactions. Active and passive user ratings of technology and partner were collected. Exploratory data analysis suggests that passive users developed perceptions of technologies based on the functioning of the technology and how the active user interacted with the technologies. Findings from this research have implications for the design of technologies in environments where active and passive users interact with technologies in different ways. Future work in this area should explore interventions that lead to enhanced affective engagement and trust calibration. PMID:22192788

  11. Food Irradiation Is Done in Grays, not Rads

    SciTech Connect

    Strom, Daniel J.

    2002-07-01

    One federal agency has chosen to use exclusively modern SI units of radiation dose in its regulations: the FDA. While not exactly hot news, this bold move by a U.S. government agency on November 26, 1997, should be noted by those who wish to encourage the switch from curies, working level months, rads, rems, and roentgens to becquerels, joule hours per cubic meter, grays, sieverts, and coulombs per kilogram. The regulation is 21 CFR 179, Irradiation in the Production, Processing, and Handling of Food. Specifically, 21 CFR 179.26 (b) 8. permits meat irradiation up to 4.5 kGy for refrigerated meat and 7.0 kGy for frozen meat. Prior to the 1997 addition, radiation doses had been quoted in grays (kGy) with rad (Mrad) values in parentheses. In the 1997 addition, the Mrads disappeared.

  12. Ejecta model development at pRad (u)

    SciTech Connect

    Buttler, William T; Oro, David M; Dimonte, Guy; Terrones, Guillermo; Morris, Christopher; Bainbridge, J R; Hogan, Gary E.; Hollander, Brian J.; Holtkamp, David B.; Kwiathowski, Kris; Marr-Lyon, Mark; Mariam, Fesseha G.; Merrill, Frank E; Nedrow, Paul; Saunders, Alexander; Schwartz, C L; Stone, B; Tupa, Dale; Vogan-McNeil, Wendy S

    2010-02-09

    In July 2009 we fielded three explosively (HE) driven Richtmyer-Meshkov instability experiments at the LANSCE Proton Radiography Facility (pRad), and in August of 2009 we fielded one flyer plate experiment on the pRad 40 mm powder gun. One HE experiment was done in vacuum, and the other two within four atmospheres of noble gasses: Xe and Ne. These two gases were chosen to study the viscous effects on ejecta formation. It is unexpected, but the viscosity {eta} of Ne is twice that of Xe, and, due to the atomic mass difference between the two, the kinematic viscosity ({eta}/{rho}) of Ne is about ten times that of Xe. The results showed that ejecta formation is sensitively linked to the gas density, which implies that the Weber number is more important in ejecta formation than the Reynolds number.

  13. Rad9 interacts with Aft1 to facilitate genome surveillance in fragile genomic sites under non-DNA damage-inducing conditions in S. cerevisiae

    PubMed Central

    Andreadis, Christos; Nikolaou, Christoforos; Fragiadakis, George S.; Tsiliki, Georgia; Alexandraki, Despina

    2014-01-01

    DNA damage response and repair proteins are centrally involved in genome maintenance pathways. Yet, little is known about their functional role under non-DNA damage-inducing conditions. Here we show that Rad9 checkpoint protein, known to mediate the damage signal from upstream to downstream essential kinases, interacts with Aft1 transcription factor in the budding yeast. Aft1 regulates iron homeostasis and is also involved in genome integrity having additional iron-independent functions. Using genome-wide expression and chromatin immunoprecipitation approaches, we found Rad9 to be recruited to 16% of the yeast genes, often related to cellular growth and metabolism, while affecting the transcription of ∼2% of the coding genome in the absence of exogenously induced DNA damage. Importantly, Rad9 is recruited to fragile genomic regions (transcriptionally active, GC rich, centromeres, meiotic recombination hotspots and retrotransposons) non-randomly and in an Aft1-dependent manner. Further analyses revealed substantial genome-wide parallels between Rad9 binding patterns to the genome and major activating histone marks, such as H3K36me, H3K79me and H3K4me. Thus, our findings suggest that Rad9 functions together with Aft1 on DNA damage-prone chromatin to facilitate genome surveillance, thereby ensuring rapid and effective response to possible DNA damage events. PMID:25300486

  14. NARAC Dispersion Model Product Integration With RadResponder

    SciTech Connect

    Aluzzi, Fernando

    2015-09-30

    Work on enhanced cooperation and interoperability of Nuclear Incident Response Teams (NIRT) is a joint effort between DHS/FEMA, DOE/NNSA and EPA. One such effort was the integration between the RadResponder Network, a resource sponsored by FEMA for the management of radiological data during an emergency, and the National Atmospheric Advisory Center (NARAC), a DOE/NNSA modeling resource whose predictions are used to aid radiological emergency preparedness and response. Working together under a FEMA-sponsored project these two radiological response assets developed a capability to read and display plume model prediction results from the NARAC computer system in the RadResponder software tool. As a result of this effort, RadResponder users have been provided with NARAC modeling predictions of contamination areas, radiological dose levels, and protective action areas (e.g., areas warranting worker protection or sheltering/evacuation) to help guide protective action decisions and field monitoring surveys, and gain key situation awareness following a radiological/nuclear accident or incident (e.g., nuclear power plant accident, radiological dispersal device incident, or improvised nuclear detonation incident). This document describes the details of this integration effort.

  15. Accounting for the MMRTG Background in MSL/RAD Data

    NASA Astrophysics Data System (ADS)

    Koehler, J.; Hassler, D.; Wimmer-Schweingruber, R. F.; Zeitlin, C. J.; Boettcher, S.; Martin, C.; Brinza, D.; Guo, J.; Böhm, E.; Buttgereit, M.; Ehresmann, B.

    2012-12-01

    To characterize the broad spectrum of the radiation field on the Martian surface is the prime science objective of Mars Science Laboratory's (MSL's) Radiation Assessment Detector (RAD). This surface radiation is a result of the interaction of solar and galactic cosmic radiation with the Martian atmosphere and soil and is a limiting factor for life on Mars or a possible future manned mission to Mars. The Martian radiation field is complex and quite different from that on Earth especially because of the presence of a strong neutral component which consists of neutrons and gammas. One of the difficulties in determining the Martian radiation field with MSL/RAD is the necessary presence of MSL's Multi-Mission Radioisotope Thermoelectric Generator (MMRTG) power source. The decay of the MMRTG's plutonium results in a large background of low-energy neutrons and gamma rays in RAD's neutral particle channels. This background needs to be accounted for by careful modeling and other available data. In this work, we will present the current status of this ongoing work.

  16. RadNet: Open network protocol for radiation data

    SciTech Connect

    Rees, B.; Olson, K.; Beckes-Talcott, J.; Kadner, S.; Wenderlich, T.; Hoy, M.; Doyle, W.; Koskelo, M.

    1998-12-31

    Safeguards instrumentation is increasingly being incorporated into remote monitoring applications. In the past, vendors of radiation monitoring instruments typically provided the tools for uploading the monitoring data to a host. However, the proprietary nature of communication protocols lends itself to increased computer support needs and increased installation expenses. As a result, a working group of suppliers and customers of radiation monitoring instruments defined an open network protocol for transferring packets on a local area network from radiation monitoring equipment to network hosts. The protocol was termed RadNet. While it is now primarily used for health physics instruments, RadNet`s flexibility and strength make it ideal for remote monitoring of nuclear materials. The incorporation of standard, open protocols ensures that future work will not render present work obsolete; because RadNet utilizes standard Internet protocols, and is itself a non-proprietary standard. The use of industry standards also simplifies the development and implementation of ancillary services, e.g. E-main generation or even pager systems.

  17. Rad51 Protein Expression and Survival in Patients with Glioblastoma Multiforme

    SciTech Connect

    Welsh, James W. Ellsworth, Ron K.; Kumar, Rachit; Fjerstad, Kyle; Martinez, Jesse; Nagel, Raymond B.; Eschbacher, Jennifer; Stea, Baldassarre

    2009-07-15

    Purpose: Treatment of glioblastoma multiforme (GBM) continues to pose a significant therapeutic challenge, with most tumors recurring within the previously irradiated tumor bed. To improve outcomes, we must be able to identify and treat resistant cell populations. Rad51, an enzyme involved in homologous recombinational repair, leads to increased resistance of tumor cells to cytotoxic treatments such as radiotherapy. We hypothesized that Rad51 might contribute to GBM's apparent radioresistance and consequently influence survival. Methods and Materials: A total of 68 patients with an initial diagnosis of GBM were retrospectively evaluated; for 10 of these patients, recurrent tumor specimens were used to construct a tissue microarray. Rad51 protein expression was then correlated with the actual and predicted survival using recursive partitioning analysis. Results: Rad51 protein was elevated in 53% of the GBM specimens at surgery. The Rad51 levels correlated directly with survival, with a median survival of 15 months for patients with elevated Rad51 compared with 9 months for patients with low or absent levels of Rad51 (p = .05). At disease recurrence, 70% of patients had additional increases in Rad51 protein. Increased Rad51 levels at disease recurrence similarly predicted for improved overall survival, with a mean survival of 16 months from the second craniotomy compared with only 4 months for patients with low Rad51 levels (p = .13). Conclusion: Elevated levels of the double-stranded DNA repair protein Rad51 predicted for an increase survival duration in patients with GBM, at both initial tumor presentation and disease recurrence.

  18. Identification and functional characterization of the Rad23 gene of the silkworm, Bombyx mori.

    PubMed

    Xu, He-ping; Xu, Yu-song; Wang, Hua-bing; He, Da; Kawasaki, Hideki

    2010-02-01

    Rad23 is an NER (nucleotide excision repair) protein and it plays an important role in the UPP (ubiquitin-proteasome pathway). In the present study, BmRad23 (a homologous gene of Rad23 from Bombyx mori) was cloned and designated as BmRad23. The ORF (open reading frame) of the BmRad23 cDNA encoded deduced 324 amino acids with a calculated molecular mass of 36.13 kDa and an estimated pI of 4.50. The deduced amino acid sequence of the BmRad23 cDNA revealed several indispensable domains for the function of the Rad23 protein family, such as one UbL (ubiquitin-like) region domain and two UBA (ubiquitin-associated) domains. UV irradiation and treatment with chemical DNA-damaging reagent increased the expression of BmRad23. The BmRad23 gene was expressed in all the examined organs, and elevated expression was observed in testis and ovary. Northern blot and immunoblot analyses showed enhanced expression of BmRad23 after day 3 of the wandering stage in the silk gland. From the present results it is suggested that BmRad23 functions in the UPP during the silkworm metamorphosis as well as participating in the NER when the genetic material is damaged by UV irradiation and other genotoxic stresses. PMID:19203347

  19. GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

    PubMed Central

    Takizawa, Yoshimasa; Qing, Yong; Takaku, Motoki; Ishida, Takako; Morozumi, Yuichi; Tsujita, Takashi; Kogame, Toshiaki; Hirota, Kouji; Takahashi, Masayuki; Shibata, Takehiko; Kurumizaka, Hitoshi; Takeda, Shunichi

    2010-01-01

    RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51–DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator. PMID:20403813

  20. Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability.

    PubMed

    Spies, Julian; Waizenegger, Anja; Barton, Olivia; Sürder, Michael; Wright, William D; Heyer, Wolf-Dietrich; Löbrich, Markus

    2016-06-16

    Never-in-mitosis A-related kinase 1 (Nek1) has established roles in apoptosis and cell cycle regulation. We show that human Nek1 regulates homologous recombination (HR) by phosphorylating Rad54 at Ser572 in late G2 phase. Nek1 deficiency as well as expression of unphosphorylatable Rad54 (Rad54-S572A) cause unresolved Rad51 foci and confer a defect in HR. Phospho-mimic Rad54 (Rad54-S572E), in contrast, promotes HR and rescues the HR defect associated with Nek1 loss. Although expression of phospho-mimic Rad54 is beneficial for HR, it causes Rad51 removal from chromatin and degradation of stalled replication forks in S phase. Thus, G2-specific phosphorylation of Rad54 by Nek1 promotes Rad51 chromatin removal during HR in G2 phase, and its absence in S phase is required for replication fork stability. In summary, Nek1 regulates Rad51 removal to orchestrate HR and replication fork stability. PMID:27264870

  1. RAD18, WRNIP1 and ATMIN promote ATM signalling in response to replication stress.

    PubMed

    Kanu, N; Zhang, T; Burrell, R A; Chakraborty, A; Cronshaw, J; DaCosta, C; Grönroos, E; Pemberton, H N; Anderton, E; Gonzalez, L; Sabbioneda, S; Ulrich, H D; Swanton, C; Behrens, A

    2016-07-28

    The DNA replication machinery invariably encounters obstacles that slow replication fork progression, and threaten to prevent complete replication and faithful segregation of sister chromatids. The resulting replication stress activates ATR, the major kinase involved in resolving impaired DNA replication. In addition, replication stress also activates the related kinase ATM, which is required to prevent mitotic segregation errors. However, the molecular mechanism of ATM activation by replication stress is not defined. Here, we show that monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA), a marker of stalled replication forks, interacts with the ATM cofactor ATMIN via WRN-interacting protein 1 (WRNIP1). ATMIN, WRNIP1 and RAD18, the E3 ligase responsible for PCNA monoubiquitination, are specifically required for ATM signalling and 53BP1 focus formation induced by replication stress, not ionising radiation. Thus, WRNIP1 connects PCNA monoubiquitination with ATMIN/ATM to activate ATM signalling in response to replication stress and contribute to the maintenance of genomic stability. PMID:26549024

  2. Characterization of the Roles of the Saccharomyces Cerevisiae Rad54 Gene and a Homologue of Rad54, Rdh54/Tid1, in Mitosis and Meiosis

    PubMed Central

    Shinohara, M.; Shita-Yamaguchi, E.; Buerstedde, J. M.; Shinagawa, H.; Ogawa, H.; Shinohara, A.

    1997-01-01

    The RAD54 gene, which encodes a protein in the SWI2/SNF2 family, plays an important role in recombination and DNA repair in Saccharomyces cerevisiae. The yeast genome project revealed a homologue of RAD54, RDH54/TID1. Properties of the rdh54/tid1 mutant and the rad54 rdh54/tid1 double mutant are shown for mitosis and meiosis. The rad54 mutant is sensitive to the alkylating agent, methyl methanesulfonate (MMS), and is defective in interchromosomal and intrachromosomal gene conversion. The rdh54/tid1 single mutant, on the other hand, does not show any significant deficiency in mitosis. However, the rad54 rdh54/tid1 mutant is more sensitive to MMS and more defective in interchromosomal gene conversion than is the rad54 mutant, but shows the same frequency of intrachromosomal gene conversion as the rad54 mutant. These results suggest that RDH54/TID1 is involved in a minor pathway of mitotic recombination in the absence of RAD54. In meiosis, both single mutants produce viable spores at slightly reduced frequency. However, only the rdh54/tid1 mutant, but not the rad54 mutant, shows significant defects in recombination: retardation of the repair of meiosis-specific double-strand breaks (DSBs) and delayed formation of physical recombinants. Furthermore, the rad54 rdh54/tid1 double mutant is completely defective in meiosis, accumulating DSBs with more recessed ends than the wild type and producing fewer physical recombinants than the wild type. These results suggest that one of the differences between the late stages of mitotic recombination and meiotic recombination might be specified by differential dependency on the Rad54 and Rdh54/Tid1 proteins. PMID:9409820

  3. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells

    PubMed Central

    Zellweger, Ralph; Dalcher, Damian; Mutreja, Karun; Berti, Matteo; Schmid, Jonas A.; Herrador, Raquel; Vindigni, Alessandro

    2015-01-01

    Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy. PMID:25733714

  4. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.

    PubMed

    Zellweger, Ralph; Dalcher, Damian; Mutreja, Karun; Berti, Matteo; Schmid, Jonas A; Herrador, Raquel; Vindigni, Alessandro; Lopes, Massimo

    2015-03-01

    Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy. PMID:25733714

  5. DmGEN, a novel RAD2 family endo-exonuclease from Drosophila melanogaster.

    PubMed

    Ishikawa, Gen; Kanai, Yoshihiro; Takata, Kei-ichi; Takeuchi, Ryo; Shimanouchi, Kaori; Ruike, Tatsushi; Furukawa, Tomoyuki; Kimura, Seisuke; Sakaguchi, Kengo

    2004-01-01

    A novel endo-exonuclease, DmGEN (Drosophila Melanogaster XPG-like endonuclease), was identified in D.melanogaster. DmGEN is composed of five exons and four introns, and the open reading frame encodes a predicted product of 726 amino acid residues with a molecular weight of 82.5 kDa and a pI of 5.36. The gene locus on Drosophila polytene chromosomes was detected at 64C9 on the left arm of chromosome 3 as a single site. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nucleases, especially XPG. Although the XPG-N- and XPG-I-domains are highly conserved in sequence, locations of the domains are similar to those of FEN-1 and EXO-1, and the molecular weight of the protein is close to that of EXO-1. In vitro, DmGEN showed endonuclease and 3'-5' exonuclease activities with both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), but the endonuclease action with dsDNA was quite specific: 5'-3' exonuclease activity was found to occur with nicked DNA, while dsDNA was endonucleolytically cut at 3-4 bp from the 5' end. Homologs are widely found in mammals and higher plants. The data suggest that DmGEN belongs to a new class of RAD2 nuclease. PMID:15576351

  6. Session: Hard Rock Penetration

    SciTech Connect

    Tennyson, George P. Jr.; Dunn, James C.; Drumheller, Douglas S.; Glowka, David A.; Lysne, Peter

    1992-01-01

    This session at the Geothermal Energy Program Review X: Geothermal Energy and the Utility Market consisted of five presentations: ''Hard Rock Penetration - Summary'' by George P. Tennyson, Jr.; ''Overview - Hard Rock Penetration'' by James C. Dunn; ''An Overview of Acoustic Telemetry'' by Douglas S. Drumheller; ''Lost Circulation Technology Development Status'' by David A. Glowka; ''Downhole Memory-Logging Tools'' by Peter Lysne.

  7. Hardness Tester for Polyur

    NASA Technical Reports Server (NTRS)

    Hauser, D. L.; Buras, D. F.; Corbin, J. M.

    1987-01-01

    Rubber-hardness tester modified for use on rigid polyurethane foam. Provides objective basis for evaluation of improvements in foam manufacturing and inspection. Typical acceptance criterion requires minimum hardness reading of 80 on modified tester. With adequate correlation tests, modified tester used to measure indirectly tensile and compressive strengths of foam.

  8. The hard metal diseases.

    PubMed

    Cugell, D W

    1992-06-01

    Hard metal is a mixture of tungsten carbide and cobalt, to which small amounts of other metals may be added. It is widely used for industrial purposes whenever extreme hardness and high temperature resistance are needed, such as for cutting tools, oil well drilling bits, and jet engine exhaust ports. Cobalt is the component of hard metal that can be a health hazard. Respiratory diseases occur in workers exposed to cobalt--either in the production of hard metal, from machining hard metal parts, or from other sources. Adverse pulmonary reactions include asthma, hypersensitivity pneumonitis, and interstitial fibrosis. A peculiar, almost unique form of lung fibrosis, giant cell interstitial pneumonia, is closely linked with cobalt exposure. PMID:1511554

  9. The role of repair protein Rad51 in synergistic cytotoxicity and mutagenicity induced by epidermal growth factor receptor inhibitor (Gefitinib, Iressa{sup R}) and benzo[a]pyrene in human lung cancer

    SciTech Connect

    Ko, J.-C.; Hong, J.-H.; Wang, L.-H.; Lin, Y.-W.

    2008-05-01

    Rad51 protein is essential for homologous recombination repair of DNA damage, and is over-expressed in chemo- or radioresistant carcinomas. The polycyclic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) affects MAPKs transduction pathways. Gefitinib (Iressa{sup R}, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that blocks growth factor-mediated cell proliferation and ERK1/2 activation. We hypothesized that gefitinib enhances B[a]P-mediated cytotoxicity by decreasing ERK1/2 activation. Exposure of human lung cancer cells to gefitinib decreased B[a]P-elicited ERK1/2 activation and induced Rad51 protein expression. Gefitinib and B[a]P co-treatment decreased Rad51 protein stability by triggering degradation via a 26S proteasome-dependent pathway. Expression of constitutive active MKK1/2 vectors (MKK1/2-CA) rescues the decreased ERK1/2 activity, and restores Rad51 protein level and stability under gefitinib and B[a]P co-treatment. Gefitinib enhances B[a]P-induced growth inhibition, cytotoxicity and mutagenicity. Co-treatment with gefitinib and B[a]P can further inhibit cell growth significantly after depletion of endogenous Rad51 by siRad51 RNA transfection. Enhancement of ERK1/2 activation by MKK1-CA expression decrease B[a]P- and gefitinib-induced cytotoxicity, and B[a]P-induced mutagenicity. Rad51 protein protects lung cancer cells from synergistic cytotoxic and mutagenic effects induced by gefitinib and B[a]P. Suppression of Rad51 protein expression may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib.

  10. Disparate requirements for the Walker A and B ATPase motifs ofhuman RAD51D in homologous recombination

    SciTech Connect

    Wiese, Claudia; Hinz, John M.; Tebbs, Robert S.; Nham, Peter B.; Urbin, Salustra S.; Collins, David W.; Thompson, Larry H.; Schild, David

    2006-04-21

    In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks. Ectopic expression of wild type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs.

  11. Genomic evolution in Barrett’s adenocarcinoma cells: critical roles of elevated hsRAD51, homologous recombination and Alu sequences in the genome

    PubMed Central

    Pal, J; Bertheau, R; Buon, L; Qazi, A; Batchu, RB; Bandyopadhyay, S; Ali-Fehmi, R; Beer, DG; Weaver, DW; Reis, RJ Shmookler; Goyal, RK; Huang, Q; Munshi, NC; Shammas, MA

    2012-01-01

    A prominent feature of most cancers including Barrett’s adenocarcinoma (BAC) is genetic instability, which is associated with development and progression of disease. In this study, we investigated the role of recombinase (hsRAD51), a key component of homologous recombination (HR)/repair, in evolving genomic changes and growth of BAC cells. We show that the expression of RAD51 is elevated in BAC cell lines and tissue specimens, relative to normal cells. HR activity is also elevated and significantly correlates with RAD51 expression in BAC cells. The suppression of RAD51 expression, by short hairpin RNA (shRNA) specifically targeting this gene, significantly prevented BAC cells from acquiring genomic changes to either copy number or heterozygosity (P<0.02) in several independent experiments employing single-nucleotide polymorphism arrays. The reduction in copy-number changes, following shRNA treatment, was confirmed by Comparative Genome Hybridization analyses of the same DNA samples. Moreover, the chromosomal distributions of mutations correlated strongly with frequencies and locations of Alu interspersed repetitive elements on individual chromosomes. We conclude that the hsRAD51 protein level is systematically elevated in BAC, contributes significantly to genomic evolution during serial propagation of these cells and correlates with disease progression. Alu sequences may serve as substrates for elevated HR during cell proliferation in vitro, as they have been reported to do during the evolution of species, and thus may provide additional targets for prevention or treatment of this disease. PMID:21423218

  12. Effects of the rad52 gene on recombination in Saccharomyces cerevisiae

    SciTech Connect

    Prakash, S.; Prakash, L.; Burke, W.; Montelone, B.A.

    1980-01-01

    Effects of the rad 52 mutation in Saccharomyces cerevisiae on meiotic, ..gamma..-ray-induced, uv-induced and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Both intra and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the his1-1/his1-315 and trp-5-2/trp5-48 heteroalleles. Gene-centromere recombination also was not observed in rad/52/rad52 diploids. No ..gamma..-ray- or uv-induced intragenic mitotic recombination is seen in rad52/rad52 diploids. The rate of spontaneous mitotic recombination is lowered five-fold at the his1-1/his1-315 and leu1-c/leu1-12 heteroalleles. Spontaneous reversion rates of both his1-1 and his1-315 were elevated 10 to 20 fold in rad52/rad52 diploids. The RAD52 gene function is required for spontaneous mitotic recombination, uv- and ..gamma..-ray-induced mitotic recombination and mitotic recombination.

  13. Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins.

