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Sample records for rad-51 filament disassembly

  1. Overlapping mechanisms promote postsynaptic RAD-51 filament disassembly during meiotic double-strand break repair.

    PubMed

    Ward, Jordan D; Muzzini, Diego M; Petalcorin, Mark I R; Martinez-Perez, Enrique; Martin, Julie S; Plevani, Paolo; Cassata, Giuseppe; Marini, Federica; Boulton, Simon J

    2010-01-29

    Homologous recombination (HR) is essential for repair of meiotic DNA double-strand breaks (DSBs). Although the mechanisms of RAD-51-DNA filament assembly and strand exchange are well characterized, the subsequent steps of HR are less well defined. Here, we describe a synthetic lethal interaction between the C. elegans helicase helq-1 and RAD-51 paralog rfs-1, which results in a block to meiotic DSB repair after strand invasion. Whereas RAD-51-ssDNA filaments assemble at meiotic DSBs with normal kinetics in helq-1, rfs-1 double mutants, persistence of RAD-51 foci and genetic interactions with rtel-1 suggest a failure to disassemble RAD-51 from strand invasion intermediates. Indeed, purified HELQ-1 and RFS-1 independently bind to and promote the disassembly of RAD-51 from double-stranded, but not single-stranded, DNA filaments via distinct mechanisms in vitro. These results indicate that two compensating activities are required to promote postsynaptic RAD-51 filament disassembly, which are collectively essential for completion of meiotic DSB repair.

  2. Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament

    PubMed Central

    Ma, Chu Jian; Gibb, Bryan; Kwon, YoungHo; Sung, Patrick; Greene, Eric C.

    2017-01-01

    Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. PMID:27903895

  3. Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination

    PubMed Central

    Taylor, Martin R.G.; Špírek, Mário; Chaurasiya, Kathy R.; Ward, Jordan D.; Carzaniga, Raffaella; Yu, Xiong; Egelman, Edward H.; Collinson, Lucy M.; Rueda, David; Krejci, Lumir; Boulton, Simon J.

    2015-01-01

    Summary Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, “open,” and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex. PMID:26186187

  4. Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination.

    PubMed

    Taylor, Martin R G; Špírek, Mário; Chaurasiya, Kathy R; Ward, Jordan D; Carzaniga, Raffaella; Yu, Xiong; Egelman, Edward H; Collinson, Lucy M; Rueda, David; Krejci, Lumir; Boulton, Simon J

    2015-07-16

    Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, "open," and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling.

    PubMed

    Taylor, Martin R G; Špírek, Mário; Jian Ma, Chu; Carzaniga, Raffaella; Takaki, Tohru; Collinson, Lucy M; Greene, Eric C; Krejci, Lumir; Boulton, Simon J

    2016-12-01

    Central to homologous recombination in eukaryotes is the RAD51 recombinase, which forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) and catalyzes strand invasion with homologous duplex DNA. Various regulatory proteins assist this reaction including the RAD51 paralogs. We recently discovered that a RAD51 paralog complex from C. elegans, RFS-1/RIP-1, functions predominantly downstream of filament assembly by binding and remodeling RAD-51-ssDNA filaments to a conformation more proficient for strand exchange. Here, we demonstrate that RFS-1/RIP-1 acts by shutting down RAD-51 dissociation from ssDNA. Using stopped-flow experiments, we show that RFS-1/RIP-1 confers this dramatic stabilization by capping the 5' end of RAD-51-ssDNA filaments. Filament end capping propagates a stabilizing effect with a 5'→3' polarity approximately 40 nucleotides along individual filaments. Finally, we discover that filament capping and stabilization are dependent on nucleotide binding, but not hydrolysis by RFS-1/RIP-1. These data define the mechanism of RAD51 filament remodeling by RAD51 paralogs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. 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

  7. 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.

  8. FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells.

    PubMed

    Simandlova, Jitka; Zagelbaum, Jennifer; Payne, Miranda J; Chu, Wai Kit; Shevelev, Igor; Hanada, Katsuhiro; Chatterjee, Sujoy; Reid, Dylan A; Liu, Ying; Janscak, Pavel; Rothenberg, Eli; Hickson, Ian D

    2013-11-22

    Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.

  9. High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy

    PubMed Central

    Short, Judith M.; Liu, Yang; Chen, Shaoxia; Soni, Neelesh; Madhusudhan, Mallur S.; Shivji, Mahmud K.K.; Venkitaraman, Ashok R.

    2016-01-01

    Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5–5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51–DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling suggests that changes in filament pitch mask or expose one binding site with filament-inhibitory potential, rationalizing the paradoxical ability of the BRC repeats to either stabilize or inhibit filament formation at different steps during HR. Collectively, our findings provide fresh insight into the structural mechanism of HR and its dysregulation in human disease. PMID:27596592

  10. Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51-DNA filaments.

    PubMed

    Sanchez, Humberto; Kertokalio, Aryandi; van Rossum-Fikkert, Sari; Kanaar, Roland; Wyman, Claire

    2013-07-09

    Essential genome transactions, such as homologous recombination, are achieved by concerted and dynamic interactions of multiple protein components with DNA. Which proteins do what and how, will be reflected in their relative arrangements. However, obtaining high-resolution structural information on the variable arrangements of these complex assemblies is a challenge. Here we demonstrate the versatility of a combined total internal reflection fluorescence and scanning force microscope (TIRF-SFM) to pinpoint fluorescently labeled human homologous recombination protein RAD54 interacting with presynaptic (ssDNA) and postsynaptic (dsDNA) human recombinase RAD51 nucleoprotein filaments. Labeled proteins were localized by superresolution imaging on complex structures in the SFM image with high spatial accuracy. We observed some RAD54 at RAD51 filament ends, as expected. More commonly, RAD54 interspersed along RAD51-DNA filaments. RAD54 promotes RAD51-mediated DNA strand exchange and has been described to both stabilize and destabilize RAD51-DNA filaments. The different architectural arrangements we observe for RAD54 with RAD51-DNA filaments may reflect the diverse roles of this protein in homologous recombination.

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

    SciTech Connect

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

    2010-09-03

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

  12. FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5'-DNA end.

    PubMed

    Sato, Koichi; Shimomuki, Mayo; Katsuki, Yoko; Takahashi, Daisuke; Kobayashi, Wataru; Ishiai, Masamichi; Miyoshi, Hiroyuki; Takata, Minoru; Kurumizaka, Hitoshi

    2016-12-15

    The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork.

  13. 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.

  14. FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5′-DNA end

    PubMed Central

    Sato, Koichi; Shimomuki, Mayo; Katsuki, Yoko; Takahashi, Daisuke; Kobayashi, Wataru; Ishiai, Masamichi; Miyoshi, Hiroyuki; Takata, Minoru; Kurumizaka, Hitoshi

    2016-01-01

    The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork. PMID:27694619

  15. Homologous Pairing Activities of Two Rice RAD51 Proteins, RAD51A1 and RAD51A2

    PubMed Central

    Ikawa, Shukuko; Mimida, Naozumi; Shimizu, Takeshi; Toki, Seiichi; Ichikawa, Hiroaki; Shibata, Takehiko; Kurumizaka, Hitoshi

    2013-01-01

    In higher eukaryotes, RAD51 functions as an essential protein in homologous recombination and recombinational repair of DNA double strand breaks. During these processes, RAD51 catalyzes homologous pairing between single-stranded DNA and double-stranded DNA. Japonica cultivars of rice (Oryza sativa) encode two RAD51 proteins, RAD51A1 and RAD51A2, whereas only one RAD51 exists in yeast and mammals. However, the functional differences between RAD51A1 and RAD51A2 have not been elucidated, because their biochemical properties have not been characterized. In the present study, we purified RAD51A1 and RAD51A2, and found that RAD51A2 robustly promotes homologous pairing in vitro. RAD51A1 also possesses homologous-pairing activity, but it is only about 10% of the RAD51A2 activity. Both RAD51A1 and RAD51A2 bind to ssDNA and dsDNA, and their DNA binding strictly requires ATP, which modulates the polymer formation activities of RAD51A1 and RAD51A2. These findings suggest that although both RAD51A1 and RAD51A2 have the potential to catalyze homologous pairing, RAD51A2 may be the major recombinase in rice. PMID:24124491

  16. 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.

  17. The RAD51-stimulatory compound RS-1 can exploit the RAD51 overexpression that exists in cancer cells and tumors

    PubMed Central

    Mason, Jennifer M; Logan, Hillary L.; Budke, Brian; Wu, Megan; Pawlowski, Michal; Weichselbaum, Ralph R.; Kozikowski, Alan P.; Bishop, Douglas K.; Connell, Philip P.

    2014-01-01

    RAD51 is the central protein that catalyzes DNA repair via homologous recombination (HR), a process that ensures genomic stability. RAD51 protein is commonly expressed at high levels in cancer cells relative to their non-cancerous precursors. High levels of RAD51 expression can lead to the formation of genotoxic RAD51 protein complexes on undamaged chromatin. We developed a therapeutic approach that exploits this potentially toxic feature of malignancy, using compounds that stimulate the DNA binding activity of RAD51 to promote cancer cell death. A panel of immortalized cell lines was challenged with the RAD51-stimulatory compound RS-1. Resistance to RS-1 tended to occur in cells with higher levels of RAD54L and RAD54B, which are Swi2/Snf2-related translocases known to dissociate RAD51 filaments from double-stranded DNA. In PC3 prostate cancer cells, RS-1 induced lethality was accompanied by the formation of microscopically visible RAD51 nuclear protein foci occurring in the absence of any DNA-damaging treatment. Treatment with RS-1 promoted significant anti-tumor responses in a mouse model, providing proof of principle for this novel therapeutic strategy. PMID:24753542

  18. Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

    PubMed Central

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

    2015-01-01

    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 Mec1ATR/Tel1ATM-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

  19. The Chromatin Assembly Factor 1 Promotes Rad51-Dependent Template Switches at Replication Forks by Counteracting D-Loop Disassembly by the RecQ-Type Helicase Rqh1

    PubMed Central

    Hardy, Julien; Costes, Audrey; Iraqui, Ismail; Ochsenbein, Françoise; Lambert, Sarah A.E.

    2014-01-01

    At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom's syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop. PMID:25313826

  20. The chromatin assembly factor 1 promotes Rad51-dependent template switches at replication forks by counteracting D-loop disassembly by the RecQ-type helicase Rqh1.

    PubMed

    Pietrobon, Violena; Fréon, Karine; Hardy, Julien; Costes, Audrey; Iraqui, Ismail; Ochsenbein, Françoise; Lambert, Sarah A E

    2014-10-01

    At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom's syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop.

  1. 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.

  2. 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.

  3. Novel insights into RAD51 activity and regulation during homologous recombination and DNA replication

    PubMed Central

    Godin, Stephen K.; Sullivan, Meghan R.; Bernstein, Kara A.

    2016-01-01

    In this review we focus on new insights that challenge our understanding of homologous recombination (HR) and Rad51 regulation. Recent advances using high resolution microscopy and single molecule techniques have broadened our knowledge of Rad51 filament formation and strand invasion at double-strand break (DSB) sites and at replication forks, which are one of most physiologically relevant forms of HR from yeast to humans. Rad51 filament formation and strand invasion is regulated by many mediator proteins such as the Rad51 paralogues and the Shu complex, consisting of a Shu2/SWS1 family member and additional Rad51 paralogues. Importantly, a novel RAD-51 paralogue was discovered in C. elegans and its in vitro characterization has demonstrated a new function for the worm RAD-51 paralogues during HR. Conservation of the human RAD51 paralogues function during HR and repair of replicative damage demonstrate how the RAD51 mediators play a critical role in human health and genomic integrity. Together, these new findings provide a framework for understanding RAD51 and its mediators in DNA repair during multiple cellular contexts. PMID:27224545

  4. Roles of XRCC2, RAD51B and RAD51D in RAD51-Independent SSA Recombination

    PubMed Central

    Serra, Heïdi; Da Ines, Olivier; Degroote, Fabienne; Gallego, Maria E.; White, Charles I.

    2013-01-01

    The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence or not on strand invasion catalysed by the RAD51 recombinase. Working with the plant Arabidopsis thaliana, we present here an unexpected role in recombination for the Arabidopsis RAD51 paralogues XRCC2, RAD51B and RAD51D in the RAD51-independent single-strand annealing pathway. The roles of these proteins are seen in spontaneous and in DSB-induced recombination at a tandem direct repeat recombination tester locus, both of which are unaffected by the absence of RAD51. Individual roles of these proteins are suggested by the strikingly different severities of the phenotypes of the individual mutants, with the xrcc2 mutant being the most affected, and this is confirmed by epistasis analyses using multiple knockouts. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2, RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination. PMID:24278037

  5. Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase.

    PubMed

    Kelso, Andrew A; Goodson, Steven D; Chavan, Suchitra; Say, Amanda F; Turchick, Audrey; Sharma, Deepti; Ledford, LeAnna L; Ratterman, Erin; Leskoske, Kristin; King, Ada V; Attaway, Christopher C; Bandera, Yura; Foulger, Stephen H; Mazin, Alexander V; Temesvari, Lesly A; Sehorn, Michael G

    The protozoan parasite responsible for human amoebiasis is Entamoeba histolytica. An important facet of the life cycle of E. histolytica involves the conversion of the mature trophozoite to a cyst. This transition is thought to involve homologous recombination (HR), which is dependent upon the Rad51 recombinase. Here, a biochemical characterization of highly purified ehRad51 protein is presented. The ehRad51 protein preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. Although the homologous DNA pairing activity of ehRad51 is weak, it is strongly enhanced by the presence of two HR accessory cofactors, calcium and Hop2-Mnd1. The biochemical system described herein was used to demonstrate the potential for targeting ehRad51 with two small molecule inhibitors of human RAD51. We show that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited ehRad51 by interfering with DNA binding and attenuated encystation in Entamoeba invadens, while B02 had no effect on ehRad51 strand exchange activity. These results provide insight into the underlying mechanism of homology-directed DNA repair in E. histolytica. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. 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.

  7. 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.

  8. Cofilin cooperates with fascin to disassemble filopodial actin filaments

    PubMed Central

    Breitsprecher, Dennis; Koestler, Stefan A.; Chizhov, Igor; Nemethova, Maria; Mueller, Jan; Goode, Bruce L.; Small, J. Victor; Rottner, Klemens; Faix, Jan

    2011-01-01

    Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin. PMID:21940796

  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. Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange

    PubMed Central

    Solinger, Jachen Armon; Heyer, Wolf-Dietrich

    2001-01-01

    Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. As previously shown, Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Here we demonstrate that Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depended on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins. PMID:11459988

  11. RI-1: a chemical inhibitor of RAD51 that disrupts homologous recombination in human cells

    PubMed Central

    Budke, Brian; Logan, Hillary L.; Kalin, Jay H.; Zelivianskaia, Anna S.; Cameron McGuire, William; Miller, Luke L.; Stark, Jeremy M.; Kozikowski, Alan P.; Bishop, Douglas K.; Connell, Philip P.

    2012-01-01

    Homologous recombination serves multiple roles in DNA repair that are essential for maintaining genomic stability. We here describe RI-1, a small molecule that inhibits the central recombination protein RAD51. RI-1 specifically reduces gene conversion in human cells while stimulating single strand annealing. RI-1 binds covalently to the surface of RAD51 protein at cysteine 319 that likely destabilizes an interface used by RAD51 monomers to oligomerize into filaments on DNA. Correspondingly, the molecule inhibits the formation of subnuclear RAD51 foci in cells following DNA damage, while leaving replication protein A focus formation unaffected. Finally, it potentiates the lethal effects of a DNA cross-linking drug in human cells. Given that this inhibitory activity is seen in multiple human tumor cell lines, RI-1 holds promise as an oncologic drug. Furthermore, RI-1 represents a unique tool to dissect the network of reaction pathways that contribute to DNA repair in cells. PMID:22573178

  12. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Arabidopsis RAD51, RAD51C and XRCC3 proteins form a complex and facilitate RAD51 localization on chromosomes for meiotic recombination

    PubMed Central

    Lin, Juan; Ma, Hong

    2017-01-01

    Meiotic recombination is required for proper homologous chromosome segregation in plants and other eukaryotes. The eukaryotic RAD51 gene family has seven ancient paralogs with important roles in mitotic and meiotic recombination. Mutations in mammalian RAD51 homologs RAD51C and XRCC3 lead to embryonic lethality. In the model plant Arabidopsis thaliana, RAD51C and XRCC3 homologs are not essential for vegetative development but are each required for somatic and meiotic recombination, but the mechanism of RAD51C and XRCC3 in meiotic recombination is unclear. The non-lethal Arabidopsis rad51c and xrcc3 null mutants provide an opportunity to study their meiotic functions. Here, we show that AtRAD51C and AtXRCC3 are components of the RAD51-dependent meiotic recombination pathway and required for normal AtRAD51 localization on meiotic chromosomes. In addition, AtRAD51C interacts with both AtRAD51 and AtXRCC3 in vitro and in vivo, suggesting that these proteins form a complex (es). Comparison of AtRAD51 foci in meiocytes from atrad51, atrad51c, and atxrcc3 single, double and triple heterozygous mutants further supports an interaction between AtRAD51C and AtXRCC3 that enhances AtRAD51 localization. Moreover, atrad51c-/+ atxrcc3-/+ double and atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygous mutants have defects in meiotic recombination, suggesting the role of the AtRAD51C-AtXRCC3 complex in meiotic recombination is in part AtRAD51-dependent. Together, our results support a model in which direct interactions between the RAD51C-XRCC3 complex and RAD51 facilitate RAD51 localization on meiotic chromosomes and RAD51-dependent meiotic recombination. Finally, we hypothesize that maintenance of RAD51 function facilitated by the RAD51C-XRCC3 complex could be highly conserved in eukaryotes. PMID:28562599

  14. Arabidopsis RAD51, RAD51C and XRCC3 proteins form a complex and facilitate RAD51 localization on chromosomes for meiotic recombination.

    PubMed

    Su, Hang; Cheng, Zhihao; Huang, Jiyue; Lin, Juan; Copenhaver, Gregory P; Ma, Hong; Wang, Yingxiang

    2017-05-01

    Meiotic recombination is required for proper homologous chromosome segregation in plants and other eukaryotes. The eukaryotic RAD51 gene family has seven ancient paralogs with important roles in mitotic and meiotic recombination. Mutations in mammalian RAD51 homologs RAD51C and XRCC3 lead to embryonic lethality. In the model plant Arabidopsis thaliana, RAD51C and XRCC3 homologs are not essential for vegetative development but are each required for somatic and meiotic recombination, but the mechanism of RAD51C and XRCC3 in meiotic recombination is unclear. The non-lethal Arabidopsis rad51c and xrcc3 null mutants provide an opportunity to study their meiotic functions. Here, we show that AtRAD51C and AtXRCC3 are components of the RAD51-dependent meiotic recombination pathway and required for normal AtRAD51 localization on meiotic chromosomes. In addition, AtRAD51C interacts with both AtRAD51 and AtXRCC3 in vitro and in vivo, suggesting that these proteins form a complex (es). Comparison of AtRAD51 foci in meiocytes from atrad51, atrad51c, and atxrcc3 single, double and triple heterozygous mutants further supports an interaction between AtRAD51C and AtXRCC3 that enhances AtRAD51 localization. Moreover, atrad51c-/+ atxrcc3-/+ double and atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygous mutants have defects in meiotic recombination, suggesting the role of the AtRAD51C-AtXRCC3 complex in meiotic recombination is in part AtRAD51-dependent. Together, our results support a model in which direct interactions between the RAD51C-XRCC3 complex and RAD51 facilitate RAD51 localization on meiotic chromosomes and RAD51-dependent meiotic recombination. Finally, we hypothesize that maintenance of RAD51 function facilitated by the RAD51C-XRCC3 complex could be highly conserved in eukaryotes.

  15. Actin-filament disassembly: it takes two to shrink them fast.

    PubMed

    Winterhoff, Moritz; Faix, Jan

    2015-06-01

    Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level.

  16. A Distinct Replication Fork Protection Pathway Connects Fanconi Anemia Tumor Suppressors to RAD51-BRCA1/2

    PubMed Central

    Schlacher, Katharina; Wu, Hong; Jasin, Maria

    2014-01-01

    SUMMARY Genes mutated in patients with Fanconi anemia (FA) interact with the DNA repair genes BRCA1 and BRCA2/FANCD1 to suppress tumorigenesis, but the molecular functions ascribed to them cannot fully explain all of their cellular roles. Here, we show a repair-independent requirement for FA genes, including FANCD2, and BRCA1 in protecting stalled replication forks from degradation. Fork protection is surprisingly rescued in FANCD2-deficient cells by elevated RAD51 levels or stabilized RAD51 filaments. Moreover, FANCD2-mediated fork protection is epistatic with RAD51 functions, revealing an unanticipated fork protection pathway that connects FA genes to RAD51 and the BRCA1/2 breast cancer suppressors. Collective results imply a unified molecular mechanism for repair-independent functions of FA, RAD51, and BRCA1/2 proteins in preventing genomic instability and suppressing tumorigenesis. PMID:22789542

  17. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2.

    PubMed

    Schlacher, Katharina; Wu, Hong; Jasin, Maria

    2012-07-10

    Genes mutated in patients with Fanconi anemia (FA) interact with the DNA repair genes BRCA1 and BRCA2/FANCD1 to suppress tumorigenesis, but the molecular functions ascribed to them cannot fully explain all of their cellular roles. Here, we show a repair-independent requirement for FA genes, including FANCD2, and BRCA1 in protecting stalled replication forks from degradation. Fork protection is surprisingly rescued in FANCD2-deficient cells by elevated RAD51 levels or stabilized RAD51 filaments. Moreover, FANCD2-mediated fork protection is epistatic with RAD51 functions, revealing an unanticipated fork protection pathway that connects FA genes to RAD51 and the BRCA1/2 breast cancer suppressors. Collective results imply a unified molecular mechanism for repair-independent functions of FA, RAD51, and BRCA1/2 proteins in preventing genomic instability and suppressing tumorigenesis.

  18. 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.

  19. 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

  20. Contribution of Germline Mutations in the RAD51B, RAD51C, and RAD51D Genes to Ovarian Cancer in the Population

    PubMed Central

    Song, Honglin; Dicks, Ed; Ramus, Susan J.; Tyrer, Jonathan P.; Intermaggio, Maria P.; Hayward, Jane; Edlund, Christopher K.; Conti, David; Harrington, Patricia; Fraser, Lindsay; Philpott, Susan; Anderson, Christopher; Rosenthal, Adam; Gentry-Maharaj, Aleksandra; Bowtell, David D.; Alsop, Kathryn; Cicek, Mine S.; Cunningham, Julie M.; Fridley, Brooke L.; Alsop, Jennifer; Jimenez-Linan, Mercedes; Høgdall, Estrid; Høgdall, Claus K.; Jensen, Allan; Kjaer, Susanne Krüger; Lubiński, Jan; Huzarski, Tomasz; Jakubowska, Anna; Gronwald, Jacek; Poblete, Samantha; Lele, Shashi; Sucheston-Campbell, Lara; Moysich, Kirsten B.; Odunsi, Kunle; Goode, Ellen L.; Menon, Usha; Jacobs, Ian J.; Gayther, Simon A.; Pharoah, Paul D.P.

    2015-01-01

    Purpose The aim of this study was to estimate the contribution of deleterious mutations in the RAD51B, RAD51C, and RAD51D genes to invasive epithelial ovarian cancer (EOC) in the population and in a screening trial of individuals at high risk of ovarian cancer. Patients and Methods The coding sequence and splice site boundaries of the three RAD51 genes were sequenced and analyzed in germline DNA from a case-control study of 3,429 patients with invasive EOC and 2,772 controls as well as in 2,000 unaffected women who were BRCA1/BRCA2 negative from the United Kingdom Familial Ovarian Cancer Screening Study (UK_FOCSS) after quality-control analysis. Results In the case-control study, we identified predicted deleterious mutations in 28 EOC cases (0.82%) compared with three controls (0.11%; P < .001). Mutations in EOC cases were more frequent in RAD51C (14 occurrences, 0.41%) and RAD51D (12 occurrences, 0.35%) than in RAD51B (two occurrences, 0.06%). RAD51C mutations were associated with an odds ratio of 5.2 (95% CI, 1.1 to 24; P = .035), and RAD51D mutations conferred an odds ratio of 12 (95% CI, 1.5 to 90; P = .019). We identified 13 RAD51 mutations (0.65%) in unaffected UK_FOCSS participants (RAD51C, n = 7; RAD51D, n = 5; and RAD51B, n = 1), which was a significantly greater rate than in controls (P < .001); furthermore, RAD51 mutation carriers were more likely than noncarriers to have a family history of ovarian cancer (P < .001). Conclusion These results confirm that RAD51C and RAD51D are moderate ovarian cancer susceptibility genes and suggest that they confer levels of risk of EOC that may warrant their use alongside BRCA1 and BRCA2 in routine clinical genetic testing. PMID:26261251

  1. Contribution of Germline Mutations in the RAD51B, RAD51C, and RAD51D Genes to Ovarian Cancer in the Population.

    PubMed

    Song, Honglin; Dicks, Ed; Ramus, Susan J; Tyrer, Jonathan P; Intermaggio, Maria P; Hayward, Jane; Edlund, Christopher K; Conti, David; Harrington, Patricia; Fraser, Lindsay; Philpott, Susan; Anderson, Christopher; Rosenthal, Adam; Gentry-Maharaj, Aleksandra; Bowtell, David D; Alsop, Kathryn; Cicek, Mine S; Cunningham, Julie M; Fridley, Brooke L; Alsop, Jennifer; Jimenez-Linan, Mercedes; Høgdall, Estrid; Høgdall, Claus K; Jensen, Allan; Kjaer, Susanne Krüger; Lubiński, Jan; Huzarski, Tomasz; Jakubowska, Anna; Gronwald, Jacek; Poblete, Samantha; Lele, Shashi; Sucheston-Campbell, Lara; Moysich, Kirsten B; Odunsi, Kunle; Goode, Ellen L; Menon, Usha; Jacobs, Ian J; Gayther, Simon A; Pharoah, Paul D P

    2015-09-10

    The aim of this study was to estimate the contribution of deleterious mutations in the RAD51B, RAD51C, and RAD51D genes to invasive epithelial ovarian cancer (EOC) in the population and in a screening trial of individuals at high risk of ovarian cancer. The coding sequence and splice site boundaries of the three RAD51 genes were sequenced and analyzed in germline DNA from a case-control study of 3,429 patients with invasive EOC and 2,772 controls as well as in 2,000 unaffected women who were BRCA1/BRCA2 negative from the United Kingdom Familial Ovarian Cancer Screening Study (UK_FOCSS) after quality-control analysis. In the case-control study, we identified predicted deleterious mutations in 28 EOC cases (0.82%) compared with three controls (0.11%; P < .001). Mutations in EOC cases were more frequent in RAD51C (14 occurrences, 0.41%) and RAD51D (12 occurrences, 0.35%) than in RAD51B (two occurrences, 0.06%). RAD51C mutations were associated with an odds ratio of 5.2 (95% CI, 1.1 to 24; P = .035), and RAD51D mutations conferred an odds ratio of 12 (95% CI, 1.5 to 90; P = .019). We identified 13 RAD51 mutations (0.65%) in unaffected UK_FOCSS participants (RAD51C, n = 7; RAD51D, n = 5; and RAD51B, n = 1), which was a significantly greater rate than in controls (P < .001); furthermore, RAD51 mutation carriers were more likely than noncarriers to have a family history of ovarian cancer (P < .001). These results confirm that RAD51C and RAD51D are moderate ovarian cancer susceptibility genes and suggest that they confer levels of risk of EOC that may warrant their use alongside BRCA1 and BRCA2 in routine clinical genetic testing. © 2015 by American Society of Clinical Oncology.

