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. 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.

  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. 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.

  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

    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

  3. 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

  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. 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

  1. 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/

  2. 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/

  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. 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

  6. 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

  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. 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

  11. 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

  12. 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.

  13. 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

  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. 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.

  18. 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

  19. 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.

  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. 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.

  2. 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.

  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. 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

  5. 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

  6. 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

  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. 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.

  10. 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

  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. 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.

  16. 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

  17. 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).

  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 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.

  1. 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

  2. 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

  3. 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

  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. 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

  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. 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

  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. 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

  11. 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

  12. 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

  13. 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

  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. 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.

  8. 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.

  9. 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

  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. 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.

  19. 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

  20. 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.

  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. 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

  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. 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.

  7. 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

  8. 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

  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. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  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. 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

  7. 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

  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. 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

  18. 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.

  19. 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

  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 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

  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. 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.

  7. 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

  8. 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

  9. 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

  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. 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

  14. 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

  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 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

  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 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.

  3. 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

  4. 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

  5. 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

  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. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  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