    PubMed

    Bankmann, M; Prakash, L; Prakash, S

    1992-02-01

    Xeroderma pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G. Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines. PMID:1741034

  14. Hardness Evolution of Gamma-Irradiated Polyoxymethylene

    NASA Astrophysics Data System (ADS)

    Hung, Chuan-Hao; Harmon, Julie P.; Lee, Sanboh

    2016-04-01

    This study focuses on analyzing hardness evolution in gamma-irradiated polyoxymethylene (POM) exposed to elevated temperatures after irradiation. Hardness increases with increasing annealing temperature and time, but decreases with increasing gamma ray dose. Hardness changes are attributed to defects generated in the microstructure and molecular structure. Gamma irradiation causes a decrease in the glass transition temperature, melting point, and extent of crystallinity. The kinetics of defects resulting in hardness changes follow a first-order structure relaxation. The rate constant adheres to an Arrhenius equation, and the corresponding activation energy decreases with increasing dose due to chain scission during gamma irradiation. The structure relaxation of POM has a lower energy barrier in crystalline regions than in amorphous ones. The hardness evolution in POM is an endothermic process due to the semi-crystalline nature of this polymer.

  15. Synthesis, spectroscopic and biological activities studies of acyclic and macrocyclic mono and binuclear metal complexes containing a hard-soft Schiff base

    NASA Astrophysics Data System (ADS)

    Abou-Hussein, Azza A. A.; Linert, Wolfgang

    Mono- and bi-nuclear acyclic and macrocyclic complexes with hard-soft Schiff base, H2L, ligand derived from the reaction of 4,6-diacetylresorcinol and thiocabohydrazide, in the molar ratio 1:2 have been prepared. The H2L ligand reacts with Co(II), Ni(II), Cu(II), Zn(II), Mn(II) and UO2(VI) nitrates, VO(IV) sulfate and Ru(III) chloride to get acyclic binuclear complexes except for VO(IV) and Ru(III) which gave acyclic mono-nuclear complexes. Reaction of the acyclic mono-nuclear VO(IV) and Ru(III) complexes with 4,6-diacetylresorcinol afforded the corresponding macrocyclic mono-nuclear VO(IV) and Ru(IIII) complexes. Template reactions of the 4,6-diacetylresorcinol and thiocarbohydrazide with either VO(IV) or Ru(III) salts afforded the macrocyclic binuclear VO(IV) and Ru(III) complexes. The Schiff base, H2L, ligand acts as dibasic with two NSO-tridentate sites and can coordinate with two metal ions to form binuclear complexes after the deprotonation of the hydrogen atoms of the phenolic groups in all the complexes, except in the case of the acyclic mononuclear Ru(III) and VO(IV) complexes, where the Schiff base behaves as neutral tetradentate chelate with N2S2 donor atoms. The ligands and the metal complexes were characterized by elemental analysis, IR, UV-vis 1H-NMR, thermal gravimetric analysis (TGA) and ESR, as well as the measurements of conductivity and magnetic moments at room temperature. Electronic spectra and magnetic moments of the complexes indicate the geometries of the metal centers are either tetrahedral, square planar or octahedral. Kinetic and thermodynamic parameters were calculated using Coats-Redfern equation, for the different thermal decomposition steps of the complexes. The ligands and the metal complexes were screened for their antimicrobial activity against Staphylococcus aureus as Gram-positive bacteria, and Pseudomonas fluorescens as Gram-negative bacteria in addition to Fusarium oxysporum fungus. Most of the complexes exhibit mild

  16. Dose Calibration of the ISS-RAD Fast Neutron Detector

    NASA Technical Reports Server (NTRS)

    Zeitlin, C.

    2015-01-01

    The ISS-RAD instrument has been fabricated by Southwest Research Institute and delivered to NASA for flight to the ISS in late 2015 or early 2016. ISS-RAD is essentially two instruments that share a common interface to ISS. The two instruments are the Charged Particle Detector (CPD), which is very similar to the MSL-RAD detector on Mars, and the Fast Neutron Detector (FND), which is a boron-loaded plastic scintillator with readout optimized for the 0.5 to 10 MeV energy range. As the FND is completely new, it has been necessary to develop methodology to allow it to be used to measure the neutron dose and dose equivalent. This talk will focus on the methods developed and their implementation using calibration data obtained in quasi-monoenergetic (QMN) neutron fields at the PTB facility in Braunschweig, Germany. The QMN data allow us to determine an approximate response function, from which we estimate dose and dose equivalent contributions per detected neutron as a function of the pulse height. We refer to these as the "pSv per count" curves for dose equivalent and the "pGy per count" curves for dose. The FND is required to provide a dose equivalent measurement with an accuracy of ?10% of the known value in a calibrated AmBe field. Four variants of the analysis method were developed, corresponding to two different approximations of the pSv per count curve, and two different implementations, one for real-time analysis onboard ISS and one for ground analysis. We will show that the preferred method, when applied in either real-time or ground analysis, yields good accuracy for the AmBe field. We find that the real-time algorithm is more susceptible to chance-coincidence background than is the algorithm used in ground analysis, so that the best estimates will come from the latter.

  17. Organizing Your Hard Disk.

    ERIC Educational Resources Information Center

    Stocker, H. Robert; Hilton, Thomas S. E.

    1991-01-01

    Suggests strategies that make hard disk organization easy and efficient, such as making, changing, and removing directories; grouping files by subject; naming files effectively; backing up efficiently; and using PATH. (JOW)

  18. Characterization of an ultra-hard CMOS 64K static ram

    SciTech Connect

    Jenkins, W.C.; Martin, R.L.; Hughes, H.L.

    1987-12-01

    The authors irradiated radiation-hard 64K CMOS Static RAMs in the /sup 60/Co pool at dose rates of 3 and 70 Rads (SiO/sub 2/)/sec to simulate a space radiation environment. The devices failed due to write-failure at a total gamma dose in some cases greater than 50 MRads(SiO/sub 2/). Test Transistors were also irradiated and measured.

  19. MAXI/GSC detection of a recent low-level X-ray activity and a bright hard X-ray flare from V404 Cyg

    NASA Astrophysics Data System (ADS)

    Sugimoto, J.; Negoro, H.; Kawai, N.; Tomida, H.; Nakahira, S.; Ishikawa, M.; Nakagawa, Y. E.; Mihara, T.; Sugizaki, M.; Serino, M.; Shidatsu, M.; Takagi, T.; Matsuoka, M.; Arimoto, M.; Yoshii, T.; Tachibana, Y.; Ono, Y.; Fujiwara, T.; Yoshida, A.; Sakamoto, T.; Kawakubo, Y.; Ohtsuki, H.; Tsunemi, H.; Imatani, R.; Nakajima, M.; Tanaka, K.; Masumitsu, T.; Ueda, Y.; Kawamuro, T.; Hori, T.; Tanimoto, A.; Tsuboi, Y.; Kanetou, S.; Nakamura, Y.; Sasaki, R.; Yamauchi, M.; Itoh, D.; Furuya, K.; Yamaoka, K.; Morii, M.

    2016-01-01

    Since 2015 December 23, a series of hard X-rays detections of the Galactic black hole candidate V404 Cyg (GS 2023+338) were reported with Swift-BAT (Barthelmy et al 2015 GCN #18716; ATel #8455), Fermi-GBM (Jenke at al 2015, GCN #18719; ATel #8457), and INTEGRAL-IBIS/ISGRI (Malyshev et al. ATel #8458).

  20. Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica.

    PubMed

    Charcas-Lopez, Ma del Socorro; Garcia-Morales, Lorena; Pezet-Valdez, Marisol; Lopez-Camarillo, Cesar; Zamorano-Carrillo, Absalom; Marchat, Laurence A

    2014-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan. PMID:24534563

  1. Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica

    PubMed Central

    del Socorro Charcas-Lopez, Ma.; Garcia-Morales, Lorena; Pezet-Valdez, Marisol; Lopez-Camarillo, Cesar; Zamorano-Carrillo, Absalom; Marchat, Laurence A.

    2014-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan. PMID:24534563

  2. How 'hard' are hard-rock deformations?

    NASA Astrophysics Data System (ADS)

    van Loon, A. J.

    2003-04-01

    The study of soft-rock deformations has received increasing attention during the past two decades, and much progress has been made in the understanding of their genesis. It is also recognized now that soft-rock deformations—which show a wide variety in size and shape—occur frequently in sediments deposited in almost all types of environments. In spite of this, deformations occurring in lithified rocks are still relatively rarely attributed to sedimentary or early-diagenetic processes. Particularly faults in hard rocks are still commonly ascribed to tectonics, commonly without a discussion about a possible non-tectonic origin at a stage that the sediments were still unlithified. Misinterpretations of both the sedimentary and the structural history of hard-rock successions may result from the negligence of a possible soft-sediment origin of specific deformations. It is therefore suggested that a re-evaluation of these histories, keeping the present-day knowledge about soft-sediment deformations in mind, may give new insights into the geological history of numerous sedimentary successions in which the deformations have not been studied from both a sedimentological and a structural point of view.

  3. RadSearch: a RIS/PACS integrated query tool

    NASA Astrophysics Data System (ADS)

    Tsao, Sinchai; Documet, Jorge; Moin, Paymann; Wang, Kevin; Liu, Brent J.

    2008-03-01

    Radiology Information Systems (RIS) contain a wealth of information that can be used for research, education, and practice management. However, the sheer amount of information available makes querying specific data difficult and time consuming. Previous work has shown that a clinical RIS database and its RIS text reports can be extracted, duplicated and indexed for searches while complying with HIPAA and IRB requirements. This project's intent is to provide a software tool, the RadSearch Toolkit, to allow intelligent indexing and parsing of RIS reports for easy yet powerful searches. In addition, the project aims to seamlessly query and retrieve associated images from the Picture Archiving and Communication System (PACS) in situations where an integrated RIS/PACS is in place - even subselecting individual series, such as in an MRI study. RadSearch's application of simple text parsing techniques to index text-based radiology reports will allow the search engine to quickly return relevant results. This powerful combination will be useful in both private practice and academic settings; administrators can easily obtain complex practice management information such as referral patterns; researchers can conduct retrospective studies with specific, multiple criteria; teaching institutions can quickly and effectively create thorough teaching files.

  4. Sturgeon conservation genomics: SNP discovery and validation using RAD sequencing.

    PubMed

    Ogden, R; Gharbi, K; Mugue, N; Martinsohn, J; Senn, H; Davey, J W; Pourkazemi, M; McEwing, R; Eland, C; Vidotto, M; Sergeev, A; Congiu, L

    2013-06-01

    Caviar-producing sturgeons belonging to the genus Acipenser are considered to be one of the most endangered species groups in the world. Continued overfishing in spite of increasing legislation, zero catch quotas and extensive aquaculture production have led to the collapse of wild stocks across Europe and Asia. The evolutionary relationships among Adriatic, Russian, Persian and Siberian sturgeons are complex because of past introgression events and remain poorly understood. Conservation management, traceability and enforcement suffer a lack of appropriate DNA markers for the genetic identification of sturgeon at the species, population and individual level. This study employed RAD sequencing to discover and characterize single nucleotide polymorphism (SNP) DNA markers for use in sturgeon conservation in these four tetraploid species over three biological levels, using a single sequencing lane. Four population meta-samples and eight individual samples from one family were barcoded separately before sequencing. Analysis of 14.4 Gb of paired-end RAD data focused on the identification of SNPs in the paired-end contig, with subsequent in silico and empirical validation of candidate markers. Thousands of putatively informative markers were identified including, for the first time, SNPs that show population-wide differentiation between Russian and Persian sturgeons, representing an important advance in our ability to manage these cryptic species. The results highlight the challenges of genotyping-by-sequencing in polyploid taxa, while establishing the potential genetic resources for developing a new range of caviar traceability and enforcement tools. PMID:23473098

  5. CIRs Observed by MSL/RAD on the Martian Surface

    NASA Astrophysics Data System (ADS)

    Lohf, Henning; Zeitlin, Cary; Rafkin, Scot; Koehler, Jan; Posner, Arik; Hassler, Donald M.; Heber, Bernd; Ehresmann, Bent; Wimmer-Schweingruber, Robert; Guo, Jingnan; Appel, Jan Kristoffer

    2016-07-01

    Co-rotating Interaction Regions (CIRs) are recurrent Stream Interaction Regions in the solar wind which are stable transient plasma structures lasting several solar rotations. They can modulate Galactic Cosmic Rays (GCRs) and to some extent result in a modulation of GCR induced secondary energetic particles on the Martian surface. The Mars Science Laboratory/ Radiation Assessment Detector (MSL/RAD) has been measuring the Martian Surface Radiation Environment for more than three years and observes this modulation effect. We will show that the effect of CIRs can be measured on the Martian surface with MSL/RAD and this can be used to derive the arrival times of CIRs at Mars. These can provide (limited) solar wind plasma properties in the vicinity of Mars and thus serve as important constraints for modeling atmospheric response to variations in the solar wind. We use multi spacecraft observations of the solar wind and compare them with the heliospheric MHD Model ENLIL to verify that a certain class of dose rate variation we see on the Martian surface is due to CIRs. We use ballistic back-mapping as well as a time-shift algorithm to map the plasma properties measured at individual spacecraft locations and times to Mars. We compare these predictions with those of the CCMC ENLIL heliospheric MHD simulations.

  6. Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance

    PubMed Central

    Jablonski, Angela M.; Lamitina, Todd; Liachko, Nicole F.; Sabatella, Mariangela; Lu, Jiayin; Zhang, Lei; Ostrow, Lyle W.; Gupta, Preetika; Wu, Chia-Yen; Doshi, Shachee; Mojsilovic-Petrovic, Jelena; Lans, Hannes; Wang, Jiou; Kraemer, Brian

    2015-01-01

    Misfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate in amyotrophic lateral sclerosis. Loss of RAD-23 suppresses the locomotor deficit of Caenorhabditis elegans engineered to express mutTDP-43 or mutSOD1 and also protects against aging and proteotoxic insults. Knockdown of RAD-23 is further neuroprotective against the toxicity of SOD1 and TDP-43 expression in mammalian neurons. Biochemical investigation indicates that RAD-23 modifies mutTDP-43 and mutSOD1 abundance, solubility, and turnover in association with altering the ubiquitination status of these substrates. In human amyotrophic lateral sclerosis spinal cord, we find that RAD-23 abundance is increased and RAD-23 is mislocalized within motor neurons. We propose a novel pathophysiological function for RAD-23 in the stabilization of mutated proteins that cause neurodegeneration. SIGNIFICANCE STATEMENT In this work, we identify RAD-23, a component of the protein homeostasis network and nucleotide excision repair pathway, as a modifier of the toxicity of two disease-causing, misfolding-prone proteins, SOD1 and TDP-43. Reducing the abundance of RAD-23 accelerates the degradation of mutant SOD1 and TDP-43 and reduces the cellular content of the toxic species. The existence of endogenous proteins that act as “anti-chaperones” uncovers new and general targets for therapeutic intervention. PMID:26490867

  7. Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52.

    PubMed

    Di Primio, Cristina; Galli, Alvaro; Cervelli, Tiziana; Zoppè, Monica; Rainaldi, Giuseppe

    2005-01-01

    When exogenous DNA is stably introduced in mammalian cells, it is typically integrated in random positions, and only a minor fraction enters a pathway of homologous recombination (HR). The complex Rad51/Rad52 is a major player in the management of exogenous DNA in eukaryotic organisms and plays a critical role in the choice of repair system. In Saccharomyces cerevisiae, the pathway of choice is HR, mediated by Rad52 (ScRad52), which differs slightly from its human homologue. Here, we present an approach that utilizes ScRad52 to enhance HR in human cells containing a specific substrate for recombination. Clones of HeLa cells were produced expressing functional ScRad52. These cells showed enhanced resistance to DNA damaging treatments and revealed a different distribution of Rad51 foci (a marker of recombination complex formation). More significantly, ScRad52 expression resulted in an up to 37-fold increase in gene targeting by HR. In the same cells, random integration of exogenous DNA was significantly reduced, consistent with the view that HR and non-homologous end joining are alternative competing pathways. Expression of ScRad52 could offer a major improvement for experiments requiring gene targeting by HR, both in basic research and in gene therapy studies. PMID:16106043

  8. Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52

    PubMed Central

    Di Primio, Cristina; Galli, Alvaro; Cervelli, Tiziana; Zoppè, Monica; Rainaldi, Giuseppe

    2005-01-01

    When exogenous DNA is stably introduced in mammalian cells, it is typically integrated in random positions, and only a minor fraction enters a pathway of homologous recombination (HR). The complex Rad51/Rad52 is a major player in the management of exogenous DNA in eukaryotic organisms and plays a critical role in the choice of repair system. In Saccharomyces cerevisiae, the pathway of choice is HR, mediated by Rad52 (ScRad52), which differs slightly from its human homologue. Here, we present an approach that utilizes ScRad52 to enhance HR in human cells containing a specific substrate for recombination. Clones of HeLa cells were produced expressing functional ScRad52. These cells showed enhanced resistance to DNA damaging treatments and revealed a different distribution of Rad51 foci (a marker of recombination complex formation). More significantly, ScRad52 expression resulted in an up to 37-fold increase in gene targeting by HR. In the same cells, random integration of exogenous DNA was significantly reduced, consistent with the view that HR and non-homologous end joining are alternative competing pathways. Expression of ScRad52 could offer a major improvement for experiments requiring gene targeting by HR, both in basic research and in gene therapy studies. PMID:16106043

  9. Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart

    PubMed Central

    Somyajit, Kumar; Saxena, Sneha; Babu, Sharath; Mishra, Anup; Nagaraju, Ganesh

    2015-01-01

    Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Taken together, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis. PMID:26354865

  10. A peptide nucleic acid targeting nuclear RAD51 sensitizes multiple myeloma cells to melphalan treatment.

    PubMed

    Alagpulinsa, David Abasiwani; Yaccoby, Shmuel; Ayyadevara, Srinivas; Shmookler Reis, Robert Joseph

    2015-01-01

    RAD51-mediated recombinational repair is elevated in multiple myeloma (MM) and predicts poor prognosis. RAD51 has been targeted to selectively sensitize and/or kill tumor cells. Here, we employed a peptide nucleic acid (PNA) to inhibit RAD51 expression in MM cells. We constructed a PNA complementary to a unique segment of the RAD51 gene promoter, spanning the transcription start site, and conjugated it to a nuclear localization signal (PKKKRKV) to enhance cellular uptake and nuclear delivery without transfection reagents. This synthetic construct, (PNArad51_nls), significantly reduced RAD51 transcripts in MM cells, and markedly reduced the number and intensity of de novo and melphalan-induced nuclear RAD51 foci, while increasing the level of melphalan-induced γH2AX foci. Melphalan alone markedly induced the expression of 5 other genes involved in homologous-recombination repair, yet suppression of RAD51 by PNArad51_nls was sufficient to synergize with melphalan, producing significant synthetic lethality of MM cells in vitro. In a SCID-rab mouse model mimicking the MM bone marrow microenvironment, treatment with PNArad51_nls ± melphalan significantly suppressed tumor growth after 2 weeks, whereas melphalan plus control PNArad4µ_nls was ineffectual. This study highlights the importance of RAD51 in myeloma growth and is the first to demonstrate that anti-RAD51 PNA can potentiate conventional MM chemotherapy. PMID:25996477

  11. Mdt1, a Novel Rad53 FHA1 Domain-Interacting Protein, Modulates DNA Damage Tolerance and G2/M Cell Cycle Progression in Saccharomyces cerevisiae

    PubMed Central

    Pike, Brietta L.; Yongkiettrakul, Suganya; Tsai, Ming-Daw; Heierhorst, Jörg

    2004-01-01

    The Rad53 kinase plays a central role in yeast DNA damage checkpoints. Rad53 contains two FHA phosphothreonine-binding domains that are required for Rad53 activation and possibly downstream signaling. Here we show that the N-terminal Rad53 FHA1 domain interacts with the RNA recognition motif, coiled-coil, and SQ/TQ cluster domain-containing protein Mdt1 (YBl051C). The interaction of Rad53 and Mdt1 depends on the structural integrity of the FHA1 phosphothreonine-binding site as well as threonine-305 of Mdt1. Mdt1 is constitutively threonine phosphorylated and hyperphosphorylated in response to DNA damage in vivo. DNA damage-dependent Mdt1 hyperphosphorylation depends on the Mec1 and Tel1 checkpoint kinases, and Mec1 can directly phosphorylate a recombinant Mdt1 SQ/TQ domain fragment. MDT1 overexpression is synthetically lethal with a rad53 deletion, whereas mdt1 deletion partially suppresses the DNA damage hypersensitivity of checkpoint-compromised strains and generally improves DNA damage tolerance. In the absence of DNA damage, mdt1 deletion leads to delayed anaphase completion, with an elongated cell morphology reminiscent of that of G2/M cell cycle mutants. mdt1-dependent and DNA damage-dependent cell cycle delays are not additive, suggesting that they act in the same pathway. The data indicate that Mdt1 is involved in normal G2/M cell cycle progression and is a novel target of checkpoint-dependent cell cycle arrest pathways. PMID:15024067

  12. Phase II Study of Temozolomide (TMZ) and Everolimus (RAD001) Therapy for Metastatic Melanoma

    PubMed Central

    Dronca, Roxana S.; Allred, Jacob B.; Perez, Domingo G.; Nevala, Wendy K.; Lieser, Elizabeth A.T.; Thompson, Michael; Maples, William J.; Creagan, Edward T.; Pockaj, Barbara A.; Kaur, Judith S.; Moore, Timothy D.; Marchello, Benjamin T.; Markovic, Svetomir N.

    2014-01-01

    Objective Mammalian target of rapamycin (mTOR) pathway is activated in malignant melanoma and in situ lesions as opposed to benign nevi. Inhibition of PI3K-Akt-mTOR signaling is implicated in sensitization of melanoma cells to alkylating agents [temozolomide (TMZ)] and inhibition of tumor angiogenesis. Methods We conducted a single-arm phase II multi-institution cooperative group study to assess the antitumor activity and safety profile of the combination of TMZ and the rapamycin derivative everolimus in patients with metastatic unresectable malignant melanoma. Patients received 10 mg/d of RAD001 for 5 of 7 days (ie, 50 mg/ wk) and 200 mg/m2/d of TMZ for 5 days each cycle. Results Of the first 39 eligible patients, 17 were PFS-9 successes, for a predetermined threshold of 18/39 patients for a positive trial. Overall, 21 of 48 patients were progression free at 9 weeks, for an event-free survival rate of 44% (95% confidence interval, 29%–59%). The median progression-free survival was 2.4 months and the median overall survival was 8.6 months. Four patients achieved a partial response; the median duration of response was 15.1 months. No complete remissions were observed. Treatment was in general well tolerated with only 1 patient discontinuing therapy due to toxicity (hyperlipidemia). Conclusions The combination of TMZ and RAD001 was well tolerated but failed to meet/exceed our study threshold for promising clinical activity in patients with metastatic melanoma. PMID:23357973

  13. Differential roles of XRCC2 in S-phase RAD51 focus formation induced by DNA replication inhibitors

    SciTech Connect

    Lim, C; Liu, N

    2004-05-14

    RAD51 proteins accumulate in discrete nuclear foci in response to DNA damage. Previous studies demonstrated that human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) are essential for the assembly of RAD51 foci induced by ionizing radiation and cross-linking agents. Here we report that XRCC2 also plays important roles in RAD51 focus formation induced by replication arrest during S-phase of cell cycle. In wild-type hamster V79 cells treated with hydroxyurea (HU), RAD51 protein form punctuate nuclear foci, accompanied by increased RAD51 protein level in both cytoplasmic and nuclear fractions, and increased association of RAD51 with chromatin. In contrast, xrcc2 hamster mutant irs1 cells are deficient in the formation of RAD51 foci after HU treatment, suggesting that the function of XRCC2 is required for the assembly of RAD51 at HU-induced stalled replication forks. Interestingly, we found that irs1 cells are able to form intact RAD51 foci in S-phase cells treated with thymidine (TR) or aphidicolin, although irs1 cells are hypersensitive to both HU and TR. Our findings suggest that there may be two distinct pathways (XRCC2-dependent or XRCC2-independent) involved in loading of RAD51 onto stalled replication forks, probably depending upon the structure of DNA lesions.