  2. Rad51 activates polyomavirus JC early transcription.

    PubMed

    White, Martyn K; Kaminski, Rafal; Khalili, Kamel; Wollebo, Hassen S

    2014-01-01

    The human neurotropic polyomavirus JC (JCV) causes the fatal CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection is very common and after primary infection, the virus is able to persist in an asymptomatic state. Rarely, and usually only under conditions of immune impairment, JCV re-emerges to actively replicate in the astrocytes and oligodendrocytes of the brain causing PML. The regulatory events involved in the reactivation of active viral replication in PML are not well understood but previous studies have implicated the transcription factor NF-κB acting at a well-characterized site in the JCV noncoding control region (NCCR). NF-κB in turn is regulated in a number of ways including activation by cytokines such as TNF-α, interactions with other transcription factors and epigenetic events involving protein acetylation--all of which can regulate the transcriptional activity of JCV. Active JCV infection is marked by the occurrence of rapid and extensive DNA damage in the host cell and the induction of the expression of cellular proteins involved in DNA repair including Rad51, a major component of the homologous recombination-directed double-strand break DNA repair machinery. Here we show that increased Rad51 expression activates the JCV early promoter. This activation is co-operative with the stimulation caused by NF-κB p65, abrogated by mutation of the NF-κB binding site or siRNA to NFκB p65 and enhanced by the histone deacetylase inhibitor sodium butyrate. These data indicate that the induction of Rad51 resulting from infection with JCV acts through NF-κB via its binding site to stimulate JCV early transcription. We suggest that this provides a novel positive feedback mechanism to enhance viral gene expression during the early stage of JCV infection.

  3. Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation

    PubMed Central

    Hyppa, Randy W.; Benko, Zsigmond; Misova, Ivana; Schleiffer, Alexander; Smith, Gerald R.; Gregan, Juraj

    2016-01-01

    To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species. PMID:27304859

  4. Distinct binding of BRCA2 BRC repeats to RAD51 generates differential DNA damage sensitivity

    PubMed Central

    Chatterjee, Gouri; Jimenez-Sainz, Judit; Presti, Thomas; Nguyen, Tiffany; Jensen, Ryan B.

    2016-01-01

    BRCA2 is a multi-faceted protein critical for the proper regulation of homology-directed repair of DNA double-strand breaks. Elucidating the mechanistic features of BRCA2 is crucial for understanding homologous recombination and how patient-derived mutations impact future cancer risk. Eight centrally located BRC repeats in BRCA2 mediate binding and regulation of RAD51 on resected DNA substrates. Herein, we dissect the biochemical and cellular features of the BRC repeats tethered to the DNA binding domain of BRCA2. To understand how the BRC repeats and isolated domains of BRCA2 contribute to RAD51 binding, we analyzed both the biochemical and cellular properties of these proteins. In contrast to the individual BRC repeat units, we find that the BRC5–8 region potentiates RAD51-mediated DNA strand pairing and provides complementation functions exceeding those of BRC repeats 1–4. Furthermore, BRC5–8 can efficiently repair nuclease-induced DNA double-strand breaks and accelerate the assembly of RAD51 repair complexes upon DNA damage. These findings highlight the importance of the BRC5–8 domain in stabilizing the RAD51 filament and promoting homology-directed repair under conditions of cellular DNA damage. PMID:27084934

  5. The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA.

    PubMed

    Rossi, Matthew J; Mazina, Olga M; Bugreev, Dmitry V; Mazin, Alexander V

    2011-04-19

    The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.

  6. TODRA, a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair.

    PubMed

    Gazy, Inbal; Zeevi, David A; Renbaum, Paul; Zeligson, Sharon; Eini, Lital; Bashari, Dana; Smith, Yoav; Lahad, Amnon; Goldberg, Michal; Ginsberg, Doron; Levy-Lahad, Ephrat

    2015-01-01

    Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51

  7. Promotion of RAD51-mediated Homologous DNA Pairing by the RAD51AP1-UAF1 Complex

    PubMed Central

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

    2017-01-01

    Summary 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 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

  8. Variation in the RAD51 gene and familial breast cancer

    PubMed Central

    Lose, Felicity; Lovelock, Paul; Chenevix-Trench, Georgia; Mann, Graham J; Pupo, Gulietta M; Spurdle, Amanda B

    2006-01-01

    Introduction Human RAD51 is a homologue of the Escherichia coli RecA protein and is known to function in recombinational repair of double-stranded DNA breaks. Mutations in the lower eukaryotic homologues of RAD51 result in a deficiency in the repair of double-stranded DNA breaks. Loss of RAD51 function would therefore be expected to result in an elevated mutation rate, leading to accumulation of DNA damage and, hence, to increased cancer risk. RAD51 interacts directly or indirectly with a number of proteins implicated in breast cancer, such as BRCA1 and BRCA2. Similar to BRCA1 mice, RAD51-/- mice are embryonic lethal. The RAD51 gene region has been shown to exhibit loss of heterozygosity in breast tumours, and deregulated RAD51 expression in breast cancer patients has also been reported. Few studies have investigated the role of coding region variation in the RAD51 gene in familial breast cancer, with only one coding region variant – exon 6 c.449G>A (p.R150Q) – reported to date. Methods All nine coding exons of the RAD51 gene were analysed for variation in 46 well-characterised, BRCA1/2-negative breast cancer families using denaturing high-performance liquid chromatography. Genotyping of the exon 6 p.R150Q variant was performed in an additional 66 families. Additionally, lymphoblastoid cell lines from breast cancer patients were subjected to single nucleotide primer extension analysis to assess RAD51 expression. Results No coding region variation was found, and all intronic variation detected was either found in unaffected controls or was unlikely to have functional consequences. Single nucleotide primer extension analysis did not reveal any allele-specific changes in RAD51 expression in all lymphoblastoid cell lines tested. Conclusion Our study indicates that RAD51 is not a major familial breast cancer predisposition gene. PMID:16762046

  9. 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.

  10. RAD51-dependent break-induced replication differs in kinetics and checkpoint responses from RAD51-mediated gene conversion.

    PubMed

    Malkova, Anna; Naylor, Maria L; Yamaguchi, Miyuki; Ira, Grzegorz; Haber, James E

    2005-02-01

    Diploid Saccharomyces cells experiencing a double-strand break (DSB) on one homologous chromosome repair the break by RAD51-mediated gene conversion >98% of the time. However, when extensive homologous sequences are restricted to one side of the DSB, repair can occur by both RAD51-dependent and RAD51-independent break-induced replication (BIR) mechanisms. Here we characterize the kinetics and checkpoint dependence of RAD51-dependent BIR when the DSB is created within a chromosome. Gene conversion products appear within 2 h, and there is little, if any, induction of the DNA damage checkpoint; however, RAD51-dependent BIR occurs with a further delay of 2 to 4 h and cells arrest in response to the G(2)/M DNA damage checkpoint. RAD51-dependent BIR does not require special facilitating sequences that are required for a less efficient RAD51-independent process. RAD51-dependent BIR occurs efficiently in G(2)-arrested cells. Once repair is initiated, the rate of repair replication during BIR is comparable to that of normal DNA replication, as copying of >100 kb is completed less than 30 min after repair DNA synthesis is detected close to the DSB.

  11. Cellular Dynamics of Rad51 and Rad54 in Response to Postreplicative Stress and DNA Damage in HeLa Cells

    PubMed Central

    Choi, Eui-Hwan; Yoon, Seobin; Hahn, Yoonsoo; Kim, Keun P.

    2017-01-01

    Homologous recombination (HR) is necessary for maintenance of genomic integrity and prevention of various mutations in tumor suppressor genes and proto-oncogenes. Rad51 and Rad54 are key HR factors that cope with replication stress and DNA breaks in eukaryotes. Rad51 binds to single-stranded DNA (ssDNA) to form the presynaptic filament that promotes a homology search and DNA strand exchange, and Rad54 stimulates the strand-pairing function of Rad51. Here, we studied the molecular dynamics of Rad51 and Rad54 during the cell cycle of HeLa cells. These cells constitutively express Rad51 and Rad54 throughout the entire cell cycle, and the formation of foci immediately increased in response to various types of DNA damage and replication stress, except for caffeine, which suppressed the Rad51-dependent HR pathway. Depletion of Rad51 caused severe defects in response to postreplicative stress. Accordingly, HeLa cells were arrested at the G2–M transition although a small amount of Rad51 was steadily maintained in HeLa cells. Our results suggest that cell cycle progression and proliferation of HeLa cells can be tightly controlled by the abundance of HR proteins, which are essential for the rapid response to postreplicative stress and DNA damage stress. PMID:28190324

  12. Interaction of p53 with the human Rad51 protein.

    PubMed Central

    Buchhop, S; Gibson, M K; Wang, X W; Wagner, P; Stürzbecher, H W; Harris, C C

    1997-01-01

    p53 is thought to function in the maintenance of genomic stability by modulating transcription and interacting with cellular proteins to influence the cell cycle, DNA repair and apoptosis. p53 mutations occur in >50% of human cancers, and cells which lack wild type p53 accumulate karyotypic abnormalities such as amplifications, deletions, inversions and translocations. We propose that p53 hinders these promiscuous recombinational events by interacting with cellular recombination and repair machinery. We recently reported that p53 can directly bind in vivo to human Rad51 (hRad51) protein and in vitro to its bacterial homologue RecA. We used GST-fusion and his-tagged protein systems to further investigate the physical interaction between p53 and hRad51, homologue of the yeast Rad51 protein that is involved in recombination and DNA double strand repair. The hRad51 binds to wild-type p53 and to a lesser extent, point mutants 135Y, 249S and 273H. This binding is not mediated by a DNA or RNA intermediate. Mapping studies using a panel of p53 deletion mutants indicate that hRad51 could bind to two regions of p53; one between amino acids 94 and 160 and a second between 264 and 315. Addition of anti-p53 antibody PAb421 (epitope 372-381 amino acids) inhibited the interaction with hRad51. In contrast, p53 interacts with the region between aa 125 and 220 of hRad51, which is highly conserved among Rad51 related proteins from bacteria to human. In Escherichia coli ecA protein, this region is required for homo-oligomerization, suggesting that p53 might disrupt the interaction between RecA and Rad51 subunits, thus inhibiting biochemical functions of Rad51 like proteins. These data are consistent with the hypothesis that p53 interaction with hRAD51 may influence DNA recombination and repair and that additional modifications of p53 by mutation and protein binding may affect this interaction. PMID:9380510

  13. Down-Regulation of Rad51 Activity during Meiosis in Yeast Prevents Competition with Dmc1 for Repair of Double-Strand Breaks

    PubMed Central

    Liu, Yan; Gaines, William A.; Callender, Tracy; Busygina, Valeria; Oke, Ashwini; Sung, Patrick; Fung, Jennifer C.; Hollingsworth, Nancy M.

    2014-01-01

    Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs. PMID:24465215

  14. Overexpression of Rad51C splice variants in colorectal tumors.

    PubMed

    Kalvala, Arjun; Gao, Li; Aguila, Brittany; Reese, Tyler; Otterson, Gregory A; Villalona-Calero, Miguel A; Duan, Wenrui

    2015-04-20

    Functional alterations in Rad51C are the cause of the Fanconi anemia complementation group O (FANCO) gene disorder. We have identified novel splice variants of Rad51C mRNA in colorectal tumors and cells. The alternatively spliced transcript variants are formed either without exon-7 (variant 1), without exon 6 and 7 (variant 2) or without exon 7 and 8 (variant 3). Real time PCR analysis of nine pair-matched colorectal tumors and non-tumors showed that variant 1 was overexpressed in tumors compared to matched non-tumors. Among 38 colorectal tumor RNA samples analyzed, 18 contained variant 1, 12 contained variant 2, 14 contained variant 3, and eight expressed full length Rad51C exclusively. Bisulfite DNA sequencing showed promoter methylation of Rad51C in tumor cells. 5-azacytidine treatment of LS-174T cells caused a 14 fold increase in variant 1, a 4.8 fold increase for variant 3 and 3.4 fold for variant 2 compared to 2.5 fold increase in WT. Expression of Rad51C variants is associated with FANCD2 foci positive colorectal tumors and is associated with microsatellite stability in those tumors. Further investigation is needed to elucidate differential function of the Rad51C variants to evaluate potential effects in drug resistance and DNA repair.

  15. Vps4 disassembles an ESCRT-III filament by global unfolding and processive translocation

    PubMed Central

    Yang, Bei; Stjepanovic, Goran; Shen, Qingtao; Martin, Andreas; Hurley, James H.

    2015-01-01

    The AAA+ ATPase Vps4 disassembles ESCRT-III and is essential for HIV-1 budding and other pathways. Vps4 is a paradigmatic member of a class of hexameric AAA+ ATPases that disassemble protein complexes without degradation. To distinguish between local displacement versus global unfolding mechanisms for complex disassembly, we carried out hydrogen-deuterium exchange during Saccharomyces cerevisiae Vps4 disassembly of of a chimeric Vps24-2 ESCRT-III filament. EX1 exchange behavior shows that Vps4 completely unfolds ESCRT-III substrates on a time scale consistent with the disassembly reaction. The established unfoldase ClpX showed the same pattern, demonstrating a common unfolding mechanism. Vps4 hexamers containing a single cysteine residue in the pore loops were cross-linked to ESCRT-III subunits containing unique cysteine within the folded core domain. These data support a mechanism in which Vps4 disassembles its substrates by completely unfolding them and threading them through the central pore. PMID:25938660

  16. Dynamic unwrapping of nucleosomes by HsRAD51 that includes sliding and rotational motion of histone octamers

    PubMed Central

    Senavirathne, Gayan; Mahto, Santosh K.; Hanne, Jeungphill; O'Brian, Daniel; Fishel, Richard

    2017-01-01

    Wrapping of genomic DNA into nucleosomes poses thermodynamic and kinetic barriers to biological processes such as replication, transcription, repair and recombination. Previous biochemical studies have demonstrated that in the presence of adenosine triphosphate (ATP) the human RAD51 (HsRAD51) recombinase can form a nucleoprotein filament (NPF) on double-stranded DNA (dsDNA) that is capable of unwrapping the nucleosomal DNA from the histone octamer (HO). Here, we have used single molecule Förster Resonance Energy Transfer (smFRET) to examine the real time nucleosome dynamics in the presence of the HsRAD51 NPF. We show that oligomerization of HsRAD51 leads to stepwise, but stochastic unwrapping of the DNA from the HO in the presence of ATP. The highly reversible dynamics observed in single-molecule trajectories suggests an antagonistic mechanism between HsRAD51 binding and rewrapping of the DNA around the HO. These stochastic dynamics were independent of the nucleosomal DNA sequence or the asymmetry created by the presence of a linker DNA. We also observed sliding and rotational oscillations of the HO with respect to the nucleosomal DNA. These studies underline the dynamic nature of even tightly associated protein–DNA complexes such as nucleosomes. PMID:27738136

  17. Rad54 functions as a heteroduplex DNA pump modulated by its DNA substrates and Rad51 during D loop formation.

    PubMed

    Wright, William Douglass; Heyer, Wolf-Dietrich

    2014-02-06

    The displacement loop (D loop) is the product of homology search and DNA strand invasion, constituting a central intermediate in homologous recombination (HR). In eukaryotes, the Rad51 DNA strand exchange protein is assisted in D loop formation by the Rad54 motor protein. Curiously, Rad54 also disrupts D loops. How these opposing activities are coordinated toward productive recombination is unknown. Moreover, a seemingly disparate function of Rad54 is removal of Rad51 from heteroduplex DNA (hDNA) to allow HR-associated DNA synthesis. Here, we uncover features of D loop formation/dissociation dynamics, employing Rad51 filaments formed on ssDNAs that mimic the physiological length and structure of in vivo substrates. The Rad54 motor is activated by Rad51 bound to synapsed DNAs and guided by a ssDNA-binding domain. We present a unified model wherein Rad54 acts as an hDNA pump that drives D loop formation while simultaneously removing Rad51 from hDNA, consolidating both ATP-dependent activities of Rad54 into a single mechanistic step.

  18. 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

  19. 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.

  20. Cloning the RAD51 homologue of Schizosaccharomyces pombe.

    PubMed Central

    Muris, D F; Vreeken, K; Carr, A M; Broughton, B C; Lehmann, A R; Lohman, P H; Pastink, A

    1993-01-01

    The RAD51 gene of Saccharomyces cerevisiae encodes a RecA like protein, which is involved in the recombinational repair of double strand breaks. We have isolated the RAD51 homologue, rhp51+, of the distantly related yeast strain Schizosaccharomyces pombe by heterologous hybridization. DNA sequence analysis of the rhp51+ gene revealed an open reading frame of 365 amino acids. Comparison of the amino acid sequences of RAD51 and rhp51+ showed a high level of conservation: 69% identical amino acids. There are two Mlul sites in the upstream region which may be associated with cell cycle regulation of the rhp51+ gene. The rhp51+ null allele, constructed by disruption of the coding region, is extremely sensitive to X-rays, indicating that the rhp51+ gene, like RAD51, is also involved in the repair of X-ray damage. The structural and functional homology between rhp51+ and RAD51 suggests evolutionary conservation of certain steps in the recombinational repair pathway. Images PMID:8233794

  1. Disruption of Brca2-Rad51 Complex in Breast Cancer Cells: Therapeutic Implications

    DTIC Science & Technology

    2006-09-01

    complexes include the Rad51 paralogs family members such as (Rad51, Rad52, Rad54, Rad51B, Rad51C, Rad51D,Xrcc2and Xrcc3 ) and the breast cancer...DNA recombination from the structure of a RAD51-BRCA2 complex. Nature 2002, 420, 287-293. 5. Xu ZY, Loignon M, Han FY, Panasci L, Aloyz R.. Xrcc3 ...Pharmacol Exp Ther. 2005, 314:495-505. 6. Xu ZY, Loignon M, Han FY, Panasci L, Aloyz R. Xrcc3 induces cisplatin resistance by stimulation of Rad51

  2. Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break.

    PubMed

    Brown, M Scott; Grubb, Jennifer; Zhang, Annie; Rust, Michael J; Bishop, Douglas K

    2015-12-01

    The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs). Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments.

  3. RAD51C-deficient cancer cells are highly sensitive to the PARP inhibitor olaparib.

    PubMed

    Min, Ahrum; Im, Seock-Ah; Yoon, Young-Kwang; Song, Sang-Hyun; Nam, Hyun-Jin; Hur, Hyung-Seok; Kim, Hwang-Phill; Lee, Kyung-Hun; Han, Sae-Won; Oh, Do-Youn; Kim, Tae-You; O'Connor, Mark J; Kim, Woo-Ho; Bang, Yung-Jue

    2013-06-01

    A PARP inhibitor is a rationally designed targeted therapy for cancers with impaired DNA repair abilities. RAD51C is a paralog of RAD51 that has an important role in the DNA damage response. We found that cell lines sensitive to a novel oral PARP inhibitor, olaparib, had low levels of RAD51C expression using microarray analysis, and we therefore hypothesized that low expression of RAD51C may hamper the DNA repair process, resulting in increased sensitivity to olaparib. Compared with the cells with normal RAD51C expression levels, RAD51C-deficient cancer cells were more sensitive to olaparib, and a higher proportion underwent cell death by inducing G2-M cell-cycle arrest and apoptosis. The restoration of RAD51C in a sensitive cell line caused attenuation of olaparib sensitivity. In contrast, silencing of RAD51C in a resistant cell line enhanced the sensitivity to olaparib, and the number of RAD51 foci decreased with ablated RAD51C expression. We also found the expression of RAD51C was downregulated in cancer cells due to epigenetic changes and RAD51C expression was low in some gastric cancer tissues. Furthermore, olaparib significantly suppressed RAD51C-deficient tumor growth in a xenograft model. In summary, RAD51C-deficient cancer cells are highly sensitive to olaparib and offer preclinical proof-of-principle that RAD51C deficiency may be considered a biomarker for predicting the antitumor effects of olaparib. ©2013 AACR

  4. Association between RAD 51 rs1801320 and susceptibility to glioblastoma.

    PubMed

    Franceschi, S; Tomei, S; Mazzanti, C M; Lessi, F; Aretini, P; La Ferla, M; De Gregorio, V; Pasqualetti, F; Zavaglia, K; Bevilacqua, G; Naccarato, A G

    2016-01-01

    Glioblastoma is the most common and aggressive malignant primary brain tumor. Despite decades of research and the advent of new therapies, patients with glioblastoma continue to have a very poor prognosis. Radiation therapy has a major role as adjuvant treatment for glioblastoma following surgical resection. Many studies have shown that polymorphisms of genes involved in pathways of DNA repair may affect the sensitivity of the cells to treatment. Although the role of these polymorphisms has been investigated in relation to response to radiotherapy, their role as predisposing factors to glioblastoma has not been clarified yet. In the present study, we evaluated the association between polymorphisms in DNA repair genes, namely: XRCC1 rs25487, XRCC3 rs861539 and RAD51 rs1801320, with the susceptibility to develop glioblastoma. Eighty-five glioblastoma patients and 70 matched controls were recruited for this study. Data from the 1000 Genomes Project (98 Tuscans) were also downloaded and used for the association analysis. Subjects carrying RAD51 rs1801320 GC genotype showed an increased risk of glioblastoma (GC vs GG, χ(2) = 10.75; OR 3.0087; p = 0.0010). The C allele was also significantly associated to glioblastoma (χ(2) = 8.66; OR 2.5674; p = 0.0032). Moreover, RAD51 rs1801320 C allele increased the risk to develop glioblastoma also when combined to XRCC1 rs25487 G allele and XRCC3 rs861539 C allele (χ(2) = 6.558; p = 0.0053).

  5. Distinct Roles of FANCO/RAD51C Protein in DNA Damage Signaling and Repair

    PubMed Central

    Somyajit, Kumar; Subramanya, Shreelakshmi; Nagaraju, Ganesh

    2012-01-01

    RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G2/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor. PMID:22167183

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

    PubMed Central

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

    2015-01-01

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

  7. A novel role for the DNA repair gene Rad51 in Netrin-1 signalling

    PubMed Central

    Glendining, K. A.; Markie, D.; Gardner, R. J. M.; Franz, E. A.; Robertson, S. P.; Jasoni, C. L.

    2017-01-01

    Mutations in RAD51 have recently been linked to human Congenital Mirror Movements (CMM), a developmental disorder of the motor system. The only gene previously linked to CMM encodes the Netrin-1 receptor DCC, which is important for formation of corticospinal and callosal axon tracts. Thus, we hypothesised that Rad51 has a novel role in Netrin-1-mediated axon development. In mouse primary motor cortex neurons, Rad51 protein was redistributed distally down the axon in response to Netrin-1, further suggesting a functional link between the two. We next manipulated Rad51 expression, and assessed Netrin-1 responsiveness. Rad51 siRNA knockdown exaggerated Netrin-1-mediated neurite branching and filopodia formation. RAD51 overexpression inhibited these responses, whereas overexpression of the CMM-linked R250Q mutation, a predicted loss-of-function, had no effect. Thus, Rad51 appears to negatively regulate Netrin-1 signalling. Finally, we examined whether Rad51 might operate by modulating the expression of the Unc5 family, known negative regulators of Netrin-1-responsiveness. Unc5b and Unc5c transcripts were downregulated in response to Rad51 knockdown, and upregulated with RAD51 overexpression, but not R250Q. Thus, Rad51 negatively regulates Netrin-1 signalling, at least in part, by modulating the expression of Unc5s. Imbalance of positive and negative influences is likely to lead to aberrant motor system development resulting in CMMs. PMID:28057929

  8. Rad51 Protein Expression and Survival in Patients with Glioblastoma Multiforme

    SciTech Connect

    Welsh, James W. Ellsworth, Ron K.; Kumar, Rachit; Fjerstad, Kyle; Martinez, Jesse; Nagel, Raymond B.; Eschbacher, Jennifer; Stea, Baldassarre

    2009-07-15

    Purpose: Treatment of glioblastoma multiforme (GBM) continues to pose a significant therapeutic challenge, with most tumors recurring within the previously irradiated tumor bed. To improve outcomes, we must be able to identify and treat resistant cell populations. Rad51, an enzyme involved in homologous recombinational repair, leads to increased resistance of tumor cells to cytotoxic treatments such as radiotherapy. We hypothesized that Rad51 might contribute to GBM's apparent radioresistance and consequently influence survival. Methods and Materials: A total of 68 patients with an initial diagnosis of GBM were retrospectively evaluated; for 10 of these patients, recurrent tumor specimens were used to construct a tissue microarray. Rad51 protein expression was then correlated with the actual and predicted survival using recursive partitioning analysis. Results: Rad51 protein was elevated in 53% of the GBM specimens at surgery. The Rad51 levels correlated directly with survival, with a median survival of 15 months for patients with elevated Rad51 compared with 9 months for patients with low or absent levels of Rad51 (p = .05). At disease recurrence, 70% of patients had additional increases in Rad51 protein. Increased Rad51 levels at disease recurrence similarly predicted for improved overall survival, with a mean survival of 16 months from the second craniotomy compared with only 4 months for patients with low Rad51 levels (p = .13). Conclusion: Elevated levels of the double-stranded DNA repair protein Rad51 predicted for an increase survival duration in patients with GBM, at both initial tumor presentation and disease recurrence.