  14. Hard tissue laser procedures.

    PubMed

    Gimbel, C B

    2000-10-01

    A more conservative, less invasive treatment of the carious lesion has intrigued researchers and clinicians for decades. With over 170 million restorations placed worldwide each year, many of which could be treated using a laser, there exists an increasing need for understanding hard tissue laser procedures. An historical review of past scientific and clinical hard research, biophysics, and histology are discussed. A complete review of present applications and procedures along with their capabilities and limitations will give the clinician a better understanding. Clinical case studies, along with guidelines for tooth preparation and hard tissue laser applications and technological advances for diagnosis and treatment will give the clinician a look into the future. PMID:11048281

  15. Biochemical characterization of plant Rad52 protein from rice (Oryza sativa).

    PubMed

    Nair, Anuradha; Agarwal, Rachna; Chittela, Rajani Kant

    2016-09-01

    DNA damage in living cells is repaired by two main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Of all the genes promoting HR, Rad52 (Radiation sensitive 52) is an important gene which is found to be highly conserved across different species. It was believed that RAD52 is absent in plant systems until lately. However, recent genetic studies have shown the presence of RAD52 homologues in plants. Rad52 homologues in plant systems have not yet been characterized biochemically. In the current study, we bring out the biochemical properties of rice Rad52-2a protein. OsRad52-2a was over-expressed in Escherichia coli BL21 (DE3) cells and the protein was purified. The identity of purified OsRad52-2a protein was confirmed via peptide mass fingerprinting. Gel filtration and native PAGE analysis indicated that the OsRad52-2a protein in its native state probably formed an undecameric structure. Purified OsRad52-2a protein showed binding to single stranded DNA, double stranded DNA. Protein also mediated the renaturation of complementary single strands into duplex DNA in both agarose gel and FRET based assays. Put together, OsRad52-2a forms oligomeric structures and binds to ssDNA/dsDNA for mediating an important function like renaturation during homologous recombination. This study represents the first report on biochemical properties of OsRad52-2a protein from important crop like rice. This information will help in dissecting the recombination and repair machinery in plant systems. PMID:27156135

  16. Polymorphisms of homologous recombination RAD51, RAD51B, XRCC2, and XRCC3 genes and the risk of prostate cancer.

    PubMed

    Nowacka-Zawisza, Maria; Wiśnik, Ewelina; Wasilewski, Andrzej; Skowrońska, Milena; Forma, Ewa; Bryś, Magdalena; Różański, Waldemar; Krajewska, Wanda M

    2015-01-01

    Genetic polymorphisms in DNA repair genes may induce individual variations in DNA repair capacity, which may in turn contribute to the risk of cancer developing. Homologous recombination repair (HRR) plays a critical role in maintaining chromosomal integrity and protecting against carcinogenic factors. The aim of the present study was to evaluate the relationship between prostate cancer risk and the presence of single nucleotide polymorphisms (SNPs) in the genes involved in HRR, that is, RAD51 (rs1801320 and rs1801321), RAD51B (rs10483813 and rs3784099), XRCC2 (rs3218536), and XRCC3 (rs861539). Polymorphisms were analyzed by PCR-RFLP and Real-Time PCR in 101 patients with prostate adenocarcinoma and 216 age- and sex-matched controls. A significant relationship was detected between the RAD51 gene rs1801320 polymorphism and increased prostate cancer risk. Our results indicate that the RAD51 gene rs1801320 polymorphism may contribute to prostate cancer susceptibility in Poland. PMID:26339569

  17. Synthesis and in vitro anticancer evaluation of some 4,6-diamino-1,3,5-triazine-2-carbohydrazides as Rad6 ubiquitin conjugating enzyme inhibitors.

    PubMed

    Kothayer, Hend; Spencer, Sebastian M; Tripathi, Kaushlendra; Westwell, Andrew D; Palle, Komaraiah

    2016-04-15

    Series of 4-amino-6-(arylamino)-1,3,5-triazine-2-carbohydrazides (3a-e) and N'-phenyl-4,6-bis(arylamino)-1,3,5-triazine-2-carbohydrazides (6a-e), for ease of readership, we will abbreviate our compound names as 'new triazines', have been synthesized, based on the previously reported Rad6B-inhibitory diamino-triazinylmethyl benzoate anticancer agents TZ9 and 4-amino-N'-phenyl-6-(arylamino)-1,3,5-triazine-2-carbohydrazides. Synthesis of the target compounds was readily accomplished in two steps from either bis-aryl/aryl biguanides via reaction of phenylhydrazine or hydrazinehydrate with key 4-amino-6-bis(arylamino)/(arylamino)-1,3,5-triazine-2-carboxylate intermediates. These new triazine derivatives were evaluated for their abilities to inhibit Rad6B ubiquitin conjugation and in vitro anticancer activity against several human cancer cell lines: ovarian (OV90 and A2780), lung (H1299 and A549), breast (MCF-7 and MDA-MB231) and colon (HT29) cancer cells by MTS assays. All the 10 new triazines exhibited superior Rad6B inhibitory activities in comparison to selective Rad6 inhibitor TZ9 that was reported previously. Similarly, new triazines also showed better IC50 values in survival assays of various tumor cell lines. Particularly, new triazines 6a-c, exhibited lower IC50 (3.3-22μM) values compared to TZ9. PMID:26965855

  18. Space experiment "Cellular Responses to Radiation in Space (CellRad)": Hardware and biological system tests.

    PubMed

    Hellweg, Christine E; Dilruba, Shahana; Adrian, Astrid; Feles, Sebastian; Schmitz, Claudia; Berger, Thomas; Przybyla, Bartos; Briganti, Luca; Franz, Markus; Segerer, Jürgen; Spitta, Luis F; Henschenmacher, Bernd; Konda, Bikash; Diegeler, Sebastian; Baumstark-Khan, Christa; Panitz, Corinna; Reitz, Günther

    2015-11-01

    HEK cells to the β-rays emitted by the radiation source dose-dependently decreased cell growth and increased NF-κB activation. The signal of the fluorescent proteins after formaldehyde fixation was stable for at least six months after fixation, allowing storage of the MPUs after fixation for several months before the transport back to Earth and evaluation of the fluorescence intensity. In conclusion, these tests show the feasibility of CellRad on the ISS with the currently available transport mechanisms. PMID:26553641

  19. Identification of Laying-Related SNP Markers in Geese Using RAD Sequencing

    PubMed Central

    Yu, ShiGang; Chu, WeiWei; Zhang, LiFan; Han, HouMing; Zhao, RongXue; Wu, Wei; Zhu, JiangNing; Dodson, Michael V.; Wei, Wei; Liu, HongLin; Chen, Jie

    2015-01-01

    Laying performance is an important economical trait of goose production. As laying performance is of low heritability, it is of significance to develop a marker-assisted selection (MAS) strategy for this trait. Definition of sequence variation related to the target trait is a prerequisite of quantitating MAS, but little is presently known about the goose genome, which greatly hinders the identification of genetic markers for the laying traits of geese. Recently developed restriction site-associated DNA (RAD) sequencing is a possible approach for discerning large-scale single nucleotide polymorphism (SNP) and reducing the complexity of a genome without having reference genomic information available. In the present study, we developed a pooled RAD sequencing strategy for detecting geese laying-related SNP. Two DNA pools were constructed, each consisting of equal amounts of genomic DNA from 10 individuals with either high estimated breeding value (HEBV) or low estimated breeding value (LEBV). A total of 139,013 SNP were obtained from 42,291,356 sequences, of which 18,771,943 were for LEBV and 23,519,413 were for HEBV cohorts. Fifty-five SNP which had different allelic frequencies in the two DNA pools were further validated by individual-based AS-PCR genotyping in the LEBV and HEBV cohorts. Ten out of 55 SNP exhibited distinct allele distributions in these two cohorts. These 10 SNP were further genotyped in a goose population of 492 geese to verify the association with egg numbers. The result showed that 8 of 10 SNP were associated with egg numbers. Additionally, liner regression analysis revealed that SNP Record-111407, 106975 and 112359 were involved in a multiplegene network affecting laying performance. We used IPCR to extend the unknown regions flanking the candidate RAD tags. The obtained sequences were subjected to BLAST to retrieve the orthologous genes in either ducks or chickens. Five novel genes were cloned for geese which harbored the candidate laying

  20. Identification of Laying-Related SNP Markers in Geese Using RAD Sequencing.

    PubMed

    Yu, ShiGang; Chu, WeiWei; Zhang, LiFan; Han, HouMing; Zhao, RongXue; Wu, Wei; Zhu, JiangNing; Dodson, Michael V; Wei, Wei; Liu, HongLin; Chen, Jie

    2015-01-01

    Laying performance is an important economical trait of goose production. As laying performance is of low heritability, it is of significance to develop a marker-assisted selection (MAS) strategy for this trait. Definition of sequence variation related to the target trait is a prerequisite of quantitating MAS, but little is presently known about the goose genome, which greatly hinders the identification of genetic markers for the laying traits of geese. Recently developed restriction site-associated DNA (RAD) sequencing is a possible approach for discerning large-scale single nucleotide polymorphism (SNP) and reducing the complexity of a genome without having reference genomic information available. In the present study, we developed a pooled RAD sequencing strategy for detecting geese laying-related SNP. Two DNA pools were constructed, each consisting of equal amounts of genomic DNA from 10 individuals with either high estimated breeding value (HEBV) or low estimated breeding value (LEBV). A total of 139,013 SNP were obtained from 42,291,356 sequences, of which 18,771,943 were for LEBV and 23,519,413 were for HEBV cohorts. Fifty-five SNP which had different allelic frequencies in the two DNA pools were further validated by individual-based AS-PCR genotyping in the LEBV and HEBV cohorts. Ten out of 55 SNP exhibited distinct allele distributions in these two cohorts. These 10 SNP were further genotyped in a goose population of 492 geese to verify the association with egg numbers. The result showed that 8 of 10 SNP were associated with egg numbers. Additionally, liner regression analysis revealed that SNP Record-111407, 106975 and 112359 were involved in a multiplegene network affecting laying performance. We used IPCR to extend the unknown regions flanking the candidate RAD tags. The obtained sequences were subjected to BLAST to retrieve the orthologous genes in either ducks or chickens. Five novel genes were cloned for geese which harbored the candidate laying

  1. Running in Hard Times

    ERIC Educational Resources Information Center

    Berry, John N., III

    2009-01-01

    Roberta Stevens and Kent Oliver are campaigning hard for the presidency of the American Library Association (ALA). Stevens is outreach projects and partnerships officer at the Library of Congress. Oliver is executive director of the Stark County District Library in Canton, Ohio. They have debated, discussed, and posted web sites, Facebook pages,…

  2. Diffractive hard scattering

    SciTech Connect

    Berger, E.L.; Collins, J.C.; Soper, D.E.; Sterman, G.

    1986-03-01

    I discuss events in high energy hadron collisions that contain a hard scattering, in the sense that very heavy quarks or high P/sub T/ jets are produced, yet are diffractive, in the sense that one of the incident hadrons is scattered with only a small energy loss. 8 refs.

  3. Involvement of budding yeast Rad5 in translesion DNA synthesis through physical interaction with Rev1

    PubMed Central

    Xu, Xin; Lin, Aiyang; Zhou, Cuiyan; Blackwell, Susan R.; Zhang, Yiran; Wang, Zihao; Feng, Qianqian; Guan, Ruifang; Hanna, Michelle D.; Chen, Zhucheng; Xiao, Wei

    2016-01-01

    DNA damage tolerance (DDT) is responsible for genomic stability and cell viability by bypassing the replication block. In Saccharomyces cerevisiae DDT employs two parallel branch pathways to bypass the DNA lesion, namely translesion DNA synthesis (TLS) and error-free lesion bypass, which are mediated by sequential modifications of PCNA. Rad5 has been placed in the error-free branch of DDT because it contains an E3 ligase domain required for PCNA polyubiquitination. Rad5 is a multi-functional protein and may also play a role in TLS, since it interacts with the TLS polymerase Rev1. In this study we mapped the Rev1-interaction domain in Rad5 to the amino acid resolution and demonstrated that Rad5 is indeed involved in TLS possibly through recruitment of Rev1. Genetic analyses show that the dual functions of Rad5 can be separated and reconstituted. Crystal structure analysis of the Rad5–Rev1 interaction reveals a consensus RFF motif in the Rad5 N-terminus that binds to a hydrophobic pocket within the C-terminal domain of Rev1 that is highly conserved in eukaryotes. This study indicates that Rad5 plays a critical role in pathway choice between TLS and error-free DDT. PMID:27001510

  4. Targeted disruption of the Rad51 gene leads to lethality in embryonic mice.

    PubMed Central

    Tsuzuki, T; Fujii, Y; Sakumi, K; Tominaga, Y; Nakao, K; Sekiguchi, M; Matsushiro, A; Yoshimura, Y; MoritaT

    1996-01-01

    The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8692798

  5. Involvement of budding yeast Rad5 in translesion DNA synthesis through physical interaction with Rev1.

    PubMed

    Xu, Xin; Lin, Aiyang; Zhou, Cuiyan; Blackwell, Susan R; Zhang, Yiran; Wang, Zihao; Feng, Qianqian; Guan, Ruifang; Hanna, Michelle D; Chen, Zhucheng; Xiao, Wei

    2016-06-20

    DNA damage tolerance (DDT) is responsible for genomic stability and cell viability by bypassing the replication block. In Saccharomyces cerevisiae DDT employs two parallel branch pathways to bypass the DNA lesion, namely translesion DNA synthesis (TLS) and error-free lesion bypass, which are mediated by sequential modifications of PCNA. Rad5 has been placed in the error-free branch of DDT because it contains an E3 ligase domain required for PCNA polyubiquitination. Rad5 is a multi-functional protein and may also play a role in TLS, since it interacts with the TLS polymerase Rev1. In this study we mapped the Rev1-interaction domain in Rad5 to the amino acid resolution and demonstrated that Rad5 is indeed involved in TLS possibly through recruitment of Rev1. Genetic analyses show that the dual functions of Rad5 can be separated and reconstituted. Crystal structure analysis of the Rad5-Rev1 interaction reveals a consensus RFF motif in the Rad5 N-terminus that binds to a hydrophobic pocket within the C-terminal domain of Rev1 that is highly conserved in eukaryotes. This study indicates that Rad5 plays a critical role in pathway choice between TLS and error-free DDT. PMID:27001510

  6. High RAD54B expression: an independent predictor of postoperative distant recurrence in colorectal cancer patients.

    PubMed

    Nagai, Yuzo; Yamamoto, Yoko; Yasuhara, Takaaki; Hata, Keisuke; Nishikawa, Takeshi; Tanaka, Toshiaki; Tanaka, Junichiro; Kiyomatsu, Tomomichi; Kawai, Kazushige; Nozawa, Hiroaki; Kazama, Shinsuke; Yamaguchi, Hironori; Ishihara, Soichiro; Sunami, Eiji; Yamanaka, Takeharu; Miyagawa, Kiyoshi; Watanabe, Toshiaki

    2015-08-28

    We recently reported a specific mechanism that RAD54B, an important factor in homologous recombination, promotes genomic instability via the degradation of p53 protein in vitro. However, clinical significance of RAD54Bin colorectal cancer (CRC) remains unclear. Thus we analyzed RAD54B geneexpression in CRC patients. Using the training set (n = 123), the optimal cut-off value for stratification was determined, and validated in another cohort (n = 89). Kaplan-Meier plots showed that distant recurrence free survival was significantly lesser in high RAD54B expression group compared with that of low expression group in both training (P = 0.0013) and validation (P = 0.024) set. Multivariate analysis using Cox proportional-hazards model showed that high RAD54B expression was an independent predictor in both training (hazard ratio, 4.31; 95% CI, 1.53-13.1; P = 0.0060) and validation (hazard ratio, 3.63; 95% CI, 1.23-10.7; P = 0.021) set. In addition, a negative significant correlation between RAD54B and CDKN1A, a target gene of p53, was partially confirmed, suggesting that RAD54B functions via the degradation of p53 protein even in clinical samples. This study first demonstrated RAD54B expression has potential to serve as a novel prognostic biomarker, particularly for distant recurrence in CRC patients. PMID:26046797

  7. High RAD54B expression: an independent predictor of postoperative distant recurrence in colorectal cancer patients

    PubMed Central

    Nagai, Yuzo; Yamamoto, Yoko; Yasuhara, Takaaki; Hata, Keisuke; Nishikawa, Takeshi; Tanaka, Toshiaki; Tanaka, Junichiro; Kiyomatsu, Tomomichi; Kawai, Kazushige; Nozawa, Hiroaki; Kazama, Shinsuke; Yamaguchi, Hironori; Ishihara, Soichiro; Sunami, Eiji; Yamanaka, Takeharu; Miyagawa, Kiyoshi; Watanabe, Toshiaki

    2015-01-01

    We recently reported a specific mechanism that RAD54B, an important factor in homologous recombination, promotes genomic instability via the degradation of p53 protein in vitro. However, clinical significance of RAD54Bin colorectal cancer (CRC) remains unclear. Thus we analyzed RAD54B geneexpression in CRC patients. Using the training set (n = 123), the optimal cut-off value for stratification was determined, and validated in another cohort (n = 89). Kaplan–Meier plots showed that distant recurrence free survival was significantly lesser in high RAD54B expression group compared with that of low expression group in both training (P = 0.0013) and validation (P = 0.024) set. Multivariate analysis using Cox proportional-hazards model showed that high RAD54B expression was an independent predictor in both training (hazard ratio, 4.31; 95% CI, 1.53–13.1; P = 0.0060) and validation (hazard ratio, 3.63; 95% CI, 1.23–10.7; P = 0.021) set. In addition, a negative significant correlation between RAD54B and CDKN1A, a target gene of p53, was partially confirmed, suggesting that RAD54B functions via the degradation of p53 protein even in clinical samples. This study first demonstrated RAD54B expression has potential to serve as a novel prognostic biomarker, particularly for distant recurrence in CRC patients. PMID:26046797

  8. Cyclic hypoxia does not alter RAD51 expression or PARP inhibitor cell kill in tumor cells.

    PubMed

    Kumareswaran, Ramya; Chaudary, Naz; Jaluba, Karolina; Meng, Alice; Sykes, Jenna; Borhan, Asm; Hill, Richard P; Bristow, Robert G

    2015-09-01

    Solid tumors contain regions of chronic and cyclic hypoxia. Chronic hypoxia can downregulate RAD51 and sensitize cells to PARP inhibition. Herein, we show that RAD51 expression, cell survival and toxicity to PARP inhibition is not affected under cyclic hypoxic conditions. This suggests that PARP inhibition may be selectively toxic in tumor sub-regions associated with chronic hypoxia. PMID:25842967

  9. Structure of the human DNA-repair protein RAD52 containing surface mutations.

    PubMed

    Saotome, Mika; Saito, Kengo; Onodera, Keiichi; Kurumizaka, Hitoshi; Kagawa, Wataru

    2016-08-01

    The Rad52 protein is a eukaryotic single-strand DNA-annealing protein that is involved in the homologous recombinational repair of DNA double-strand breaks. The isolated N-terminal half of the human RAD52 protein (RAD52(1-212)) forms an undecameric ring structure with a surface that is mostly positively charged. In the present study, it was found that RAD52(1-212) containing alanine mutations of the charged surface residues (Lys102, Lys133 and Glu202) is highly amenable to crystallization. The structure of the mutant RAD52(1-212) was solved at 2.4 Å resolution. The structure revealed an association between the symmetry-related RAD52(1-212) rings, in which a partially unfolded, C-terminal region of RAD52 extended into the DNA-binding groove of the neighbouring ring in the crystal. The alanine mutations probably reduced the surface entropy of the RAD52(1-212) ring and stabilized the ring-ring association observed in the crystal. PMID:27487923

  10. RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates

    SciTech Connect

    Reynolds, P.; Weber, S.; Prakash, L.

    1985-01-01

    The RAD6 gene of Saccharomyces cerevisiae is required for postreplication repair of UV-damaged DNA, for induced mutagenesis, and for sporulation. The authors have mapped the transcripts and determined the nucleotide sequence of the cloned RAD6 gene. The RAD6 gene encodes two transcripts of 0.98 and 0.86 kilobases which differ only in their 3' termini. The transcribed region contains an open reading frame of 516 nucleotides. The rad6-1 and rad6-3 mutant alleles, which the authors have cloned and sequenced, introduce amber and ochre nonsense mutations, respectively into the open reading frame, proving that it encodes the RAD6 protein. The RAD6 protein predicted by the nucleotide sequence is 172 amino acids long, has a molecular weight of 19,704, and contains 23.3% acidic and 11.6% basic residues. Its most striking feature is the highly acidic carboxyl terminus: 20 of the 23 terminal amino acids are acidic, including 13 consecutive aspartates. RAD6 protein thus resembles high mobility group proteins HMG-1 and HMG-2, which each contain a carboxyl-proximal tract of acidic amino acids. 48 references, 6 figures.

  11. Radiation Hardness Assurance (RHA) Guideline

    NASA Technical Reports Server (NTRS)

    Campola, Michael J.

    2016-01-01

    Radiation Hardness Assurance (RHA) consists of all activities undertaken to ensure that the electronics and materials of a space system perform to their design specifications after exposure to the mission space environment. The subset of interests for NEPP and the REAG, are EEE parts. It is important to register that all of these undertakings are in a feedback loop and require constant iteration and updating throughout the mission life. More detail can be found in the reference materials on applicable test data for usage on parts.

  12. RadSTAR L-Band Imaging Scatterometer: Performance Assessment

    NASA Technical Reports Server (NTRS)

    Rincon, Rafael; Hildebrand, Peter; Hilliard, Larry

    2007-01-01

    RadSTAR is an instrument development program aimed at combining a radiometer and a scatterometer system into a highly compact configuration that uses a single, electronically scanned antenna to provide co-located and simultaneous measurements of emission and backscatter for airborne and spaceborne applications [I]. The program was designed to map soil moisture and ocean salinity, both important components of the water cycle, and to map sea ice density and thickness, an important factor in ocean-atmosphere heat exchange in Polar Regions. The accuracy in estimation of these and a number of other Earth science parameters can be greatly enhanced by providing the co-aligned radar/radiometer microwave measurements. For instance, radiometer estimates of soil moisture from soil emission are affected by emission from vegetation, and from the roughness of the surface. Complementary measurements using the scatterometer can be used to evaluate the vegetation and surface roughness effects. Hence, the combined observations can provide an improved estimate. As with soil moisture, the ocean salinity is a function of the microwave emission from the sea surface temperature (SST) and sea roughness. There, the addition of radar backscatter measurements of sea roughness enables the correction of the emissivity and provide more accurate estimates of ocean salinity. Similar arguments can be made for other important Earth science parameters. This paper discusses the RadSTAR program, the radar system design, calibration, and digital beamforming techniques, and presents preliminary analysis of the data collected during the test flights. The data sets obtained during the flights and during the radar calibration in the anechoic chamber are also employed to asses the performance of the radar. The paper also discusses the Digital Beamforming Synthetic Aperture Radar (DBSAR) processor, a real-time processor recently developed for the LIS instrument which enables beam synthesis, fine resolutions

  13. The ras-related protein rad associates with the cytoskeleton in a non-lipid-dependent manner.

    PubMed

    Bilan, P J; Moyers, J S; Kahn, C R

    1998-08-01

    Rad is the prototypic member of a new family of Ras-related proteins (Rad, Gem, and Kir) which lack typical C-terminal amino acid motifs for isoprenylation. In mouse C2C12 muscle cell lines about 50% of Rad protein resides in the cytosol and behaves as a hydrophilic protein partitioning away from TX-114. The remainder of Rad is associated with plasma and internal membranes. The association of Rad with the membrane does not occur through the lipid bilayer, but instead depends on the interaction of Rad with the cytoskeleton or membrane skeleton. In contrast to Ras, biosynthetic labeling of cellular proteins in C2Cl2 cells with [3H]palmitic acid demonstrates that Rad is not modified with this fatty acid, and inhibition of isoprenylation with lovastatin treatment has no effect on Rad subcellular distribution. Furthermore, removal of the C-terminal 11 amino acids that are precisely conserved in all three Rad family members has no effect on Rad subcellular distribution. Addition of the 9 amino acids from the C-terminus of H-Ras to the truncated Rad protein results in a redistribution of Rad from the cytosol to the membrane skeleton without the presence of any detectable lipid modification of the chimeric protein. These data suggest that Rad possesses unique cellular localization signals which, in contrast to other Ras-related family members, do not depend on the lipid modification of the C-terminus. PMID:9683526

  14. Pleiotropic defects caused by loss of the proteasome-interacting factors Rad23 and Rpn10 of Saccharomyces cerevisiae.

    PubMed Central

    Lambertson, D; Chen, L; Madura, K

    1999-01-01

    Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant. A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered. PMID:10471701

  15. Mutations in RAD21 Disrupt Regulation of APOB in Patients with Chronic Intestinal Pseudo-obstruction

    PubMed Central

    Bonora, Elena; Bianco, Francesca; Cordeddu, Lina; Bamshad, Michael; Francescatto, Ludmila; Dowless, Dustin; Stanghellini, Vincenzo; Cogliandro, Rosanna F.; Lindberg, Greger; Mungan, Zeynel; Cefle, Kivanc; Ozcelik, Tayfun; Palanduz, Sukru; Ozturk, Sukru; Gedikbasi, Asuman; Gori, Alessandra; Pippucci, Tommaso; Graziano, Claudio; Volta, Umberto; Caio, Giacomo; Barbara, Giovanni; D'Amato, Mauro; Seri, Marco; Katsanis, Nicholas; Romeo, Giovanni; De Giorgio, Roberto

    2015-01-01

    Background & Aims Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimicks a mechanical sub-occlusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and indentify potential biomarkers. Methods We performed whole-exome sequencing of genomic DNA from patients with familial CIPO syndrome. Blood and lymphoblastoid cells were collected from patients and controls (individuals without CIPO); levels of mRNA and proteins were analyzed by quantitative reverse transcription PCR, immunoblot, and mobility shift assays. cDNAs were transfected into HEK293 cells. Expression of rad21 was suppressed in zebrafish embryos using a splice-blocking morpholino (rad21a MO). Gut tissues were collected and analyzed. Results We identified a homozygous mutation (p.622, encodes Ala>Thr) in RAD21 in patients from a consanguineous family with CIPO. Expression of RUNX1, a target of RAD21, was reduced in cells from patients with CIPO compared with controls. In zebrafish, suppression of rad21a reduced expression of runx1; this phenotype was corrected by injection of human RAD21 mRNA, but not with the mRNA from the mutated p.622 allele. rad21a MO zebrafish had delayed intestinal transit and greatly reduced numbers of enteric neurons, similar to patients with CIPO. This defect was greater in zebrafish with suppressed expression of ret and rad21, indicating their interaction in regulation of gut neurogenesis. The promoter region of APOB bound RAD21 but not RAD21 p.622 Ala>Thr; expression of wild-type RAD21 in HEK293 cells repressed expression of APOB, compared with control vector. The gut-specific isoform of APOB (APOB48) is overexpressed in sera from patients with CIPO who carry the RAD21 mutation. APOB48 is also overexpressed in sporadic CIPO in sera and gut biopsies. Conclusions Some patients with CIPO carry mutations in RAD21 that disrupt the ability of

  16. Hardness of irradiated poly(methyl methacrylate) at elevated temperatures

    SciTech Connect

    Lu, K.-P.; Lee, Sanboh; Cheng, Cheu Pyeng

    2001-08-15

    The decrease in hardness induced by gamma irradiation in poly(methyl methacrylate) (PMMA) has been investigated. The hardness is assumed to decrease linearly with the concentration of radiation-induced defects. Annealing at high temperatures induces defect annihilation as tracked by an increase in hardness. The annihilation follows first-order kinetics during isothermal annealing. The dependence of hardness on the reciprocal of the time constant satisfies the Arrhenius equation, and the corresponding activation energy of the kinetic process decreases with increasing dose. The hardness of postannealed PMMA decreases linearly with increasing dose. {copyright} 2001 American Institute of Physics.

  17. Basic features of low-temperature plasma formation in the course of composite coating synthesis at the active faces of complex contoured hard tools

    NASA Astrophysics Data System (ADS)

    Brzhozovsky, B. M.; Zimnyakov, D. A.; Zinina, E. P.; Martynov, V. V.; Pleshakova, E. S.; Yuvchenko, S. A.

    2016-04-01

    Basic features of combined-discharge low-temperature plasma formation around the surfaces of complex-contoured metal units are considered. It is shown that it makes the possibilities for synthesis of hardened high-durable coatings of hard tools appropriate for material processing in extreme load-temperature conditions. Experimental study of the coating formation was carried out in combination with the analysis of emission spectra of a low-temperature plasma cloud. Some practical examples of the coating applications are presented.

  18. Panasonic dosimetry system performance testing and results at nuclear accident dose levels 500 rad to 10,000 rad

    SciTech Connect

    Klueber, M.R.

    1998-04-06

    Panasonic thermoluminescent dosimeters (TLDs) are used as the photon dose assessment part of the personal nuclear accident dosimeter (PNAD) and may be used for the same purpose with the fixed nuclear accident dosimeter (FNAD). To demonstrate compliance with 10CFR835.1304 (and, its predecessor, DOE Order 5480.11), several sets of dosimeters were irradiated to photon doses above the upper limit of the DOELAP testing standard, DOE/EH-0026 and DOE/EH-0027. The upper range of the test was 10,000 rads, using both low energy (70 keV) and high energy (662 keV and 1,332 keV) sources. The testing indicated that the Panasonic TLD system is capable of meeting the requirements of 10CFR835.1304 and DOE Order 5480.11.

  19. CLIC-ACM: generic modular rad-hard data acquisition system based on CERN GBT versatile link

    NASA Astrophysics Data System (ADS)

    Bielawski, B.; Locci, F.; Magnoni, S.

    2015-01-01

    CLIC is a world-wide collaboration to study the next ``terascale'' lepton collider, relying upon a very innovative concept of two-beam-acceleration. This accelerator, currently under study, will be composed of the subsequence of 21000 two-beam-modules. Each module requires more than 300 analogue and digital signals which need to be acquired and controlled in a synchronous way. CLIC-ACM (Acquisition and Control Module) is the 'generic' control and acquisition module developed to accommodate the controls of all these signals for various sub-systems and related specification in term of data bandwidth, triggering and timing synchronization. This paper describes the system architecture with respect to its radiation-tolerance, power consumption and scalability.

  20. A Library of Rad Hard Mixed-Voltage/Mixed-Signal Building Blocks for Integration of Avionics Systems for Deep Space

    NASA Technical Reports Server (NTRS)

    Mojarradi, M. M.; Blaes, B.; Kolawa, E. A.; Blalock, B. J.; Li, H. W.; Buck, K.; Houge, D.

    2001-01-01

    To build the sensor intensive system-on-a-chip for the next generation spacecrafts for deep space, Center for Integration of Space Microsystems at JPL (CISM) takes advantage of the lower power rating and inherent radiation resistance of Silicon on Insulator technology (SOI). We are developing a suite of mixed-voltage and mixed-signal building blocks in Honeywell's SOI process that can enable the rapid integration of the next generation avionics systems with lower power rating, higher reliability, longer life, and enhanced radiation tolerance for spacecrafts such as the Europa Orbiter and Europa Lander. The mixed-voltage building blocks are predominantly for design of adaptive power management systems. Their design centers around an LDMOS structure that is being developed by Honeywell, Boeing Corp, and the University of Idaho. The mixed-signal building blocks are designed to meet the low power, extreme radiation requirement of deep space applications. These building blocks are predominantly used to interface analog sensors to the digital CPU of the next generation avionics system on a chip. Additional information is contained in the original extended abstract.

  1. Rad-hard vertical JFET switch for the HV-MUX system of the ATLAS upgrade Inner Tracker

    NASA Astrophysics Data System (ADS)

    Fernández-Martínez, P.; Ullán, M.; Flores, D.; Hidalgo, S.; Quirion, D.; Lynn, D.

    2016-01-01

    This work presents a new silicon vertical JFET (V-JFET) device, based on the trenched 3D-detector technology developed at IMB-CNM, to be used as a switch for the High-Voltage powering scheme of the ATLAS upgrade Inner Tracker. The optimization of the device characteristics is performed by 2D and 3D TCAD simulations. Special attention has been paid to the on-resistance and the switch-off and breakdown voltages to meet the specific requirements of the system. In addition, a set of parameter values has been extracted from the simulated curves to implement a SPICE model of the proposed V-JFET transistor. As these devices are expected to operate under very high radiation conditions during the whole experiment life-time, a study of the radiation damage effects and the expected degradation of the device performance is also presented at the end of the paper.

  2. Determination of radon concentration in water using RAD7 with RAD H{sub 2}O accessories

    SciTech Connect

    Malik, M. F. I.; Rabaiee, N. A.; Jaafar, M. S.

    2015-04-24

    In the last decade, the radon issue has become one of the major problems of radiation protection. Radon exposure occurs when using water for showering, washing dishes, cooking and drinking water. RAD7 and Rad H20 accessories were used in order to measure radon concentration in water sample. In this study, four types of water were concerns which are reverse osmosis (drinking water), mineral water, tap water and well water. Reverse osmosis (drinking water) and mineral water were bought from the nearest supermarket while tap water and well water were taken from selected areas of Pulau Pinang and Kedah. Total 20 samples were taken with 5 samples for each type of water. The measured radon concentration ranged from 2.9±2.9 to 79.5±17 pCi/L, 2.9±2.9 to 67.8±16 pCi/L, 15.97±7 to 144.25±24 pCi/L and 374.89±37 to 6409.03±130 pCi/L in reverse osmosis (drinking water), mineral water, tap water and well water. Well water has the highest radon compared to others. It was due to their geological element such as granite. Results for all types of water are presented and compared with maximum contamination limit (MCL) recommended by United State Environmental Protection Agency (USEPA) which is 300pCi/L. Reverse osmosis water, mineral water and tap water were fall below MCL. However, well water was exceeded maximum level that was recommended. Thus, these findings were suggested that an action should be taken to reduce radon concentration level in well water as well as reduce a health risk towards the public.

  3. Sen1, the yeast homolog of human senataxin, plays a more direct role than Rad26 in transcription coupled DNA repair.

    PubMed

    Li, Wentao; Selvam, Kathiresan; Rahman, Sheikh A; Li, Shisheng

    2016-08-19

    Rad26, a DNA dependent ATPase that is homologous to human CSB, has been well known to play an important role in transcription coupled DNA repair (TCR) in the yeast Saccharomyces cerevisiae Sen1, a DNA/RNA helicase that is essential for yeast cell viability and homologous to human senataxin, has been known to be required for transcriptional termination of short noncoding RNA genes and for a fail-safe transcriptional termination mechanism of protein-coding genes. Sen1 has also been shown to protect the yeast genome from transcription-associated recombination by resolving RNA:DNA hybrids naturally formed during transcription. Here, we show that the N-terminal non-essential region of Sen1 plays an important role in TCR, whereas the C-terminal nonessential region and the helicase activity of Sen1 are largely dispensable for the repair. Unlike Rad26, which becomes completely dispensable for TCR in cells lacking the TCR repressor Spt4, Sen1 is still required for efficient TCR in the absence of Spt4. Also unlike Rad26, which is important for repair at many but not all damaged sites in the transcribed strand of a gene, Sen1 is required for efficient repair at essentially all the damaged sites. Our results indicate that Sen1 plays a more direct role than Rad26 in TCR. PMID:27179024

  4. Human DNA Helicase B Functions in Cellular Homologous Recombination and Stimulates Rad51-Mediated 5′-3′ Heteroduplex Extension In Vitro

    PubMed Central

    Liu, Hanjian; Yan, Peijun; Fanning, Ellen

    2015-01-01

    Homologous recombination is involved in the repair of DNA damage and collapsed replication fork, and is critical for the maintenance of genomic stability. Its process involves a network of proteins with different enzymatic activities. Human DNA helicase B (HDHB) is a robust 5′-3′ DNA helicase which accumulates on chromatin in cells exposed to DNA damage. HDHB facilitates cellular recovery from replication stress, but its role in DNA damage response remains unclear. Here we report that HDHB silencing results in reduced sister chromatid exchange, impaired homologous recombination repair, and delayed RPA late-stage foci formation induced by ionizing radiation. Ectopically expressed HDHB colocalizes with Rad51, Rad52, RPA, and ssDNA. In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5′-3′ direction. A helicase-defective mutant HDHB failed to promote this reaction. Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5′-3′ heteroduplex extension during Rad51-mediated strand exchange in vitro. PMID:25617833

  5. Hard Times Hit Schools

    ERIC Educational Resources Information Center

    McNeil, Michele

    2008-01-01

    Hard-to-grasp dollar amounts are forcing real cuts in K-12 education at a time when the cost of fueling buses and providing school lunches is increasing and the demands of the federal No Child Left Behind Act still loom larger over states and districts. "One of the real challenges is to continue progress in light of the economy," said Gale Gaines,…

  6. Work Hard. Be Nice

    ERIC Educational Resources Information Center

    Mathews, Jay

    2009-01-01

    In 1994, fresh from a two-year stint with Teach for America, Mike Feinberg and Dave Levin inaugurated the Knowledge Is Power Program (KIPP) in Houston with an enrollment of 49 5th graders. By this Fall, 75 KIPP schools will be up and running, setting children from poor and minority families on a path to college through a combination of hard work,…

  7. SUPER HARD SURFACED POLYMERS

    SciTech Connect

    Mansur, Louis K; Bhattacharya, R; Blau, Peter Julian; Clemons, Art; Eberle, Cliff; Evans, H B; Janke, Christopher James; Jolly, Brian C; Lee, E H; Leonard, Keith J; Trejo, Rosa M; Rivard, John D

    2010-01-01

    High energy ion beam surface treatments were applied to a selected group of polymers. Of the six materials in the present study, four were thermoplastics (polycarbonate, polyethylene, polyethylene terephthalate, and polystyrene) and two were thermosets (epoxy and polyimide). The particular epoxy evaluated in this work is one of the resins used in formulating fiber reinforced composites for military helicopter blades. Measures of mechanical properties of the near surface regions were obtained by nanoindentation hardness and pin on disk wear. Attempts were also made to measure erosion resistance by particle impact. All materials were hardness tested. Pristine materials were very soft, having values in the range of approximately 0.1 to 0.5 GPa. Ion beam treatment increased hardness by up to 50 times compared to untreated materials. For reference, all materials were hardened to values higher than those typical of stainless steels. Wear tests were carried out on three of the materials, PET, PI and epoxy. On the ion beam treated epoxy no wear could be detected, whereas the untreated material showed significant wear.

  8. Rad51C deficiency destabilizes XRCC3, impairs recombination and radiosensitizes S/G2-phase cells

    SciTech Connect

    Lio, Yi-Ching; Schild, David; Brenneman, Mark A.; Redpath, J. Leslie; Chen, David J.

    2004-05-01

    The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, XRCC3) are expressed in mitotically growing cells, and are thought to play mediating roles in homologous recombination, though their precise functions remain unclear. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA cross-linking agent mitomycin C, and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G{sub 2}/M phases of the cell cycle but not in G{sub 1} phase. Together, these results provide direct cellular evidence for the importance of human Rad51C in homologous recombinational repair.

  9. Without Binding ATP, Human Rad51 Does Not Form Helical Filaments on ssDNA.

    PubMed

    Schay, Gusztáv; Borka, Bálint; Kernya, Linda; Bulyáki, Éva; Kardos, József; Fekete, Melinda; Fidy, Judit

    2016-03-10

    Construction of the presynaptic filament (PSF) of proper helical structure by Rad51 recombinases is a prerequisite of the progress of homologous recombination repair. We studied the contribution of ATP-binding to this structure of wt human Rad51 (hRad51). We exploited the protein-dissociation effect of high hydrostatic pressure to determine the free energy of dissociation of the protomer interfaces in hRad51 oligomer states and used electron microscopy to obtain topological parameters. Without cofactors ATP and Ca(2+) and template DNA, hRad51 did not exist in monomer form, but it formed rodlike long filaments without helical order. ΔG(diss) indicated a strong inherent tendency of aggregation. Binding solely ssDNA left the filament unstructured with slightly increased ΔG(diss). Adding only ATP and Ca(2+) to the buffer disintegrated the self-associated rods into rings and short helices of further increased ΔG(diss). Rad51 binding to ssDNA only with ATP and Ca bound could lead to ordered helical filament formation of proper pitch size with interface contacts of K(d) ∼ 2 × 10(-11) M, indicating a structure of outstanding stability. ATP/Ca binding increased the ΔG(diss) of protomer contacts in the filament by 16 kJ/mol. The results emphasize that ATP-binding in the PSF of hRad51 has an essential, yet purely structural, role. PMID:26890079

  10. The role of recombination and RAD52 in mutation of chromosomal DNA transformed into yeast.

    PubMed Central

    Larionov, V; Graves, J; Kouprina, N; Resnick, M A

    1994-01-01

    While transformation is a prominent tool for genetic analysis and genome manipulation in many organisms, transforming DNA has often been found to be unstable relative to established molecules. We determined the potential for transformation-associated mutations in a 360 kb yeast chromosome III composed primarily of unique DNA. Wild-type and rad52 Saccharomyces cerevisiae strains were transformed with either a homologous chromosome III or a diverged chromosome III from S. carlsbergensis. The host strain chromosome III had a conditional centromere allowing it to be lost on galactose medium so that recessive mutations in the transformed chromosome could be identified. Following transformation of a RAD+ strain with the homologous chromosome, there were frequent changes in the incoming chromosome, including large deletions and mutations that do not lead to detectable changes in chromosome size. Based on results with the diverged chromosome, interchromosomal recombinational interactions were the source of many of the changes. Even though rad52 exhibits elevated mitotic mutation rates, the percentage of transformed diverged chromosomes incapable of substituting for the resident chromosome was not increased in rad52 compared to the wild-type strain, indicating that the mutator phenotype does not extend to transforming chromosomal DNA. Based on these results and our previous observation that the incidence of large mutations is reduced during the cloning of mammalian DNA into a rad52 as compared to a RAD+ strain, a rad52 host is well-suited for cloning DNA segments in which gene function must be maintained. Images PMID:7937151

  11. SUB-M-RAD ANGULAR STABILITY MEASUREMENTS BY USE OF LONG TRACE PROFILER BASED SYSTEMS.

    SciTech Connect

    QIAN,S.

    1999-07-23

    High accuracy angle measurement at the sub-{mu}rad level requires extremely high instrument stability. In order to reach sub-{mu}rad stability (0.1 arc second or less) over long time periods, it is necessary to maintain the test object and almost all of the optical components in the measuring instrument in very steady positions. However, mechanical force relaxation, thermal expansion, and asymmetric structures produce angular and linear displacements in the system resulting in angular measurement error. A Long-Trace-Profiler (LTP)-based stable equipment is used to test precision angular stability with sub-{mu}rad resolution. Long term stability over 15 hours has been measured on different kind of mechanical structures. Temperature monitoring during the tests is extremely important. Some test results showing the effects of thermal variations are presented, which indicate that temperature stability on the order of 0.1 C is absolutely necessary for repeatable sub-{mu}rad measurements. The optical method, using optics with an even number of reflecting surfaces (for example, a right angle prism, pentaprism, or rhomboid prism) to reduce the influence of existing angular displacement, is introduced and the comparison measurement is presented. An optical fiber transfer line is able to reduce the laser angular shift from about 10 {mu}rad to a level of 0.3 {mu}rad rms. Careful system configuration, design and operation are very important for the sub-{mu}rad angle stability.

  12. Survival of the Replication Checkpoint Deficient Cells Requires MUS81-RAD52 Function

    PubMed Central

    Murfuni, Ivana; Basile, Giorgia; Subramanyam, Shyamal; Malacaria, Eva; Bignami, Margherita; Spies, Maria; Franchitto, Annapaola; Pichierri, Pietro

    2013-01-01

    In checkpoint-deficient cells, DNA double-strand breaks (DSBs) are produced during replication by the structure-specific endonuclease MUS81. The mechanism underlying MUS81-dependent cleavage, and the effect on chromosome integrity and viability of checkpoint deficient cells is only partly understood, especially in human cells. Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate. Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81. Moreover, in CHK1-deficient cells depletion of RAD52, but not of MUS81, rescues chromosome instability observed after replication fork stalling. However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion. Our findings reveal a novel RAD52/MUS81-dependent mechanism that promotes cell viability and genome integrity in checkpoint-deficient cells, and disclose the involvement of MUS81 to multiple processes after replication stress. PMID:24204313

  13. [Cloning, expression and protective efficacy evaluation of radiation sensitive protein 23 (RAD23) from Schistosoma japonicum].

    PubMed

    Li, Changjian; Zhang, Min; Hong, Yang; Han, Yanhui; Cao, Xiaodan; Han, Hongxiao; Fu, Zhiqiang; Zhu, Chuangang; Lu, Ke; Li, Hao; Lin, Jiaojiao

    2014-11-01

    Radiation sensitive protein 23 (RAD23) is a nucleotide excision repair (NER) protein that plays an important role in Ubiquitin-proteasome pathway (UPP). Schistosoma japonicum radiation sensitive protein23 (SjRAD23) cDNA sequences were amplified by PCR and cloned into pET28a (+) vector to construct recombinant expression plasmid pET28a(+)-SjRAD23. The recombinant protein was expressed as both inclusion bodies and the supernatant in Escherichia coli BL21 (DE3) cell. Immunofluorescence observation shows that SjRAD23 was mainly distributed on the tegument surface of the worms. ELISA assay reveals that specific IgG, IgG1 and IgG2a antibodies could be detected in the sera of rSjRAD23 immunized mice. Western blotting analysis shows that the recombinant SjRAD23 could be recognized by serum specific to soluble adult worm antigen of S. japonicum. BALB/c mice vaccinated with rSjRAD23 combined with 206 adjuvant revealed 35.94% worm reduction and 40.59% liver egg reduction when compared with that of the adjuvant control PMID:25985518

  14. Germline mutations in RAD51D confer susceptibility to ovarian cancer.

    PubMed

    Loveday, Chey; Turnbull, Clare; Ramsay, Emma; Hughes, Deborah; Ruark, Elise; Frankum, Jessica R; Bowden, Georgina; Kalmyrzaev, Bolot; Warren-Perry, Margaret; Snape, Katie; Adlard, Julian W; Barwell, Julian; Berg, Jonathan; Brady, Angela F; Brewer, Carole; Brice, Glen; Chapman, Cyril; Cook, Jackie; Davidson, Rosemarie; Donaldson, Alan; Douglas, Fiona; Greenhalgh, Lynn; Henderson, Alex; Izatt, Louise; Kumar, Ajith; Lalloo, Fiona; Miedzybrodzka, Zosia; Morrison, Patrick J; Paterson, Joan; Porteous, Mary; Rogers, Mark T; Shanley, Susan; Walker, Lisa; Eccles, Diana; Evans, D Gareth; Renwick, Anthony; Seal, Sheila; Lord, Christopher J; Ashworth, Alan; Reis-Filho, Jorge S; Antoniou, Antonis C; Rahman, Nazneen

    2011-09-01

    Recently, RAD51C mutations were identified in families with breast and ovarian cancer. This observation prompted us to investigate the role of RAD51D in cancer susceptibility. We identified eight inactivating RAD51D mutations in unrelated individuals from 911 breast-ovarian cancer families compared with one inactivating mutation identified in 1,060 controls (P = 0.01). The association found here was principally with ovarian cancer, with three mutations identified in the 59 pedigrees with three or more individuals with ovarian cancer (P = 0.0005). The relative risk of ovarian cancer for RAD51D mutation carriers was estimated to be 6.30 (95% CI 2.86-13.85, P = 4.8 × 10(-6)). By contrast, we estimated the relative risk of breast cancer to be 1.32 (95% CI 0.59-2.96, P = 0.50). These data indicate that RAD51D mutation testing may have clinical utility in individuals with ovarian cancer and their families. Moreover, we show that cells deficient in RAD51D are sensitive to treatment with a PARP inhibitor, suggesting a possible therapeutic approach for cancers arising in RAD51D mutation carriers. PMID:21822267

  15. Differential hRad17 expression by histologic subtype of ovarian cancer

    PubMed Central

    2011-01-01

    Background In the search for unique ovarian cancer biomarkers, ovarian specific cDNA microarray analysis identified hRad17, a cell cycle checkpoint protein, as over-expressed in ovarian cancer. The aim of this study was to validate this expression. Methods Immunohistochemistry was performed on 72 serous, 19 endometrioid, 10 clear cell, and 6 mucinous ovarian cancers, 9 benign ovarian tumors, and 6 normal ovarian tissue sections using an anti-hRad17 antibody. Western blot analysis and quantitative PCR were performed using cell lysates and total RNA prepared from 17 ovarian cancer cell lines and 6 normal ovarian epithelial cell cultures (HOSE). Results Antibody staining confirmed upregulation of hRad17 in 49.5% of ovarian cancer cases. Immunohistochemistry demonstrated that only 42% of serous and 47% of endometrioid subtypes showed overexpression compared to 80% of clear cell and 100% of mucinous cancers. Western blot confirmed overexpression of hRad17 in cancer cell lines compared to HOSE. Quantitative PCR demonstrated an upregulation of hRad17 RNA by 1.5-7 fold. hRad17 RNA expression differed by subtype. Conclusions hRad17 is over-expressed in ovarian cancer. This over-expression varies by subtype suggesting a role in the pathogenesis of these types. Functional studies are needed to determine the potential role of this protein in ovarian cancer. PMID:21450056

  16. Ultrasonic characterization of materials hardness

    PubMed

    Badidi Bouda A; Benchaala; Alem

    2000-03-01

    In this paper, an experimental technique has been developed to measure velocities and attenuation of ultrasonic waves through a steel with a variable hardness. A correlation between ultrasonic measurements and steel hardness was investigated. PMID:10829663

  17. Budding yeast Rad50, Mre11, Xrs2, and Hdf1, but not Rad52, are involved in the formation of deletions on a dicentric plasmid.