  9. Overexpression of Rad51 Predicts Poor Prognosis in Colorectal Cancer: Our Experience with 54 Patients

    PubMed Central

    Xu, Feng; Liao, Dian-ying; Xie, Li; Wang, Jin; Luo, Feng

    2017-01-01

    Background Aberrant Rad51 expression is implicated in the progression of human malignancies. However, the role of Rad51 in colorectal cancer (CRC) remains undefined. This study aimed to establish a relationship between Rad51 and clinicopathologic features of CRC. Methods We retrospectively examined the paraffin-embedded tissue samples obtained from 54 patients with CRC who had received surgical therapies at our institution during 2006–2008. Rad51 expression in adenocarcinoma, paracancerous tissue, and normal colonic tissue was determined by immunohistochemistry. The correlation between Rad51 immunoreactivity and clinicopathologic features of these patients was evaluated. Results Rad51 immunoreactivity was detected in 67% of adenocarcinoma, 48% of paracancerous tissue, and 27% of normal colonic mucosa. Rad51 expression in adenocarcinoma was significantly higher than normal colonic tissue (p < 0.05). Rad51 was also overexpressed in poorly differentiated tumors and tumor samples from patients with lymph node metastasis (p < 0.05). Patients with Rad51 overexpression had a 69% two-year survival, 49% three-year survival, and 16% five-year survival, considerably worse than patients with negative Rad51 expression (p < 0.05). Conclusion Our data suggest that Rad51 overexpression is correlated with malignant phenotypes of CRC and may predict poor prognosis for these patients. PMID:28099437

  10. Extracellular Inhibitors, Repellents, and Semaphorin/Plexin/MICAL-mediated Actin Filament Disassembly

    PubMed Central

    Hung, Ruei-Jiun; Terman, Jonathan R.

    2011-01-01

    Multiple extracellular signals have been identified that regulate actin dynamics within motile cells, but how these instructive cues present on the cell surface exert their precise effects on the internal actin cytoskeleton is still poorly understood. One particularly interesting class of these cues is a group of extracellular proteins that negatively alter the movement of cells and their processes. Over the years, these types of events have been described using a variety of terms and herein we provide an overview of inhibitory/repulsive cellular phenomena and highlight the largest known protein family of repulsive extracellular cues, the Semaphorins. Specifically, the Semaphorins (Semas) utilize Plexin cell-surface receptors to dramatically collapse the actin cytoskeleton and we summarize what is known of the direct molecular and biochemical mechanisms of Sema-triggered actin filament (F-actin) disassembly. We also discuss new observations from our lab that reveal that the multi-domain oxidoreductase (Redox) enzyme MICAL, an important mediator of Sema/Plexin repulsion, is a novel F-actin disassembly factor. Our results indicate that MICAL triggers Sema/Plexin-mediated reorganization of the F-actin cytoskeleton and suggest a role for specific Redox signaling events in regulating actin dynamics. PMID:21800438

  11. Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly

    PubMed Central

    Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

    2015-01-01

    Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells. DOI: http://dx.doi.org/10.7554/eLife.06126.001 PMID:26652273

  12. RAD51, XRCC3, and XRCC2 mutation screening in Finnish breast cancer families.

    PubMed

    Pelttari, Liisa M; Kiiski, Johanna I; Ranta, Salla; Vilske, Sara; Blomqvist, Carl; Aittomäki, Kristiina; Nevanlinna, Heli

    2015-01-01

    Majority of the known breast cancer susceptibility genes have a role in DNA repair and the most important high-risk genes BRCA1 and BRCA2 are specifically involved in the homologous recombination repair (HRR) of DNA double-strand breaks. A central player in HRR is RAD51 that binds DNA at the damage site. The RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 facilitate the binding of RAD51 to DNA. While germline mutations in RAD51C and RAD51D are associated with high ovarian cancer risk and RAD51B polymorphisms with breast cancer, the contribution of RAD51, XRCC3, and XRCC2 is more unclear. To investigate the role of RAD51, XRCC3, and XRCC2 in breast cancer predisposition and to identify putative recurrent founder mutations in the Finnish population where such mutations have been observed in most of the currently known susceptibility genes, we screened 182 familial Finnish breast or ovarian cancer patients for germline variation in the RAD51and XRCC3 genes and 342 patients for variation in XRCC2, with a subset of the patients selected on the basis of decreased RAD51 protein expression on tumors. We also performed haplotype analyses for 1516 breast cancer cases and 1234 controls to assess the common variation in these genes. No pathogenic mutations were detected in any of the genes and the distribution of haplotypes was similar between cases and controls. Our results suggest that RAD51, XRCC3, and XRCC2 do not substantially contribute to breast cancer predisposition in the Finnish population.

  13. RAD51AP1-deficiency in vertebrate cells impairs DNA replication.

    PubMed

    Parplys, Ann C; Kratz, Katja; Speed, Michael C; Leung, Stanley G; Schild, David; Wiese, Claudia

    2014-12-01

    RAD51-associated protein 1 (RAD51AP1) is critical for homologous recombination (HR) by interacting with and stimulating the activities of the RAD51 and DMC1 recombinases. In human somatic cells, knockdown of RAD51AP1 results in increased sensitivity to DNA damaging agents and to impaired HR, but the formation of DNA damage-induced RAD51 foci is unaffected. Here, we generated a genetic model system, based on chicken DT40 cells, to assess the phenotype of fully inactivated RAD51AP1 in vertebrate cells. Targeted inactivation of both RAD51AP1 alleles has no effect on either viability or doubling-time in undamaged cells, but leads to increased levels of cytotoxicity after exposure to cisplatin or to ionizing radiation. Interestingly, ectopic expression of GgRAD51AP1, but not of HsRAD51AP1 is able to fully complement in cell survival assays. Notably, in RAD51AP1-deficient DT40 cells the resolution of DNA damage-induced RAD51 foci is greatly slowed down, while their formation is not impaired. We also identify, for the first time, an important role for RAD51AP1 in counteracting both spontaneous and DNA damage-induced replication stress. In human and in chicken cells, RAD51AP1 is required to maintain wild type speed of replication fork progression, and both RAD51AP1-depleted human cells and RAD51AP1-deficient DT40 cells respond to replication stress by a slow-down of replication fork elongation rates. However, increased firing of replication origins occurs in RAD51AP1-/- DT40 cells, likely to ensure the timely duplication of the entire genome. Taken together, our results may explain why RAD51AP1 commonly is overexpressed in tumor cells and tissues, and we speculate that the disruption of RAD51AP1 function could be a promising approach in targeted tumor therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation

    PubMed Central

    Zelensky, Alex N.; Sanchez, Humberto; Ristic, Dejan; Vidic, Iztok; van Rossum-Fikkert, Sari E.; Essers, Jeroen; Wyman, Claire; Kanaar, Roland

    2013-01-01

    Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair. PMID:23666627

  15. A phosphorylation-deubiquitination cascade regulates the BRCA2-RAD51 axis in homologous recombination.

    PubMed

    Luo, Kuntian; Li, Lei; Li, Yunhui; Wu, Chenming; Yin, Yujiao; Chen, Yuping; Deng, Min; Nowsheen, Somaira; Yuan, Jian; Lou, Zhenkun

    2016-12-01

    Homologous recombination (HR) is one of the major DNA double-strand break (DSB) repair pathways in mammalian cells. Defects in HR trigger genomic instability and result in cancer predisposition. The defining step of HR is homologous strand exchange directed by the protein RAD51, which is recruited to DSBs by BRCA2. However, the regulation of the BRCA2-RAD51 axis remains unclear. Here we report that ubiquitination of RAD51 hinders RAD51-BRCA2 interaction, while deubiquitination of RAD51 facilitates RAD51-BRCA2 binding and RAD51 recruitment and thus is critical for proper HR. Mechanistically, in response to DNA damage, the deubiquitinase UCHL3 is phosphorylated and activated by ATM. UCHL3, in turn, deubiquitinates RAD51 and promotes the binding between RAD51 and BRCA2. Overexpression of UCHL3 renders breast cancer cells resistant to radiation and chemotherapy, while depletion of UCHL3 sensitizes cells to these treatments, suggesting a determinant role of UCHL3 in cancer therapy. Overall, we identify UCHL3 as a novel regulator of DNA repair and reveal a model in which a phosphorylation-deubiquitination cascade dynamically regulates the BRCA2-RAD51 pathway. © 2016 Luo et al.; Published by Cold Spring Harbor Laboratory Press.

  16. DrRad51 is required for chiasmata formation in meiosis in planarian Dugesia ryukyuensis.

    PubMed

    Chinone, Ayako; Matsumoto, Midori

    2014-05-01

    Rad51, a conserved eukaryotic protein, mediates the homologous-recombination repair of DNA double-strand breaks that occur during both mitosis and meiosis. During prophase I of meiosis, homologous recombination enhances the linkage between homologous chromosomes to increase the accuracy of segregation at anaphase I. In polyploidy situations, however, difficulties with homologous chromosome segregation often disrupt meiosis. Yet, triploid individuals of the planarian Dugesia ryukyuensis are able to produce functional gametes through a specialized form of meiosis. To shed light on the molecular mechanisms that promote successful meiosis in triploid D. ryukyuensis, we investigated rad51 gene function. We isolated three genes of the Rad51 family, the Rad51 homolog Dr-rad51 and the Rad51 paralogs Dr-rad51B and Dr-rad51C. Dr-rad51 was expressed in germ-line and presumably in somatic stem cells, but was not necessary for the regeneration of somatic tissue. RNA-interference (RNAi) depletion of Dr-rad51 during sexualization did not affect chromosome behavior in zygotene oocytes, but did result in the loss of chiasmata at the diplotene stage. Thus, homologous recombination does not appear to be necessary for synapsis, but is needed for crossover and proper segregation in D. ryukyuensis.

  17. Role for Caspase-Mediated Cleavage of Rad51 in Induction of Apoptosis by DNA Damage

    PubMed Central

    Huang, YinYin; Nakada, Shuji; Ishiko, Takatoshi; Utsugisawa, Taiju; Datta, Rakesh; Kharbanda, Surender; Yoshida, Kiyotsugu; Talanian, Robert V.; Weichselbaum, Ralph; Kufe, Donald; Yuan, Zhi-Min

    1999-01-01

    We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage. PMID:10082566

  18. Disparate requirements for the Walker A and B ATPase motifs ofhuman RAD51D in homologous recombination

    SciTech Connect

    Wiese, Claudia; Hinz, John M.; Tebbs, Robert S.; Nham, Peter B.; Urbin, Salustra S.; Collins, David W.; Thompson, Larry H.; Schild, David

    2006-04-21

    In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks. Ectopic expression of wild type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs.

  19. Functions of Human Rad51 and Other Recombination Factors in DNA Double-Strand Break Repair

    DTIC Science & Technology

    2002-06-01

    cells express five Rad5l-like proteins - XRCC2, XRCC3 , Rad51B, Rad51C and Rad51D. These Rad51 paralogs are important for homologous recombination and...complex with 42-kD Rad5 IC species was detected. The observed sizes XRCC3 (Schild et al. 2000; Kurumizaka et al. 2001; Mas- A kIa a-Rad51B kDa a-RadSIC...RadS1C XRCC3 or that the XRCC3 -Rad51C complex was not A x-Rad5l B retained on the Q column. 07a 0- The results above indicated that Rad5lB and Rad5lC

  20. Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51.

    PubMed

    Wu, L; Davies, S L; Levitt, N C; Hickson, I D

    2001-06-01

    Bloom's syndrome (BS) is an autosomal recessive disorder that predisposes individuals to a wide range of cancers. The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases. The precise role played by these enzymes in the cell remains to be determined. However, genome-wide hyper-recombination is a feature of many RecQ helicase-deficient cells. In eukaryotes, a central step in homologous recombination is catalyzed by the RAD51 protein. In response to agents that induce DNA double-strand breaks, RAD51 accumulates in nuclear foci that are thought to correspond to sites of recombinational repair. Here, we report that purified BLM and human RAD51 interact in vitro and in vivo, and that residues in the N- and C-terminal domains of BLM can independently mediate this interaction. Consistent with these observations, BLM localizes to a subset of RAD51 nuclear foci in normal human cells. Moreover, the number of BLM foci and the extent to which BLM and RAD51 foci co-localize increase in response to ionizing radiation. Nevertheless, the formation of RAD51 foci does not require functional BLM. Indeed, in untreated BS cells, an abnormally high proportion of the cells contain RAD51 nuclear foci. Exogenous expression of BLM markedly reduces the fraction of cells containing RAD51 foci. The interaction between BLM and RAD51 appears to have been evolutionarily conserved since the C-terminal domain of Sgs1, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Rad51. Furthermore, genetic analysis reveals that the SGS1 and RAD51 genes are epistatic indicating that they operate in a common pathway. Potential roles for BLM in the RAD51 recombinational repair pathway are discussed.

  1. Prolonged exposure to particulate chromate inhibits RAD51 nuclear import mediator proteins.

    PubMed

    Browning, Cynthia L; Wise, John Pierce

    2017-09-15

    Particulate hexavalent chromium (Cr(VI)) is a human lung carcinogen and a human health concern. The induction of structural chromosome instability is considered to be a driving mechanism of Cr(VI)-induced carcinogenesis. Homologous recombination repair protects against Cr(VI)-induced chromosome damage, due to its highly accurate repair of Cr(VI)-induced DNA double strand breaks. However, recent studies demonstrate Cr(VI) inhibits homologous recombination repair through the misregulation of RAD51. RAD51 is an essential protein in HR repair that facilitates the search for a homologous sequence. Recent studies show prolonged Cr(VI) exposure prevents proper RAD51 subcellular localization, causing it to accumulate in the cytoplasm. Since nuclear import of RAD51 is crucial to its function, this study investigated the effect of Cr(VI) on the RAD51 nuclear import mediators, RAD51C and BRCA2. We show acute (24h) Cr(VI) exposure induces the proper localization of RAD51C and BRCA2. In contrast, prolonged (120h) exposure increased the cytoplasmic localization of both proteins, although RAD51C localization was more severely impaired. These results correlate temporally with the previously reported Cr(VI)-induced RAD51 cytoplasmic accumulation. In addition, we found Cr(VI) does not inhibit interaction between RAD51 and its nuclear import mediators. Altogether, our results suggest prolonged Cr(VI) exposure inhibits the nuclear import of RAD51C, and to a lesser extent, BRCA2, which results in the cytoplasmic accumulation of RAD51. Cr(VI)-induced inhibition of nuclear import may play a key role in its carcinogenic mechanism since the nuclear import of many tumor suppressor proteins and DNA repair proteins is crucial to their function. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Disruption of Brca2-Rad51 Complex in Breast Cancer Cells: Therapeutic Implications

    DTIC Science & Technology

    2005-09-01

    Rad51B, Rad51 C, Rad51D,Xrcc2and Xrcc3 ) and the breast cancer associated proteins,BRCAI and BRCA2. Defective cell lines in each of the above...Aloyz recombination from the structure of a RAD5 I-BRCA2 complex. Nature 2002, 420, 287-293. 5. Xu ZY, Loignon M, Han FY, Panasci L, Aloyz R.. Xrcc3

  3. Germline mutation in the RAD51B gene confers predisposition to breast cancer

    PubMed Central

    2013-01-01

    Background Most currently known breast cancer predisposition genes play a role in DNA repair by homologous recombination. Recent studies conducted on RAD51 paralogs, involved in the same DNA repair pathway, have identified rare germline mutations conferring breast and/or ovarian cancer predisposition in the RAD51C, RAD51D and XRCC2 genes. The present study analysed the five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) to estimate their contribution to breast and ovarian cancer predisposition. Methods The study was conducted on 142 unrelated patients with breast and/or ovarian cancer either with early onset or with a breast/ovarian cancer family history. Patients were referred to a French family cancer clinic and had been previously tested negative for a BRCA1/2 mutation. Coding sequences of the five genes were analysed by EMMA (Enhanced Mismatch Mutation Analysis). Detected variants were characterized by Sanger sequencing analysis. Results Three splicing mutations and two likely deleterious missense variants were identified: RAD51B c.452 + 3A > G, RAD51C c.706-2A > G, RAD51C c.1026 + 5_1026 + 7del, RAD51B c.475C > T/p.Arg159Cys and XRCC3 c.448C > T/p.Arg150Cys. No RAD51D and XRCC2 gene mutations were detected. These mutations and variants were detected in families with both breast and ovarian cancers, except for the RAD51B c.475C > T/p.Arg159Cys variant that occurred in a family with 3 breast cancer cases. Conclusions This study identified the first RAD51B mutation in a breast and ovarian cancer family and is the first report of XRCC3 mutation analysis in breast and ovarian cancer. It confirms that RAD51 paralog mutations confer breast and ovarian cancer predisposition and are rare events. In view of the low frequency of RAD51 paralog mutations, international collaboration of family cancer clinics will be required to more accurately estimate their penetrance and establish clinical guidelines in carrier individuals. PMID

  4. A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity.

    PubMed

    Uringa, Evert-Jan; Baldeyron, Céline; Odijk, Hanny; Wassenaar, Evelyne; van Cappellen, Wiggert A; Maas, Alex; Hoeijmakers, Jan H J; Baarends, Willy M; Kanaar, Roland; Essers, Jeroen

    2015-01-01

    Accurate DNA double-strand break repair through homologous recombination is essential for preserving genome integrity. Disruption of the gene encoding RAD51, the protein that catalyzes DNA strand exchange during homologous recombination, results in lethality of mammalian cells. Proteins required for homologous recombination, also play an important role during DNA replication. To explore the role of RAD51 in DNA replication and DSB repair, we used a knock-in strategy to express a carboxy-terminal fusion of green fluorescent protein to mouse RAD51 (mRAD51-GFP) in mouse embryonic stem cells. Compared to wild-type cells, heterozygous mRad51(+/wt-GFP) embryonic stem cells showed increased sensitivity to DNA damage induced by ionizing radiation and mitomycin C. Moreover, gene targeting was found to be severely impaired in mRad51(+/wt-GFP) embryonic stem cells. Furthermore, we found that mRAD51-GFP foci were not stably associated with chromatin. From these experiments we conclude that this mRad51-GFP allele is an antimorphic allele. When this allele is present in a heterozygous condition over wild-type mRad51, embryonic stem cells are proficient in DNA replication but display defects in homologous recombination and DNA damage repair. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart

    PubMed Central

    Somyajit, Kumar; Saxena, Sneha; Babu, Sharath; Mishra, Anup; Nagaraju, Ganesh

    2015-01-01

    Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Taken together, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis. PMID:26354865

  6. Estrogen induces RAD51C expression and localization to sites of DNA damage.

    PubMed

    Alayev, Anya; Salamon, Rachel S; Manna, Subrata; Schwartz, Naomi S; Berman, Adi Y; Holz, Marina K

    2016-12-01

    Homologous recombination (HR) is a conserved process that maintains genome stability and cell survival by repairing DNA double-strand breaks (DSBs). The RAD51-related family of proteins is involved in repair of DSBs; consequently, deregulation of RAD51 causes chromosomal rearrangements and stimulates tumorigenesis. RAD51C has been identified as a potential tumor suppressor and a breast and ovarian cancer susceptibility gene. Recent studies have also implicated estrogen as a DNA-damaging agent that causes DSBs. We found that in ERα-positive breast cancer cells, estrogen transcriptionally regulates RAD51C expression in ERα-dependent mechanism. Moreover, estrogen induces RAD51C assembly into nuclear foci at DSBs, which is a precursor to RAD51 complex recruitment to the nucleus. Additionally, disruption of ERα signaling by either anti-estrogens or siRNA prevented estrogen induced upregulation of RAD51C. We have also found an association of a worse clinical outcome between RAD51C expression and ERα status of tumors. These findings provide insight into the mechanism of genomic instability in ERα-positive breast cancer and suggest that individuals with mutations in RAD51C that are exposed to estrogen would be more susceptible to accumulation of DNA damage, leading to cancer progression.

  7. A peptide nucleic acid targeting nuclear RAD51 sensitizes multiple myeloma cells to melphalan treatment

    PubMed Central

    Alagpulinsa, David Abasiwani; Yaccoby, Shmuel; Ayyadevara, Srinivas; Shmookler Reis, Robert Joseph

    2015-01-01

    RAD51-mediated recombinational repair is elevated in multiple myeloma (MM) and predicts poor prognosis. RAD51 has been targeted to selectively sensitize and/or kill tumor cells. Here, we employed a peptide nucleic acid (PNA) to inhibit RAD51 expression in MM cells. We constructed a PNA complementary to a unique segment of the RAD51 gene promoter, spanning the transcription start site, and conjugated it to a nuclear localization signal (PKKKRKV) to enhance cellular uptake and nuclear delivery without transfection reagents. This synthetic construct, (PNArad51_nls), significantly reduced RAD51 transcripts in MM cells, and markedly reduced the number and intensity of de novo and melphalan-induced nuclear RAD51 foci, while increasing the level of melphalan-induced γH2AX foci. Melphalan alone markedly induced the expression of 5 other genes involved in homologous-recombination repair, yet suppression of RAD51 by PNArad51_nls was sufficient to synergize with melphalan, producing significant synthetic lethality of MM cells in vitro. In a SCID-rab mouse model mimicking the MM bone marrow microenvironment, treatment with PNArad51_nls ± melphalan significantly suppressed tumor growth after 2 weeks, whereas melphalan plus control PNArad4µ_nls was ineffectual. This study highlights the importance of RAD51 in myeloma growth and is the first to demonstrate that anti-RAD51 PNA can potentiate conventional MM chemotherapy. PMID:25996477

  8. Polymorphisms of Homologous Recombination RAD51, RAD51B, XRCC2, and XRCC3 Genes and the Risk of Prostate Cancer

    PubMed Central

    Nowacka-Zawisza, Maria; Wiśnik, Ewelina; Wasilewski, Andrzej; Skowrońska, Milena; Forma, Ewa; Bryś, Magdalena; Różański, Waldemar; Krajewska, Wanda M.

    2015-01-01

    Genetic polymorphisms in DNA repair genes may induce individual variations in DNA repair capacity, which may in turn contribute to the risk of cancer developing. Homologous recombination repair (HRR) plays a critical role in maintaining chromosomal integrity and protecting against carcinogenic factors. The aim of the present study was to evaluate the relationship between prostate cancer risk and the presence of single nucleotide polymorphisms (SNPs) in the genes involved in HRR, that is, RAD51 (rs1801320 and rs1801321), RAD51B (rs10483813 and rs3784099), XRCC2 (rs3218536), and XRCC3 (rs861539). Polymorphisms were analyzed by PCR-RFLP and Real-Time PCR in 101 patients with prostate adenocarcinoma and 216 age- and sex-matched controls. A significant relationship was detected between the RAD51 gene rs1801320 polymorphism and increased prostate cancer risk. Our results indicate that the RAD51 gene rs1801320 polymorphism may contribute to prostate cancer susceptibility in Poland. PMID:26339569

  9. Polymorphisms of homologous recombination RAD51, RAD51B, XRCC2, and XRCC3 genes and the risk of prostate cancer.

    PubMed

    Nowacka-Zawisza, Maria; Wiśnik, Ewelina; Wasilewski, Andrzej; Skowrońska, Milena; Forma, Ewa; Bryś, Magdalena; Różański, Waldemar; Krajewska, Wanda M

    2015-01-01

    Genetic polymorphisms in DNA repair genes may induce individual variations in DNA repair capacity, which may in turn contribute to the risk of cancer developing. Homologous recombination repair (HRR) plays a critical role in maintaining chromosomal integrity and protecting against carcinogenic factors. The aim of the present study was to evaluate the relationship between prostate cancer risk and the presence of single nucleotide polymorphisms (SNPs) in the genes involved in HRR, that is, RAD51 (rs1801320 and rs1801321), RAD51B (rs10483813 and rs3784099), XRCC2 (rs3218536), and XRCC3 (rs861539). Polymorphisms were analyzed by PCR-RFLP and Real-Time PCR in 101 patients with prostate adenocarcinoma and 216 age- and sex-matched controls. A significant relationship was detected between the RAD51 gene rs1801320 polymorphism and increased prostate cancer risk. Our results indicate that the RAD51 gene rs1801320 polymorphism may contribute to prostate cancer susceptibility in Poland.

  10. Differential roles of XRCC2 in S-phase RAD51 focus formation induced by DNA replication inhibitors

    SciTech Connect

    Lim, C; Liu, N

    2004-05-14

    RAD51 proteins accumulate in discrete nuclear foci in response to DNA damage. Previous studies demonstrated that human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) are essential for the assembly of RAD51 foci induced by ionizing radiation and cross-linking agents. Here we report that XRCC2 also plays important roles in RAD51 focus formation induced by replication arrest during S-phase of cell cycle. In wild-type hamster V79 cells treated with hydroxyurea (HU), RAD51 protein form punctuate nuclear foci, accompanied by increased RAD51 protein level in both cytoplasmic and nuclear fractions, and increased association of RAD51 with chromatin. In contrast, xrcc2 hamster mutant irs1 cells are deficient in the formation of RAD51 foci after HU treatment, suggesting that the function of XRCC2 is required for the assembly of RAD51 at HU-induced stalled replication forks. Interestingly, we found that irs1 cells are able to form intact RAD51 foci in S-phase cells treated with thymidine (TR) or aphidicolin, although irs1 cells are hypersensitive to both HU and TR. Our findings suggest that there may be two distinct pathways (XRCC2-dependent or XRCC2-independent) involved in loading of RAD51 onto stalled replication forks, probably depending upon the structure of DNA lesions.