    PubMed

    Tsukamoto, Y; Kato, J; Ikeda, H

    1997-08-01

    We have previously shown that the RAD50, RAD52, MRE11, XRS2, and HDF1 genes of Saccharomyces cervisiae are involved in the formation of deletions by illegitimate recombination on a monocentric plasmid. In this study, we investigated the effects of mutations of these genes on formation of deletions of a dicentric plasmid, in which DNA double-strand breaks are expected to occur frequently because the two centromeres are pulled to opposite poles in mitosis. We transformed yeast cells with a dicentric plasmid, and after incubation for a few division cycles, cells carrying deleted plasmids were detected using negative selection markers. Deletions occurred at a higher frequency than on the monocentric plasmid and there were short regions of homology at the recombination junctions as observed on the monocentric plasmid. In rad50, mre11, xrs2, and hdf1 mutants, the frequency of occurrence of deletions was reduced by about 50-fold, while in the rad52 mutant, it was comparable to that in the wild-type strain. The end-joining functions of Rad50, Mre11, Xrs2, and Hdf1, suggest that these proteins play important roles in the joining of DNA ends produced on the dicentric plasmid during mitosis. PMID:9294039

  18. Phytoplasma effector SAP54 hijacks plant reproduction by degrading MADS-box proteins and promotes insect colonization in a RAD23-dependent manner.

    PubMed

    MacLean, Allyson M; Orlovskis, Zigmunds; Kowitwanich, Krissana; Zdziarska, Anna M; Angenent, Gerco C; Immink, Richard G H; Hogenhout, Saskia A

    2014-04-01

    Pathogens that rely upon multiple hosts to complete their life cycles often modify behavior and development of these hosts to coerce them into improving pathogen fitness. However, few studies describe mechanisms underlying host coercion. In this study, we elucidate the mechanism by which an insect-transmitted pathogen of plants alters floral development to convert flowers into vegetative tissues. We find that phytoplasma produce a novel effector protein (SAP54) that interacts with members of the MADS-domain transcription factor (MTF) family, including key regulators SEPALLATA3 and APETALA1, that occupy central positions in the regulation of floral development. SAP54 mediates degradation of MTFs by interacting with proteins of the RADIATION SENSITIVE23 (RAD23) family, eukaryotic proteins that shuttle substrates to the proteasome. Arabidopsis rad23 mutants do not show conversion of flowers into leaf-like tissues in the presence of SAP54 and during phytoplasma infection, emphasizing the importance of RAD23 to the activity of SAP54. Remarkably, plants with SAP54-induced leaf-like flowers are more attractive for colonization by phytoplasma leafhopper vectors and this colonization preference is dependent on RAD23. An effector that targets and suppresses flowering while simultaneously promoting insect herbivore colonization is unprecedented. Moreover, RAD23 proteins have, to our knowledge, no known roles in flower development, nor plant defence mechanisms against insects. Thus SAP54 generates a short circuit between two key pathways of the host to alter development, resulting in sterile plants, and promotes attractiveness of these plants to leafhopper vectors helping the obligate phytoplasmas reproduce and propagate (zombie plants). PMID:24714165

  19. Stimulation of Sister Chromatid Exchanges and Mutation by Aflatoxin B1-DNA Adducts in Saccharomyces cerevisiae Requires MEC1 (ATR), RAD53, and DUN1

    PubMed Central

    Fasullo, Michael; Sun, Mingzeng; Egner, Patricia

    2008-01-01

    The hepatocarcinogen aflatoxin B1 (AFB1) is a potent recombinagen but weak mutagen in the yeast Saccharomyces cerevisiae. AFB1 exposure induces DNA damage-inducible genes, such as RAD51 and those encoding ribonucleotide reductase (RNR), through a MEC1 (ATR homolog)-dependent pathway. Previous studies have indicated that MEC1 is required for both AFB1-associated recombination and mutation, and suggested that AFB1-DNA adducts are common substrates for recombination and mutagenesis. However, little is known about the downstream effectors of MEC1 required for genotoxic events associated with AFB1 exposure. Here we show that AFB1 exposure increases frequencies of RAD51-dependent unequal sister chromatid exchange (SCE) and activates Rad53 (CHK2). We found that MEC1, RAD53, and DUN1 are required for both AFB1-associated mutation and SCE. Deletion of SML1, which encodes an inhibitor of RNR, did not suppress the DUN1-dependent requirement for AFB1-associated genetic events, indicating that higher dNTP levels could not suppress the dun1 phenotype. We identified AFB1-DNA adducts and show that approximately the same number of adducts are obtained in both wild type and rad53 mutants. Since DUN1 is not required for UV-associated mutation and recombination, these studies define a distinct role for DUN1 in AFB1-associated mutagenesis and recombination. We speculate that AFB1-associated DNA adducts stall DNA replication, a consequence of which can either be mutation or recombination. PMID:18228255

  20. Phytoplasma Effector SAP54 Hijacks Plant Reproduction by Degrading MADS-box Proteins and Promotes Insect Colonization in a RAD23-Dependent Manner

    PubMed Central

    MacLean, Allyson M.; Orlovskis, Zigmunds; Kowitwanich, Krissana; Zdziarska, Anna M.; Angenent, Gerco C.; Immink, Richard G. H.; Hogenhout, Saskia A.

    2014-01-01

    Pathogens that rely upon multiple hosts to complete their life cycles often modify behavior and development of these hosts to coerce them into improving pathogen fitness. However, few studies describe mechanisms underlying host coercion. In this study, we elucidate the mechanism by which an insect-transmitted pathogen of plants alters floral development to convert flowers into vegetative tissues. We find that phytoplasma produce a novel effector protein (SAP54) that interacts with members of the MADS-domain transcription factor (MTF) family, including key regulators SEPALLATA3 and APETALA1, that occupy central positions in the regulation of floral development. SAP54 mediates degradation of MTFs by interacting with proteins of the RADIATION SENSITIVE23 (RAD23) family, eukaryotic proteins that shuttle substrates to the proteasome. Arabidopsis rad23 mutants do not show conversion of flowers into leaf-like tissues in the presence of SAP54 and during phytoplasma infection, emphasizing the importance of RAD23 to the activity of SAP54. Remarkably, plants with SAP54-induced leaf-like flowers are more attractive for colonization by phytoplasma leafhopper vectors and this colonization preference is dependent on RAD23. An effector that targets and suppresses flowering while simultaneously promoting insect herbivore colonization is unprecedented. Moreover, RAD23 proteins have, to our knowledge, no known roles in flower development, nor plant defence mechanisms against insects. Thus SAP54 generates a short circuit between two key pathways of the host to alter development, resulting in sterile plants, and promotes attractiveness of these plants to leafhopper vectors helping the obligate phytoplasmas reproduce and propagate (zombie plants). PMID:24714165

  1. Control of Floret Symmetry by RAY3, SvDIV1B, and SvRAD in the Capitulum of Senecio vulgaris1[OPEN

    PubMed Central

    2016-01-01

    All members of Asteraceae, the largest flowering family, have a unique compressed inflorescence known as a capitulum, which resembles a solitary flower. The capitulum often consists of bilateral (zygomorphic) ray florets and radial (actinomorphic) disc florets. In Antirrhinum majus, floral zygomorphy is established by the interplay between dorsal petal identity genes, CYCLOIDEA (CYC) and RADIALIS (RAD), and a ventral gene DIVARICATA (DIV). To investigate the role of CYC, RAD, and DIV in the development of ray and disc florets within a capitulum, we isolated homologs of these genes from an Asteraceae species, Senecio vulgaris (common groundsel). After initial uniform expression of RAY3 (CYC), SvRAD, and SvDIV1B in ray florets only, RAY3 and SvRAD were exclusively expressed in the ventral petals of the ray florets. Our functional analysis further showed that RAY3 promotes and SvDIV1B represses petal growth, confirming their roles in floral zygomorphy. Our results highlight that while floral symmetry genes such as RAY3 and SvDIV1B appear to have a conserved role in petal growth in both Senecio and Antirrhinum, the regulatory relationships and expression domains are divergent, allowing ventral petal elongation in Senecio versus dorsal petal elongation in Antirrhinum. In S. vulgaris, diversification of CYC genes has led to novel interactions; SvDIV1B inhibits RAY3 and SvRAD, and may activate RAY2. This highlights how recruitment of floral symmetry regulators into dynamic networks was crucial for creating a complex and elaborate structure such as the capitulum. PMID:27208229

  2. Hard-pan soils - Management

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hard pans, hard layers, or compacted horizons, either surface or subsurface, are universal problems that limit crop production. Hard layers can be caused by traffic or soil genetic properties that result in horizons with high density or cemented soil particles; these horizons have elevated penetrati...

  3. A practical primer on PI-RADS version 2: a pictorial essay.

    PubMed

    Horn, Gary Lloyd; Hahn, Peter Florin; Tabatabaei, Shahin; Harisinghani, Mukesh

    2016-05-01

    Multiparametric magnetic resonance imaging has become an established method for evaluating the prostate for clinically significant prostate adenocarcinoma. Criteria have been developed for categorizing MRI findings, the most frequently used of which is the PI-RADS system. The PI-RADS V2 document provides separate image interpretation and clinical grading sections. Within this article we give an overview of the integrated, algorithmic way, we approach prostate MRI, show images corresponding to each PI-RADS category, and provide several illustrative cases. PMID:26984751

  4. Schizosaccharomyces pombe rad32 protein: a phosphoprotein with an essential phosphoesterase motif required for repair of DNA double strand breaks.

    PubMed

    Wilson, S; Tavassoli, M; Watts, F Z

    1998-12-01

    The Schizosaccharomyces pombe Rad32 protein is required for repair of DNA double strand breaks, minichromosome stability and meiotic recombination. We show here that the Rad32 protein is phosphorylated in a cell cycle-dependent manner and during meiosis. The phosphorylation is not dependent on the checkpoint protein Rad3. Analysis of a partially purified protein preparation indicates that Rad32 is likely to act in a complex. Characterisation of the rad32-1 mutation and site-directed mutagenesis indicate that three aspartate residues in the conserved phosphoesterase motifs are important for both mitotic and meiotic functions, namely response to UV and ionising radiation and spore viability. PMID:9826747

  5. Hard metal composition

    DOEpatents

    Sheinberg, Haskell

    1986-01-01

    A composition of matter having a Rockwell A hardness of at least 85 is formed from a precursor mixture comprising between 3 and 10 weight percent boron carbide and the remainder a metal mixture comprising from 70 to 90 percent tungsten or molybdenum, with the remainder of the metal mixture comprising nickel and iron or a mixture thereof. The composition has a relatively low density of between 7 to 14 g/cc. The precursor is preferably hot pressed to yield a composition having greater than 100% of theoretical density.

  6. Hard metal composition

    DOEpatents

    Sheinberg, H.

    1983-07-26

    A composition of matter having a Rockwell A hardness of at least 85 is formed from a precursor mixture comprising between 3 and 10 wt % boron carbide and the remainder a metal mixture comprising from 70 to 90% tungsten or molybdenum, with the remainder of the metal mixture comprising nickel and iron or a mixture thereof. The composition has a relatively low density of between 7 and 14 g/cc. The precursor is preferably hot pressed to yield a composition having greater than 100% of theoretical density.

  7. Xe-bearing hydrocarbon ions: Observation of Xe.acetylene+rad and Xe.benzene+rad radical cations and calculations of their ground state structures

    NASA Astrophysics Data System (ADS)

    Cui, Zhong-hua; Attah, Isaac K.; Platt, Sean P.; Aziz, Saadullah G.; Kertesz, Miklos; El-Shall, M. S.

    2016-04-01

    This work reports evidence for novel types of Xe-bearing hydrocarbon radical cations. The Xe.acetylene+rad radical cation adduct is observed at nearly room temperature using the mass-selected drift cell technique. The irreversible addition of the Xe atom and the lack of back dissociation to HCCH+rad + Xe is consistent with the calculated binding energy of 0.85 eV to be contrasted with the metastable nature of the neutral Xe.acetylene adduct. The observed Xe.benzene+rad radical cation appears to be a weakly bound complex stabilized mainly by ion-induced dipole interaction consistent with a calculated binding energy in the range of 0.14-0.17 eV.

  8. Active standoff detection of CH4 and N2O leaks using hard-target backscattered light using an open-path quantum cascade laser sensor

    NASA Astrophysics Data System (ADS)

    Diaz, Adrian; Thomas, Benjamin; Castillo, Paulo; Gross, Barry; Moshary, Fred

    2016-05-01

    Fugitive gas emissions from agricultural or industrial plants and gas pipelines are an important environmental concern as they contribute to the global increase of greenhouse gas concentrations. Moreover, they are also a security and safety concern because of possible risk of fire/explosion or toxicity. This study presents standoff detection of CH4 and N2O leaks using a quantum cascade laser open-path system that retrieves path-averaged concentrations by collecting the backscattered light from a remote hard target. It is a true standoff system and differs from other open-path systems that are deployed as point samplers or long-path transmission systems that use retroreflectors. The measured absorption spectra are obtained using a thermal intra-pulse frequency chirped DFB quantum cascade laser at ~7.7 µm wavelength range with ~200 ns pulse width. Making fast time resolved observations, the system simultaneously realizes high spectral resolution and range to the target, resulting in path-averaged concentration retrieval. The system performs measurements at high speed ~15 Hz and sufficient range (up to 45 m, ~148 feet) achieving an uncertainty of 3.1 % and normalized sensitivity of 3.3 ppm m Hz-1/2 for N2O and 9.3 % and normalized sensitivity of 30 ppm m Hz-1/2 for CH4 with a 0.31 mW average power QCL. Given these characteristics, this system is promising for mobile or multidirectional search and remote detection of gas leaks.

  9. Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent Plasmodium falciparum SSB as a Reporter for Single-Stranded DNA.

    PubMed

    Davenport, Eric Parker; Harris, Derek F; Origanti, Sofia; Antony, Edwin

    2016-01-01

    Single-stranded DNA binding (SSB) proteins coordinate DNA replication, repair, and recombination and are critical for maintaining genomic integrity. SSB binds to single-stranded DNA (ssDNA) rapidly and with very high affinity making it a useful molecular tool to detect free ssDNA in solution. We have labeled SSB from Plasmodium falciparum (Pf-SSB) with the MDCC (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)-carbonyl)coumarin) fluorophore which yields a four-fold increase in fluorescence upon binding to ssDNA. Pf-SSBMDCC binding to DNA is unaffected by NaCl or Mg2+ concentration and does not display salt-dependent changes in DNA binding modes or cooperative binding on long DNA substrates. These features are unique to Pf-SSB, making it an ideal tool to probe the presence of free ssDNA in any biochemical reaction. Using this Pf-SSBMDCC probe as a sensor for free ssDNA, we have investigated the clearing of preformed yeast Rad51 nucleoprotein filaments by the Srs2 helicase during HR. Our studies provide a rate for the disassembly of the Rad51 filament by full length Srs2 on long ssDNA substrates. Mutations in the conserved 2B domain in the homologous bacterial UvrD, Rep and PcrA helicases show an enhancement of DNA unwinding activity, but similar mutations in Srs2 do not affect its DNA unwinding or Rad51 clearing properties. These studies showcase the utility of the Pf-SSB probe in mechanistic investigation of enzymes that function in DNA metabolism. PMID:27416037

  10. Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent Plasmodium falciparum SSB as a Reporter for Single-Stranded DNA

    PubMed Central

    Davenport, Eric Parker; Harris, Derek F.; Origanti, Sofia

    2016-01-01

    Single-stranded DNA binding (SSB) proteins coordinate DNA replication, repair, and recombination and are critical for maintaining genomic integrity. SSB binds to single-stranded DNA (ssDNA) rapidly and with very high affinity making it a useful molecular tool to detect free ssDNA in solution. We have labeled SSB from Plasmodium falciparum (Pf-SSB) with the MDCC (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)-carbonyl)coumarin) fluorophore which yields a four-fold increase in fluorescence upon binding to ssDNA. Pf-SSBMDCC binding to DNA is unaffected by NaCl or Mg2+ concentration and does not display salt-dependent changes in DNA binding modes or cooperative binding on long DNA substrates. These features are unique to Pf-SSB, making it an ideal tool to probe the presence of free ssDNA in any biochemical reaction. Using this Pf-SSBMDCC probe as a sensor for free ssDNA, we have investigated the clearing of preformed yeast Rad51 nucleoprotein filaments by the Srs2 helicase during HR. Our studies provide a rate for the disassembly of the Rad51 filament by full length Srs2 on long ssDNA substrates. Mutations in the conserved 2B domain in the homologous bacterial UvrD, Rep and PcrA helicases show an enhancement of DNA unwinding activity, but similar mutations in Srs2 do not affect its DNA unwinding or Rad51 clearing properties. These studies showcase the utility of the Pf-SSB probe in mechanistic investigation of enzymes that function in DNA metabolism. PMID:27416037

  11. Hard Metal Disease

    PubMed Central

    Bech, A. O.; Kipling, M. D.; Heather, J. C.

    1962-01-01

    In Great Britain there have been no published reports of respiratory disease occurring amongst workers in the hard metal (tungsten carbide) industry. In this paper the clinical and radiological findings in six cases and the pathological findings in one are described. In two cases physiological studies indicated mild alveolar diffusion defects. Histological examination in a fatal case revealed diffuse pulmonary interstitial fibrosis with marked peribronchial and perivascular fibrosis and bronchial epithelial hyperplasia and metaplasia. Radiological surveys revealed the sporadic occurrence and low incidence of the disease. The alterations in respiratory mechanics which occurred in two workers following a day's exposure to dust are described. Airborne dust concentrations are given. The industrial process is outlined and the literature is reviewed. The toxicity of the metals is discussed, and our findings are compared with those reported from Europe and the United States. We are of the opinion that the changes which we would describe as hard metal disease are caused by the inhalation of dust at work and that the component responsible may be cobalt. Images PMID:13970036

  12. Spins, phonons, and hardness

    SciTech Connect

    Gilman, J.J.

    1996-12-31

    In crystals (and/or glasses) with localized sp{sup 3} or spd-bonding orbitals, dislocations have very low mobilities, making the crystals very hard. Classical Peierls-Nabarro theory does not account for the low mobility. The breaking of spin-pair bonds which creates internal free-radicals must be considered. Therefore, a theory based on quantum mechanics has been proposed (Science, 261, 1436 (1993)). It has been applied successfully to diamond, Si, Ge, SiC, and with a modification to TiC and WC. It has recently been extended to account for the temperature independence of the hardness of silicon at low temperatures together with strong softening at temperatures above the Debye temperature. It is quantitatively consistent with the behaviors of the Group 4 elements (C, Si, Ge, Sn) when their Debye temperatures are used as normalizing factors; and appears to be consistent with data for TiC if an Einstein temperature for carbon is used. Since the Debye temperature marks the approximate point at which phonons of atomic wavelengths become excited (as contrasted with collective acoustic waves), this confirms the idea that the process which limits dislocation mobility is localized to atomic dimensions (sharp kinks).

  13. 11 MeV low-energy magnifying pRad at CAEP

    NASA Astrophysics Data System (ADS)

    Li, Yiding; Yang, Guojun; Zhang, Xiaoding; Wei, Tao; Jiang, Xiaoguo

    2016-04-01

    To make a further improvement of resolution, the 11 MeV low-energy pRad beamline at CAEP was rebuilt into a 1:3 magnifying imaging beamline recently. The principle of low-energy pRad and the design of magnifying pRad line are described in detail. By using magnetic imaging lens of magnification three, images are spread over larger area at image plane and the effect of optical system on spatial resolution limitation is weakened. According to the radiographing results, for 10 μm thick aluminum object, spatial resolution less than 30 μm is achieved on the new magnifying pRad beamline.

  14. Onset Times of Solar Particle Events Observed by MSL/RAD - Constraints on Particle Transport

    NASA Astrophysics Data System (ADS)

    Wimmer-Schweingruber, R. F.; Hassler, D. M.; Boettcher, S.; Martin, C.; Zeitlin, C.; Brinza, D.; Koehler, J.; Guo, J.; Posner, A.; Appel, J.

    2012-12-01

    En route to Mars, Mars Science Laboratory's (MSL's) Radiation Assessment Detector (RAD) was already operational and observed a number of solar particle events. These were also seen by an array of spacecraft in the heliosphere. This constellation provides an unique opportunity to investigate particle propagation in the ecliptic plane and thus constrain particle propagation models. RAD measures energetic ions up to approximately 100 MeV/nuc, but was buried deep inside the Mars Science Laboratory (MSL). Due to the significant shielding provided by the MSL cruise stage, back-shell, heat shield, and skycrane, RAD was sensitive to particles in a significantly higher and somewhat uncertain energy range. Even behind the aforementioned shielding, onset times of particle events can be clearly identified. In this work, we will provide them for the particle events observed by RAD in 2012. We will also relate them to other data and solar/interplanetary acceleration sites.

  15. Hard X-ray astrophysics

    NASA Technical Reports Server (NTRS)

    Rothschild, R. E.

    1981-01-01

    Past hard X-ray and lower energy satellite instruments are reviewed and it is shown that observation above 20 keV and up to hundreds of keV can provide much valuable information on the astrophysics of cosmic sources. To calculate possible sensitivities of future arrays, the efficiencies of a one-atmosphere inch gas counter (the HEAO-1 A-2 xenon filled HED3) and a 3 mm phoswich scintillator (the HEAO-1 A-4 Na1 LED1) were compared. Above 15 keV, the scintillator was more efficient. In a similar comparison, the sensitivity of germanium detectors did not differ much from that of the scintillators, except at high energies where the sensitivity would remain flat and not rise with loss of efficiency. Questions to be addressed concerning the physics of active galaxies and the diffuse radiation background, black holes, radio pulsars, X-ray pulsars, and galactic clusters are examined.

  16. Chemogenetic profiling identifies RAD17 as synthetically lethal with checkpoint kinase inhibition

    PubMed Central

    Shen, John Paul; Srivas, Rohith; Gross, Andrew; Li, Jianfeng; Jaehnig, Eric J.; Sun, Su Ming; Bojorquez-Gomez, Ana; Licon, Katherine; Sivaganesh, Vignesh; Xu, Jia L.; Klepper, Kristin; Yeerna, Huwate; Pekin, Daniel; Qiu, Chu Ping; van Attikum, Haico; Sobol, Robert W.; Ideker, Trey

    2015-01-01

    Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function. PMID:26437225

  17. Chemogenetic profiling identifies RAD17 as synthetically lethal with checkpoint kinase inhibition.

    PubMed

    Shen, John Paul; Srivas, Rohith; Gross, Andrew; Li, Jianfeng; Jaehnig, Eric J; Sun, Su Ming; Bojorquez-Gomez, Ana; Licon, Katherine; Sivaganesh, Vignesh; Xu, Jia L; Klepper, Kristin; Yeerna, Huwate; Pekin, Daniel; Qiu, Chu Ping; van Attikum, Haico; Sobol, Robert W; Ideker, Trey

    2015-11-01

    Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function. PMID:26437225

  18. Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast.

    PubMed

    Xu, Yong-Jie

    2016-04-01

    DNA replication checkpoint is a highly conserved cellular signaling pathway critical for maintaining genome integrity in eukaryotes. It is activated when DNA replication is perturbed. In Schizosaccharomyces pombe, perturbed replication forks activate the sensor kinase Rad3 (ATR/Mec1), which works cooperatively with mediator Mrc1 and the 9-1-1 checkpoint clamp to phosphorylate the effector kinase Cds1 (CHK2/Rad53). Phosphorylation of Cds1 promotes autoactivation of the kinase. Activated Cds1 diffuses away from the forks and stimulates most of the checkpoint responses under replication stress. Although this signaling pathway has been well understood in fission yeast, how the signaling is initiated and thus regulated remains incompletely understood. Previous studies have shown that deletion of lem2(+) sensitizes cells to the inhibitor of ribonucleotide reductase, hydroxyurea. However, the underlying mechanism is still not well understood. This study shows that in the presence of hydroxyurea, Lem2 facilitates Rad3-mediated checkpoint signaling for Cds1 activation. Without Lem2, all known Rad3-dependent phosphorylations critical for replication checkpoint signaling are seriously compromised, which likely causes the aberrant mitosis and drug sensitivity observed in this mutant. Interestingly, the mutant is not very sensitive to DNA damage and the DNA damage checkpoint remains largely intact, suggesting that the main function of Lem2 is to facilitate checkpoint signaling in response to replication stress. Since Lem2 is an inner nuclear membrane protein, these results also suggest that the replication checkpoint may be spatially regulated inside the nucleus, a previously unknown mechanism. PMID:26746798

  19. Uncovering Cryptic Asexuality in Daphnia magna by RAD Sequencing.

    PubMed

    Svendsen, Nils; Reisser, Celine M O; Dukić, Marinela; Thuillier, Virginie; Ségard, Adeline; Liautard-Haag, Cathy; Fasel, Dominique; Hürlimann, Evelin; Lenormand, Thomas; Galimov, Yan; Haag, Christoph R

    2015-11-01

    The breeding systems of many organisms are cryptic and difficult to investigate with observational data, yet they have profound effects on a species' ecology, evolution, and genome organization. Genomic approaches offer a novel, indirect way to investigate breeding systems, specifically by studying the transmission of genetic information from parents to offspring. Here we exemplify this method through an assessment of self-fertilization vs. automictic parthenogenesis in Daphnia magna. Self-fertilization reduces heterozygosity by 50% compared to the parents, but under automixis, whereby two haploid products from a single meiosis fuse, the expected heterozygosity reduction depends on whether the two meiotic products are separated during meiosis I or II (i.e., central vs. terminal fusion). Reviewing the existing literature and incorporating recombination interference, we derive an interchromosomal and an intrachromosomal prediction of how to distinguish various forms of automixis from self-fertilization using offspring heterozygosity data. We then test these predictions using RAD-sequencing data on presumed automictic diapause offspring of so-called nonmale producing strains and compare them with "self-fertilized" offspring produced by within-clone mating. The results unequivocally show that these offspring were produced by automixis, mostly, but not exclusively, through terminal fusion. However, the results also show that this conclusion was only possible owing to genome-wide heterozygosity data, with phenotypic data as well as data from microsatellite markers yielding inconclusive or even misleading results. Our study thus demonstrates how to use the power of genomic approaches for elucidating breeding systems, and it provides the first demonstration of automictic parthenogenesis in Daphnia. PMID:26341660

  20. Overexpressed of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

    SciTech Connect

    Schild, David; Wiese, Claudia

    2009-10-15

    RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the 'recombination mediators'. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as 'recombination co-mediators'. Defects in either recombination mediators or comediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic restabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51.

  1. Rad52 forms DNA repair and recombination centers during S phase

    PubMed Central

    Lisby, Michael; Rothstein, Rodney; Mortensen, Uffe H.

    2001-01-01

    Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by γ-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions. PMID:11459964

  2. ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage.

    PubMed

    McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

    2005-07-01

    Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions. PMID:15933716

  3. ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

    PubMed Central

    McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

    2005-01-01

    Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions. PMID:15933716

  4. High-throughput polymorphism detection and genotyping in Brassica napus using next-generation RAD sequencing

    PubMed Central

    2012-01-01

    Background The complex genome of rapeseed (Brassica napus) is not well understood despite the economic importance of the species. Good knowledge of sequence variation is needed for genetics approaches and breeding purposes. We used a diversity set of B. napus representing eight different germplasm types to sequence genome-wide distributed restriction-site associated DNA (RAD) fragments for polymorphism detection and genotyping. Results More than 113,000 RAD clusters with more than 20,000 single nucleotide polymorphisms (SNPs) and 125 insertions/deletions were detected and characterized. About one third of the RAD clusters and polymorphisms mapped to the Brassica rapa reference sequence. An even distribution of RAD clusters and polymorphisms was observed across the B. rapa chromosomes, which suggests that there might be an equal distribution over the Brassica oleracea chromosomes, too. The representation of Gene Ontology (GO) terms for unigenes with RAD clusters and polymorphisms revealed no signature of selection with respect to the distribution of polymorphisms within genes belonging to a specific GO category. Conclusions Considering the decreasing costs for next-generation sequencing, the results of our study suggest that RAD sequencing is not only a simple and cost-effective method for high-density polymorphism detection but also an alternative to SNP genotyping from transcriptome sequencing or SNP arrays, even for species with complex genomes such as B. napus. PMID:22726880

  5. Assessment and Management of Challenging BI-RADS Category 3 Mammographic Lesions.

    PubMed

    Michaels, Aya Y; Birdwell, Robyn L; Chung, Chris SungWon; Frost, Elisabeth P; Giess, Catherine S

    2016-01-01

    Breast Imaging Reporting and Data System (BI-RADS) category 3 lesions are probably benign by definition and are recommended for short-interval follow-up after a diagnostic workup has been completed. Although the original lexicon-derived BI-RADS category 3 definition applied to lesions without prior imaging studies (when stability could not be determined), in clinical practice, many lesions with prior images may be assigned to BI-RADS category 3. Although the BI-RADS fifth edition specifically delineates lesions that are appropriate for categorization as probably benign, it also specifies that the interpreting radiologist may use his or her discretion and experience to justify a "watchful waiting" approach for lesions that do not meet established criteria. Examples of such lesions include evolving masses or calcifications suggestive of prior trauma and instances when stability cannot be ascertained because of image quality. Although interval change is an important feature of malignancy, many benign lesions also change over time; thus, use of prior imaging studies and ongoing imaging surveillance to demonstrate the evolution of a probably benign lesion is justified. Some examples of common pitfalls associated with inappropriate BI-RADS category 3 assessment include failure to use proper BI-RADS descriptors, failure to perform a complete diagnostic workup, and overreliance on negative ultrasonographic findings. When appropriately used, short-interval follow-up saves many patients from undergoing biopsy of benign lesions, without decreasing the rate of cancer detection. (©)RSNA, 2016. PMID:27541437

  6. Targeting homologous recombination, new pre-clinical and clinical therapeutic combinations inhibiting RAD51.

    PubMed

    Ward, Ambber; Khanna, Kum Kum; Wiegmans, Adrian P

    2015-01-01

    The DNA damage response (DDR) is essential for maintaining genomic stability and cell survival. However, when tumour cells with deficiencies in HR are faced with radio- and chemotherapies they are forced to rely on error-prone, alternative repair pathways or aberrant HR for survival; threatening genome integrity and driving further mutation. Accurate therapeutic targeting of the key drivers of DNA repair can circumvent survival pathways and avoid aggressive therapy resistant mutants. Several studies have identified that stabilization of the cancer genome in HR deficient cells can be achieved by overexpression of the recombinase RAD51. Radio- and chemotherapeutic resistance is associated with overactive HR repair mechanisms. However no clinical trials have directly targeted RAD51, despite RAD51 displaying synergy in several drug screens against multiple cancer types. Currently synthetic lethality targeting the DDR pathways and HR deficiency has had clinical success with BRCA1 functional loss and PARP inhibition. In this review we suggest that clinical outcomes could be improved by additionally targeting RAD51. We examine the latest developments in directly and indirectly targeting RAD51. We scrutinize the potential treatment efficacy and future clinical applications of RAD51 inhibitors as single agents and in combination with other therapies and consider the best therapeutic options. PMID:25467108

  7. ROLE OF THE HOMOLOGOUS RECOMBINATION GENES RAD51 and RAD59 IN THE RESISTANCE OF Candida albicans TO UV LIGHT, RADIOMIMETIC AND ANTI-TUMOR COMPOUNDS AND OXIDIZING AGENTS

    PubMed Central

    García-Prieto, Fátima; Gómez-Raja, Jonathan; Andaluz, Encarnación; Calderone, Richard; Larriba, Germán

    2010-01-01

    We have cloned and characterized the RAD51 and RAD59 orthologues of the pathogenic fungus Candida albicans. CaRad51 exhibited more than 50% identity with several other eukaryotes and the conserved the catalytic domain of a bacterial RecA. As compared to the parental strain, null strains of rad51 exhibited a filamentous morphology, had a decreased grow rate and exhibited a moderate sensitivity to UV light, oxidizing agents, and compounds that cause double-strand breaks (DSB), indicating a role in DNA repair. By comparison, the rad52 null had a higher percentage of filaments, a more severe growth defect and a greater sensitivity to DNA-damaging compounds. Null strains of rad59 showed a UV-sensitive phenotype but behaved similarly to the parental strain in the rest of the assays. As compared to S. cerevisiae, C. albicans was much more resistant to bleomycin and the same was true for their respective homologous recombination (HR) mutants. These results indicate that, as described in S. cerevisiae, RAD52 plays a more prominent role than RAD51 in the repair of DSBs in C. albicans and suggest the existence of at least two Rad52-dependent HR pathways, one dependent and one independent of Rad51. PMID:20206282

  8. Overexpression of Rad in muscle worsens diet-induced insulin resistance and glucose intolerance and lowers plasma triglyceride level

    NASA Astrophysics Data System (ADS)

    Ilany, Jacob; Bilan, Philip J.; Kapur, Sonia; Caldwell, James S.; Patti, Mary-Elizabeth; Marette, Andre; Kahn, C. Ronald

    2006-03-01

    Rad is a low molecular weight GTPase that is overexpressed in skeletal muscle of some patients with type 2 diabetes mellitus and/or obesity. Overexpression of Rad in adipocytes and muscle cells in culture results in diminished insulin-stimulated glucose uptake. To further elucidate the potential role of Rad in vivo, we have generated transgenic (tg) mice that overexpress Rad in muscle using the muscle creatine kinase (MCK) promoter-enhancer. Rad tg mice have a 6- to 12-fold increase in Rad expression in muscle as compared to wild-type littermates. Rad tg mice grow normally and have normal glucose tolerance and insulin sensitivity, but have reduced plasma triglyceride levels. On a high-fat diet, Rad tg mice develop more severe glucose intolerance than the wild-type mice; this is due to increased insulin resistance in muscle, as exemplified by a rightward shift in the dose-response curve for insulin stimulated 2-deoxyglucose uptake. There is also a unexpected further reduction of the plasma triglyceride levels that is associated with increased levels of lipoprotein lipase in the Rad tg mice. These results demonstrate a potential synergistic interaction between increased expression of Rad and high-fat diet in creation of insulin resistance and altered lipid metabolism present in type 2 diabetes. diabetes mellitus | glucose transport | RGK GTPase | transgenic mouse

  9. Regulated expression and dynamic changes in subnuclear localization of mammalian Rad18 under normal and genotoxic conditions.

    PubMed

    Masuyama, Sadaharu; Tateishi, Satoshi; Yomogida, Kentaro; Nishimune, Yoshitake; Suzuki, Keiichiro; Sakuraba, Yoshiyuki; Inoue, Hirokazu; Ogawa, Michio; Yamaizumi, Masaru

    2005-08-01

    Rad18 plays a crucial role in postreplication repair in both lower eukaryotes and higher eukaryotes. However, regulation of the Rad18 expression in higher eukaryotes is largely unknown. We found that the RAD18 transcript is expressed ubiquitously in various tissues and very highly in the testis in mammals. Although human RAD18 (hRAD18) transcription levels fluctuate during the cell cycle, being maximal in the late S and minimal in the early G1, the protein levels remain constant throughout the cell cycle. Following UV-irradiation, hRAD18 transcription levels decrease significantly, but Rad18 protein levels change little. The protein levels are maintained at least in part by enhanced translation rates. hRad18 localizes in the nucleus in two forms: a diffused form and a condensed form forming nuclear dots. These nuclear dots disperse rapidly in the nucleoplasm after treatments with various genotoxic agents, resulting in an enhancement of the intranuclear Rad18 concentration of the diffused form. No de novo protein synthesis is required for this process. These results suggest that in higher eukaryotes, the maintenance and dynamic translocation of Rad18 protein is important for postreplication repair. PMID:16098139

  10. RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity.

    PubMed

    Yard, Brian D; Reilly, Nicole M; Bedenbaugh, Michael K; Pittman, Douglas L

    2016-06-01

    The RAD51 family is integral for homologous recombination (HR) mediated DNA repair and maintaining chromosome integrity. RAD51D, the fourth member of the family, is a known ovarian cancer susceptibility gene and required for the repair of interstrand crosslink DNA damage and preserving chromosomal stability. In this report, we describe the RNF138 E3 ubiquitin ligase that interacts with and ubiquitinates the RAD51D HR protein. RNF138 is a member of an E3 ligase family that contains an amino-terminal RING finger domain and a putative carboxyl-terminal ubiquitin interaction motif. In mammalian cells, depletion of RNF138 increased the stability of the RAD51D protein, suggesting that RNF138 governs ubiquitin-proteasome-mediated degradation of RAD51D. However, RNF138 depletion conferred sensitivity to DNA damaging agents, reduced RAD51 focus formation, and increased chromosomal instability. Site-specific mutagenesis of the RNF138 RING finger domain demonstrated that it was necessary for RAD51D ubiquitination. Presence of RNF138 also enhanced the interaction between RAD51D and a known interacting RAD51 family member XRCC2 in a yeast three-hybrid assay. Therefore, RNF138 is a newly identified regulatory component of the HR mediated DNA repair pathway that has implications toward understanding how ubiquitination modifies the functions of the RAD51 paralog protein complex. PMID:27161866

  11. Prospective validation of an ultrasound-based thyroid imaging reporting and data system (TI-RADS) on 3980 thyroid nodules

    PubMed Central

    Zhang, Jing; Liu, Bo-Ji; Xu, Hui-Xiong; Xu, Jun-Mei; Zhang, Yi-Feng; Liu, Chang; Wu, Jian; Sun, Li-Ping; Guo, Le-Hang; Liu, Lin-Na; Xu, Xiao-Hong; Qu, Shen

    2015-01-01

    Objective: Ultrasound (US) features of solidity, hypoechogenicity or marked hypoechogenicity, microlobulated or irregular margins, microcalcifications, and taller-than-wide shape are suspicious characteristics for thyroid nodules. An US based Thyroid Imaging Reporting and Data System (TI-RADS) is classified based on the number of aforesaid features. TI-RADS category 3 included nodules without any suspicious features, and categories 4a, 4b, 4c, and 5 included nodules with one, two, three or four, or five suspicious US features. The purpose of the study was to prospectively validate the effectiveness of the TI-RADS. Methods: From October 2011 to June 2013, we prospectively categorized 3980 thyroid nodules (3752 benign and 228 malignant lesions) in 2921 patients using TI-RADS classification. TI-RADS categories 2 and 3 were considered as benign whereas TI-RADS categories 4 and 5 as malignant. The sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and accuracy were calculated. Results: Of the 3980 nodules, 2953 nodules were TI-RADS category 2 (0% malignancy), 466 nodules TI-RADS category 3 (1.3% malignancy), 186 nodules TI-RADS category 4a (4.8% malignancy), 165 nodules TI-RADS category 4b (30.3% malignancy), 188 nodules TI-RADS category 4c (75.5% malignancy), and 22 nodules TI-RADS category 5 (95.5% malignancy). The sensitivity, specificity, PPV, NPV and accuracy were 97%, 90%, 40%, 99%, and 91%, respectively. Conclusions: TI-RADS classification had great diagnostic value in diagnosing thyroid nodules. The actual probability of malignancy was in accord with the theory risk of malignancy. PMID:26131184

  12. Enhancing cytochrome P450-mediated conversions in P. pastoris through RAD52 over-expression and optimizing the cultivation conditions.

    PubMed

    Wriessnegger, Tamara; Moser, Sandra; Emmerstorfer-Augustin, Anita; Leitner, Erich; Müller, Monika; Kaluzna, Iwona; Schürmann, Martin; Mink, Daniel; Pichler, Harald

    2016-04-01

    Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6. PMID:26898115

  13. BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine.

    PubMed

    Jones, Rebecca M; Kotsantis, Panagiotis; Stewart, Grant S; Groth, Petra; Petermann, Eva

    2014-10-01

    Replication inhibitors cause replication fork stalling and double-strand breaks (DSB) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitize cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here, we investigate why, paradoxically, mutations in HR genes protect cells from killing by gemcitabine. Using DNA replication and DNA damage assays in mammalian cells, we show that even short gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression, and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at gemcitabine-stalled forks are converted into DSBs and thus contribute to gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumors. PMID:25053826

  14. Extending RAD tag analysis to microbial ecology: a comparison between MultiLocus Sequence Typing and 2b-RAD to investigate Listeria monocytogenes genetic structure.

    PubMed

    Pauletto, Marianna; Carraro, Lisa; Babbucci, Massimiliano; Lucchini, Rosaria; Bargelloni, Luca; Cardazzo, Barbara

    2016-05-01

    The advent of next-generation sequencing (NGS) has dramatically changed bacterial typing technologies, increasing our ability to differentiate bacterial isolates. Despite it is now possible to sequence a bacterial genome in a few days and at reasonable costs, most genetic analyses do not require whole-genome sequencing, which also remains impractical for large population samples due to the cost of individual library preparation and bioinformatics. More traditional sequencing approaches, however, such as MultiLocus Sequence Typing (mlst) are quite laborious and time-consuming, especially for large-scale analyses. In this study, a genotyping approach based on restriction site-associated (RAD) tag sequencing, 2b-RAD, was applied to characterize Listeria monocytogenes strains. To verify the feasibility of the method, an in silico analysis was performed on 30 available complete genomes. For the same set of strains, in silico mlst analysis was conducted as well. Subsequently, 2b-RAD and mlst analyses were experimentally carried out on 58 isolates collected from food samples or food-processing sites. The obtained results demonstrate that 2b-RAD predicts mlst types and often provides more detailed information on population structure than mlst. Moreover, the majority of variants differentiating identical sequence type isolates mapped against accessory fragments, thus providing additional information to characterize strains. Although mlst still represents a reliable typing method, large-scale studies on molecular epidemiology and public health, as well as bacterial phylogenetics, population genetics and biosafety could benefit of a low cost and fast turnaround time approach such as the 2b-RAD analysis proposed here. PMID:26613186

  15. Hard-on-hard lubrication in the artificial hip under dynamic loading conditions.

    PubMed

    Sonntag, Robert; Reinders, Jörn; Rieger, Johannes S; Heitzmann, Daniel W W; Kretzer, J Philippe

    2013-01-01

    The tribological performance of an artificial hip joint has a particularly strong influence on its success. The principle causes for failure are adverse short- and long-term reactions to wear debris and high frictional torque in the case of poor lubrication that may cause loosening of the implant. Therefore, using experimental and theoretical approaches models have been developed to evaluate lubrication under standardized conditions. A steady-state numerical model has been extended with dynamic experimental data for hard-on-hard bearings used in total hip replacements to verify the tribological relevance of the ISO 14242-1 gait cycle in comparison to experimental data from the Orthoload database and instrumented gait analysis for three additional loading conditions: normal walking, climbing stairs and descending stairs. Ceramic-on-ceramic bearing partners show superior lubrication potential compared to hard-on-hard bearings that work with at least one articulating metal component. Lubrication regimes during the investigated activities are shown to strongly depend on the kinematics and loading conditions. The outcome from the ISO gait is not fully confirmed by the normal walking data and more challenging conditions show evidence of inferior lubrication. These findings may help to explain the differences between the in vitro predictions using the ISO gait cycle and the clinical outcome of some hard-on-hard bearings, e.g., using metal-on-metal. PMID:23940772

  16. A Radiation-Hard Analog Memory In The AVLSI-RA Process

    SciTech Connect

    Britton, C.L. Jr.; Wintenberg, A.L.; Read, K.F.; Simpson, M.L.; Young, G.R.; Clonts, L.G., Kennedy, E.J., Smith, R.S., Swann, B.K.; Musser, J.A.

    1995-12-31

    A radiation hardened analog memory for an Interpolating Pad Camber has been designed at Oak Ridge National Laboratory and fabricated by Harris Semiconductor in the AVLSI-RA CMOS process. The goal was to develop a rad-hard analog pipeline that would deliver approximately 9-bit performance, a readout settling time of 500ns following read enable, an input and output dynamic range of +/-2.25V, a corrected rms pedestal of approximately 5mV or less, and a power dissipation of less than 10mW/channel. The pre- and post-radiation measurements to 5MRad are presented.

  17. Histogramming of the Charged Particle Measurements with MSL/RAD - Comparison of Histogram Data with Simulations

    NASA Astrophysics Data System (ADS)

    Ehresmann, B.; Zeitlin, C.; Hassler, D. M.; Wimmer-Schweingruber, R. F.; Boettcher, S.; Koehler, J.; Martin, C.; Brinza, D.; Rafkin, S. C.

    2012-12-01

    The Radiation Assessment Detector (RAD) on-board the Mars Science Laboratory (MSL) is designed to measure a broad range of energetic particle radiation. A significant part of this radiation consists of charged particles, which mainly stem from cosmic background radiation, Solar particle events, and secondaries created by the interaction of these particles with the Martian atmosphere and soil. To measure charged particles RAD is equipped with a set of detectors: a particle telescope consisting of three silicon Solid-State Detectors (SSDs), a CsI scintillator and a plastic scintillator, as well as a further plastic scintillator used as anti-coincidence. RAD uses an elaborate post-processing logic to analyze if a measured event qualifies as a charged particle, as well as to distinguish between particles stopping in any one of the detectors and particles penetrating the whole detector stack. RAD then arranges these qualifying events in an appropriate stopping or penetrating charged particle histogram, reducing the data volume necessary to maintain crucial information about the measured particle. For ground-based data analysis it is of prime importance to derive information, such as particle species or energy, from the data in the downloaded histograms. Here, we will present how the chosen binning of these histograms enables us to derive this information. Pre-flight, we used the Monte-Carlo code GEANT4 to simulate the expected particle radiation and its interactions with a full model of the RAD sensor head. By mirroring the on-board processing logic, we derived statistics of which particle species and energies populate any one bin in the set of charged particle histograms. Finally, we will compare the resulting histogram data from RAD cruise and surface observations with simulations. RAD is supported by NASA (HEOMD) under JPL subcontract #1273039 to SwRI, and by DLR in Germany under contract to Christian-Albrechts-Universitaet zu Kiel (CAU).

  18. Liver imaging reporting and data system (LI-RADS) version 2014: understanding and application of the diagnostic algorithm

    PubMed Central

    An, Chansik; Rakhmonova, Gulbahor; Choi, Jin-Young; Kim, Myeong-Jin

    2016-01-01

    Liver Imaging Reporting and Data System (LI-RADS) is a system for interpreting and reporting of computed tomography and magnetic resonance imaging of the liver in patients at risk for hepatocellular carcinoma (HCC). LI-RADS has been developed to address the limitations of prior imaging-based criteria including the lack of established consensus regarding the exact definitions of imaging features, binary categorization (either definite or not definite HCC), and failure to consider non-HCC malignancies. One of the most important goals of LI-RADS is to facilitate clear communication between all the personnel involved in the diagnosis and treatment of HCC, such as radiologists, hepatologists, surgeons, and pathologists. Therefore, clinicians should also be familiar with LI-RADS. This article reviews the LI-RADS diagnostic algorithm, and the definitions and management implications of LI-RADS categories. PMID:27304548

  19. Measuring the Hardness of Minerals

    ERIC Educational Resources Information Center

    Bushby, Jessica

    2005-01-01

    The author discusses Moh's hardness scale, a comparative scale for minerals, whereby the softest mineral (talc) is placed at 1 and the hardest mineral (diamond) is placed at 10, with all other minerals ordered in between, according to their hardness. Development history of the scale is outlined, as well as a description of how the scale is used…

  20. “How hard could it be?” A descriptive analysis of errors made on a validated lifetime physical activity questionnaire

    PubMed Central

    Anderton, Natalie; Newhouse, Megan E.; Ainsworth, Barbara E.; Nygaard, Ingrid E.; Egger, Marlene J.; Shaw, Janet M.

    2015-01-01

    Background Measuring historical physical activity in epidemiologic research depends on self-report. We aimed to describe data reporting errors women made in completing two validated questionnaires – Lifetime Physical Activity Questionnaire (LPAQ) and Occupational Questionnaire (OQ). Methods Participants, 229 women aged 38 – 65 years, completed questionnaires on paper (n=160) or by web interface (n=69). One research assistant collected questionnaire data, identified potential errors and contacted participants to trouble-shoot errors. Results Women made mean 9.7 (SD 11.2) errors on paper and 7.1 (SD 6.2) errors on electronic versions of the LPAQ and 2.6 (SD 3.8) and 1.1 (SD 1.4) errors on paper and electronic versions of the OQ, respectively. Fewer mistakes were made on electronic versions of both questionnaires combined (8.5±6.1) when compared to the paper versions (12.7±13.1). Only ~2% of the sample completed all questionnaires without detectable errors. The most common errors were reporting activities or frequencies inconsistently between past year survey and the current age epoch, reporting more years than allowed by age epoch and missing information. Conclusions Despite the implications of “self-report” questionnaires, we recommend researchers provide participants with additional instructions, either verbally or as written tip sheet or both, and follow-up after questionnaire completion to correct mistakes as needed. PMID:24809450

  1. Cyclic strength of hard metals

    SciTech Connect

    Sereda, N.N.; Gerikhanov, A.K.; Koval'chenko, M.S.; Pedanov, L.G.; Tsyban', V.A.