  11. A novel small molecule RAD51 inactivator overcomes imatinib-resistance in chronic myeloid leukaemia.

    PubMed

    Zhu, Jiewen; Zhou, Longen; Wu, Guikai; Konig, Heiko; Lin, Xiaoqin; Li, Guideng; Qiu, Xiao-Long; Chen, Chi-Fen; Hu, Chun-Mei; Goldblatt, Erin; Bhatia, Ravi; Chamberlin, A Richard; Chen, Phang-Lang; Lee, Wen-Hwa

    2013-03-01

    RAD51 recombinase activity plays a critical role for cancer cell proliferation and survival, and often contributes to drug-resistance. Abnormally elevated RAD51 function and hyperactive homologous recombination (HR) rates have been found in a panel of cancers, including breast cancer and chronic myeloid leukaemia (CML). Directly targeting RAD51 and attenuating the deregulated RAD51 activity has therefore been proposed as an alternative and supplementary strategy for cancer treatment. Here we show that a newly identified small molecule, IBR2, disrupts RAD51 multimerization, accelerates proteasome-mediated RAD51 protein degradation, reduces ionizing radiation-induced RAD51 foci formation, impairs HR, inhibits cancer cell growth and induces apoptosis. In a murine imatinib-resistant CML model bearing the T315I Bcr-abl mutation, IBR2, but not imatinib, significantly prolonged animal survival. Moreover, IBR2 effectively inhibits the proliferation of CD34(+) progenitor cells from CML patients resistant to known BCR-ABL inhibitors. Therefore, small molecule inhibitors of RAD51 may suggest a novel class of broad-spectrum therapeutics for difficult-to-treat cancers. Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

  12. Meiotic Recombination in Arabidopsis Is Catalysed by DMC1, with RAD51 Playing a Supporting Role

    PubMed Central

    Da Ines, Olivier; Degroote, Fabienne; Goubely, Chantal; Amiard, Simon; Gallego, Maria E.; White, Charles I.

    2013-01-01

    Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis. PMID:24086145

  13. Targeted disruption of the Rad51 gene leads to lethality in embryonic mice.

    PubMed Central

    Tsuzuki, T; Fujii, Y; Sakumi, K; Tominaga, Y; Nakao, K; Sekiguchi, M; Matsushiro, A; Yoshimura, Y; MoritaT

    1996-01-01

    The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8692798

  14. 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.

  15. 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

  16. Meiotic recombination in Arabidopsis is catalysed by DMC1, with RAD51 playing a supporting role.

    PubMed

    Da Ines, Olivier; Degroote, Fabienne; Goubely, Chantal; Amiard, Simon; Gallego, Maria E; White, Charles I

    2013-01-01

    Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis.

  17. Berberine Radiosensitizes Human Esophageal Cancer Cells by Downregulating Homologous Recombination Repair Protein RAD51

    PubMed Central

    Liu, Zhaojian; Wang, Yu; Zhao, Minnan; Hao, Chunyan; Feng, Shuai; Guo, Haiyang; Xu, Bing; Yang, Qifeng; Gong, Yaoqin; Shao, Changshun

    2011-01-01

    Background Esophageal squamous cell carcinomas (ESCC) have poor prognosis. While combined modality of chemotherapy and radiotherapy increases survival, most patients die within five years. Development of agents that confer cancer cell-specific chemo- and radiosensitivity may improve the therapy of ESCC. We here reported the discovery of berberine as a potent radiosensitizing agent on ESCC cells. Principal Findings Berberine at low concentrations (<15 µM) substantially radiosensitized ESCC cells. X-ray induced DNA double-strand breaks (DSBs) persist longer in ESCC cells pretreated with berberine. Berberine pretreatment led to a significant downregulation of RAD51, a key player in homologous recombination repair, in ESCC cells, but not in non-malignant human cells. Downregulation of RAD51 by RNA interference similarly radiosensitized the cancer cells, and, conversely, introduction of exogenous RAD51 was able to significantly counteract the radiosensitizing effect of berberine, thus establishing RAD51 as a key determinant in radiation sensitivity. We also observed that RAD51 was commonly overexpressed in human ESCC tissues, suggesting that it is necessary to downregulate RAD51 to achieve high radio- or chemotherapeutic efficacy of ESCC in clinic, because overexpression of RAD51 is known to confer radio- and chemoresistance. Conclusions/Significance Berberine can effectively downregulate RAD51 in conferring radiosensitivity on esophageal cancer cells. Its clinical application as an adjuvant in chemotherapy and radiotherapy of esophageal cancers should be explored. PMID:21858113

  18. 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.

  19. Saccharomyces cerevisiae Dmc1 and Rad51 proteins preferentially function with Tid1 and Rad54 proteins, respectively, to promote DNA strand invasion during genetic recombination.

    PubMed

    Nimonkar, Amitabh V; Dombrowski, Christopher C; Siino, Joseph S; Stasiak, Alicja Z; Stasiak, Andrzej; Kowalczykowski, Stephen C

    2012-08-17

    The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

  20. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner*

    PubMed Central

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-01-01

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. PMID:26747606

  1. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

    PubMed

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-03-04

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Secondary Somatic Mutations Restoring RAD51C and RAD51D Associated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma

    PubMed Central

    Kondrashova, Olga; Nguyen, Minh; Shield-Artin, Kristy; Tinker, Anna V.; Teng, Nelson N.H.; Harrell, Maria I.; Kuiper, Michael J.; Ho, Gwo-Yaw; Barker, Holly; Jasin, Maria; Prakash, Rohit; Kass, Elizabeth M.; Sullivan, Meghan R.; Brunette, Gregory J.; Bernstein, Kara A.; Coleman, Robert L.; Floquet, Anne; Friedlander, Michael; Kichenadasse, Ganessan; O'Malley, David M.; Oza, Amit; Sun, James; Robillard, Liliane; Maloney, Lara; Giordano, Heidi; Wakefield, Matthew J.; Kaufmann, Scott H.; Simmons, Andrew D.; Harding, Thomas C.; Raponi, Mitch; McNeish, Iain A.; Swisher, Elizabeth M.; Lin, Kevin K.; Scott, Clare L.

    2017-01-01

    High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations. Significance Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. PMID:28588062

  3. 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).

  4. Characterisation of the promoter region of the human DNA-repair gene Rad51.

    PubMed

    Hasselbach, L; Haase, S; Fischer, D; Kolberg, H C; Stürzbecher, H W

    2005-01-01

    Regulatory elements of the 5'-flanking region of the DNA-repair gene Rad51 were analysed to characterise pathological alterations of Rad51 mRNA expression during tumour development. Various fragments of the Rad51 promoter were cloned into the pGL3 reporter vector and the respective promoter activity was determined by luciferase assays in transfected U2-OS cells. Transcription factor binding was identified using Protein/DNA arrays. The region encompassing base pairs -204 to -58 was identified as crucial for Rad51 gene transcription. Down regulator sequences are present upstream (-305 to -204) and downstream (-48 and +204) of this core promoter element. Promoter activity is significantly enhanced by substituting G at the polymorphic positions +135 and +172 for C and T, respectively. Transcription factors Ets1/PEA3, E2F1, p53, EGR1, and Stat5 were identified as relevant for regulating expression of Rad51. We identified three separate cis-sequence elements within the Rad51 transcriptional promoter, one ensuring basal levels of expression and two elements limiting expression to relatively low levels. The characterisation of transcription factor binding might help to explain high-level expression of Rad51 in a variety of solid tumours. The polymorphic sites appear important for the increased risk of breast and/or ovarian cancer for BRCA2 mutation carriers.

  5. RAD51 genotype and triple-negative breast cancer (TNBC) risk in Polish women.

    PubMed

    Smolarz, Beata; Zadrożny, Marek; Duda-Szymańska, Joanna; Makowska, Marianna; Samulak, Dariusz; Michalska, Magdalena M; Mojs, Ewa; Bryś, Magdalena; Forma, Ewa; Romanowicz-Makowska, Hanna

    2013-04-01

    The most lethal damage for the cell among all damage is double-strand breaks (DSB) of DNA. DSB cause development of cancer diseases including the triple-negative molecular subtype of breast cancer. The aim of this work was to evaluate the single nucleotide polymorphism -135G>C (rs1801320) of the RAD51 gene encoding DNA repair proteins by homologous recombination (HR) in triple-negative breast cancer (TNBC). We assessed the RAD51 -135G>C polymorphism in 50 women with triple-negative breast cancer and in 50 women from the control group. RAD51 polymorphism was analysed by the PCR-RFLP (restriction fragment length polymorphism) technique. Our results demonstrated a significant positive association between the RAD51 C/C genotype and TNBC, with an adjusted odds ratio (OR) of 5.95 (p = 0.002). The homozygous C/C genotype was found in 68% of breast cancer cases and 20% of controls. The variant 135C allele of RAD51 increased TNBC risk. This is the first study linking single nucleotide polymorphisms of the RAD51 gene with TNBC incidence in the population of Polish women. In conclusion, RAD51 polymorphisms may be regarded as predictive factors of triple-negative breast cancer in the female population. Large studies are needed to confirm our findings.

  6. Modulation of Saccharomyces Cerevisiae DNA Double-Strand Break Repair by Srs2 and Rad51

    PubMed Central

    Milne, G. T.; Ho, T.; Weaver, D. T.

    1995-01-01

    RAD52 function is required for virtually all DNA double-strand break repair and recombination events in Saccharomyces cerevisiae. To gain greater insight into the mechanism of RAD52-mediated repair, we screened for genes that suppress partially active alleles of RAD52 when mutant or overexpressed. Described here is the isolation of a phenotypic null allele of SRS2 that suppressed multiple alleles of RAD52 (rad52B, rad52D, rad52-1 and KlRAD52) and RAD51 (KlRAD51) but failed to suppress either a rad52δ or a rad51δ. These results indicate that SRS2 antagonizes RAD51 and RAD52 function in recombinational repair. The mechanism of suppression of RAD52 alleles by srs2 is distinct from that which has been previously described for RAD51 overexpression, as both conditions were shown to act additively with respect to the rad52B allele. Furthermore, overexpression of either RAD52 or RAD51 enhanced the recombination-dependent sensitivity of an srs2δ RAD52 strain, suggesting that RAD52 and RAD51 positively influence recombinational repair mechanisms. Thus, RAD52-dependent recombinational repair is controlled both negatively and positively. PMID:7768432

  7. The host factor RAD51 is involved in mungbean yellow mosaic India virus (MYMIV) DNA replication.

    PubMed

    Suyal, Geetika; Mukherjee, Sunil K; Choudhury, Nirupam R

    2013-09-01

    Geminiviruses replicate their single-stranded genomes with the help of only a few viral factors and various host cellular proteins primarily by rolling-circle replication (RCR) and/or recombination-dependent replication. AtRAD51 has been identified, using the phage display technique, as a host factor that potentially interacts with the Rep protein of mungbean yellow mosaic India virus (MYMIV), a member of the genus Begomovirus. In this study, we demonstrate the interaction between MYMIV Rep and a host factor, AtRAD51, using yeast two-hybrid and β-galactosidase assays, and this interaction was confirmed using a co-immunoprecipitation assay. The AtRAD51 protein complemented the rad51∆ mutation of Saccharomyces cerevisiae in an ex vivo yeast-based geminivirus DNA replication restoration assay. The semiquantitative RT-PCR and northern hybridization data revealed a higher level of expression of the Rad51 transcript in MYMIV-infected mungbean than in uninfected, healthy plants. Our findings provide evidence for a possible cross-talk between RAD51 and MYMIV Rep, which essentially controls viral DNA replication in plants, presumably in conjunction with other host factors. The present study demonstrates for the first time the involvement of a eukaryotic RAD51 protein in MYMIV replication, and this is expected to shed light on the machinery involved in begomovirus DNA replication.

  8. Arabidopsis RAD51C gene is important for homologous recombination in meiosis and mitosis.

    PubMed

    Abe, Kiyomi; Osakabe, Keishi; Nakayama, Shigeki; Endo, Masaki; Tagiri, Akemi; Todoriki, Setsuko; Ichikawa, Hiroaki; Toki, Seiichi

    2005-10-01

    Rad51 is a homolog of the bacterial RecA recombinase, and a key factor in homologous recombination in eukaryotes. Rad51 paralogs have been identified from yeast to vertebrates. Rad51 paralogs are thought to play an important role in the assembly or stabilization of Rad51 that promotes homologous pairing and strand exchange reactions. We previously characterized two RAD51 paralogous genes in Arabidopsis (Arabidopsis thaliana) named AtRAD51C and AtXRCC3, which are homologs of human RAD51C and XRCC3, respectively, and described the interaction of their products in a yeast two-hybrid system. Recent studies showed the involvement of AtXrcc3 in DNA repair and functional role in meiosis. To determine the role of RAD51C in meiotic and mitotic recombination in higher plants, we characterized a T-DNA insertion mutant of AtRAD51C. Although the atrad51C mutant grew normally during vegetative developmental stage, the mutant produced aborted siliques, and their anthers did not contain mature pollen grains. Crossing of the mutant with wild-type plants showed defective male and female gametogeneses as evidenced by lack of seed production. Furthermore, meiosis was severely disturbed in the mutant. The atrad51C mutant also showed increased sensitivity to gamma-irradiation and cisplatin, which are known to induce double-strand DNA breaks. The efficiency of homologous recombination in somatic cells in the mutant was markedly reduced relative to that in wild-type plants.

  9. 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

  10. 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.

  11. Detection and sequence analysis of the DNA repair gene RAD51 in the Korean spider Callobius koreanus (Amaurobiidae).

    PubMed

    Kim, J Y; Park, Y C

    2015-11-19

    We identified a partial sequence (483 bp) of the RAD51 gene from the Korean spider Callobius koreanus. Sequence variation was found at one position during alignment with the human RAD51 gene sequence. This partial sequence included the region corresponding to exon 4 in the human RAD51 gene, which encodes 39 amino acids. These results show that the RAD51 gene is highly conserved between human and spiders.

  12. Endogenous levels of Rad51 and Brca2 are required for homologous recombination and regulated by homeostatic re-balancing.

    PubMed

    Magwood, Alissa C; Malysewich, Michael J; Cealic, Iulia; Mundia, Maureen M; Knapp, Jennifer; Baker, Mark D

    2013-12-01

    Stable expression of Rad51 siRNA was used to generate mouse hybridoma cell lines in which endogenous Rad51 levels were depleted by as much as 60%. Stable Rad51 knockdowns feature reduced homologous recombination responses. The relative ease with which stable Rad51 knockdowns were recovered was surprising, given the embryonic lethality of Rad51 ablation. Interestingly, Rad51-depleted hybridoma cell lines are characterized by reduced levels of p53 protein. Completely unexpected, was the finding that Rad51-depleted hybridoma cell lines are also reduced for the breast cancer susceptibility 2 (Brca2) protein. Additionally, hybridoma cell lines that are siRNA depleted for mouse Brca2 show a corresponding reduction in Rad51 and p53 proteins. Furthermore, cellular levels of Rad51, Brca2 and p53 can be elevated in these cell lines by ectopic expression of wild-type human Rad51 and wild-type human BRCA2. In marked contrast, hybridoma cell lines that are siRNA depleted for mouse p53 feature relatively normal Rad51 and Brca2 levels. These results suggest that cellular levels of Brca2 and Rad51 are mutually dependent on each other, and that low levels of these proteins provide selective pressure for reduction of p53, which permits cell growth.

  13. Rad51C deficiency destabilizes XRCC3, impairs recombination and radiosensitizes S/G2-phase cells

    SciTech Connect

    Lio, Yi-Ching; Schild, David; Brenneman, Mark A.; Redpath, J. Leslie; Chen, David J.

    2004-05-01

    The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, XRCC3) are expressed in mitotically growing cells, and are thought to play mediating roles in homologous recombination, though their precise functions remain unclear. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA cross-linking agent mitomycin C, and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G{sub 2}/M phases of the cell cycle but not in G{sub 1} phase. Together, these results provide direct cellular evidence for the importance of human Rad51C in homologous recombinational repair.

  14. Characterisation of the novel deleterious RAD51C p.Arg312Trp variant and prioritisation criteria for functional analysis of RAD51C missense changes.

    PubMed

    Gayarre, Javier; Martín-Gimeno, Paloma; Osorio, Ana; Paumard, Beatriz; Barroso, Alicia; Fernández, Victoria; de la Hoya, Miguel; Rojo, Alejandro; Caldés, Trinidad; Palacios, José; Urioste, Miguel; Benítez, Javier; García, María J

    2017-08-22

    Despite a high prevalence of deleterious missense variants, most studies of RAD51C ovarian cancer susceptibility gene only provide in silico pathogenicity predictions of missense changes. We identified a novel deleterious RAD51C missense variant (p.Arg312Trp) in a high-risk family, and propose a criteria to prioritise RAD51C missense changes qualifying for functional analysis. To evaluate pathogenicity of p.Arg312Trp variant we used sequence homology, loss of heterozygosity (LOH) and segregation analysis, and a comprehensive functional characterisation. To define a functional-analysis prioritisation criteria, we used outputs for the known functionally confirmed deleterious and benign RAD51C missense changes from nine pathogenicity prediction algorithms. The p.Arg312Trp variant failed to correct mitomycin and olaparib hypersensitivity and to complement abnormal RAD51C foci formation according to functional assays, which altogether with LOH and segregation data demonstrated deleteriousness. Prioritisation criteria were based on the number of predictors providing a deleterious output, with a minimum of 5 to qualify for testing and a PredictProtein score greater than 33 to assign high-priority indication. Our study points to a non-negligible number of RAD51C missense variants likely to impair protein function, provides a guideline to prioritise and encourage their selection for functional analysis and anticipates that reference laboratories should have available resources to conduct such assays.British Journal of Cancer advance online publication: 22 August 2017; doi:10.1038/bjc.2017.286 www.bjcancer.com.

  15. Overexpressed of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

    SciTech Connect

    Schild, David; Wiese, Claudia

    2009-10-15

    RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the 'recombination mediators'. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as 'recombination co-mediators'. Defects in either recombination mediators or comediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic restabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51.

  16. Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells.

    PubMed

    Collis, S J; Tighe, A; Scott, S D; Roberts, S A; Hendry, J H; Margison, G P

    2001-04-01

    The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to gamma-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of approximately 2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the alpha parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.

  17. Overexpression of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

    PubMed Central

    Schild, David; Wiese, Claudia

    2010-01-01

    RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA-binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the ‘recombination mediators’. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as ‘recombination co-mediators’. Defects in either recombination mediators or co-mediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic re-stabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51 expression. PMID:19942681

  18. ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

    PubMed Central

    McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

    2005-01-01

    Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions. PMID:15933716

  19. NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection

    PubMed Central

    Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

    2017-01-01

    ABSTRACT Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51. PMID:27892797

  20. An optimized RAD51 inhibitor that disrupts homologous recombination without requiring Michael acceptor reactivity

    PubMed Central

    Budke, Brian; Kalin, Jay H.; Pawlowski, Michal; Zelivianskaia, Anna S.; Wu, Megan; Kozikowski, Alan P.; Connell, Philip P.

    2013-01-01

    Homologous recombination (HR) is an essential process in cells that provides repair of DNA double-strand breaks and lesions that block DNA replication. RAD51 is an evolutionarily conserved protein that is central to HR. Overexpression of RAD51 protein is common in cancer cells and represents a potential therapeutic target in oncology. We previously described a chemical inhibitor of RAD51, called RI-1 (referred to as compound 1 in this report). The chloromaleimide group of this compound is thought to act as a Michael acceptor and react with the thiol group on C319 of RAD51, using a conjugate addition-elimination mechanism. In order to reduce the likelihood of off-target effects and to improve compound stability in biological systems, we developed an analog of compound 1 that lacks maleimide-based reactivity but retains RAD51 inhibitory activity. This compound, 1-(3,4-dichlorophenyl)-3-(4-methoxyphenyl)-4-morpholino-1H-pyrrole-2,5-dione, named RI-2 (referred to as compound 7a in this report), appears to bind reversibly to the same site on the RAD51 protein as does compound 1. Like compound 1, compound 7a specifically inhibits HR repair in human cells. PMID:23231413

  1. Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells

    PubMed Central

    Collis, S. J.; Tighe, A.; Scott, S. D.; Roberts, S. A.; Hendry, J. H.; Margison, G. P.

    2001-01-01

    The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20–50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to γ-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of ∼2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the α parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation. PMID:11266555

  2. ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage.

    PubMed

    McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

    2005-07-06

    Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.

  3. Rad51C-ATXN7 fusion gene expression in colorectal tumors.

    PubMed

    Kalvala, Arjun; Gao, Li; Aguila, Brittany; Dotts, Kathleen; Rahman, Mohammad; Nana-Sinkam, Serge P; Zhou, Xiaoping; Wang, Qi-En; Amann, Joseph; Otterson, Gregory A; Villalona-Calero, Miguel A; Duan, Wenrui

    2016-06-13

    Fusion proteins have unique oncogenic properties and their identification can be useful either as diagnostic or therapeutic targets. Next generation sequencing data have previously shown a fusion gene formed between Rad51C and ATXN7 genes in the MCF7 breast cancer cell line. However, the existence of this fusion gene in colorectal patient tumor tissues is largely still unknown. We evaluated for the presence of Rad51C-ATXN7 fusion gene in colorectal tumors and cells by RT-PCR, PCR, Topo TA cloning, Real time PCR, immunoprecipitation and immunoblotting techniques. We identified two forms of fusion mRNAs between Rad51C and ATXN7 in the colorectal tumors, including a Variant 1 (fusion transcript between Rad51C exons 1-7 and ATXN7 exons 6-13), and a Variant 2 (between Rad51C exons 1-6 and ATXN7 exons 6-13). In silico analysis showed that the Variant 1 produces a truncated protein, whereas the Variant 2 was predicted to produce a fusion protein with molecular weight of 110 KDa. Immunoprecipitation and Western blot analysis further showed a 110 KDa protein in colorectal tumors. 5-Azacytidine treatment of LS-174 T cells caused a 3.51-fold increase in expression of the fusion gene (Variant 2) as compared to no treatment controls evaluated by real time PCR. In conclusion we found a fusion gene between DNA repair gene Rad51C and neuro-cerebral ataxia Ataxin-7 gene in colorectal tumors. The in-frame fusion transcript of Variant 2 results in a fusion protein with molecular weight of 110 KDa. In addition, we found that expression of fusion gene is associated with functional impairment of Fanconi Anemia (FA) DNA repair pathway in colorectal tumors. The expression of Rad51C-ATXN7 in tumors warrants further investigation, as it suggests the potential of the fusion gene in treatment and predictive value in colorectal cancers.

  4. The role of Rad51 in safeguarding mitochondrial activity during the meiotic cell cycle in mammalian oocytes

    PubMed Central

    Kim, Kyeoung-Hwa; Park, Ji-Hoon; Kim, Eun-Young; Ko, Jung-Jae; Park, Kyung-Soon; Lee, Kyung-Ah

    2016-01-01

    Rad51 is a conserved eukaryotic protein that mediates the homologous recombination repair of DNA double-strand breaks that occur during mitosis and meiosis. In addition, Rad51 promotes mitochondrial DNA synthesis when replication stress is increased. Rad51 also regulates cell cycle progression by preserving the G2/M transition in embryonic stem cells. In this study, we report a novel function of Rad51 in regulating mitochondrial activity during in vitro maturation of mouse oocytes. Suppression of Rad51 by injection of Rad51 dsRNA into germinal vesicle-stage oocytes resulted in arrest of meiosis in metaphase I. Rad51-depleted oocytes showed chromosome misalignment and failures in spindle aggregation, affecting the completion of cytokinesis. We found that Rad51 depletion was accompanied by decreased ATP production and mitochondrial membrane potential and increased DNA degradation. We further demonstrated that the mitochondrial defect activated autophagy in Rad51-depleted oocytes. Taken together, we concluded that Rad51 functions to safeguard mitochondrial integrity during the meiotic maturation of oocytes. PMID:27677401

  5. Effects of XRCC2 and RAD51B mutations on somatic and meiotic recombination in Arabidopsis thaliana.

    PubMed

    Da Ines, Olivier; Degroote, Fabienne; Amiard, Simon; Goubely, Chantal; Gallego, Maria E; White, Charles I

    2013-06-01

    Homologous recombination is key to the maintenance of genome integrity and the creation of genetic diversity. At the mechanistic level, recombination involves the invasion of a homologous DNA template by broken DNA ends, repair of the break and exchange of genetic information between the two DNA molecules. Invasion of the template in eukaryotic cells is catalysed by the RAD51 and DMC1 recombinases, assisted by a number of accessory proteins, including the RAD51 paralogues. Eukaryotic genomes encode a variable number of RAD51 paralogues, ranging from two in yeast to five in animals and plants. The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilization of the RAD51-DNA nucleofilament. Somatic recombination assays and immunocytology confirm that the three 'non-meiotic' paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and that they are not required for the formation of radioinduced RAD51 foci. Given the presence of all five proteins in meiotic cells, the apparent absence of a meiotic role for RAD51B, RAD51D and XRCC2 is surprising, and perhaps simply the result of a more subtle meiotic phenotype in the mutants. Analysis of meiotic recombination confirms this, showing that the absence of XRCC2, and to a lesser extent RAD51B, but not RAD51D, increases rates of meiotic crossing over. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  6. TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity

    PubMed Central

    Moudry, Pavel; Watanabe, Kenji; Wolanin, Kamila M.; Bartkova, Jirina; Wassing, Isabel E.; Watanabe, Sugiko; Strauss, Robert; Troelsgaard Pedersen, Rune; Oestergaard, Vibe H.; Lisby, Michael; Andújar-Sánchez, Miguel; Maya-Mendoza, Apolinar; Esashi, Fumiko; Lukas, Jiri

    2016-01-01

    Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase–mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1’s role in HR, with potential clinical implications for cancer treatment. PMID:26811421

  7. Targeting human Rad51 by specific DNA aptamers induces inhibition of homologous recombination.

    PubMed

    Martinez, Susan F; Renodon-Cornière, Axelle; Nomme, Julian; Eveillard, Damien; Fleury, Fabrice; Takahashi, Masayuki; Weigel, Pierre

    2010-12-01

    Human Rad51 (HsRad51), a key element of the homologous recombination repair pathway, is related to the resistance of cancer cells to chemo- and radio-therapies. This protein is thus a good target for the development of anti-cancer treatments. We have searched for new inhibitors directed against HsRad51 using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach. We have selected three aptamers displaying strong effects on strand exchange activity. Analysis by circular dichroism shows that they are highly structured DNA molecules. Our results also show that they affect the first step of the strand exchange reaction by promoting the dissociation of DNA from the ATP/HsRad51/DNA complex. Moreover, these inhibitors bind only weakly to RecA, a prokaryotic ortholog of HsRad51. Both the specificity and the efficiency of their inhibition of recombinase activity offer an analytical tool based on molecular recognition and the prospect of developing new therapeutic agents.