    1986-02-01

    The authors study the strength of hard-metal specimens and structural elements under conditions of cyclic loading since many elements of processing plants, equipment, and machines are made of hard metals. Fatigue tests were conducted on KTS-1N, KTSL-1, and KTNKh-70 materials, which are titanium carbide hard metals cemented with nickel-molybdenum, nickelcobalt-chromium, and nickel-chromium alloys, respectively. As a basis of comparison, the standard VK-15 (WC+15% Co) alloy was used. Some key physicomechanical characteristics of the materials investigated are presented. On time bases not exceeding 10/sup 6/ cycles, titanium carbide hard metals are comparable in fatigue resistance to the standard tungstencontaining hard metals.

  2. Distinct binding of BRCA2 BRC repeats to RAD51 generates differential DNA damage sensitivity

    PubMed Central

    Chatterjee, Gouri; Jimenez-Sainz, Judit; Presti, Thomas; Nguyen, Tiffany; Jensen, Ryan B.

    2016-01-01

    BRCA2 is a multi-faceted protein critical for the proper regulation of homology-directed repair of DNA double-strand breaks. Elucidating the mechanistic features of BRCA2 is crucial for understanding homologous recombination and how patient-derived mutations impact future cancer risk. Eight centrally located BRC repeats in BRCA2 mediate binding and regulation of RAD51 on resected DNA substrates. Herein, we dissect the biochemical and cellular features of the BRC repeats tethered to the DNA binding domain of BRCA2. To understand how the BRC repeats and isolated domains of BRCA2 contribute to RAD51 binding, we analyzed both the biochemical and cellular properties of these proteins. In contrast to the individual BRC repeat units, we find that the BRC5–8 region potentiates RAD51-mediated DNA strand pairing and provides complementation functions exceeding those of BRC repeats 1–4. Furthermore, BRC5–8 can efficiently repair nuclease-induced DNA double-strand breaks and accelerate the assembly of RAD51 repair complexes upon DNA damage. These findings highlight the importance of the BRC5–8 domain in stabilizing the RAD51 filament and promoting homology-directed repair under conditions of cellular DNA damage. PMID:27084934

  3. Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

    PubMed Central

    Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; Park, Beomseok; Shim, Yoonjung; Kim, Youngchang; Liu, Lili; Van Houten, Bennett; He, Chuan; Ansari, Anjum; Min, Jung-Hyun

    2015-01-01

    The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites. PMID:25562780

  4. RadNotes: a novel software development tool for radiology education.

    PubMed

    Baxter, A B; Klein, J S; Oesterle, E V

    1997-01-01

    RadNotes is a novel software development tool that enables physicians to develop teaching materials incorporating text and images in an intelligent, highly usable format. Projects undertaken in the RadNotes environment require neither programming expertise nor the assistance of a software engineer. The first of these projects, Thoracic Imaging, integrates image teaching files, concise disease and topic summaries, references, and flash card quizzes into a single program designed to provide an overview of chest radiology. RadNotes is intended to support the academic goals of teaching radiologists by enabling authors to create, edit, and electronically distribute image-oriented presentations. RadNotes also supports the educational goals of physicians who wish to quickly review selected imaging topics, as well as to develop a visual vocabulary of corresponding radiologic anatomy and pathologic conditions. Although Thoracic Imaging was developed with the aim of introducing chest radiology to residents, RadNotes can be used to develop tutorials and image-based tests for all levels; create corresponding World Wide Web sites; and organize notes, images, and references for individual use. PMID:9153710

  5. Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation

    PubMed Central

    Hyppa, Randy W.; Benko, Zsigmond; Misova, Ivana; Schleiffer, Alexander; Smith, Gerald R.; Gregan, Juraj

    2016-01-01

    To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species. PMID:27304859

  6. Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

    DOE PAGESBeta

    Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; Park, Beomseok; Shim, Yoonjung; Kim, Youngchang; Liu, Lili; Van Houten, Bennett; He, Chuan; Ansari, Anjum; et al

    2015-01-06

    The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformations similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivitymore » arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump pertubation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.« less

  7. Efficient Strand Transfer by the RadA Recombinase from the Hyperthermophilic Archaeon Desulfurococcus amylolyticus

    PubMed Central

    Kil, Yuri V.; Baitin, Dmitry M.; Masui, Ryoji; Bonch-Osmolovskaya, Elizaveta A.; Kuramitsu, Seiki; Lanzov, Vladislav A.

    2000-01-01

    The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65°C (catalytic rate constant of 1.2 to 2.5 min−1 at 80 to 95°C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90°C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92°C). PMID:10613871

  8. Monitoring the Heliospheric Conditions at Mars Using MSL/RAD Measurements

    NASA Astrophysics Data System (ADS)

    Guo, J.; Wimmer-Schweingruber, R. F.; Zeitlin, C. J.; Rafkin, S. C.; Hassler, D.; Posner, A.

    2015-12-01

    The Radiation Assessment Detector (RAD), on board Mars Science Laboratory's (MSL) rover Curiosity, measures the radiation dose rate as well as the energy spectra of energetic charged and neutral particles at the surface of Mars. With these first-ever measurements of GCR fluxes on the Martian surface, RAD can be used as a monitor for heliospheric modulation at Mars location, similar to neutron monitors at Earth. We do this by first correlating the GCR dose rate measurements at Mars and solar modulations at Earth when there is a good magnetic connection between the two planets. With the thus obtained correlation we obtain an empirical function for the dependence of the modulation parameter at Mars on RAD dose rate. This function can in turn help to calibrate the heliospheric modulation at Mars throughout the MSL/RAD mission period. The resulting solar modulation at Mars and at Earth over three years (>1000 sols) is then compared. In order to verify our 'prediction' method, we use the local modulation parameter at Mars as an input for Badhwar O'Neil model providing the primary spectra for PLANETOCOSMIC simulations which eventually model the surface particle spectra that can be compared with RAD measurements of the spectra.

  9. Monte Carlo calculation of the energy response characteristics of a RadFET radiation detector

    NASA Astrophysics Data System (ADS)

    Belicev, P.; Spasic Jokic, V.; Mayer, S.; Milosevic, M.; Ilic, R.; Pesic, M.

    2010-07-01

    The Metal -Oxide Semiconductor Field-Effect-Transistor (MOSFET, RadFET) is frequently used as a sensor of ionizing radiation in nuclear-medicine, diagnostic-radiology, radiotherapy quality-assurance and in the nuclear and space industries. We focused our investigations on calculating the energy response of a p-type RadFET to low-energy photons in range from 12 keV to 2 MeV and on understanding the influence of uncertainties in the composition and geometry of the device in calculating the energy response function. All results were normalized to unit air kerma incident on the RadFET for incident photon energy of 1.1 MeV. The calculations of the energy response characteristics of a RadFET radiation detector were performed via Monte Carlo simulations using the MCNPX code and for a limited number of incident photon energies the FOTELP code was also used for the sake of comparison. The geometry of the RadFET was modeled as a simple stack of appropriate materials. Our goal was to obtain results with statistical uncertainties better than 1% (fulfilled in MCNPX calculations for all incident energies which resulted in simulations with 1 - 2×109 histories.

  10. Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

    NASA Astrophysics Data System (ADS)

    Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; Park, Beomseok; Shim, Yoonjung; Kim, Youngchang; Liu, Lili; van Houten, Bennett; He, Chuan; Ansari, Anjum; Min, Jung-Hyun

    2015-01-01

    The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

  11. Conservatism in SRS Criticality Alarm System 12 Rad Zone Calculations - How Much is Enough?

    SciTech Connect

    Yates, K.R.

    2002-06-13

    Savannah River Site (SRS) uses two methods (i.e., Approximate Method and MCNP) of calculating the 12-rad zone. The reasons for the two-tier approach are described in Ref. 1 and 2. Lately, there have been occasions in which the use of either the Approximate Method (AM) or MCNP3 calculations indicated potential facility impacts. For example, one or both methods may indicate that a 12-rad zone extends outside of relatively thick shielding, or extends to the roof of a facility, or extends through shielding to part of a stairwell. In such cases, a criticality alarm system may have to be installed to protect workers in a small, localized area from a potential dose that is not substantially greater than 12 rad in air. But, is the potential dose really greater than 12 rad in air? A subcommittee was appointed to look into the two 12-rad zone calculation methods for the purpose of identifying items contributing to over-conservatism and under-conservatism, and to recommend a path forward.

  12. Recent results from the Radiometric Calibration Test Site (RadCaTS) at Railroad Valley, Nevada

    NASA Astrophysics Data System (ADS)

    Czapla-Myers, Jeffrey S.; Leisso, Nathan P.

    2010-09-01

    The Radiometric Calibration Test Site (RadCaTS) is an automated approach to ground-based vicarious calibration that does not require on-site personnel during the overpass of an airborne or spaceborne sensor. The concept originates as an attempt to increase the amount of ground-based data that are collected throughout the year. All-weather instruments are used to measure atmospheric and surface conditions. The data are used in an automated processing scheme to produce top-of-atmosphere spectral radiance, which are then compared to the sensor under test. RadCaTS has been located at Railroad Valley, Nevada, since 2004, but the concept is applicable to any site that is suitable for vicarious calibration. Railroad Valley was chosen to test the RadCaTS concept because it has been used by the Remote Sensing Group (RSG) for over 15 years and is well understood. This work describes the RadCaTS automated concept, and outlines the automated processing scheme that is used to determine the surface reflectance. A description of the instrumentation used to measure the surface reflectance and atmosphere is presented, followed by a discussion of their placement on the site, and also their calibration. Finally, the RadCaTS ground-based results are compared to those from Aqua and Terra MODIS in 2008, and Landsat 7 ETM+ in 2009.

  13. Performance comparison of quantitative semantic features and lung-RADS in the National Lung Screening Trial

    NASA Astrophysics Data System (ADS)

    Li, Qian; Balagurunathan, Yoganand; Liu, Ying; Schabath, Matthew; Gillies, Robert J.

    2016-03-01

    Background: Lung-RADS is the new oncology classification guideline proposed by American College of Radiology (ACR), which provides recommendation for further follow up in lung cancer screening. However, only two features (solidity and size) are included in this system. We hypothesize that additional sematic features can be used to better characterize lung nodules and diagnose cancer. Objective: We propose to develop and characterize a systematic methodology based on semantic image traits to more accurately predict occurrence of cancerous nodules. Methods: 24 radiological image traits were systematically scored on a point scale (up to 5) by a trained radiologist, and lung-RADS was independently scored. A linear discriminant model was used on the semantic features to access their performance in predicting cancer status. The semantic predictors were then compared to lung-RADS classification in 199 patients (60 cancers, 139 normal controls) obtained from the National Lung Screening Trial. Result: There were different combinations of semantic features that were strong predictors of cancer status. Of these, contour, border definition, size, solidity, focal emphysema, focal fibrosis and location emerged as top candidates. The performance of two semantic features (short axial diameter and contour) had an AUC of 0.945, and was comparable to that of lung-RADS (AUC: 0.871). Conclusion: We propose that a semantics-based discrimination approach may act as a complement to the lung-RADS to predict cancer status.

  14. TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity

    PubMed Central

    Moudry, Pavel; Watanabe, Kenji; Wolanin, Kamila M.; Bartkova, Jirina; Wassing, Isabel E.; Watanabe, Sugiko; Strauss, Robert; Troelsgaard Pedersen, Rune; Oestergaard, Vibe H.; Lisby, Michael; Andújar-Sánchez, Miguel; Maya-Mendoza, Apolinar; Esashi, Fumiko; Lukas, Jiri

    2016-01-01

    Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase–mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1’s role in HR, with potential clinical implications for cancer treatment. PMID:26811421

  15. Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation.

    PubMed

    Polakova, Silvia; Molnarova, Lucia; Hyppa, Randy W; Benko, Zsigmond; Misova, Ivana; Schleiffer, Alexander; Smith, Gerald R; Gregan, Juraj

    2016-06-01

    To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species. PMID:27304859

  16. Distinct binding of BRCA2 BRC repeats to RAD51 generates differential DNA damage sensitivity.

    PubMed

    Chatterjee, Gouri; Jimenez-Sainz, Judit; Presti, Thomas; Nguyen, Tiffany; Jensen, Ryan B

    2016-06-20

    BRCA2 is a multi-faceted protein critical for the proper regulation of homology-directed repair of DNA double-strand breaks. Elucidating the mechanistic features of BRCA2 is crucial for understanding homologous recombination and how patient-derived mutations impact future cancer risk. Eight centrally located BRC repeats in BRCA2 mediate binding and regulation of RAD51 on resected DNA substrates. Herein, we dissect the biochemical and cellular features of the BRC repeats tethered to the DNA binding domain of BRCA2. To understand how the BRC repeats and isolated domains of BRCA2 contribute to RAD51 binding, we analyzed both the biochemical and cellular properties of these proteins. In contrast to the individual BRC repeat units, we find that the BRC5-8 region potentiates RAD51-mediated DNA strand pairing and provides complementation functions exceeding those of BRC repeats 1-4. Furthermore, BRC5-8 can efficiently repair nuclease-induced DNA double-strand breaks and accelerate the assembly of RAD51 repair complexes upon DNA damage. These findings highlight the importance of the BRC5-8 domain in stabilizing the RAD51 filament and promoting homology-directed repair under conditions of cellular DNA damage. PMID:27084934

  17. TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity.

    PubMed

    Moudry, Pavel; Watanabe, Kenji; Wolanin, Kamila M; Bartkova, Jirina; Wassing, Isabel E; Watanabe, Sugiko; Strauss, Robert; Troelsgaard Pedersen, Rune; Oestergaard, Vibe H; Lisby, Michael; Andújar-Sánchez, Miguel; Maya-Mendoza, Apolinar; Esashi, Fumiko; Lukas, Jiri; Bartek, Jiri

    2016-02-01

    Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase-mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1's role in HR, with potential clinical implications for cancer treatment. PMID:26811421

  18. Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

    SciTech Connect

    Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; Park, Beomseok; Shim, Yoonjung; Kim, Youngchang; Liu, Lili; Van Houten, Bennett; He, Chuan; Ansari, Anjum; Min, Jung -Hyun

    2015-01-06

    The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformations similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump pertubation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

  19. MISTIC: Radiation hard ECRIS

    NASA Astrophysics Data System (ADS)

    Labrecque, F.; Lecesne, N.; Bricault, P.

    2008-10-01

    The ISAC RIB facility at TRIUMF utilizes up to 100 μA from the 500 MeV H- cyclotron to produce RIB using the isotopic separation on line (ISOL) method. In the moment, we are mainly using a hot surface ion source and a laser ion source to produce our RIB. A FEBIAD ion source has been recently tested at ISAC, but these ion sources are not suitable for gaseous elements like N, O, F, Ne, … , A new type of ion source is then necessary. By combining a high frequency electromagnetic wave and a magnetic confinement, the ECRIS [R. Geller, Electron Cyclotron Resonance Ion Source and ECR Plasmas, Institute of Physics Publishing, Bristol, 1996], [1] (electron cyclotron resonance ion source) can produce high energy electrons essential for efficient ionization of those elements. To this end, a prototype ECRIS called MISTIC (monocharged ion source for TRIUMF and ISAC complex) has been built at TRIUMF using a design similar to the one developed at GANIL [GANIL (Grand Accélérateur National d'Ions Lourds), www.ganil.fr], [2] The high level radiation caused by the proximity to the target prevented us to use a conventional ECRIS. To achieve a radiation hard ion source, we used coils instead of permanent magnets to produce the magnetic confinement. Each coil is supplied by 1000 A-15 V power supply. The RF generator cover a frequency range from 2 to 8 GHz giving us all the versatility we need to characterize the ionization of the following elements: He, Ne, Ar, Kr, Xe, C, O, N, F. Isotopes of these elements are involved in star thermonuclear cycles and, consequently, very important for researches in nuclear astrophysics. Measures of efficiency, emittance and ionization time will be performed for each of those elements. Preliminary tests show that MISTIC is very stable over a large range of frequency, magnetic field and pressure.

  20. Hard X rays and low-energy gamma rays from the Moon: Dependence of the continuum on the regolith composition and the solar activity

    NASA Astrophysics Data System (ADS)

    Banerjee, D.; Gasnault, O.

    2008-07-01

    The primary aim of the high-energy X-ray spectrometer (HEX) experiment on the Chandrayaan-1 mission to the Moon is to characterize the movement of volatiles on the lunar surface through the detection of the 46.5 keV line from 210Pb, a decay product of 222Rn. An important consideration for design and operation of HEX is to estimate the continuum background signal expected from the lunar surface, as well as its dependence on solar activity and lunar composition. We have developed a Monte Carlo code utilizing Geant4 for simulating the interaction of cosmic rays in the lunar regolith, and we estimated the variation in the continuum background in the energy region of interest for various lunar compositions. Dependence of the continuum background on solar activity was also evaluated considering ferroan anorthositic (FAN) composition. Our results suggest the viability of inferring lithologic characteristics of planetary surfaces based on a study of low-energy gamma ray emission.

  1. Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular fluorescence complementation analysis

    SciTech Connect

    Min, Jaewon; Park, Pil-gu; Ko, Eunkyong; Choi, Eunhee; Lee, Hyunsook

    2007-11-03

    BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair.

  2. Rad51 promoter-targeted gene therapy is effective for in vivo visualization and treatment of cancer.

    PubMed

    Hine, Christopher M; Seluanov, Andrei; Gorbunova, Vera

    2012-02-01

    Rad51 protein is overexpressed in a wide range of human cancers. Our previous in vitro studies demonstrated that a construct comprised Rad51 promoter driving expression of the diphtheria toxin A gene (pRad51-diphtheria toxin A (DTA)) destroys a variety of human cancer cell lines, with minimal to no toxicity to normal human cells. Here we delivered Rad51 promoter-based constructs in vivo using linear polyethylenimine nanoparticles, in vivo jetPEI, to visualize and treat tumors in mice with HeLa xenografts. For tumor detection, we used pRad51-Luc, a construct containing the firefly luciferase under the Rad51 promoter, administered by intraperitoneal (IP) injection. Tumors were detected with an in vivo bioluminescent camera. All mice with cancer displayed strong bioluminescence, while mice without cancer displayed no detectable bioluminescence. Treatment with pRad51-DTA/jetPEI decreased tumor mass of subcutaneous (SC) and IP tumors by sixfold and fourfold, respectively, along with the strong reduction of malignant ascites. Fifty percent of the mice with SC tumors were cancer-free after six pRad51-DTA/jetPEI injections, and for the mice with IP tumors, mean survival time increased by 90% compared to control mice. This study demonstrates the clinical potential of pRad51-based constructs delivered by nanoparticles for the diagnostics and treatment of a wide range of cancers. PMID:22008909

  3. Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA

    SciTech Connect

    Guzder, S.N.; Sung, P.; Prakash, S. ); Prakash, L. )

    1993-06-15

    Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A--G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. The authors have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14. As determined by atomic emission spectroscopy, RAD14 contains one zinc atom. They also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions. In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA. Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites. These findings indicate that RAD14 functions in damage recognition during excision repair. 37 refs., 4 figs.

  4. Foreword to the Special Issue on the 11th Specialist Meeting on Microwave Radiometry and Remote Sensing Applications (MicroRad 2010)

    NASA Technical Reports Server (NTRS)

    Le Vine, David M; Jackson, Thomas J.; Kim, Edward J.; Lang, Roger H.

    2011-01-01

    The Specialist Meeting on Microwave Radiometry and Remote Sensing of the Environment (MicroRad 2010) was held in Washington, DC from March 1 to 4, 2010. The objective of MicroRad 2010 was to provide an open forum to report and discuss recent advances in the field of microwave radiometry, particularly with application to remote sensing of the environment. The meeting was highly successful, with more than 200 registrations representing 48 countries. There were 80 oral presentations and more than 100 posters. MicroRad has become a venue for the microwave radiometry community to present new research results, instrument designs, and applications to an audience that is conversant in these issues. The meeting was divided into 16 sessions (listed in order of presentation): 1) SMOS Mission; 2) Future Passive Microwave Remote Sensing Missions; 3) Theory and Physical Principles of Electromagnetic Models; 4) Field Experiment Results; 5) Soil Moisture and Vegetation; 6) Snow and Cryosphere; 7) Passive/Active Microwave Remote Sensing Synergy; 8) Oceans; 9) Atmospheric Sounding and Assimilation; 10) Clouds and Precipitation; 11) Instruments and Advanced Techniques I; 12) Instruments and Advanced Techniques II; 13) Cross Calibration of Satellite Radiometers; 14) Calibration Theory and Methodology; 15) New Technologies for Microwave Radiometry; 16) Radio Frequency Interference.

  5. Assessment of DNA binding to human Rad51 protein by using quartz crystal microbalance and atomic force microscopy: effects of ADP and BRC4-28 peptide inhibitor.

    PubMed

    Esnault, Charles; Renodon-Cornière, Axelle; Takahashi, Masayuki; Casse, Nathalie; Delorme, Nicolas; Louarn, Guy; Fleury, Fabrice; Pilard, Jean-François; Chénais, Benoît

    2014-12-01

    The interaction of human Rad51 protein (HsRad51) with single-stranded deoxyribonucleic acid (ssDNA) was investigated by using quartz crystal microbalance (QCM) monitoring and atomic force microscopy (AFM) visualization. Gold surfaces for QCM and AFM were modified by electrografting of the in situ generated aryldiazonium salt from the sulfanilic acid to obtain the organic layer Au-ArSO3 H. The Au-ArSO3 H layer was activated by using a solution of PCl5 in CH2 Cl2 to give a Au-ArSO2 Cl layer. The modified surface was then used to immobilize long ssDNA molecules. The results obtained showed that the presence of adenosine diphosphate promotes the protein autoassociation rather than nucleation around DNA. In addition, when the BRC4-28 peptide inhibitor was used, both QCM and AFM confirmed the inhibitory effect of BRC4-28 toward HsRad51 autoassociation. Altogether these results show the suitability of this modified surface to investigate the kinetics and structure of DNA-protein interactions and for the screening of inhibitors. PMID:25208912

  6. Relationship of DNA degradation by Saccharomyces cerevisiae Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection

    PubMed Central

    Cannavo, Elda; Cejka, Petr; Kowalczykowski, Stephen C.

    2013-01-01

    Homologous recombination is a major pathway for repair of DNA double-strand breaks. This repair process is initiated by resection of the 5′-terminated strand at the break site. In yeast, resection is carried out by three nucleolytic complexes: Mre11-Rad50-Xrs2, which functions at the initial step and also stimulates the two processive pathways, Sgs1-Dna2 and Exonuclease 1 (Exo1). Here we investigated the relationship between the three resection pathways with a focus on Exo1. Exo1 preferentially degrades the 5′-terminal stand of duplex DNA that is single stranded at the 3′ end, in agreement with its role downstream of the Mre11-Rad50-Xrs2 complex. Replication protein A (RPA) stimulates DNA end resection by Exo1 by both preventing nonspecific binding of Exo1 to and preventing degradation of single-stranded DNA. Nucleolytic degradation of DNA by Exo1 is inhibited by the helicase-deficient Sgs1 K706A mutant protein and, reciprocally, the nuclease-deficient Exo1 D173A mutant protein inhibits DNA unwinding by Sgs1. Thus, the activities of Sgs1 and Exo1 at DNA ends are mutually exclusive, establishing biochemically that both machineries function independently in DNA end processing. We also reconstituted Sgs1-Top3-Rmi1-RPA-Dna2 and Exo1 resection reactions both individually and combined, either with or without the Mre11-Rad50-Xrs2 complex. We show that the yeast Sgs1-Dna2 and Exo1 pathways do not stimulate one another and function as independent and separate DNA end-processing machineries, even in the presence of the stimulatory Mre11-Rad50-Xrs2 complex. PMID:23589858

  7. Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of saccharomyces cerevisiae and schizosaccharomyces pombe

    SciTech Connect

    MacInnes, M.A.; Dickson, J.A.; Hernandez, R.R.; Lin, G.Y.; Park, M.S.; Schauer, S.; Reynolds, R.J.; Strniste, G.F. ); Learmonth, D. ); Mudgett, J.S. ); Yu, J.Y. )

    1993-10-01

    Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. The authors have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Their available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5[prime]-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Their evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed for several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeast, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Their analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B. 69 refs., 6 figs., 1 tab.