  8. Resolving RAD51C function in late stages of homologous recombination

    PubMed Central

    Sharan, Shyam K; Kuznetsov, Sergey G

    2007-01-01

    DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction. PMID:17547768

  9. Suppression of OsRAD51D results in defects in reproductive development in rice (Oryza sativa L.).

    PubMed

    Byun, Mi Young; Kim, Woo Taek

    2014-07-01

    The cellular roles of RAD51 paralogs in somatic and reproductive growth have been extensively described in a wide range of animal systems and, to a lesser extent, in Arabidopsis, a dicot model plant. Here, the OsRAD51D gene was identified and characterized in rice (Oryza sativa L.), a monocot model crop. In the rice genome, three alternative OsRAD51D mRNA splicing variants, OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3, were predicted. Yeast two-hybrid studies, however, showed that only OsRAD51D.1 interacted with OsRAD51B and OsRAD51C paralogs, suggesting that OsRAD51D.1 is a functional OsRAD51D protein in rice. Loss-of-function osrad51d mutant rice plants displayed normal vegetative growth. However, the mutant plants were defective in reproductive growth, resulting in sterile flowers. Homozygous osrad51d mutant flowers exhibited impaired development of lemma and palea and contained unusual numbers of stamens and stigmas. During early meiosis, osrad51d pollen mother cells (PMCs) failed to form normal homologous chromosome pairings. In subsequent meiotic progression, mutant PMCs represented fragmented chromosomes. The osrad51d pollen cells contained numerous abnormal micro-nuclei that resulted in malfunctioning pollen. The abnormalities of heterozygous mutant and T2 Ubi:RNAi-OsRAD51D RNAi-knock-down transgenic plants were intermediate between those of wild type and homozygous mutant plants. The osrad51d and Ubi:RNAi-OsRAD51D plants contained longer telomeres compared with wild type plants, indicating that OsRAD51D is a negative factor for telomere lengthening. Overall, these results suggest that OsRAD51D plays a critical role in reproductive growth in rice. This essential function of OsRAD51D is distinct from Arabidopsis, in which AtRAD51D is not an essential factor for meiosis or reproductive development. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  10. RAD51B Activity and Cell Cycle Regulation in Response to DNA Damage in Breast Cancer Cell Lines

    PubMed Central

    Lee, Phoebe S; Fang, Jun; Jessop, Lea; Myers, Timothy; Raj, Preethi; Hu, Nan; Wang, Chaoyu; Taylor, Philip R; Wang, Jianjun; Khan, Javed; Jasin, Maria; Chanock, Stephen J

    2014-01-01

    Common genetic variants mapping to two distinct regions of RAD51B, a paralog of RAD51, have been associated with breast cancer risk in genome-wide association studies (GWAS). RAD51B is a plausible candidate gene because of its established role in the homologous recombination (HR) process. How germline genetic variation in RAD51B confers susceptibility to breast cancer is not well understood. Here, we investigate the molecular function of RAD51B in breast cancer cell lines by knocking down RAD51B expression by small interfering RNA and treating cells with DNA-damaging agents, namely cisplatin, hydroxyurea, or methyl-methanesulfonate. Our results show that RAD51B-depleted breast cancer cells have increased sensitivity to DNA damage, reduced efficiency of HR, and altered cell cycle checkpoint responses. The influence of RAD51B on the cell cycle checkpoint is independent of its role in HR and further studies are required to determine whether these functions can explain the RAD51B breast cancer susceptibility alleles. PMID:25368520

  11. RAD51 and XRCC3 gene polymorphisms and the risk of developing acute myeloid leukemia.

    PubMed

    Hamdy, Mona S; El-Haddad, Alaa M; Bahaa El-Din, Neveen M; Makhlouf, Manal Mohamed; Abdel-Hamid, Samah M

    2011-10-01

    RAD51 (Rec A homolog of E. coli) is a polymorphic gene and one of the central proteins in homologous recombination-DNA-double-stand breaks (HR-DNA-DSB) repair pathway, which is vital in maintaining genetic stability within a cell. The x-ray repair cross complementing (XRCC3) protein also functions in HR-DNA-DSB repair pathway and directly interacts with and stabilizes RAD51 and the closely related RAD51C. The aim of this study was to determine the prevalence of the RAD51 and XRCC3 repair gene polymorphisms among acute myeloid leukemia (AML) patients and to define their role in development of AML and its correlation with the clinical presentation, laboratory data as well as treatment outcome using polymerase chain reaction-restriction fragment length polymorphism assay in 50 de novo AML patients as well as 30 healthy subjects as a control group. Our study revealed that RAD51 G135C and XRCC3 Thr241Met alleles were associated with increased risk of AML with odds ratio (OR) of 2.833 and 2.909 and 95% confidence interval (CI) of 1.527 to 8.983 and 1.761 to 9.788, respectively. Moreover, when combining the 2 genes polymorphisms, a significant elevation of the risk of AML was found with OR of 3.124 and 95% CI of 1.872 to 11.243. As regards treatment outcome, a highly statistical significant difference was found between XRCC3 genotypes with P value of 0.001, whereas no significant difference was present between RAD51 genotypes with P value of 0.29. This clarifies that XRCC3 gene polymorphisms was found to have a significant impact on the risk of treatment failure with OR of 3.560 and 95% CI of 1.167 to 10.875; however, RAD51 gene polymorphism was not found to have an equivalent effect with OR of 2.813 and 95% CI of 0.933 to 10.828. So XRCC3 gene polymorphism might be considered as a prognostic marker in AML. In conclusion, RAD51 and XRCC3 genes polymorphisms may play an important role in the development of AML.

  12. RAD51 as a potential surrogate marker for DNA repair capacity in solid malignancies.

    PubMed

    Gachechiladze, Mariam; Škarda, Josef; Soltermann, Alex; Joerger, Markus

    2017-10-01

    Targeting deficient mechanisms of cellular DNA repair still represents the basis for the treatment of the majority of solid tumors, and increased DNA repair capacity is a hallmark mechanism of resistance not only to DNA-damaging treatments such as cytotoxic drugs and radiotherapy, but also to small molecule targeted drugs such as inhibitors of poly-ADP ribose polymerase (PARP). Hence, there is substantial medical need for potent and convenient biomarkers of individual response to DNA-targeted treatment in personalized cancer care. RAD51 is a highly conserved protein that catalyzes DNA repair via homologous recombination, a major DNA repair pathway which directly modulates cellular sensitivity to DNA-damaging treatments. The clinical and biological significance of RAD51 protein expression is still under investigation. Pre-clinical studies consistently show the important role of nuclear RAD51 immunoreactivity in chemo- and radioresistance. Validating data from clinical trials however is limited at present, and some clinical studies show controversial results. This review gives a comprehensive overview on the current knowledge about the prognostic and predictive value of RAD51 protein expression and genetic variability in patients with solid malignancies. © 2017 UICC.

  13. Suppression of mutagenesis by Rad51D-mediated homologous recombination

    SciTech Connect

    Hinz, J M; Tebbs, R S; Wilson, P F; Nham, P B; Salazar, E P; Nagasawa, H; Urbin, S S; Thompson, L H

    2005-11-15

    Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute in a nonessential but critical manner to HRR efficiency. We constructed and characterized a Rad51D knockout cell line in widely studied CHO cells. The rad51d mutant (51D1) displays sensitivity to a wide spectrum of induced DNA damage, indicating the broad relevance of HRR to genotoxicity. Untreated 51D1 cells exhibit {approx}5-fold elevated chromosomal breaks, a 12-fold increased rate of hprt mutation, and 4- to 10-fold increased rates of gene amplification at the dhfr and CAD loci, respectively. These results explicitly show the quantitative importance of HHR in preventing these types genetic alterations, which are associated with carcinogenesis. Thus, HRR copes in an error-free manner with spontaneous DNA damage encountered during DNA replication, and Rad51D is essential for this fidelity.

  14. 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.

  15. RAD51 Gene 135G/C polymorphism and ovarian cancer risk: a meta-analysis.

    PubMed

    Hu, Xingzhong; Sun, Suyu

    2015-01-01

    Previous studies suggest that the RAD51 gene 135G/C polymorphism could be potentially associated with the risk of ovarian cancer. However, results from observational studies are conflicting rather than conclusive. We performed a meta-analysis of the literature aiming to clarify the relationship between the polymorphism of RAD51 gene 135G/C polymorphism and the risk of ovarian cancer. Summary odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. We identified five eligible articles, 2336 ovarian cancer cases and 3548 controls. Meta-analysis results showed no significant association between 135G/C polymorphism in the RAD51 gene and ovarian cancer risk (GG vs CC: OR=0.42, 95% CI 0.16-1.06; GC vs CC: OR=0.37, 95% CI 0.12-1.16; Dominant model: OR=0.38, 95% CI 0.13-1.06; Recessive model: OR=1.20, 95% CI 0.91-1.58). No publication bias was found in the present study. This meta-analysis suggests that the RAD51 gene 135G/C polymorphism was not associated with risk of ovarian cancer. Further large and well-designed studies are needed to confirm this conclusion.

  16. Differing Requirements for RAD51 and DMC1 in Meiotic Pairing of Centromeres and Chromosome Arms in Arabidopsis thaliana

    PubMed Central

    Da Ines, Olivier; Abe, Kiyomi; Goubely, Chantal; Gallego, Maria Eugenia; White, Charles I.

    2012-01-01

    During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains. PMID:22532804

  17. Differing requirements for RAD51 and DMC1 in meiotic pairing of centromeres and chromosome arms in Arabidopsis thaliana.

    PubMed

    Da Ines, Olivier; Abe, Kiyomi; Goubely, Chantal; Gallego, Maria Eugenia; White, Charles I

    2012-01-01

    During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains.

  18. Stimulation of the Human RAD51 Nucleofilament Restricts HIV-1 Integration In Vitro and in Infected Cells

    PubMed Central

    Cosnefroy, O.; Tocco, A.; Lesbats, P.; Thierry, S.; Calmels, C.; Wiktorowicz, T.; Reigadas, S.; Kwon, Y.; De Cian, A.; Desfarges, S.; Bonot, P.; San Filippo, J.; Litvak, S.; Le Cam, E.; Rethwilm, A.; Fleury, H.; Connell, P. P.; Sung, P.; Delelis, O.; Andréola, M. L.

    2012-01-01

    Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51. PMID:22013044

  19. A Small Molecule Inhibitor of Human RAD51 Potentiates Breast Cancer Cell Killing by Therapeutic Agents in Mouse Xenografts

    PubMed Central

    Huang, Fei; Mazin, Alexander V.

    2014-01-01

    The homologous recombination pathway is responsible for the repair of DNA double strand breaks. RAD51, a key homologous recombination protein, promotes the search for homology and DNA strand exchange between homologous DNA molecules. RAD51 is overexpressed in a variety of cancer cells. Downregulation of RAD51 by siRNA increases radio- or chemo-sensitivity of cancer cells. We recently developed a specific RAD51 small molecule inhibitor, B02, which inhibits DNA strand exchange activity of RAD51 in vitro. In this study, we used human breast cancer cells MDA-MB-231 to investigate the ability of B02 to inhibit RAD51 and to potentiate an anti-cancer effect of chemotherapeutic agents including doxorubicin, etoposide, topotecan, and cisplatin. We found that the combination of B02 with cisplatin has the strongest killing effect on the cancer cells. We then tested the effect of B02 and cisplatin on the MDA-MB-231 cell proliferation in mouse xenografts. Our results showed that B02 significantly enhances the therapeutic effect of cisplatin on tumor cells in vivo. Our current data demonstrate that use of RAD51-specific small molecule inhibitor represents a feasible strategy of a combination anti-cancer therapy. PMID:24971740

  20. Rad51 and Rad54 promote noncrossover recombination between centromere repeats on the same chromatid to prevent isochromosome formation

    PubMed Central

    Onaka, Atsushi T.; Toyofuku, Naoko; Inoue, Takahiro; Okita, Akiko K.; Sagawa, Minami; Su, Jie; Shitanda, Takeshi; Matsuyama, Rei; Zafar, Faria; Takahashi, Tatsuro S.; Masukata, Hisao; Nakagawa, Takuro

    2016-01-01

    Centromeres consist of DNA repeats in many eukaryotes. Non-allelic homologous recombination (HR) between them can result in gross chromosomal rearrangements (GCRs). In fission yeast, Rad51 suppresses isochromosome formation that occurs between inverted repeats in the centromere. However, how the HR enzyme prevents homology-mediated GCRs remains unclear. Here, we provide evidence that Rad51 with the aid of the Swi/Snf-type motor protein Rad54 promotes non-crossover recombination between centromere repeats to prevent isochromosome formation. Mutations in Rad51 and Rad54 epistatically increased the rates of isochromosome formation and chromosome loss. In sharp contrast, these mutations decreased gene conversion between inverted repeats in the centromere. Remarkably, analysis of recombinant DNAs revealed that rad51 and rad54 increase the proportion of crossovers. In the absence of Rad51, deletion of the structure-specific endonuclease Mus81 decreased both crossovers and isochromosomes, while the cdc27/pol32-D1 mutation, which impairs break-induced replication, did not. We propose that Rad51 and Rad54 promote non-crossover recombination between centromere repeats on the same chromatid, thereby suppressing crossover between non-allelic repeats on sister chromatids that leads to chromosomal rearrangements. Furthermore, we found that Rad51 and Rad54 are required for gene silencing in centromeres, suggesting that HR also plays a role in the structure and function of centromeres. PMID:27697832

  1. DNA RECOMBINATION. Base triplet stepping by the Rad51/RecA family of recombinases.

    PubMed

    Lee, Ja Yil; Terakawa, Tsuyoshi; Qi, Zhi; Steinfeld, Justin B; Redding, Sy; Kwon, YoungHo; Gaines, William A; Zhao, Weixing; Sung, Patrick; Greene, Eric C

    2015-08-28

    DNA strand exchange plays a central role in genetic recombination across all kingdoms of life, but the physical basis for these reactions remains poorly defined. Using single-molecule imaging, we found that bacterial RecA and eukaryotic Rad51 and Dmc1 all stabilize strand exchange intermediates in precise three-nucleotide steps. Each step coincides with an energetic signature (0.3 kBT) that is conserved from bacteria to humans. Triplet recognition is strictly dependent on correct Watson-Crick pairing. Rad51, RecA, and Dmc1 can all step over mismatches, but only Dmc1 can stabilize mismatched triplets. This finding provides insight into why eukaryotes have evolved a meiosis-specific recombinase. We propose that canonical Watson-Crick base triplets serve as the fundamental unit of pairing interactions during DNA recombination. Copyright © 2015, American Association for the Advancement of Science.

  2. Mechanism of RAD51-dependent DNA interstrand cross-link repair.

    PubMed

    Long, David T; Räschle, Markus; Joukov, Vladimir; Walter, Johannes C

    2011-07-01

    DNA interstrand cross-links (ICLs) are toxic DNA lesions whose repair in S phase of eukaryotic cells is incompletely understood. In Xenopus egg extracts, ICL repair is initiated when two replication forks converge on the lesion. Dual incisions then create a DNA double-strand break (DSB) in one sister chromatid, whereas lesion bypass restores the other sister. We report that the broken sister chromatid is repaired via RAD51-dependent strand invasion into the regenerated sister. Recombination acts downstream of FANCI-FANCD2, yet RAD51 binds ICL-stalled replication forks independently of FANCI-FANCD2 and before DSB formation. Our results elucidate the functional link between the Fanconi anemia pathway and the recombination machinery during ICL repair. In addition, they demonstrate the complete repair of a DSB via homologous recombination in vitro.

  3. Inhibition of Rad51 sensitizes breast cancer cells with wild-type PTEN to olaparib.

    PubMed

    Zhao, Qian; Guan, Jiawei; Zhang, Zhiwei; Lv, Jian; Wang, Yulu; Liu, Likun; Zhou, Qi; Mao, Weifeng

    2017-10-01

    PTEN is a tumor suppressor gene well characterized as a phosphatase. However, more evidences demonstrate PTEN functions in DNA repair independent of its phosphatase activity, which affects the efficacy of DNA damage anti-tumoral drugs in treating cancer cells with PTEN variations. Using BT549 breast cancer cells, we studied the roles of PTEN in DNA repair and in sensitization of breast cancer cells to olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor. Comet assay showed PTEN promoted DNA repair. PTEN-deficient BT549 cells are sensitive to olaparib, which shows the synthetic lethality between PTEN and PARP1. We expressed PTEN in BT549 cells and found PTEN-proficient BT549 cells resist to olaparib. Western blot showed that PTEN up-regulated Rad51 expression, suggesting PTEN promotes DNA repair through Rad51-dependnent homologous recombination. We used 5μM olaparib or 5μM RI-1, a Rad51 inhibitor, to treat PTEN-proficient BT549 cells respectively. The immunofluorescent analysis showed the combination of olaparib and RI-1 induced more than 4-fold of γH2AX foci than either of them. MTT assay showed 5μM RI-1 did not change the survival of PTEN-proficient BT549 cells, however, this dose of RI-1 sensitized PTEN-proficient BT549 cells to olaparib. Consequently, these results demonstrate that inhibition of Rad51 can sensitize BT549 cells with wild type PTEN to olaparib, which would contribute to using PARP inhibitors in individual treatment of breast cancer patients with PTEN variations. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  4. Rad51 and BRCA2--New molecular targets for sensitizing glioma cells to alkylating anticancer drugs.

    PubMed

    Quiros, Steve; Roos, Wynand Paul; Kaina, Bernd

    2011-01-01

    First line chemotherapeutics for brain tumors (malignant gliomas) are alkylating agents such as temozolomide and nimustine. Despite growing knowledge of how these agents work, patients suffering from this malignancy still face a dismal prognosis. Alkylating agents target DNA, forming the killing lesion O(6)-alkylguanine, which is converted into DNA double-strand breaks (DSBs) that trigger apoptosis. Here we assessed whether inhibiting repair of DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ) is a reasonable strategy for sensitizing glioma cells to alkylating agents. For down-regulation of HR in glioma cells, we used an interference RNA (iRNA) approach targeting Rad51 and BRCA2, and for NHEJ we employed the DNA-PK inhibitor NU7026. We also assessed whether inhibition of poly(ADP)ribosyltransferase (PARP) by olaparib would enhance the killing effect. The data show that knockdown of Rad51 or BRCA2 greatly sensitizes cells to DSBs and the induction of cell death following temozolomide and nimustine (ACNU). It did not sensitize to ionizing radiation (IR). The expression of O(6)-methylguanine-DNA methyltransferase (MGMT) abolished all these effects, indicating that O(6)-alkylguanine induced by these drugs is the primary lesion responsible for the formation of DSBs and increased sensitivity of glioma cells following knockdown of Rad51 and BRCA2. Inhibition of DNA-PK only slightly sensitized to temozolomide whereas a significant effect was observed with IR. A triple strategy including siRNA and the PARP inhibitor olaparib further improved the killing effect of temozolomide. The data provides evidence that down-regulation of Rad51 or BRCA2 is a reasonable strategy for sensitizing glioma cells to killing by O(6)-alkylating anti-cancer drugs. The data also provide proof of principle that a triple strategy involving down-regulation of HR, PARP inhibition and MGMT depletion may greatly enhance the therapeutic effect of temozolomide.

  5. Analysis of the impact of the absence of RAD51 strand exchange activity in Arabidopsis meiosis

    PubMed Central

    Singh, Gunjita; Da Ines, Olivier; Gallego, Maria Eugenia

    2017-01-01

    The ploidy of eukaryote gametes must be halved to avoid doubling of numbers of chromosomes with each generation and this is carried out by meiosis, a specialized cell division in which a single chromosomal replication phase is followed by two successive nuclear divisions. With some exceptions, programmed recombination ensures the proper pairing and distribution of homologous pairs of chromosomes in meiosis and recombination defects thus lead to sterility. Two highly related recombinases are required to catalyse the key strand-invasion step of meiotic recombination and it is the meiosis-specific DMC1 which is generally believed to catalyse the essential non-sister chromatid crossing-over, with RAD51 catalysing sister-chromatid and non-cross-over events. Recent work in yeast and plants has however shown that in the absence of RAD51 strand-exchange activity, DMC1 is able to repair all meiotic DNA breaks and surprisingly, that this does not appear to affect numbers of meiotic cross-overs. In this work we confirm and extend this conclusion. Given that more than 95% of meiotic homologous recombination in Arabidopsis does not result in inter-homologue crossovers, Arabidopsis is a particularly sensitive model for testing the relative importance of the two proteins—even minor effects on the non-crossover event population should produce detectable effects on crossing-over. Although the presence of RAD51 protein provides essential support for the action of DMC1, our results show no significant effect of the absence of RAD51 strand-exchange activity on meiotic crossing-over rates or patterns in different chromosomal regions or across the whole genome of Arabidopsis, strongly supporting the argument that DMC1 catalyses repair of all meiotic DNA breaks, not only non-sister cross-overs. PMID:28797117

  6. Nascent DNA synthesis during homologous recombination is synergistically promoted by the rad51 recombinase and DNA homology.

    PubMed

    Mundia, Maureen M; Desai, Vatsal; Magwood, Alissa C; Baker, Mark D

    2014-05-01

    In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (3' extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 3' extension for homology lengths of 500 bp to 2 kb. At values below ∼500 bp, the frequency of 3' extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 3' extension. Overexpression of wild-type Rad51 stimulated the frequency of 3' extension by ∼3-fold for homology lengths <900 bp, but when homology was >2 kb, 3' extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 3' extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 3' extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 3' extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1-8, rescues the 3' extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 3' extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization.

  7. Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

    PubMed Central

    Mundia, Maureen M.; Desai, Vatsal; Magwood, Alissa C.; Baker, Mark D.

    2014-01-01

    In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (3′ extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 3′ extension for homology lengths of 500 bp to 2 kb. At values below ∼500 bp, the frequency of 3′ extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 3′ extension. Overexpression of wild-type Rad51 stimulated the frequency of 3′ extension by ∼3-fold for homology lengths <900 bp, but when homology was >2 kb, 3′ extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 3′ extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 3′ extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 3′ extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1–8, rescues the 3′ extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 3′ extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization. PMID:24583581

  8. BRCA2 Acts as RAD51 Loader to Facilitate Telomere Replication and Capping

    PubMed Central

    Badie, Sophie; Escandell, Jose M.; Bouwman, Peter; Carlos, Ana Rita; Thanasoula, Maria; Gallardo, Maria M.; Suram, Anitha; Jaco, Isabel; Benitez, Javier; Herbig, Utz; Blasco, Maria A.; Jonkers, Jos; Tarsounas, Madalena

    2010-01-01

    BRCA2 is a key component of the homologous recombination (HR) pathway of DNA repair, acting as the loader of RAD51 recombinase at sites of double-strand breaks. Here, we demonstrate that BRCA2 associates with telomeres during S/G2 and facilitates RAD51 loading onto telomeres. Conditional Brca2 deletion and Rad51 inhibition in mouse embryonic fibroblasts (MEFs), but not Brca1 inactivation, led to telomere shortening and accumulation of fragmented telomeric signals, a hallmark of telomere fragility associated with replication defects. This suggests that BRCA2-mediated HR reactions contribute to telomere length maintenance by facilitating telomere replication and implies an essential role for BRCA2 in telomere integrity during unchallenged cell proliferation. Mouse mammary tumors lacking Brca2 accumulated telomere dysfunction-induced foci. BRCA2-mutated human breast tumors had shorter telomeres than BRCA1-mutated ones, suggesting that the genomic instability observed in BRCA2-deficient tumors is due in part to telomere dysfunction. PMID:21076401

  9. Polymorphisms of RAD51B are associated with rheumatoid arthritis and erosion in rheumatoid arthritis patients

    PubMed Central

    Zhi, Liqiang; Yao, Shuxin; Ma, Wenlong; Zhang, Weijie; Chen, Honggan; Li, Meng; Ma, Jianbing

    2017-01-01

    Rheumatoid arthritis (RA) is a common, chronic autoimmune disease affecting 0.5–1.0% of adults worldwide, including approximately 4.5–5.0 million patients in China. The genetic etiology and pathogenesis of RA have not yet been fully elucidated. Recently, one new RA susceptibility gene (RAD51B) has been identified in Korean and European populations. In this study, we designed a two-stage case-control study to further assess the relationship of common variants in the RAD51B gene with increased risk of RA in a total of 965 RA patients and 2,511 unrelated healthy controls of Han Chinese ancestry. We successfully identified a common variant, rs911263, as being significantly associated with the disease status of RA (P = 4.8 × 10−5, OR = 0.64). In addition, this SNP was shown to be related to erosion, a clinical assessment of disease severity in RA (P = 2.89 × 10−5, OR = 0.52). These findings shed light on the role of RAD51B in the onset and severity of RA. More research in the future is needed to clarify the underlying functional link between rs911263 and the disease. PMID:28361912

  10. A novel Fanconi anaemia subtype associated with a dominant-negative mutation in RAD51

    PubMed Central

    Ameziane, Najim; May, Patrick; Haitjema, Anneke; van de Vrugt, Henri J.; van Rossum-Fikkert, Sari E.; Ristic, Dejan; Williams, Gareth J.; Balk, Jesper; Rockx, Davy; Li, Hong; Rooimans, Martin A.; Oostra, Anneke B.; Velleuer, Eunike; Dietrich, Ralf; Bleijerveld, Onno B.; Maarten Altelaar, A. F.; Meijers-Heijboer, Hanne; Joenje, Hans; Glusman, Gustavo; Roach, Jared; Hood, Leroy; Galas, David; Wyman, Claire; Balling, Rudi; den Dunnen, Johan; de Winter, Johan P.; Kanaar, Roland; Gelinas, Richard; Dorsman, Josephine C.

    2015-01-01

    Fanconi anaemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. A total of 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, ‘FA-R', which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and paediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders, our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility. PMID:26681308

  11. Rad51 and Rad52 are involved in homologous recombination of replicating herpes simplex virus DNA.

    PubMed

    Tang, Ka-Wei; Norberg, Peter; Holmudden, Martin; Elias, Per; Liljeqvist, Jan-Åke

    2014-01-01

    Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells.

  12. Rad51 and Rad52 Are Involved in Homologous Recombination of Replicating Herpes Simplex Virus DNA

    PubMed Central

    Tang, Ka-Wei; Norberg, Peter; Holmudden, Martin; Elias, Per; Liljeqvist, Jan-Åke

    2014-01-01

    Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells. PMID:25365323

  13. Rad51-mediated double-strand break repair and mismatch correction of divergent substrates

    PubMed Central

    Anand, Ranjith; Beach, Annette; Li, Kevin; Haber, James

    2017-01-01

    The RecA/Rad51 family of recombinases execute the critical step in homologous recombination (HR): the search for homologous DNA to serve as the template during DNA double-strand break (DSB) repair1–7. Although budding yeast Rad51 has been extensively characterized in vitro3,4,6–9, the stringency of its search and sensitivity to mismatched sequences in vivo remain poorly defined. We analyzed Rad51-dependent break-induced replication (BIR) where the invading DSB end and its donor template share 108 bp homology and the donor carries different densities of single-bp mismatches (Fig. 1a). With every 8th bp mismatched, repair was ~14% compared to completely homologous sequences. With every 6th bp mismatched, repair was >5%. Thus completing BIR in vivo overcomes the apparent requirement for at least 6–8 consecutive paired bases inferred from in vitro studies6,8. When recombination occurs without a protruding nonhomologous 3′ tail, mismatch repair protein Msh2 does not discourage homeologous recombination. However, when the DSB end contains a 3′ protruding nonhomologous tail, Msh2 promotes rejection of mismatched substrates. Mismatch correction of strand invasion heteroduplex DNA is strongly polar, favoring correction close to the DSB end. Nearly all mismatch correction depends on the proofreading activity of DNA polymerase δ, although Msh2-Mlh1 and Exo1 influence the extent of correction. PMID:28405019

  14. Rad51-mediated double-strand break repair and mismatch correction of divergent substrates.

    PubMed

    Anand, Ranjith; Beach, Annette; Li, Kevin; Haber, James

    2017-04-20

    The Rad51 (also known as RecA) family of recombinases executes the critical step in homologous recombination: the search for homologous DNA to serve as a template during the repair of DNA double-strand breaks (DSBs). Although budding yeast Rad51 has been extensively characterized in vitro, the stringency of its search and sensitivity to mismatched sequences in vivo remain poorly defined. Here, in Saccharomyces cerevisiae, we analysed Rad51-dependent break-induced replication in which the invading DSB end and its donor template share a 108-base-pair homology region and the donor carries different densities of single-base-pair mismatches. With every eighth base pair mismatched, repair was about 14% of that of completely homologous sequences. With every sixth base pair mismatched, repair was still more than 5%. Thus, completing break-induced replication in vivo overcomes the apparent requirement for at least 6-8 consecutive paired bases that has been inferred from in vitro studies. When recombination occurs without a protruding nonhomologous 3' tail, the mismatch repair protein Msh2 does not discourage homeologous recombination. However, when the DSB end contains a 3' protruding nonhomologous tail, Msh2 promotes the rejection of mismatched substrates. Mismatch correction of strand invasion heteroduplex DNA is strongly polar, favouring correction close to the DSB end. Nearly all mismatch correction depends on the proofreading activity of DNA polymerase-δ, although the repair proteins Msh2, Mlh1 and Exo1 influence the extent of correction.

  15. Functional effects of diphosphomimetic mutations at cAbl-mediated phosphorylation sites on Rad51 recombinase activity.

    PubMed

    Alligand, Brendan; Le Breton, Magali; Marquis, Damien; Vallette, François; Fleury, Fabrice

    2017-08-01

    Homologous Recombination enables faithful repair of the deleterious double strand breaks of DNA. This pathway relies on Rad51 to catalyze homologous DNA strand exchange. Rad51 is known to be phosphorylated in a sequential manner on Y315 and then on Y54, but the effect of such phosphorylation on Rad51 function remains poorly understood. We have developed a phosphomimetic model in order to study all the phosphorylation states. With the purified phosphomimetic proteins we performed in vitro assays to determine the activity of Rad51. Here we demonstrate the inhibitory effect of the double phosphomimetic mutant and suggest that it may be due to a defect in nucleofilament formation. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  16. Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination

    PubMed Central

    Lorenz, Alexander; Mehats, Alizée; Osman, Fekret; Whitby, Matthew C.

    2014-01-01

    During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved. PMID:25414342

  17. Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination.

    PubMed

    Lorenz, Alexander; Mehats, Alizée; Osman, Fekret; Whitby, Matthew C

    2014-12-16

    During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.

  18. Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

    PubMed Central

    Chappell, William H.; Gautam, Dipendra; Ok, Suzan T.; Johnson, Bryan A.; Anacker, Daniel C.

    2015-01-01

    ABSTRACT High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for

  19. Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31.

    PubMed

    Chappell, William H; Gautam, Dipendra; Ok, Suzan T; Johnson, Bryan A; Anacker, Daniel C; Moody, Cary A

    2015-12-23

    High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral

  20. 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

    2015-08-06

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Real-time solution measurement of RAD51- and RecA-mediated strand assimilation without background annealing.

    PubMed

    Budke, Brian; Chan, Yuen-Ling; Bishop, Douglas K; Connell, Philip P

    2013-07-01

    RAD51 is the central strand exchange recombinase in somatic homologous recombination, providing genomic stability and promoting resistance to DNA damage. An important tool for mechanistic studies of RAD51 is the D-loop or strand assimilation assay, which measures the ability of RAD51-coated single-stranded DNA (ssDNA) to search for, invade and exchange ssDNA strands with a homologous duplex DNA target. As cancer cells generally overexpress RAD51, the D-loop assay has also emerged as an important tool in oncologic drug design programs for targeting RAD51. Previous studies have adapted the traditional gel-based D-loop assay by using fluorescence-based substrates, which in principle allow for use in high-throughput screening platforms. However, these existing D-loop methods depend on linear oligonucleotide DNA duplex targets, and these substrates enable recombinase-independent ssDNA annealing that can obscure the recombinase-dependent strand assimilation signal. This compelled us to fundamentally re-design this assay, using a fluorescent target substrate that consists of a covalently closed linear double-hairpin dsDNA. This new microplate-based method represents a fast, inexpensive and non-radioactive alternative to existing D-loop assays. It provides accurate kinetic analysis of strand assimilation in high-throughput and performs well with human RAD51 and Escherichia coli RecA protein. This advance will aid in both mechanistic studies of homologous recombination and drug screening programs.

  2. 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.

  3. 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.

  4. Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in plant meiotic recombination.

    PubMed

    Pradillo, Mónica; López, Eva; Linacero, Rosario; Romero, Concepción; Cuñado, Nieves; Sánchez-Morán, Eugenio; Santos, Juan L

    2012-03-01

    The eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, non-homologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.

  5. Adenoviral Vector Driven by a Minimal Rad51 Promoter Is Selective for p53-Deficient Tumor Cells

    PubMed Central

    Fong, Vincent; Osterbur, Marika; Capella, Cristina; Kim, Yo-El; Hine, Christopher; Gorbunova, Vera; Seluanov, Andrei; Dewhurst, Stephen

    2011-01-01

    Background The full length Rad51 promoter is highly active in cancer cells but not in normal cells. We therefore set out to assess whether we could confer this tumor-selectivity to an adenovirus vector. Methodology/Principal Findings Expression of an adenovirally-vectored luciferase reporter gene from the Rad51 promoter was up to 50 fold higher in cancer cells than in normal cells. Further evaluations of a panel of truncated promoter mutants identified a 447 bp minimal core promoter element that retained the full tumor selectivity and transcriptional activity of the original promoter, in the context of an adenovirus vector. This core Rad51 promoter was highly active in cancer cells that lack functional p53, but less active in normal cells and in cancer cell lines with intact p53 function. Exogenous expression of p53 in a p53 null cell line strongly suppressed activity of the Rad51 core promoter, underscoring the selectivity of this promoter for p53-deficient cells. Follow-up experiments showed that the p53-dependent suppression of the Rad51 core promoter was mediated via an indirect, p300 coactivator dependent mechanism. Finally, transduction of target cells with an adenovirus vector encoding the thymidine kinase gene under transcriptional control of the Rad51 core promoter resulted in efficient killing of p53 defective cancer cells, but not of normal cells, upon addition of ganciclovir. Conclusions/Significance Overall, these experiments demonstrated that a small core domain of the Rad51 promoter can be used to target selective transgene expression from adenoviral vectors to tumor cells lacking functional p53. PMID:22174876

  6. Adenoviral vector driven by a minimal Rad51 promoter is selective for p53-deficient tumor cells.

    PubMed

    Fong, Vincent; Osterbur, Marika; Capella, Cristina; Kim, Yo-El; Hine, Christopher; Gorbunova, Vera; Seluanov, Andrei; Dewhurst, Stephen

    2011-01-01

    The full length Rad51 promoter is highly active in cancer cells but not in normal cells. We therefore set out to assess whether we could confer this tumor-selectivity to an adenovirus vector. Expression of an adenovirally-vectored luciferase reporter gene from the Rad51 promoter was up to 50 fold higher in cancer cells than in normal cells. Further evaluations of a panel of truncated promoter mutants identified a 447 bp minimal core promoter element that retained the full tumor selectivity and transcriptional activity of the original promoter, in the context of an adenovirus vector. This core Rad51 promoter was highly active in cancer cells that lack functional p53, but less active in normal cells and in cancer cell lines with intact p53 function. Exogenous expression of p53 in a p53 null cell line strongly suppressed activity of the Rad51 core promoter, underscoring the selectivity of this promoter for p53-deficient cells. Follow-up experiments showed that the p53-dependent suppression of the Rad51 core promoter was mediated via an indirect, p300 coactivator dependent mechanism. Finally, transduction of target cells with an adenovirus vector encoding the thymidine kinase gene under transcriptional control of the Rad51 core promoter resulted in efficient killing of p53 defective cancer cells, but not of normal cells, upon addition of ganciclovir. Overall, these experiments demonstrated that a small core domain of the Rad51 promoter can be used to target selective transgene expression from adenoviral vectors to tumor cells lacking functional p53.

  7. CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51-independent gene targeting in the moss Physcomitrella patens.

    PubMed

    Collonnier, Cécile; Epert, Aline; Mara, Kostlend; Maclot, François; Guyon-Debast, Anouchka; Charlot, Florence; White, Charles; Schaefer, Didier G; Nogué, Fabien

    2017-01-01

    The ability to address the CRISPR-Cas9 nuclease complex to any target DNA using customizable single-guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single-guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2-fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end-joining (alt-EJ)-driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology-driven repair (HDR) at the target locus but also that Cas9-induced double-strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR-mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR-induced HDR is only partially mediated by the classical homologous recombination pathway. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  8. Oleandrin induces DNA damage responses in cancer cells by suppressing the expression of Rad51

    PubMed Central

    Bao, Zhengqiang; Tian, Baoping; Wang, Xiaohui; Feng, Hanrong; Liang, Ye; Chen, Zhihua; Li, Wen; Shen, Huahao; Ying, Songmin

    2016-01-01

    Oleandrin is a monomeric compound extracted from leaves and seeds of Nerium oleander. It had been reported that oleandrin could effectively inhibit the growth of human cancer cells. However, the specific mechanisms of the oleandrin-induced anti-tumor effects remain largely unclear. Genomic instability is one of the main features of cancer cells, it can be the combined effect of DNA damage and tumour-specific DNA repair defects. DNA damage plays important roles during tumorigenesis. In fact, most of the current chemotherapy agents were designed to kill cancer cells by inducing DNA damage. In this study, we found that oleandrin was effective to induce apoptosis in cancer cells, and cause rapid DNA damage response, represented by nuclear RPA (Replication Protein A, a single strand DNA binding protein) and γH2AX(a marker for DNA double strand breaks) foci formation. Interestingly, expression of RAD51, a key protein involved in homologous recombination (HR), was suppressed while XRCC1 was up-regulated in oleandrin treated cancer cells. These results suggested that XRCC1 may play a predominant role in repairing oleandrin-induced DNA damage. Collectively, oleandrin may be a potential anti-tumor agent by suppressing the expression of Rad51. PMID:27449097

  9. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells

    PubMed Central

    Zellweger, Ralph; Dalcher, Damian; Mutreja, Karun; Berti, Matteo; Schmid, Jonas A.; Herrador, Raquel; Vindigni, Alessandro

    2015-01-01

    Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy. PMID:25733714

  10. RAD51 inhibition in triple negative breast cancer cells is challenged by compensatory survival signaling and requires rational combination therapy

    PubMed Central

    Wiegmans, Adrian P.; Miranda, Mariska; Wen, Shu Wen

    2016-01-01

    The molecular rationale to induce synthetic lethality, by targeting defective homologous recombination repair in triple negative breast cancer (TNBC), has proven to have several shortcomings. Not meeting the expected minimal outcomes in clinical trials has highlighted common clinical resistance mechanisms including; increased expression of the target gene PARP1, increased expression or reversion mutation of BRCA1, or up-regulation of the compensatory homologous recombination protein RAD51. Indeed, RAD51 has been demonstrated to be an alternative synthetic lethal target in BRCA1-mutated cancers. To overcome selective pressure on DNA repair pathways, we examined new potential targets within TNBC that demonstrate synthetic lethality in association with RAD51 depletion. We confirmed complementary targets of PARP1/2 and DNA-PK as well as a new synthetic lethality combination with p38. p38 is considered a relevant target in breast cancer, as it has been implicated in resistance to chemotherapy, including tamoxifen. We show that the combination of targeting RAD51 and p38 inhibits cell proliferation both in vitro and in vivo, which was further enhanced by targeting of PARP1. Analysis of the molecular mechanisms revealed that depletion of RAD51 increased ERK1/2 and p38 signaling. Our results highlight a potential compensatory mechanism via p38 that limits DNA targeted therapy. PMID:27507046

  11. Nanoscopic exclusion between Rad51 and 53BP1 after ion irradiation in human HeLa cells

    NASA Astrophysics Data System (ADS)

    Reindl, Judith; Drexler, Guido A.; Girst, Stefanie; Greubel, Christoph; Siebenwirth, Christian; Drexler, Sophie E.; Dollinger, Günther; Friedl, Anna A.

    2015-12-01

    Many proteins involved in detection, signalling and repair of DNA double-strand breaks (DSB) accumulate in large number in the vicinity of DSB sites, forming so called foci. Emerging evidence suggests that these foci are sub-divided in structural or functional domains. We use stimulated emission depletion (STED) microscopy to investigate localization of mediator protein 53BP1 and recombination factor Rad51 after irradiation of cells with low linear energy transfer (LET) protons or high LET carbon ions. With a resolution better than 100 nm, STED microscopy and image analysis using a newly developed analyzing algorithm, the reduced product of the differences from the mean, allowed us to demonstrate that with both irradiation types Rad51 occupies spherical regions of about 200 nm diameter. These foci locate within larger 53BP1 accumulations in regions of local 53BP1 depletion, similar to what has been described for the localization of Brca1, CtIP and RPA. Furthermore, localization relative to 53BP1 and size of Rad51 foci was not different after irradiation with low and high LET radiation. As expected, 53BP1 foci induced by low LET irradiation mostly contained one Rad51 focal structure, while after high LET irradiation, most foci contained >1 Rad51 accumulation.

  12. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

    SciTech Connect

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; Groesser, Torsten; Liang, Fengshan; Maranon, David G.; Leung, Stanley G.; Grundt, Kirsten; Dray, Eloïse; Idate, Rupa; Østvold, Anne Carine; Schild, David; Sung, Patrick; Wiese, Claudia

    2015-08-31

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Finally, our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

  13. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

    DOE PAGES

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; ...

    2015-08-31

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintainingmore » wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Finally, our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.« less

  14. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability.

    PubMed

    Parplys, Ann C; Zhao, Weixing; Sharma, Neelam; Groesser, Torsten; Liang, Fengshan; Maranon, David G; Leung, Stanley G; Grundt, Kirsten; Dray, Eloïse; Idate, Rupa; Østvold, Anne Carine; Schild, David; Sung, Patrick; Wiese, Claudia

    2015-11-16

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Arabidopsis BRCA2 and RAD51 proteins are specifically involved in defense gene transcription during plant immune responses

    PubMed Central

    Wang, Shui; Durrant, Wendy E.; Song, Junqi; Spivey, Natalie W.; Dong, Xinnian

    2010-01-01

    Systemic acquired resistance (SAR) is a plant immune response associated with both transcriptional reprogramming and increased homologous DNA recombination (HR). SNI1 is a negative regulator of SAR and HR, as indicated by the increased basal expression of defense genes and HR in sni1. We found that the sni1 phenotypes are rescued by mutations in BREAST CANCER 2 (BRCA2). In humans, BRCA2 is a mediator of RAD51 in pairing of homologous DNA. Mutations in BRCA2 cause predisposition to breast/ovarian cancers; however, the role of the BRCA2–RAD51 complex in transcriptional regulation remains unclear. In Arabidopsis, both brca2 and rad51 were found to be hypersusceptible not only to genotoxic substances, but also to pathogen infections. A whole-genome microarray analysis showed that downstream of NPR1, BRCA2A is a major regulator of defense-related gene transcription. ChIP demonstrated that RAD51 is specifically recruited to the promoters of defense genes during SAR. This recruitment is dependent on the SAR signal salicylic acid (SA) and on the function of BRCA2. This study provides the molecular evidence showing that the BRCA2–RAD51 complex, known for its function in HR, also plays a direct and specific role in transcription regulation during plant immune responses. PMID:21149701

  16. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

    PubMed Central

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; Groesser, Torsten; Liang, Fengshan; Maranon, David G.; Leung, Stanley G.; Grundt, Kirsten; Dray, Eloïse; Idate, Rupa; Østvold, Anne Carine; Schild, David; Sung, Patrick; Wiese, Claudia

    2015-01-01

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression. PMID:26323318

  17. Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing.

    PubMed

    Kobayashi, Wataru; Takaku, Motoki; Machida, Shinichi; Tachiwana, Hiroaki; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi

    2016-04-07

    In eukaryotes, genomic DNA is compacted as chromatin, in which histones and DNA form the nucleosome as the basic unit. DMC1 and RAD51 are essential eukaryotic recombinases that mediate homologous chromosome pairing during homologous recombination. However, the means by which these two recombinases distinctly function in chromatin have remained elusive. Here we found that, in chromatin, the human DMC1-single-stranded DNA complex bypasses binding to the nucleosome, and preferentially promotes homologous pairing at the nucleosome-depleted regions. Consistently, DMC1 forms ternary complex recombination intermediates with the nucleosome-free DNA or the nucleosome-depleted DNA region. Surprisingly, removal of the histone tails improperly enhances the nucleosome binding by DMC1. In contrast, RAD51 does not specifically target the nucleosome-depleted region in chromatin. These are the first demonstrations that the chromatin architecture specifies the sites to promote the homologous recombination reaction by DMC1, but not by RAD51.

  18. Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing

    PubMed Central

    Kobayashi, Wataru; Takaku, Motoki; Machida, Shinichi; Tachiwana, Hiroaki; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi

    2016-01-01

    In eukaryotes, genomic DNA is compacted as chromatin, in which histones and DNA form the nucleosome as the basic unit. DMC1 and RAD51 are essential eukaryotic recombinases that mediate homologous chromosome pairing during homologous recombination. However, the means by which these two recombinases distinctly function in chromatin have remained elusive. Here we found that, in chromatin, the human DMC1-single-stranded DNA complex bypasses binding to the nucleosome, and preferentially promotes homologous pairing at the nucleosome-depleted regions. Consistently, DMC1 forms ternary complex recombination intermediates with the nucleosome-free DNA or the nucleosome-depleted DNA region. Surprisingly, removal of the histone tails improperly enhances the nucleosome binding by DMC1. In contrast, RAD51 does not specifically target the nucleosome-depleted region in chromatin. These are the first demonstrations that the chromatin architecture specifies the sites to promote the homologous recombination reaction by DMC1, but not by RAD51. PMID:27052786

  19. Induction of Rad51 protein levels by p38 MAPK decreases cytotoxicity and mutagenicity in benzo[a]pyrene-exposed human lung cancer cells

    SciTech Connect

    Chuang, S.-M.; Wang, L.-H.; Hong, J.-H.; Lin, Y.-W.

    2008-08-01

    Rad51 is an essential component of the homologous recombination repair pathway. Abnormal expression of Rad51 has been reported in various carcinomas. Benzo[a]pyrene (B[a]P), a polycyclic hydrocarbon carcinogen found in the environment, induces cancer in multiple organs. B[a]P has been shown to activate the p38 MAPK signaling pathway in mammalian cells. The prime purpose of this study was to determine how B[a]P activates the p38 MAPK signaling pathway, and how this then regulates Rad51 expression in human cancer cells. Exposure of human lung cancer cells with B[a]P increased Rad51 protein levels in a time- and dose-dependent fashion. B[a]P also induced Rad51 mRNA and protein synthesis. Blockage of p38 MAPK activation by SB202190 or small interfering RNA (si-p38) decreased B[a]P-elicited Rad51 protein levels by increasing Rad51 protein instability, but did not affect Rad51 mRNA transcription. Furthermore, enhancement of p38 MAPK signaling by constitutively active MKK6 (MKK6E) increased Rad51 protein levels and protein stability. Moreover, B[a]P-induced cytotoxicity and mutagenicity were significantly increased in cells depleted of endogenous Rad51. Taken together, these results indicate that Rad51 protein provides a critical role in inhibiting the cytotoxicity and mutagenicity of B[a]P in B[a]P-treated human lung cancer cells. Furthermore, the work points to an unexpected role of p38 MAPK signaling in the control of Rad51 protein stability in response to B[a]P exposure.

  20. Genetic polymorphisms of RAD51 and XRCC3 and acute myeloid leukemia risk: a meta-analysis.

    PubMed

    Li, Cuiping; Liu, Yun; Hu, Zhen; Zhou, Yang

    2014-06-01

    Studies on gene polymorphisms of RAD51 and X-ray repair cross-complementing group 3 (XRCC3) and acute myeloid leukemia risk (AML) are conflicting and there is no recent meta-analysis. Therefore, the purpose of this study was to evaluate the effect of RAD51 G135C and XRCC3 Thr241Met genotypes on AML susceptibility. We conducted a systematic search of three databases including PubMed and EMBASE for the period up to 20 February 2013 and identified 43 relevant studies. Six eligible studies were eventually selected for RAD51 (1764 cases and 3469 controls) and six studies for XRCC3 (1352 cases and 2582 controls). Pooled odds ratios (ORs) and 95% confidence intervals (CIs) for the risk of AML associated with RAD51 and XRCC3 were appropriately calculated based on fixed- or random-effects models. The quality of studies was evaluated using the Newcastle-Ottawa Scale (NOS). Subgroup analyses were performed among Asian, Caucasian and other populations. The pooled results showed that the leukemia risk was not significantly associated with RAD51: the same results were obtained among any subgroup analysis. No significant association was demonstrated for AML risk with XRCC3 in the total population, but elevated associations were observed in Caucasians for the homozygote and recessive comparison (Met/Met vs. Thr/Thr, OR = 1.67, 95% CI = 1.09-2.57, p = 0.019; recessive model, OR = 1.78, 95% CI = 1.19-2.65, p = 0.005). This meta-analysis provides evidence that the RAD51 and XRCC3 polymorphisms are not associated with an increased risk of AML in the total population.

  1. Brca2, Rad51 and Mre11: performing balancing acts on replication forks.

    PubMed

    Costanzo, Vincenzo

    2011-10-10

    Homologous recombination (HR) is required for faithful repair of double strand breaks (DSBs) and is believed to be important for DNA replication under stressful conditions in unicellular organisms. However, its role during DNA replication in high eukaryotes has always been elusive. In particular, due to the essential nature of its main players it has been difficult to dissect the direct role of HR in DNA replication. Recent studies revealed that some key HR factors such as Rad51 and BRCA2 play unexpected functions during DNA replication by protecting nascent DNA from Mre11 mediated degradation, which takes place at stalled replication forks. These novel functions appear to be essential to ensure smooth progression of DNA replication and to promote maintenance of genome stability.

  2. Formation of higher-order nuclear Rad51 structures is functionally linked to p21 expression and protection from DNA damage-induced apoptosis.

    PubMed

    Raderschall, Elke; Bazarov, Alex; Cao, Jiangping; Lurz, Rudi; Smith, Avril; Mann, Wolfgang; Ropers, Hans-Hilger; Sedivy, John M; Golub, Efim I; Fritz, Eberhard; Haaf, Thomas

    2002-01-01

    After exposure of mammalian cells to DNA damage, the endogenous Rad51 recombination protein is concentrated in multiple discrete foci, which are thought to represent nuclear domains for recombinational DNA repair. Overexpressed Rad51 protein forms foci and higher-order nuclear structures, even in the absence of DNA damage, in cells that do not undergo DNA replication synthesis. This correlates with increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21. Following DNA damage, constitutively Rad51-overexpressing cells show reduced numbers of DNA breaks and chromatid-type chromosome aberrations and a greater resistance to apoptosis. In contrast, Rad51 antisense inhibition reduces p21 protein levels and sensitizes cells to etoposide treatment. Downregulation of p21 inhibits Rad51 foci formation in both normal and Rad51-overexpressing cells. Collectively, our results show that Rad51 expression, Rad51 foci formation and p21 expression are interrelated, suggesting a functional link between mammalian Rad51 protein and p21-mediated cell cycle regulation. This mechanism may contribute to a highly effective recombinational DNA repair in cell cycle-arrested cells and protection against DNA damage-induced apoptosis.

  3. 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.