  8. A synthetic defect in protein degradation caused by loss of Ufd4 and Rad23

    SciTech Connect

    Ju Donghong; Xie Youming . E-mail: xiey@karmanos.org

    2006-03-10

    The UFD (ubiquitin fusion degradation) pathway is responsible for multiubiquitination of the fusion proteins that bear a 'non-removable' N-terminal ubiquitin moiety. Previous reports have shown that the UFD pathway is conserved from yeast to human. The essential elements of the UFD pathway have also been identified in Saccharomyces cerevisiae. These studies, however, are limited to use of engineered UFD substrates. The biological significance of the UFD pathway remains unknown. Here we demonstrate that Ufd4, the E3 component of the UFD pathway, is involved in controlling the degradation of Rad4, a nucleotide excision repair protein. Moreover, simultaneous loss of Ufd4 and Rad23 exhibits a synthetic inhibitory effect on Rad4 degradation, presenting First example that a UBA/UBL-domain protein functionally overlaps with a ubiquitin ligase in determining the turnover rate of a protein substrate. The current work also provides a direction for further investigation of the physiological functions of the UFD pathway.

  9. Capabilities, Calibration, and Impact of the ISS-RAD Fast Neutron Detector

    NASA Technical Reports Server (NTRS)

    Leitgab, Martin

    2015-01-01

    In the current NASA crew radiation health risk assessment framework, estimates for the neutron contributions to crew radiation exposure largely rely on simulated data with sizeable uncertainties due to the lack of experimental measurements inside the ISS. Integrated in the ISS-RAD instrument, the ISS-RAD Fast Neutron Detector (FND) will deploy to the ISS on one of the next cargo supply missions. Together with the ISS-RAD Charged Particle Detector, the FND will perform, for the first time, routine and precise direct neutron measurements inside the ISS between 0.5 and 80 MeV. The measurements will close the NASA Medical Operations Requirement to monitor neutrons inside the ISS and impact crew radiation health risk assessments by reducing uncertainties on the neutron contribution to crew exposure, enabling more efficient mission planning. The presentation will focus on the FND detection mechanism, calibration results and expectations about the FND's interaction with the mixed radiation field inside the ISS.

  10. Expression, purification, crystallization and preliminary crystallographic analysis of human Rad GTPase

    SciTech Connect

    Yanuar, Arry; Sakurai, Shigeru; Kitano, Ken; Hakoshima, Toshio

    2005-11-01

    Human Rad has been crystallized. A diffraction data set was collected to a resolution of 1.8 Å. Human Rad is a new member of the Ras GTPase superfamily and is overexpressed in human skeletal muscle of individuals with type II diabetes. The GTPase core domain was overexpressed in Escherichia coli and purified for crystallization. Crystals were obtained at 293 K by vapour diffusion using a crystallization robot. The crystals were found to belong to space group P2{sub 1}, with unit-cell parameters a = 52.2, b = 58.6, c = 53.4 Å, β = 97.9°, and contained two Rad molecules in the crystallographic asymmetric unit. A diffraction data set was collected to a resolution of 1.8 Å using synchrotron radiation at SPring-8.

  11. Initial experience with optical-CT scanning of RadBall Dosimeters.

    PubMed

    Oldham, M; Clift, C; Thomas, A; Farfan, E; Foley, T; Jannik, T; Adamovics, J; Holmes, C; Stanley, S

    2010-12-01

    The RadBall dosimeter is a novel device for providing 3-D information on the magnitude and distribution of contaminant sources of unknown radiation in a given hot cell, glovebox, or contaminated room. The device is presently under evaluation by the National Nuclear Lab (NNL, UK) and the Savannah River National Laboratory (SRNL, US), for application as a diagnostic device for such unknown contaminants in the nuclear industry. A critical component of the technique is imaging the dose distribution recorded in the RadBall using optical-CT scanning. Here we present our initial investigations using the Duke Mid-sized Optical-CT Scanner (DMOS) to image dose distributions deposited in RadBalls exposed to a variety of radiation treatments. PMID:21218190

  12. Initial experience with optical-CT scanning of RadBall Dosimeters

    PubMed Central

    Oldham, M; Clift, C; Thomas, A; Farfan, E; Foley, T; Jannik, T; Adamovics, J; Holmes, C; Stanley, S

    2010-01-01

    The RadBall dosimeter is a novel device for providing 3-D information on the magnitude and distribution of contaminant sources of unknown radiation in a given hot cell, glovebox, or contaminated room. The device is presently under evaluation by the National Nuclear Lab (NNL, UK) and the Savannah River National Laboratory (SRNL, US), for application as a diagnostic device for such unknown contaminants in the nuclear industry. A critical component of the technique is imaging the dose distribution recorded in the RadBall using optical-CT scanning. Here we present our initial investigations using the Duke Mid-sized Optical-CT Scanner (DMOS) to image dose distributions deposited in RadBalls exposed to a variety of radiation treatments. PMID:21218190

  13. Initial experience with optical-CT scanning of RadBall Dosimeters

    NASA Astrophysics Data System (ADS)

    Oldham, M.; Clift, C.; Thomas, A.; Farfan, E.; Foley, T.; Jannik, T.; Adamovics J.; Holmes, C.; Stanley, S.

    2010-11-01

    The RadBall dosimeter is a novel device for providing 3-D information on the magnitude and distribution of contaminant sources of unknown radiation in a given hot cell, glovebox, or contaminated room. The device is presently under evaluation by the National Nuclear Lab (NNL, UK) and the Savannah River National Laboratory (SRNL, US), for application as a diagnostic device for such unknown contaminants in the nuclear industry. A critical component of the technique is imaging the dose distribution recorded in the RadBall using optical-CT scanning. Here we present our initial investigations using the Duke Mid-sized Optical-CT Scanner (DMOS) to image dose distributions deposited in RadBalls exposed to a variety of radiation treatments.

  14. Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans.

    PubMed

    McClendon, T Brooke; Sullivan, Meghan R; Bernstein, Kara A; Yanowitz, Judith L

    2016-05-01

    Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSB-inducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1 Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1 We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability. PMID:26936927

  15. Predicting RAD-seq Marker Numbers across the Eukaryotic Tree of Life

    PubMed Central

    Herrera, Santiago; Reyes-Herrera, Paula H.; Shank, Timothy M.

    2015-01-01

    High-throughput sequencing of reduced representation libraries obtained through digestion with restriction enzymes—generically known as restriction site associated DNA sequencing (RAD-seq)—is a common strategy to generate genome-wide genotypic and sequence data from eukaryotes. A critical design element of any RAD-seq study is knowledge of the approximate number of genetic markers that can be obtained for a taxon using different restriction enzymes, as this number determines the scope of a project, and ultimately defines its success. This number can only be directly determined if a reference genome sequence is available, or it can be estimated if the genome size and restriction recognition sequence probabilities are known. However, both scenarios are uncommon for nonmodel species. Here, we performed systematic in silico surveys of recognition sequences, for diverse and commonly used type II restriction enzymes across the eukaryotic tree of life. Our observations reveal that recognition sequence frequencies for a given restriction enzyme are strikingly variable among broad eukaryotic taxonomic groups, being largely determined by phylogenetic relatedness. We demonstrate that genome sizes can be predicted from cleavage frequency data obtained with restriction enzymes targeting “neutral” elements. Models based on genomic compositions are also effective tools to accurately calculate probabilities of recognition sequences across taxa, and can be applied to species for which reduced representation data are available (including transcriptomes and neutral RAD-seq data sets). The analytical pipeline developed in this study, PredRAD (https://github.com/phrh/PredRAD), and the resulting databases constitute valuable resources that will help guide the design of any study using RAD-seq or related methods. PMID:26537225

  16. FERMI LARGE AREA TELESCOPE OBSERVATIONS OF THE ACTIVE GALAXY 4C +55.17: STEADY, HARD GAMMA-RAY EMISSION AND ITS IMPLICATIONS

    SciTech Connect

    McConville, W.; McEnery, J. E.; Ostorero, L.; Moderski, R.; Stawarz, L.; Cheung, C. C.; Ajello, M.; Monzani, M. E.; Bouvier, A.; Bregeon, J.; Donato, D.; Finke, J.; Furniss, A.; Williams, D. A.; Orienti, M.; Reyes, L. C.; Rossetti, A. E-mail: stawarz@astro.isas.jaxa.jp

    2011-09-10

    We report Fermi Large Area Telescope (LAT) observations and broadband spectral modeling of the radio-loud active galaxy 4C +55.17 (z = 0.896), formally classified as a flat-spectrum radio quasar. Using 19 months of all-sky survey Fermi-LAT data, we detect a {gamma}-ray continuum extending up to an observed energy of 145 GeV, and furthermore we find no evidence of {gamma}-ray variability in the source over its observed history. We illustrate the implications of these results in two different domains. First, we investigate the origin of the steady {gamma}-ray emission, where we re-examine the common classification of 4C +55.17 as a quasar-hosted blazar and consider instead its possible nature as a young radio source. We analyze and compare constraints on the source physical parameters in both blazar and young radio source scenarios by means of a detailed multiwavelength analysis and theoretical modeling of its broadband spectrum. Second, we show that the {gamma}-ray spectrum may be formally extrapolated into the very high energy (VHE, {>=}100 GeV) range at a flux level detectable by the current generation of ground-based Cherenkov telescopes. This enables us to place constraints on models of extragalactic background light within LAT energies and features the source as a promising candidate for VHE studies of the universe at an unprecedented redshift of z = 0.896.

  17. Weak solar flares with a detectable flux of hard X rays: Specific features of microwave radiation in the corresponding active regions

    NASA Astrophysics Data System (ADS)

    Grigor'eva, I. Yu.; Livshits, M. A.

    2014-12-01

    The emission of very weak flares was registered at the Suzaku X-ray observatory in 2005-2009. The photon power spectrum in the 50-110 keV range for a number of these phenomena shows that some electrons accelerate to energies higher than 100 keV. The corresponding flares originate in active regions (ARs) with pronounced sunspots. As in the case of AR 10933 in January 2007 analyzed by us previously (Grigor'eva et al., 2013), the thoroughly studied weak flares in May 2007 are related to the emergence of a new magnetic field in the AR and to the currents that originate in this case. A comparison of the Suzaku data with the RATAN-600 microwave observations indicates that a new polarized source of microwave radiation develops in the AR (or the previously existing source intensifies) one-two days before a weak flare in the emerging flux regions. Arguments in favor of recent views that fields are force-free in the AR corona are put forward. The development of weak flares is related to the fact that the free energy of the currents that flow above the field neutral line at altitudes reaching several thousand kilometers is accumulated and subsequently released.

  18. Beta Backscatter Measures the Hardness of Rubber

    NASA Technical Reports Server (NTRS)

    Morrissey, E. T.; Roje, F. N.

    1986-01-01

    Nondestructive testing method determines hardness, on Shore scale, of room-temperature-vulcanizing silicone rubber. Measures backscattered beta particles; backscattered radiation count directly proportional to Shore hardness. Test set calibrated with specimen, Shore hardness known from mechanical durometer test. Specimen of unknown hardness tested, and radiation count recorded. Count compared with known sample to find Shore hardness of unknown.

  19. The place of hard coal in energy supply pattern of Turkey

    SciTech Connect

    Yilmaz, A.O.; Aydiner, K.

    2009-07-01

    Lignite and hard coal are the major sources of domestic energy sources of Turkey. Hard coal is produced at only one district in the country. Zonguldak Hard Coal Basin is the major power for development of the Turkish steel-making industry. It is the only hard coal basin in the country and it has, to date, supplied approximately 400 million tons of run-of-mine hard coal. This article investigates the potential of hard coal as an energy source and discusses the measures to activate the region for the future energy supply objectives of the country.

  20. RadSTraM: Radiological Source Tracking and Monitoring, Phase II Final Report

    SciTech Connect

    Warren, Tracy A; Walker, Randy M; Hill, David E; Gross, Ian G; Smith, Cyrus M; Abercrombie, Robert K

    2008-12-01

    This report focuses on the technical information gained from the Radiological Source Tracking and Monitoring (RadSTraM) Phase II investigation and its implications. The intent of the RadSTraM project was to determine the feasibility of tracking radioactive materials in commerce, particularly International Atomic Energy Agency (IAEA) Category 3 and 4 materials. Specifically, Phase II of the project addressed tracking radiological medical isotopes in commerce. These categories of materials are susceptible to loss or theft but the problem is not being addressed by other agencies.

  1. MSL-RAD Dosimetry Measurements in Cruise and on Mars: Calibration and First Results

    NASA Astrophysics Data System (ADS)

    Zeitlin, C. J.; Hassler, D. M.; Wimmer-Schweingruber, R. F.

    2012-12-01

    The Radiation Assessment Detector (RAD) was the first MSL science instrument to start collecting data, with data acquisition commencing 10 days after launch and continuing until the final three weeks of the cruise phase. RAD resumed data-taking on the first sol on Mars, returning the first-ever detailed measurements of cosmic radiation from the surface of another planet. Coincidentally, but appropriately, RAD's first measurements on Mars were taken on the 100th anniversary of the balloon flight experiment by Victor Hess, from which the existence of cosmic rays was deduced. RAD is an advanced and unique flight instrument. It combines charged- and neutral-particle measurement capabilities in an extremely compact, low-mass package. RAD contains six detectors, three of which (A, B, and C) are silicon diodes arranged as a telescope, with the other three (D, E, and F) being scintillators. Two of the scintillators, E and F, are made of Bicron BC-432m plastic; the other, D, is made of CsI for efficient gamma-ray detection. To minimize RAD's telemetry requirements, the instrument processes its data in real time and populates a number of histograms, sorting events into broad categories of penetrating charged particles, stopping charged particles, and neutral particles. There is also a group of histograms referred to as the "dosimetry" histograms. These include minute-by-minute totals of energy deposition in the B and E detectors, as well as LET spectra for charged particles in the telescope field of view. In this presentation, we will describe the methodology used to turn the onboard histograms into properly normalized dosimetric quantities, and show results expressed as time series of dose rates in silicon and tissue, and dose-equivalent rates in tissue. Interpretation of the dosimetry data depends on understanding the effects of the shielding around RAD, which is substantial, both in cruise (spacecraft mass) and on the surface of Mars (atmosphere). This shielding

  2. Dose-rate and irradiation temperature dependence of BJT SPICE model rad-parameters

    SciTech Connect

    Montagner, X.; Briand, R.; Fouillat, P.; Touboul, A.; Schrimpf, R.D.; Galloway, K.F.; Calvet, M.C.; Calvel, P.

    1998-06-01

    A method to predict low dose rate degradation of bipolar transistors using high dose-rate, high temperature irradiation is evaluated, based on an analysis of four new rad-parameters that are introduced in the BJT SPICE model. This improved BJT model describes the radiation-induced excess base current with great accuracy. The low-level values of the rad-parameters are good tools for evaluating the proposed high-temperature test method because of their high sensitivity to radiation-induced degradation.

  3. Improved Methodology Application for 12-Rad Analysis in a Shielded Facility at SRS

    SciTech Connect

    Paul, P.

    2003-01-31

    The DOE Order 420.1 requires establishing 12-rad evacuation zone boundaries and installing Criticality Accident Alarm System (CAAS) per ANS-8.3 standard for facilities having a probability of criticality greater than 10-6 per year. The H-Canyon at the Savannah River Site (SRS) is one of the reprocessing facilities where SRS reactor fuels, research reactor fuels, and other fissile materials are processed and purified using a modified Purex process called H-Modified or HM Process. This paper discusses an improved methodology for 12-rad zone analysis and its implementation within this large shielded facility that has a large variety of criticality sources and scenarios.

  4. Clinical and biological significance of RAD51 expression in breast cancer: a key DNA damage response protein.

    PubMed

    Alshareeda, Alaa Tarig; Negm, Ola H; Aleskandarany, Mohammed A; Green, Andrew R; Nolan, Christopher; TigHhe, Patrick J; Madhusudan, Srinivasan; Ellis, Ian O; Rakha, Emad A

    2016-08-01

    Impaired DNA damage response (DDR) may play a fundamental role in the pathogenesis of breast cancer (BC). RAD51 is a key player in DNA double-strand break repair. In this study, we aimed to assess the biological and clinical significance of RAD51 expression with relevance to different molecular classes of BC and patients' outcome. The expression of RAD51 was assessed immunohistochemically in a well-characterised annotated series (n = 1184) of early-stage invasive BC with long-term follow-up. A subset of cases of BC from patients with known BRCA1 germline mutations was included as a control group. The results were correlated with clinicopathological and molecular parameters and patients' outcome. RAD51 protein expression level was also assayed in a panel of cell lines using reverse phase protein array (RPPA). RAD51 was expressed in the nuclei (N) and cytoplasm (C) of malignant cells. Subcellular co-localisation phenotypes of RAD51 were significantly associated with clinicopathological features and patient outcome. Cytoplasmic expression (RAD51C(+)) and lack of nuclear expression (RAD51 N(-)) were associated with features of aggressive behaviour, including larger tumour size, high grade, lymph nodal metastasis, basal-like, and triple-negative phenotypes, together with aberrant expression of key DDR biomarkers including BRCA1. All BRCA1-mutated tumours had RAD51C(+)/N(-) phenotype. RPPA confirmed IHC results and showed differential expression of RAD51 in cell lines based on ER expression and BRCA1 status. RAD51 N(+) and RAD51C(+) tumours were associated with longer and shorter breast cancer-specific survival (BCSS), respectively. The RAD51 N(+) was an independent predictor of longer BCSS (P < 0.0001). Lack of RAD51 nuclear expression is associated with poor prognostic parameters and shorter survival in invasive BC patients. The significant associations between RAD51 subcellular localisation and clinicopathological features, molecular subtype and patients

  5. Suzaku Observations of Moderately Obscured (Compton-thin) Active Galactic Nuclei Selected by Swift/BAT Hard X-ray Survey

    NASA Astrophysics Data System (ADS)

    Kawamuro, Taiki; Ueda, Yoshihiro; Tazaki, Fumie; Ricci, Claudio; Terashima, Yuichi

    2016-07-01

    We report the results obtained by a systematic, broadband (0.5–150 keV) X-ray spectral analysis of moderately obscured (Compton-thin, 22≤slant {log}{N}{{H}}\\lt 24) active galactic nuclei (AGNs) observed with Suzaku and Swift/Burst Alert Telescope (BAT). Our sample consists of 45 local AGNs at z\\lt 0.1 with {log}{L}14-195{keV}\\gt 42 detected in the Swift/BAT 70-month survey, whose Suzaku archival data are available as of 2015 December. All spectra are uniformly fit with a baseline model composed of an absorbed cutoff power-law component, reflected emission accompanied by a narrow fluorescent iron-Kα line from cold matter (torus), and scattered emission. The main results based on the above analysis are as follows. (1) The photon index is correlated with Eddington ratio, but not with luminosity or black hole mass. (2) The ratio of the luminosity of the iron-Kα line to the X-ray luminosity an indicator of the covering fraction of the torus, shows significant anticorrelation with luminosity. (3) The averaged reflection strength derived from stacked spectra above 14 keV is larger in less luminous ({log}{L}10-50{keV}≤slant 43.3, R={1.04}-0.19+0.17) or highly obscured ({log}{N}{{H}}\\gt 23, R={1.03}-0.17+0.15) AGNs than in more luminous ({log}{L}10-50{keV}\\gt 43.3, R={0.46}-0.09+0.08) or lightly obscured ({log}{N}{{H}}≤slant 23, R={0.59}-0.10+0.09) objects. (4) The ratio of the luminosity of the [{{O}} {{IV}}] 25.89 μm line to the X-ray luminosity is significantly smaller in AGNs with lower soft X-ray scattering fractions, suggesting that the former luminosity underestimates the intrinsic power of an AGN buried in a torus of small opening angle.

  6. DNA replication stress differentially regulates G1/S genes via Rad53-dependent inactivation of Nrm1

    PubMed Central

    Travesa, Anna; Kuo, Dwight; de Bruin, Robertus A M; Kalashnikova, Tatyana I; Guaderrama, Marisela; Thai, Kevin; Aslanian, Aaron; Smolka, Marcus B; Yates, John R; Ideker, Trey; Wittenberg, Curt

    2012-01-01

    MBF and SBF transcription factors regulate a large family of coordinately expressed G1/S genes required for early cell-cycle functions including DNA replication and repair. SBF is inactivated upon S-phase entry by Clb/CDK whereas MBF targets are repressed by the co-repressor, Nrm1. Using genome-wide expression analysis of cells treated with methyl methane sulfonate (MMS), hydroxyurea (HU) or camptothecin (CPT), we show that genotoxic stress during S phase specifically induces MBF-regulated genes. This occurs via direct phosphorylation of Nrm1 by Rad53, the effector checkpoint kinase, which prevents its binding to MBF target promoters. We conclude that MBF-regulated genes are distinguished from SBF-regulated genes by their sensitivity to activation by the S-phase checkpoint, thereby, providing an effective mechanism for enhancing DNA replication and repair and promoting genome stability. PMID:22333915

  7. Effect of gate oxide thickness on the radiation hardness of silicon-gate CMOS

    SciTech Connect

    Nordstrom, T.V.; Gibbon, C.F.

    1981-01-01

    Significant improvements have been made in the radiation hardness of silicon-gate CMOS by reducing the gate oxide thickness. The device studied is an 8-bit arithmetic logic unit designed with Sandia's Expanded Linear Array (ELA) standard cells. Devices with gate oxide thicknesses of 400, 570 (standard), and 700 A were fabricated. Irradiations were done at a dose rate of 2 x 10/sup 6/ rads (Si) per hour. N- and P-channel maximum threshold shifts were reduced by 0.3 and 1.2 volts, respectively, for the thinnest oxide. Approximately, a linear relationship is found for threshold shift versus thickness. The functional radiation hardness of the full integrated circuit was also measured.

  8. Rad5 Template Switch Pathway of DNA Damage Tolerance Determines Synergism between Cisplatin and NSC109268 in Saccharomyces cerevisiae

    PubMed Central

    Jain, Dilip; Siede, Wolfram

    2013-01-01

    The success of cisplatin (CP) based therapy is often hindered by acquisition of CP resistance. We isolated NSC109268 as a compound altering cellular sensitivity to DNA damaging agents. Previous investigation revealed an enhancement of CP sensitivity by NSC109268 in wild-type Saccharomyces cerevisiae and CP-sensitive and -resistant cancer cell lines that correlated with a slower S phase traversal. Here, we extended these studies to determine the target pathway(s) of NSC109268 in mediating CP sensitization, using yeast as a model. We reasoned that mutants defective in the relevant target of NSC109268 should be hypersensitive to CP and the sensitization effect by NSC109268 should be absent or strongly reduced. A survey of various yeast deletion mutants converged on the Rad5 pathway of DNA damage tolerance by template switching as the likely target pathway of NSC109268 in mediating cellular sensitization to CP. Additionally, cell cycle delays following CP treatment were not synergistically influenced by NSC109268 in the CP hypersensitive rad5Δ mutant. The involvement of the known inhibitory activities of NSC109268 on 20S proteasome and phosphatases 2Cα and 2A was tested. In the CP hypersensitive ptc2Δptc3Δpph3Δ yeast strain, deficient for 2C and 2A-type phosphatases, cellular sensitization to CP by NSC109268 was greatly reduced. It is therefore suggested that NSC109268 affects CP sensitivity by inhibiting the activity of unknown protein(s) whose dephosphorylation is required for the template switch pathway. PMID:24130896

  9. Melanoma Development and Progression Are Associated with Rad6 Upregulation and β-Catenin Relocation to the Cell Membrane

    PubMed Central

    Mehregan, Darius R.; Abrams, Judith; Haynes, Brittany; Shekhar, Malathy P. V.

    2014-01-01

    We have previously demonstrated that Rad6 and β-catenin enhance each other's expression through a positive feedback loop to promote breast cancer development/progression. While β-catenin has been implicated in melanoma pathogenesis, Rad6 function has not been investigated. Here, we examined the relationship between Rad6 and β-catenin in melanoma development and progression. Eighty-eight cutaneous tumors, 30 nevi, 29 primary melanoma, and 29 metastatic melanomas, were immunostained with anti-β-catenin and anti-Rad6 antibodies. Strong expression of Rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas. β-Catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas, and unlike Rad6, in 93% of nevi. None of the tumors expressed nuclear β-catenin. β-Catenin was exclusively localized on the cell membrane of 55% of primary, 62% of metastatic melanomas, and only 10% of nevi. Cytoplasmic β-catenin was detected in 90% of nevi, 17% of primary, and 8% of metastatic melanoma, whereas 28% of primary and 30% of metastatic melanomas exhibited β-catenin at both locations. These data suggest that melanoma development and progression are associated with Rad6 upregulation and membranous redistribution of β-catenin and that β-catenin and Rad6 play independent roles in melanoma development. PMID:24891954

  10. The Cohesin Subunit Rad21 Is Required for Synaptonemal Complex Maintenance, but Not Sister Chromatid Cohes