  4. αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

    PubMed Central

    Hansen, Scott D.; Kwiatkowski, Adam V.; Ouyang, Chung-Yueh; Liu, HongJun; Pokutta, Sabine; Watkins, Simon C.; Volkmann, Niels; Hanein, Dorit; Weis, William I.; Mullins, R. Dyche; Nelson, W. James

    2013-01-01

    The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays. PMID:24068324

  5. Spatial and temporal control of nonmuscle myosin localization: identification of a domain that is necessary for myosin filament disassembly in vivo

    PubMed Central

    1991-01-01

    Myosin null mutants of Dictyostelium are defective for cytokinesis, multicellular development, and capping of surface proteins. We have used these cells as transformation recipients for an altered myosin heavy chain gene that encodes a protein bearing a carboxy-terminal 34- kD truncation. This truncation eliminates threonine phosphorylation sites previously shown to control filament assembly in vitro. Despite restoration of growth in suspension, development, and ability to cap cell surface proteins, these delta C34-truncated myosin transformants display severe cytoskeletal abnormalities, including excessive localization of the truncated myosin to the cortical cytoskeleton, impaired cell shaped dynamics, and a temporal defect in myosin dissociation from beneath capped surface proteins. These data demonstrate that the carboxy-terminal domain of myosin plays a critical role in regulating the disassembly of the protein from contractile structures in vivo. PMID:1899668

  6. BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

    PubMed Central

    Reuter, Marcel; Zelensky, Alex; Smal, Ihor; Meijering, Erik; van Cappellen, Wiggert A.; de Gruiter, H. Martijn; van Belle, Gijsbert J.; van Royen, Martin E.; Houtsmuller, Adriaan B.; Essers, Jeroen; Kanaar, Roland

    2014-01-01

    Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2. PMID:25488918

  7. Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1

    PubMed Central

    Machida, Shinichi; Takaku, Motoki; Ikura, Masae; Sun, Jiying; Suzuki, Hidekazu; Kobayashi, Wataru; Kinomura, Aiko; Osakabe, Akihisa; Tachiwana, Hiroaki; Horikoshi, Yasunori; Fukuto, Atsuhiko; Matsuda, Ryo; Ura, Kiyoe; Tashiro, Satoshi; Ikura, Tsuyoshi; Kurumizaka, Hitoshi

    2014-01-01

    Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1. PMID:24798879

  8. (−)-Guaiol regulates RAD51 stability via autophagy to induce cell apoptosis in non-small cell lung cancer

    PubMed Central

    Yang, Qingyuan; Wu, Jianchun; Luo, Yingbin; Huang, Nan; Zhen, Ni; Zhou, Yun; Sun, Fenyong; Li, Zhi

    2016-01-01

    (−)-Guaiol, generally known as an antibacterial compound, has been found in many medicinal plants. Its roles in tumor suppression are still under investigation. In the study, we mainly focused on exploring its applications in dealing with non-small cell lung cancer (NSCLC) and the underlying mechanisms. Here, we show that (−)-Guaiol significantly inhibits cell growth of NSCLC cells both in vitro and in vivo. Further high throughput analysis reveals that RAD51, a pivotal factor in homologous recombination repair, is a potential target for it. The following mechanism studies show that (−)-Guaiol is involved in cell autophagy to regulate the expression of RAD51, leading to double-strand breaks triggered cell apoptosis. Moreover, targeting RAD51, which is highly overexpressed in the lung adenocarcinoma tissues, can significantly increase the chemosensitivity of NSCLC cells to (−)-Guaiol both in vitro and in vivo. All in all, our studies provide an attractive insight in applying (−)-Guaiol into NSCLC treatments and further suggest that knockdown of oncogenic RAD51 will greatly enhance the chemosensitivity of patients with NSCLC. PMID:27566579

  9. Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1.

    PubMed

    Machida, Shinichi; Takaku, Motoki; Ikura, Masae; Sun, Jiying; Suzuki, Hidekazu; Kobayashi, Wataru; Kinomura, Aiko; Osakabe, Akihisa; Tachiwana, Hiroaki; Horikoshi, Yasunori; Fukuto, Atsuhiko; Matsuda, Ryo; Ura, Kiyoe; Tashiro, Satoshi; Ikura, Tsuyoshi; Kurumizaka, Hitoshi

    2014-05-06

    Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.

  10. RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase.

    PubMed Central

    Le, S; Moore, J K; Haber, J E; Greider, C W

    1999-01-01

    Telomere length is maintained by the de novo addition of telomere repeats by telomerase, yet recombination can elongate telomeres in the absence of telomerase. When the yeast telomerase RNA component, TLC1, is deleted, telomeres shorten and most cells die. However, gene conversion mediated by the RAD52 pathway allows telomere lengthening in rare survivor cells. To further investigate the role of recombination in telomere maintenance, we assayed telomere length and the ability to generate survivors in several isogenic DNA recombination mutants, including rad50, rad51, rad52, rad54, rad57, xrs2, and mre11. The rad51, rad52, rad54, and rad57 mutations increased the rate of cell death in the absence of TLC1. In contrast, although the rad50, xrs2, and mre11 strains initially had short telomeres, double mutants with tlc1 did not affect the rate of cell death, and survivors were generated at later times than tlc1 alone. While none of the double mutants of recombination genes and tlc1 (except rad52 tlc1) blocked the ability to generate survivors, a rad50 rad51 tlc1 triple mutant did not allow the generation of survivors. Thus RAD50 and RAD51 define two separate pathways that collaborate to allow cells to survive in the absence of telomerase. PMID:10224249

  11. Cyclin D1 promotes BRCA2-Rad51 interaction by restricting cyclin A/B-dependent BRCA2 phosphorylation.

    PubMed

    Chalermrujinanant, C; Michowski, W; Sittithumcharee, G; Esashi, F; Jirawatnotai, S

    2016-06-02

    BRCA2 has an important role in the maintenance of genome stability by interacting with RAD51 recombinase through its C-terminal domain. This interaction is abrogated by cyclin A-CDK2-mediated phosphorylation of BRCA2 at serine 3291 (Ser3291). Recently, we showed that cyclin D1 facilitates RAD51 recruitment to BRCA2-containing DNA repair foci, and that downregulation of cyclin D1 leads to inefficient homologous-mediated DNA repair. Here, we demonstrate that cyclin D1, via amino acids 20-90, interacts with the C-terminal domain of BRCA2, and that this interaction is increased in response to DNA damage. Interestingly, CDK4-cyclin D1 does not phosphorylate Ser3291. Instead, cyclin D1 bars cyclin A from the C-terminus of BRCA2, prevents cyclin A-CDK2-dependent Ser3291 phosphorylation and facilitates RAD51 binding to the C-terminal domain of BRCA2. These findings indicate that the interplay between cyclin D1 and other cyclins such as cyclin A regulates DNA integrity through RAD51 interaction with the BRCA2 C-terminal domain.

  12. Genome stability of Arabidopsis atm, ku80 and rad51b mutants: somatic and transgenerational responses to stress.

    PubMed

    Yao, Youli; Bilichak, Andriy; Titov, Viktor; Golubov, Andrey; Kovalchuk, Igor

    2013-06-01

    DNA double-strand breaks (DSBs) can be repaired via two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). Our previous work showed that exposure to abiotic stresses resulted in an increase in point mutation frequency (PMF) and homologous recombination frequency (HRF), and these changes were heritable. We hypothesized that mutants impaired in DSB recognition and repair would also be deficient in somatic and transgenerational changes in PMF and HRF. To test this hypothesis, we analyzed the genome stability of the Arabidopsis thaliana mutants deficient in ATM (communication between DNA strand break recognition and the repair machinery), KU80 (deficient in NHEJ) and RAD51B (deficient in HR repair) genes. We found that all three mutants exhibited higher levels of DSBs. Plants impaired in ATM had a lower spontaneous PMF and HRF, whereas ku80 plants had higher frequencies. Plants impaired in RAD51B had a lower HRF. HRF in wild-type, atm and rad51b plants increased in response to several abiotic stressors, whereas it did not increase in ku80 plants. The progeny of stressed wild-type and ku80 plants exhibited an increase in HRF in response to all stresses, and the increase was higher in ku80 plants. The progeny of atm plants showed an increase in HRF only when the parental generation was exposed to cold or flood, whereas the progeny of rad51b plants completely lacked a transgenerational increase in HRF. Our experiments showed that mutants impaired in the recognition and repair of DSBs exhibited changes in the efficiency of DNA repair as reflected by changes in strand breaks, point mutation and HRF. They also showed that the HR RAD51B protein and the protein ATM that recognized damaged DNA might play an important role in transgenerational changes in HRF.

  13. DMC1 functions in a Saccharomyces cerevisiae meiotic pathway that is largely independent of the RAD51 pathway.

    PubMed

    Dresser, M E; Ewing, D J; Conrad, M N; Dominguez, A M; Barstead, R; Jiang, H; Kodadek, T

    1997-10-01

    Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains.

  14. Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair.

    PubMed

    Sonoda, Eiichiro; Zhao, Guang Yu; Kohzaki, Masaoki; Dhar, Pawan Kumar; Kikuchi, Koji; Redon, Christophe; Pilch, Duane R; Bonner, William M; Nakano, Atsushi; Watanabe, Masami; Nakayama, Tatsuo; Takeda, Shunichi; Takami, Yasunari

    2007-03-01

    One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.

  15. Combined effect of polymorphisms in Rad51 and Xrcc3 on breast cancer risk and chromosomal radiosensitivity.

    PubMed

    Vral, A; Willems, P; Claes, K; Poppe, B; Perletti, Gianpaolo; Thierens, H

    2011-01-01

    Enhanced in vitro chromosomal radiosensitivity (CRS) has been proposed as a marker for low-penetrance gene mutations predisposing to breast cancer (BC). Since the double strand break (DSB) is the most detrimental form of DNA damage induced by ionizing radiation, it is possible that mutations in genes encoding proteins involved in DSB repair affect breast cancer risk. The purpose of the present study was to examine whether five single nucleotide polymorphisms (SNPs) in Rad51 and Xrcc3 (rs1801320, rs1801321, rs1799796, rs861539 and rs1799794) exhibited an association with breast cancer susceptibility in a Belgian population of BC patients with a known or putative genetic predisposition. We also ascertained whether a relationship exists between the occurrence of the variant alleles of these variations and in vitro CRS. Blood samples were obtained from BC patients and from the control population that included healthy female individuals. Variations in the 5' UTR of Rad51 and Xrcc3 were genotyped, and statistical analysis was performed. The results showed that low-penetrant variations in Rad51 and Xrcc3, two proteins belonging to the homologous recombination DSB repair pathway, may modify BC risk in patients already carrying a pathological mutation in the highly penetrant BC genes BRCA1 and BRCA2. Combined risk genotype analysis revealed that Rad51 SNPs enhance BC risk in BRCA2 patients, whereas Xrcc3 SNPs significantly enhance BC risk in carriers of BRCA1 mutations and in patients with hereditary BC. When four putative risk genotypes of Rad51 and Xrcc3 were combined, positive significant odds ratios were obtained in the entire patient population and in patients with a hereditary history of disease. Although obtained from a limited number of patients, our data are supportive of a polygenic model whereby combinations of weak variations are responsible for an enhanced BC risk by acting jointly with high-penetrant mutations in BRCA1 or BRCA2.

  16. 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.

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

    PubMed Central

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

    2015-01-01

    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

  18. Polymorphisms in RAD51 and their relation with breast cancer in Saudi females.

    PubMed

    Tulbah, Sahar; Alabdulkarim, Huda; Alanazi, Mohammad; Parine, Narasimha Reddy; Shaik, Jilani; Pathan, Akbar Ali Khan; Al-Amri, Abdullah; Khan, Wajahatullah; Warsy, Arjumand

    2016-01-01

    The present study aimed at investigating the relationship between rs1801320 (G>C), rs1801321 (G>T), and rs2619681 (C>T) RAD51 gene polymorphisms and the risk of breast cancer development in Saudi females. The genotypes were analyzed using TaqMan genotyping assay and polymerase chain reaction-restriction fragment length polymorphism. The genotype and allele frequencies were computed using chi-square or Fisher's exact test (two-tailed) by SPSS 21 software. The results showed that rs1801321G>T GG genotype and G allele frequency were strongly (P<0.0001) related to an elevated risk of breast cancer, while the mutant T allele appeared to provide protection against breast cancer development as observed from the significantly lower (P<0.0001) frequencies of the TT and GT genotypes in cancer patients compared to the healthy controls. The variant rs1801320G>C showed no significant differences in the frequencies of the genotypes and alleles in the patients and the control groups. The CC genotype and C allele frequency of rs2619681 (C>T) variant were significantly (P=0.012) higher in cancer patients, whereas the T allele showed a protective effect against cancer development. The frequencies of the three single-nucleotide polymorphisms did not differ in cancer patients with different tumor grades and human epidermal growth factor receptor 2 status (+ or -). However, the genotype frequency of rs1801320 (135G>C) differed in the patients with estrogen receptor (ER)+ and ER-, where CC genotype showed a significantly higher prevalence in the females with ER- who were suffering from breast cancer. In addition, the frequency of C allele of rs2619681 (C>T) was also significantly higher in the breast cancer patients who were ER+ and progesterone receptor (PR)+ compared to those with ER- and PR-. In the Saudi females, rs1801320 did not show an association with risk of breast cancer. Taken together, the results suggest that RAD51 rs1801321 polymorphism may be involved in the etiology of

  19. Polymorphisms in RAD51 and their relation with breast cancer in Saudi females

    PubMed Central

    Tulbah, Sahar; Alabdulkarim, Huda; Alanazi, Mohammad; Parine, Narasimha Reddy; Shaik, Jilani; Pathan, Akbar Ali Khan; Al-Amri, Abdullah; Khan, Wajahatullah; Warsy, Arjumand

    2016-01-01

    The present study aimed at investigating the relationship between rs1801320 (G>C), rs1801321 (G>T), and rs2619681 (C>T) RAD51 gene polymorphisms and the risk of breast cancer development in Saudi females. The genotypes were analyzed using TaqMan genotyping assay and polymerase chain reaction-restriction fragment length polymorphism. The genotype and allele frequencies were computed using chi-square or Fisher’s exact test (two-tailed) by SPSS 21 software. The results showed that rs1801321G>T GG genotype and G allele frequency were strongly (P<0.0001) related to an elevated risk of breast cancer, while the mutant T allele appeared to provide protection against breast cancer development as observed from the significantly lower (P<0.0001) frequencies of the TT and GT genotypes in cancer patients compared to the healthy controls. The variant rs1801320G>C showed no significant differences in the frequencies of the genotypes and alleles in the patients and the control groups. The CC genotype and C allele frequency of rs2619681 (C>T) variant were significantly (P=0.012) higher in cancer patients, whereas the T allele showed a protective effect against cancer development. The frequencies of the three single-nucleotide polymorphisms did not differ in cancer patients with different tumor grades and human epidermal growth factor receptor 2 status (+ or −). However, the genotype frequency of rs1801320 (135G>C) differed in the patients with estrogen receptor (ER)+ and ER−, where CC genotype showed a significantly higher prevalence in the females with ER− who were suffering from breast cancer. In addition, the frequency of C allele of rs2619681 (C>T) was also significantly higher in the breast cancer patients who were ER+ and progesterone receptor (PR)+ compared to those with ER− and PR−. In the Saudi females, rs1801320 did not show an association with risk of breast cancer. Taken together, the results suggest that RAD51 rs1801321 polymorphism may be involved in the

  20. Large inverted repeats in the vicinity of a single double-strand break strongly affect repair in yeast diploids lacking Rad51.

    PubMed

    Downing, Brandon; Morgan, Rachel; VanHulle, Kelly; Deem, Angela; Malkova, Anna

    2008-10-14

    DNA double-strand breaks (DSBs) are critical lesions that can lead to cell death or chromosomal rearrangements. Rad51 is necessary for most mitotic and meiotic DSB repair events, although a number of RAD51-independent pathways exist. Previously, we described DSB repair in rad51Delta yeast diploids that was stimulated by a DNA region termed "facilitator of break-induced replication" (FBI) located approximately 30kb from the site of an HO-induced DSB. Here, we demonstrate that FBI is a large inverted DNA repeat that channels the repair of DSBs into the single-strand annealing-gross chromosomal rearrangements (SSA-GCR) pathway. Further, analysis of DSB repair in rad54Delta cells allowed us to propose that the SSA-GCR repair pathway is suppressed in the presence of Rad51p. Therefore, an additional role of Rad51 might be to protect eukaryotic genomes from instabilities by preventing chromosomal rearrangements.

  1. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. 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.

  3. Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions.

    PubMed

    Konomura, Naoto; Arai, Naoto; Shinohara, Takeshi; Kobayashi, Jun; Iwasaki, Wakana; Ikawa, Shukuko; Kusano, Kohji; Shibata, Takehiko

    2017-01-09

    RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.

  4. 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. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

    PubMed Central

    Konomura, Naoto; Arai, Naoto; Shinohara, Takeshi; Kobayashi, Jun; Iwasaki, Wakana; Ikawa, Shukuko; Kusano, Kohji; Shibata, Takehiko

    2017-01-01

    RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2–5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo. PMID:27794044

  6. HELQ promotes RAD51 paralog-dependent repair to avert germ cell attrition and tumourigenesis

    PubMed Central

    Adelman, Carrie A.; Lolo, Rafal L.; Birkbak, Nicolai J.; Murina, Olga; Matsuzaki, Kenichiro; Horejsi, Zuzana; Parmar, Kalindi; Borel, Valérie; Skehel, J. Mark; Stamp, Gordon; D’Andrea, Alan; Sartori, Alessandro A.; Swanton, Charles; Boulton, Simon J.

    2013-01-01

    Repair of interstrand crosslinks (ICLs) requires the coordinate action of the intra-S phase checkpoint and the Fanconi Anemia (FA) pathway, which promote ICL incision, translesion synthesis, and homologous recombination (reviewed in 1,2). Previous studies have implicated the 3′-5′ superfamily 2 helicase HELQ/Hel308 in ICL repair in D. melanogaster (known as Mus301 or Spn-C3) and C. elegans (known as Helq-1 or Hel-3084). While in vitro analysis suggests that HELQ preferentially unwinds synthetic replication fork substrates with 3′ ssDNA overhangs and also disrupts protein/DNA interactions while translocating along DNA5,6, little is known regarding its functions in mammalian organisms. Here we report that HELQ helicase-deficient mice exhibit subfertility, germ cell attrition, ICL sensitivity and tumour predisposition, with HelQ heterozygous mice exhibiting a similar, albeit less severe, phenotype than the null, indicative of haploinsufficiency. We establish that HELQ interacts directly with the RAD51 paralog complex, BCDX2, and functions in parallel to the FA pathway to promote efficient HR at damaged replication forks. Thus, our results reveal a critical role for HELQ in replication-coupled DNA repair, germ cell maintenance and tumour suppression in mammals. PMID:24005329

  7. Meiotic Crossover Control by Concerted Action of Rad51-Dmc1 in Homolog Template Bias and Robust Homeostatic Regulation

    PubMed Central

    Huang, Chu-Chun; Grubb, Jennifer; Thacker, Drew; Lee, Chih-Ying; Dresser, Michael E.; Hunter, Neil; Bishop, Douglas K.

    2013-01-01

    During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1's strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51's strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1's chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1's dominance in promoting strand exchange between homologs involves repression of Rad51's strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to

  8. Transient expression of RAD51 in the late G2-phase is required for cell cycle progression in synchronous Physarum cells.

    PubMed

    Le Cigne, Anthony; Menil-Philippot, Vanessa; Fleury, Fabrice; Takahashi, Masayuki; Thiriet, Christophe

    2014-10-01

    The homologous recombination factor RAD51 is highly conserved. This criterion enabled us to identify a RAD51 ortholog in Physarum polycephalum. We found that the Physarum protein presents a high homology to the human protein and cross-reacted with antibodies directed against the human RAD51. Taking advantage of the natural synchrony of millions of nuclei within a single cell of Physarum, we investigated the fluctuation of the amount of the PpRAD51 throughout the cell cycle. Our results showed that in the late G2-phase, RAD51 was transiently expressed in a large quantity. Furthermore, knocking-down RAD51 in the G2-phase abolished this transient expression before mitosis and affected cell cycle progression. These results support the idea that RAD51 plays a role in the progression of the cell cycle in the late G2-phase. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  9. Moonlighting at replication forks - a new life for homologous recombination proteins BRCA1, BRCA2 and RAD51.

    PubMed

    Kolinjivadi, Arun Mouli; Sannino, Vincenzo; de Antoni, Anna; Técher, Hervé; Baldi, Giorgio; Costanzo, Vincenzo

    2017-01-12

    Coordination between DNA replication and DNA repair ensures maintenance of genome integrity, which is lost in cancer cells. Emerging evidence has linked homologous recombination (HR) proteins RAD51, BRCA1 and BRCA2 to the stability of nascent DNA. This function appears to be distinct from double-strand break (DSB) repair and is in part due to the prevention of MRE11-mediated degradation of nascent DNA at stalled forks. The role of RAD51 in fork protection resembles the activity described for its prokaryotic orthologue RecA, which prevents nuclease-mediated degradation of DNA and promotes replication fork restart in cells challenged by DNA-damaging agents. Here, we examine the mechanistic aspects of HR-mediated fork protection, addressing the crosstalk between HR and replication proteins.

  10. Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells.

    PubMed

    Du, Li-Qing; Du, Xiao-Qing; Bai, Jian-Qiang; Wang, Yan; Yang, Qing-Shan; Wang, Xiao-Chun; Zhao, Peng; Wang, Hong; Liu, Qiang; Fan, Fei-Yue

    2012-05-01

    Methotrexate is an inhibitor of folic acid metabolism. Homologous recombination is one of the most important ways to repair double-stranded breaks in DNA and influence the radio- and chemosensitivity of tumor cells. But the relationship between methotrexate and homologous recombination repair has not been elucidated. Induction of double-strand breaks by methotrexate in HOS cells is assessed by the neutral comet assay. Inhibition of subnuclear repair foci by methotrexate is measured by immunofluorescence. Western blot and quantitative real-time PCR are conducted to detect whether methotrexate affects the expression level of genes involved in homologous recombination. In addition, we used a pCMV3xnls-I-SceI construct to determine whether methotrexate directly inhibits the process of homologous recombinational repair in cells, and the sensitivity to methotrexate in the Ku80-deficient cells is detected using clonogenic survival assays. The result showed that methotrexate can regulate the repair of DNA double-strand breaks after radiation exposure, and methotrexate inhibition caused the complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigation revealed that methotrexate led to a significant reduction in the transcription of RAD51 genes. Treatment with methotrexate resulted in a decreased ability to perform homology-directed repair of I-SceI-induced chromosome breaks. In addition, enhancement of cell death was observed in Ku mutant cells compared to wild-type cells. These results demonstrate that methotrexate can affect homologous recombination repair of DNA double-strand breaks by controlling the expression of homologous recombination-related genes and suppressing the proper assembly of homologous recombination-directed subnuclear foci.

  11. Mutant IDH1-driven cellular transformation increases RAD51-mediated homologous recombination and temozolomide resistance.

    PubMed

    Ohba, Shigeo; Mukherjee, Joydeep; See, Wendy L; Pieper, Russell O

    2014-09-01

    Isocitrate dehydrogenase 1 (IDH1) mutations occur in most lower grade glioma and not only drive gliomagenesis but are also associated with longer patient survival and improved response to temozolomide. To investigate the possible causative relationship between these events, we introduced wild-type (WT) or mutant IDH1 into immortalized, untransformed human astrocytes, then monitored transformation status and temozolomide response. Temozolomide-sensitive parental cells exhibited DNA damage (γ-H2AX foci) and a prolonged G2 cell-cycle arrest beginning three days after temozolomide (100 μmol/L, 3 hours) exposure and persisting for more than four days. The same cells transformed by expression of mutant IDH1 exhibited a comparable degree of DNA damage and cell-cycle arrest, but both events resolved significantly faster in association with increased, rather than decreased, clonogenic survival. The increases in DNA damage processing, cell-cycle progression, and clonogenicity were unique to cells transformed by mutant IDH1, and were not noted in cells transformed by WT IDH1 or an oncogenic form (V12H) of Ras. Similarly, these effects were not noted following introduction of mutant IDH1 into Ras-transformed cells or established glioma cells. They were, however, associated with increased homologous recombination (HR) and could be reversed by the genetic or pharmacologic suppression of the HR DNA repair protein RAD51. These results show that mutant IDH1 drives a unique set of transformative events that indirectly enhance HR and facilitate repair of temozolomide-induced DNA damage and temozolomide resistance. The results also suggest that inhibitors of HR may be a viable means to enhance temozolomide response in IDH1-mutant glioma.

  12. Susceptibility of XPD and RAD51 Genetic Variants to Carcinoma of Urinary Bladder in North Indian Population

    PubMed Central

    Kaur, Saranjeet; Sharma, Vijay Lakshmi; Singh, Shrawan Kumar; Hosseini, Seyed Ali; Kler, Rupinder

    2012-01-01

    For the present study, two polymorphisms, xeroderma pigmentosum, complementation group D (XPD) Lys751Gln and RAD51 135G/C were studied with regard to bladder cancer. For XPD Lys751Gln polymorphism, an increased risk of bladder cancer was found to be associated with the Gln variant allele (odds ratio [OR]=1.86, 95% confidence interval [CI]=1.27–2.73), on taking AA (Lys/Lys) as the referent genotype. In males, the XPD 751C (Gln) allele was found to be associated with a significantly increased risk (OR=2.33, 95% CI=1.52–3.56). The inhabitants of rural areas showed a significantly increased risk with the XPD Gln allele (OR=2.59, 95% CI=1.46–4.62) when compared with those of urban areas. In smokers (OR=5.30, 95% CI=2.42–11.68), alcohol drinkers (OR=4.33, 95% CI=2.17–8.70), and nonvegetarians (OR=2.21, 95% CI=1.26–3.87), the XPD Gln allele showed a significantly increased risk toward bladder cancer. For RAD51 135G/C polymorphism, no significant difference was observed in the allelic and genotypic frequencies. Even after stratification, no significant association could be seen. After stratifying histopathologically, the RAD51 CC genotype was associted with decreased risk in subjects having superficial stage (OR=0.51, 95% CI=0.27–0.99) and with those having G2 grade (OR=0.24, 95% CI=0.09–0.62) of bladder cancer. XPD polymorphism may be a predisposing factor, but the same cannot be said for RAD51 gene polymorphism. PMID:21740187

  13. Correlation between selected XRCC2, XRCC3 and RAD51 gene polymorphisms and primary breast cancer in women in Pakistan.

    PubMed

    Qureshi, Z; Mahjabeen, I; Baig, Rm; Kayani, Ma

    2014-01-01

    Genetic polymorphisms in homologous recombination repair genes cause an abnormal development of cancerous cells. In the present study we evaluated the possibility of breast cancer association with single nucleotide polymorphisms of RAD51, XRCC2 and XRCC3 genes. Polymorphisms selected in this study were RAD51 135G/C, XRCC2 Arg188His; and XRCC3 Thr241Met. Each polymorphism was genotyped using Polymerase chain reaction-restriction fragment length polymorphism in study cohort of 306 females (156 breast cancer patients and 150 controls). We observed that heterozygous variant genotype (GC) of RAD51 135 G/C polymorphism was associated with a significantly (OR=2.70; 95%CI (0.63-1.79); p<0.03) increased risk of breast cancer. In case of the XRCC3 gene we observed that frequency of heterozygous (OR=2.88; 95%CI (1.02-8.14); p<0.02) and homozygous (OR=1.46; 95%CI (0.89-2.40); p<0.04) genotype of Thr241Met polymorphism were significantly higher in breast cancer patients. For the Arg188His polymorphism of XRCC2, ~2fold increase in breast cancer risk (OR=1.6, 95%CI = 0.73-3.50) was associated with GA genotype with a p value for trend of 0.03. Our results suggest that the 135G/C polymorphism of the RAD51, Thr241Met polymorphism of XRCC3 and Arg188His polymorphism of XRCC2 can be independent markers of breast cancer risk in Pakistan.

  14. Isolation of novel human and mouse genes of the recA/RAD51 recombination-repair gene family.

    PubMed Central

    Cartwright, R; Dunn, A M; Simpson, P J; Tambini, C E; Thacker, J

    1998-01-01

    Genes from the recA/RAD51 family play essential roles in homologous recombination in all organisms. Using sequence homologies from eukaryotic members of this family we have identified fragments of two additional mammalian genes with homology to RAD51. Cloning the full-length cDNAs for both human and mouse genes showed that the sequences are highly conserved, and that the predicted proteins have characteristic features of this gene family. One of the novel genes (R51H2) occurs in two forms in human cDNA, differing extensively at the 3' end, probably due to an unusual form of alternative splicing. The new genes (R51H2 and R51H3) were mapped to human chromosomes 14q23-24 and 17q1.2, respectively. Expression studies showed that R51H2 is expressed at lower levels than R51H3 , but that expression of both genes occurs at elevated levels in the testis compared with other tissues. The combination of gene structure conservation and the transcript expression patterns suggests that these new members of the recA/RAD51 family may also function in homologous recombination-repair pathways. PMID:9512535

  15. Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci

    PubMed Central

    Reindl, Judith; Girst, Stefanie; Walsh, Dietrich W. M.; Greubel, Christoph; Schwarz, Benjamin; Siebenwirth, Christian; Drexler, Guido A.; Friedl, Anna A.; Dollinger, Günther

    2017-01-01

    The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 ± 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 ± 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened. PMID:28094292

  16. CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes.

    PubMed

    Ogiwara, Hideaki; Kohno, Takashi

    2012-01-01

    Histone acetylation at DNA double-strand break (DSB) sites by CBP and p300 histone acetyltransferases (HATs) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the BRCA1 and RAD51 genes, which are involved in homologous recombination (HR), a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.

  17. Ubiquitylation of Rad51d Mediated by E3 Ligase Rnf138 Promotes the Homologous Recombination Repair Pathway.

    PubMed

    Han, Deqiang; Liang, Junbo; Lu, Yalan; Xu, Longchang; Miao, Shiying; Lu, Lin-Yu; Song, Wei; Wang, Linfang

    2016-01-01

    Ubiquitylation has an important role as a signal transducer that regulates protein function, subcellular localization, or stability during the DNA damage response. In this study, we show that Ring domain E3 ubiquitin ligases RNF138 is recruited to DNA damage site quickly. And the recruitment is mediated through its Zinc finger domains. We further confirm that RNF138 is phosphorylated by ATM at Ser124. However, the phosphorylation was dispensable for recruitment to the DNA damage site. Our findings also indicate that RAD51 assembly at DSB sites following irradiation is dramatically affected in RNF138-deficient cells. Hence, RNF138 is likely involved in regulating homologous recombination repair pathway. Consistently, efficiency of homologous recombination decreased observably in RNF138-depleted cells. In addition, RNF138-deficient cell is hypersensitive to DNA damage insults, such as IR and MMS. And the comet assay confirmed that RNF138 directly participated in DNA damage repair. Moreover, we find that RAD51D directly interacted with RNF138. And the recruitment of RAD51D to DNA damage site is delayed and unstable in RNF138-depleted cells. Taken together, these results suggest that RNF138 promotes the homologous recombination repair pathway.

  18. Ubiquitylation of Rad51d Mediated by E3 Ligase Rnf138 Promotes the Homologous Recombination Repair Pathway

    PubMed Central

    Han, Deqiang; Liang, Junbo; Lu, Yalan; Xu, Longchang; Miao, Shiying; Lu, Lin-Yu; Song, Wei; Wang, Linfang

    2016-01-01

    Ubiquitylation has an important role as a signal transducer that regulates protein function, subcellular localization, or stability during the DNA damage response. In this study, we show that Ring domain E3 ubiquitin ligases RNF138 is recruited to DNA damage site quickly. And the recruitment is mediated through its Zinc finger domains. We further confirm that RNF138 is phosphorylated by ATM at Ser124. However, the phosphorylation was dispensable for recruitment to the DNA damage site. Our findings also indicate that RAD51 assembly at DSB sites following irradiation is dramatically affected in RNF138-deficient cells. Hence, RNF138 is likely involved in regulating homologous recombination repair pathway. Consistently, efficiency of homologous recombination decreased observably in RNF138-depleted cells. In addition, RNF138-deficient cell is hypersensitive to DNA damage insults, such as IR and MMS. And the comet assay confirmed that RNF138 directly participated in DNA damage repair. Moreover, we find that RAD51D directly interacted with RNF138. And the recruitment of RAD51D to DNA damage site is delayed and unstable in RNF138-depleted cells. Taken together, these results suggest that RNF138 promotes the homologous recombination repair pathway. PMID:27195665

  19. Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases

    PubMed Central

    Ploquin, Mickaël; Petukhova, Galina V.; Morneau, Dany; Déry, Ugo; Bransi, Ali; Stasiak, Andrzej; Camerini-Otero, R. Daniel; Masson, Jean-Yves

    2007-01-01

    Genetic analysis of fission yeast suggests a role for the spHop2–Mnd1 proteins in the Rad51 and Dmc1-dependent meiotic recombination pathways. In order to gain biochemical insights into this process, we purified Schizosaccharomyces pombe Hop2-Mnd1 to homogeneity. spHop2 and spMnd1 interact by co-immunoprecipitation and two-hybrid analysis. Electron microscopy reveals that S. pombe Hop2–Mnd1 binds single-strand DNA ends of 3′-tailed DNA. Interestingly, spHop2-Mnd1 promotes the renaturation of complementary single-strand DNA and catalyses strand exchange reactions with short oligonucleotides. Importantly, we show that spHop2-Mnd1 stimulates spDmc1-dependent strand exchange and strand invasion. Ca2+ alleviate the requirement for the order of addition of the proteins on DNA. We also demonstrate that while spHop2-Mnd1 affects spDmc1 specifically, mHop2 or mHop2-Mnd1 stimulates both the hRad51 and hDmc1 recombinases in strand exchange assays. Thus, our results suggest a crucial role for S. pombe and mouse Hop2-Mnd1 in homologous pairing and strand exchange and reveal evolutionary divergence in their specificity for the Dmc1 and Rad51 recombinases. PMID:17426123

  20. RAD51 plays a crucial role in halting cell death program induced by ionizing radiation in bovine oocytes.

    PubMed

    Kujjo, Loro L; Ronningen, Reg; Ross, Pablo; Pereira, Ricardo J G; Rodriguez, Ramon; Beyhan, Zeki; Goissis, Marcelo D; Baumann, Thomas; Kagawa, Wataru; Camsari, Cagri; Smith, George W; Kurumizaka, Hitoshi; Yokoyama, Shigeyuki; Cibelli, Jose B; Perez, Gloria I

    2012-03-01

    Reproductive health of humans and animals exposed to daily irradiants from solar/cosmic particles remains largely understudied. We evaluated the sensitivities of bovine and mouse oocytes to bombardment by krypton-78 (1 Gy) or ultraviolet B (UV-B; 100 microjoules). Mouse oocytes responded to irradiation by undergoing massive activation of caspases, rapid loss of energy without cytochrome-c release, and subsequent necrotic death. In contrast, bovine oocytes became positive for annexin-V, exhibited cytochrome-c release, and displayed mild activation of caspases and downstream DNAses but with the absence of a complete cell death program; therefore, cytoplasmic fragmentation was never observed. However, massive cytoplasmic fragmentation and increased DNA damage were induced experimentally by both inhibiting RAD51 and increasing caspase 3 activity before irradiation. Microinjection of recombinant human RAD51 prior to irradiation markedly decreased both cytoplasmic fragmentation and DNA damage in both bovine and mouse oocytes. RAD51 response to damaged DNA occurred faster in bovine oocytes than in mouse oocytes. Therefore, we conclude that upon exposure to irradiation, bovine oocytes create a physiologically indeterminate state of partial cell death, attributed to rapid induction of DNA repair and low activation of caspases. The persistence of these damaged cells may represent an adaptive mechanism with potential implications for livestock productivity and long-term health risks associated with human activity in space.

  1. Rad51c- and Trp53-double-mutant mouse model reveals common features of homologous recombination-deficient breast cancers.

    PubMed

    Tumiati, M; Munne, P M; Edgren, H; Eldfors, S; Hemmes, A; Kuznetsov, S G

    2016-09-01

    Almost half of all hereditary breast cancers (BCs) are associated with germ-line mutations in homologous recombination (HR) genes. However, the tumor phenotypes associated with different HR genes vary, making it difficult to define the role of HR in BC predisposition. To distinguish between HR-dependent and -independent features of BCs, we generated a mouse model in which an essential HR gene, Rad51c, is knocked-out specifically in epidermal tissues. Rad51c is one of the key mediators of HR and a well-known BC predisposition gene. Here, we demonstrate that deletion of Rad51c invariably requires inactivation of the Trp53 tumor suppressor (TP53 in humans) to produce mammary carcinomas in 63% of female mice. Nonetheless, loss of Rad51c shortens the latency of Trp53-deficient mouse tumors from 11 to 6 months. Remarkably, the histopathological features of Rad51c-deficient mammary carcinomas, such as expression of hormone receptors and luminal epithelial markers, faithfully recapitulate the histopathology of human RAD51C-mutated BCs. Similar to other BC models, Rad51c/p53 double-mutant mouse mammary tumors also reveal a propensity for genomic instability, but lack the focal amplification of the Met locus or distinct mutational signatures reported for other HR genes. Using the human mammary epithelial cell line MCF10A, we show that deletion of TP53 can rescue RAD51C-deficient cells from radiation-induced cellular senescence, whereas it exacerbates their centrosome amplification and nuclear abnormalities. Altogether, our data indicate that a trend for genomic instability and inactivation of Trp53 are common features of HR-mediated BCs, whereas histopathology and somatic mutation patterns are specific for different HR genes.

  2. Association between the RAD51 135 G>C Polymorphism and Risk of Cancer: A Meta-Analysis of 19,068 Cases and 22,630 Controls

    PubMed Central

    He, Xiao-Feng; Li, An-Ping; Cai, Yong-Lin; Xu, Na; Sun, Shu-Mei; Wu, Bing-Yi

    2013-01-01

    Background RAD51 135G>C can modify promoter activity and the penetrance of BRCA1/2 mutations, which plays vital roles in the etiology of various cancer. To date, previous published data on the association between RAD51 135G>C polymorphism and cancer risk remained controversial. Recent meta-analysis only analyzed RAD51 135G>C polymorphism with breast cancer risk, but the results were also inconsistent. Methods A meta-analysis based on 39 case-control studies was performed to investigate the association between cancer susceptibility and RAD51 135G>C. Odds ratios (OR) with 95% confidence intervals (CIs) were used to assess the association in different inheritance models. Heterogeneity among studies was tested and sensitivity analysis was applied. Results Overall, no significant association was found between RAD51 135G>C polymorphism and cancer susceptibility in any genetic model. In further stratified analysis, significantly elevated breast cancer risk was observed in BRCA2 mutation carriers (recessive model: OR = 4.88, 95% CI = 1.10–21.67; additive model: OR = 4.92, 95% CI = 1.11–21.83). Conclusions This meta-analysis suggests that RAD51 variant 135C homozygote is associated with elevated breast cancer risk among BRCA2 mutation carriers. Moreover, our work also points out the importance of new studies for RAD51 135G>C association in acute myeloid leukemia, especially in Caucasians, where at least some of the covariates responsible for heterogeneity could be controlled, to obtain a more conclusive understanding about the function of the RAD51 135G>C polymorphism in cancer development. PMID:24040396

  3. Endoplasmic reticulum stress-inducing drugs sensitize glioma cells to temozolomide through downregulation of MGMT, MPG, and Rad51.

    PubMed

    Xipell, Enric; Aragón, Tomás; Martínez-Velez, Naiara; Vera, Beatriz; Idoate, Miguel Angel; Martínez-Irujo, Juan José; Garzón, Antonia García; Gonzalez-Huarriz, Marisol; Acanda, Arlet M; Jones, Chris; Lang, Frederick F; Fueyo, Juan; Gomez-Manzano, Candelaria; Alonso, Marta M

    2016-08-01

    Endoplasmic reticulum (ER) stress results from protein misfolding imbalance and has been postulated as a therapeutic strategy. ER stress activates the unfolded protein response which leads to a complex cellular response, including the upregulation of aberrant protein degradation in the ER, with the goal of resolving that stress. O(6)-methylguanine DNA methyltransferase (MGMT), N-methylpurine DNA glycosylase (MPG), and Rad51 are DNA damage repair proteins that mediate resistance to temozolomide in glioblastoma. In this work we sought to evaluate whether ER stress-inducing drugs were able to downmodulate DNA damage repair proteins and become candidates to combine with temozolomide. MTT assays were performed to evaluate the cytotoxicity of the treatments. The expression of proteins was evaluated using western blot and immunofluorescence. In vivo studies were performed using 2 orthotopic glioblastoma models in nude mice to evaluate the efficacy of the treatments. All statistical tests were 2-sided. Treatment of glioblastoma cells with ER stress-inducing drugs leads to downregulation of MGMT, MPG, and Rad51. Inhibition of ER stress through pharmacological treatment resulted in rescue of MGMT, MPG, and Rad51 protein levels. Moreover, treatment of glioblastoma cells with salinomycin, an ER stress-inducing drug, and temozolomide resulted in enhanced DNA damage and a synergistic antitumor effect in vitro. Of importance, treatment with salinomycin/temozolomide resulted in a significant antiglioma effect in 2 aggressive orthotopic intracranial brain tumor models. These findings provide a strong rationale for combining temozolomide with ER stress-inducing drugs as an alternative therapeutic strategy for glioblastoma. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Endoplasmic reticulum stress-inducing drugs sensitize glioma cells to temozolomide through downregulation of MGMT, MPG, and Rad51

    PubMed Central

    Xipell, Enric; Aragón, Tomás; Martínez-Velez, Naiara; Vera, Beatriz; Idoate, Miguel Angel; Martínez-Irujo, Juan José; Garzón, Antonia García; Gonzalez-Huarriz, Marisol; Acanda, Arlet M.; Jones, Chris; Lang, Frederick F.; Fueyo, Juan; Gomez-Manzano, Candelaria; Alonso, Marta M.

    2016-01-01

    Background Endoplasmic reticulum (ER) stress results from protein misfolding imbalance and has been postulated as a therapeutic strategy. ER stress activates the unfolded protein response which leads to a complex cellular response, including the upregulation of aberrant protein degradation in the ER, with the goal of resolving that stress. O6-methylguanine DNA methyltransferase (MGMT), N-methylpurine DNA glycosylase (MPG), and Rad51 are DNA damage repair proteins that mediate resistance to temozolomide in glioblastoma. In this work we sought to evaluate whether ER stress-inducing drugs were able to downmodulate DNA damage repair proteins and become candidates to combine with temozolomide. Methods MTT assays were performed to evaluate the cytotoxicity of the treatments. The expression of proteins was evaluated using western blot and immunofluorescence. In vivo studies were performed using 2 orthotopic glioblastoma models in nude mice to evaluate the efficacy of the treatments. All statistical tests were 2-sided. Results Treatment of glioblastoma cells with ER stress-inducing drugs leads to downregulation of MGMT, MPG, and Rad51. Inhibition of ER stress through pharmacological treatment resulted in rescue of MGMT, MPG, and Rad51 protein levels. Moreover, treatment of glioblastoma cells with salinomycin, an ER stress-inducing drug, and temozolomide resulted in enhanced DNA damage and a synergistic antitumor effect in vitro. Of importance, treatment with salinomycin/temozolomide resulted in a significant antiglioma effect in 2 aggressive orthotopic intracranial brain tumor models. Conclusions These findings provide a strong rationale for combining temozolomide with ER stress-inducing drugs as an alternative therapeutic strategy for glioblastoma. PMID:26951384

  5. Genome-wide association study identifies a novel variant in RAD51B associated with male breast cancer risk

    PubMed Central

    Orr, Nick; Lemnrau, Alina; Cooke, Rosie; Fletcher, Olivia; Tomczyk, Katarzyna; Jones, Michael; Johnson, Nichola; Lord, Christopher J; Mitsopoulos, Costas; Zvelebil, Marketa; McDade, Simon S; Buck, Gemma; Blancher, Christine; Consortium, KConFab; Trainer, Alison H; James, Paul A; Bojesen, Stig E; Bokmand, Susanne; Nevanlinna, Heli; Mattson, Johanna; Friedman, Eitan; Laitman, Yael; Palli, Domenico; Masala, Giovanna; Zanna, Ines; Ottini, Laura; Giannini, Giuseppe; Hollestelle, Antoinette; van den Ouweland, Ans M.W.; Novaković, Srdjan; Krajc, Mateja; Gago-Dominguez, Manuela; Castelao, Jose Esteban; Olsson, Håkan; Hedenfalk, Ingrid; Easton, Douglas F; Pharoah, Paul DP; Dunning, Alison M; Bishop, D Timothy; Neuhausen, Susan L; Steele, Linda; Houlston, Richard S; Garcia-Closas, Montserrat; Ashworth, Alan; Swerdlow, Anthony J

    2013-01-01

    We conducted a genome-wide association study of male breast cancer using 823 cases and 2,795 controls of European ancestry with validation in independent sample sets totalling 438 cases and 474 controls. A novel variant in RAD51B (14q24.1) was significantly associated with male breast cancer risk (P = 3.02 ×10−13, odds ratio (OR) = 1.57). TOX3 (16q12.1) was also a susceptibility locus (P = 3.87 ×10−15, OR = 1.50). PMID:23001122

  6. Enhanced dependency of KRAS-mutant colorectal cancer cells on RAD51-dependent homologous recombination repair identified from genetic interactions in Saccharomyces cerevisiae.

    PubMed

    Kalimutho, Murugan; Bain, Amanda L; Mukherjee, Bipasha; Nag, Purba; Nanayakkara, Devathri M; Harten, Sarah K; Harris, Janelle L; Subramanian, Goutham N; Sinha, Debottam; Shirasawa, Senji; Srihari, Sriganesh; Burma, Sandeep; Khanna, Kum Kum

    2017-02-07

    Activating KRAS mutations drive colorectal cancer tumorigenesis and influence response to anti-EGFR-targeted therapy. Despite recent advances in understanding Ras signaling biology and the revolution in therapies for melanoma using BRAF inhibitors, no targeted agents have been effective in KRAS-mutant cancers, mainly due to activation of compensatory pathways. Here, by leveraging the largest synthetic lethal genetic interactome in yeast, we identify that KRAS-mutated colorectal cancer cells have augmented homologous recombination repair (HRR) signaling. We found that KRAS mutation resulted in slowing and stalling of the replication fork and accumulation of DNA damage. Moreover, we found that KRAS-mutant HCT116 cells have an increase in MYC-mediated RAD51 expression with a corresponding increase in RAD51 recruitment to irradiation-induced DNA double-strand breaks (DSBs) compared to genetically complemented isogenic cells. MYC depletion using RNA interference significantly reduced IR-induced RAD51 foci formation and HRR. On the contrary, overexpression of either HA-tagged wild-type (WT) MYC or phospho-mutant S62A increased RAD51 protein levels and hence IR-induced RAD51 foci. Likewise, depletion of RAD51 selectively induced apoptosis in HCT116-mutant cells by increasing DSBs. Pharmacological inhibition targeting HRR signaling combined with PARP inhibition selectivity killed KRAS-mutant cells. Interestingly, these differences were not seen in a second isogenic pair of KRAS WT and mutant cells (DLD-1), likely due to their nondependency on the KRAS mutation for survival. Our data thus highlight a possible mechanism by which KRAS-mutant-dependent cells drive HRR in vitro by upregulating MYC-RAD51 expression. These data may offer a promising therapeutic vulnerability in colorectal cancer cells harboring otherwise nondruggable KRAS mutations, which warrants further investigation in vivo.

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

    PubMed Central

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

    2015-01-01

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

  8. BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine.

    PubMed

    Jones, Rebecca M; Kotsantis, Panagiotis; Stewart, Grant S; Groth, Petra; Petermann, Eva

    2014-10-01

    Replication inhibitors cause replication fork stalling and double-strand breaks (DSB) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitize cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here, we investigate why, paradoxically, mutations in HR genes protect cells from killing by gemcitabine. Using DNA replication and DNA damage assays in mammalian cells, we show that even short gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression, and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at gemcitabine-stalled forks are converted into DSBs and thus contribute to gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumors.

  9. Somatic SNPs of the BRCA2 gene at the fragments encoding RAD51 binding sites of canine mammary tumors.

    PubMed

    Ozmen, O; Kul, S; Risvanli, A; Ozalp, G; Sabuncu, A; Kul, O

    2017-01-30

    Mammary tumors are the most common tumor type both in women and in female dogs. In women, heritable breast cancers have been linked mutations in the breast cancer susceptibility gene BRCA2 and it contains eight BRC repeats in exon 11 that bind to RAD51. In this study, we investigated the sequence variations of BRC1-BRC8 and C-terminus of canine BRCA2 gene. From a total of 64 canine patients with mammary tumors, 31 mammary tumors with benign and malign carcinomas and the 3 normal mammary glands were used for the study. In this study, 19 SNPs of exon 11 of BRCA2 in canine mammary tumors were detected for the first time. The c.2383A>C (T1425P) SNP was found to be the most probable disease-associated nsSNP. Our findings suggest that T1425P variation in BRC3 to be the most probable disease-associated nsSNP and may affect RAD51 binding strength.

  10. Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs

    PubMed Central

    Lopes, Anne; Amarir-Bouhram, Jihane; Faure, Guilhem; Petit, Marie-Agnès; Guerois, Raphaël

    2010-01-01

    Homologous recombination is a key in contributing to bacteriophages genome repair, circularization and replication. No less than six kinds of recombinase genes have been reported so far in bacteriophage genomes, two (UvsX and Gp2.5) from virulent, and four (Sak, Redβ, Erf and Sak4) from temperate phages. Using profile–profile comparisons, structure-based modelling and gene-context analyses, we provide new views on the global landscape of recombinases in 465 bacteriophages. We show that Sak, Redβ and Erf belong to a common large superfamily adopting a shortcut Rad52-like fold. Remote homologs of Sak4 are predicted to adopt a shortcut Rad51/RecA fold and are discovered widespread among phage genomes. Unexpectedly, within temperate phages, gene-context analyses also pinpointed the presence of distant Gp2.5 homologs, believed to be restricted to virulent phages. All in all, three major superfamilies of phage recombinases emerged either related to Rad52-like, Rad51-like or Gp2.5-like proteins. For two newly detected recombinases belonging to the Sak4 and Gp2.5 families, we provide experimental evidence of their recombination activity in vivo. Temperate versus virulent lifestyle together with the importance of genome mosaicism is discussed in the light of these novel recombinases. Screening for these recombinases in genomes can be performed at http://biodev.extra.cea.fr/virfam. PMID:20194117

  11. Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments.

    PubMed

    Kolinjivadi, Arun Mouli; Sannino, Vincenzo; De Antoni, Anna; Zadorozhny, Karina; Kilkenny, Mairi; Técher, Hervé; Baldi, Giorgio; Shen, Rong; Ciccia, Alberto; Pellegrini, Luca; Krejci, Lumir; Costanzo, Vincenzo

    2017-09-07

    Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51(T131P) mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Fanconi anemia complementation group D2 (FANCD2) functions independently of BRCA2- and RAD51-associated homologous recombination in response to DNA damage.

    PubMed

    Ohashi, Akihiro; Zdzienicka, Malgorzata Z; Chen, Junjie; Couch, Fergus J

    2005-04-15

    The BRCA2 breast cancer tumor suppressor is involved in the repair of double strand breaks and broken replication forks by homologous recombination through its interaction with DNA repair protein Rad51. Cells defective in BRCA2.FANCD1 are extremely sensitive to mitomycin C (MMC) similarly to cells deficient in any of the Fanconi anemia (FA) complementation group proteins (FANC). These observations suggest that the FA pathway and the BRCA2 and Rad51 repair pathway may be linked, although a functional connection between these pathways in DNA damage signaling remains to be determined. Here, we systematically investigated the interaction between these pathways. We show that in response to DNA damage, BRCA2-dependent Rad51 nuclear focus formation was normal in the absence of FANCD2 and that FANCD2 nuclear focus formation and mono-ubiquitination appeared normal in BRCA2-deficient cells. We report that the absence of BRCA2 substantially reduced homologous recombination repair of DNA breaks, whereas the absence of FANCD2 had little effect. Furthermore, we established that depletion of BRCA2 or Rad51 had a greater effect on cell survival in response to MMC than depletion of FANCD2 and that depletion of BRCA2 in FANCD2 mutant cells further sensitized these cells to MMC. Our results suggest that FANCD2 mediates double strand DNA break repair independently of Rad51-associated homologous recombination.

  13. 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

  14. CRISPR Technology Reveals RAD(51)-ical Mechanisms of Repair in Roundworms: An Educational Primer for Use with "Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans".

    PubMed

    Turcotte, Carolyn A; Andrews, Nicolas P; Sloat, Solomon A; Checchi, Paula M

    2016-11-01

    The mechanisms cells use to maintain genetic fidelity via DNA repair and the accuracy of these processes have garnered interest from scientists engaged in basic research to clinicians seeking improved treatment for cancer patients. Despite the continued advances, many details of DNA repair are still incompletely understood. In addition, the inherent complexity of DNA repair processes, even at the most fundamental level, makes it a challenging topic. This primer is meant to assist both educators and students in using a recent paper, "Promotion of homologous recombination by SWS-1 in complex with RAD-51 paralogs in Caenorhabditis elegans," to understand mechanisms of DNA repair. The goals of this primer are to highlight and clarify several key techniques utilized, with special emphasis on the clustered, regularly interspaced, short palindromic repeats technique and the ways in which it has revolutionized genetics research, as well as to provide questions for deeper in-class discussion.

  15. RAD51 135G→C Modifies Breast Cancer Risk among BRCA2 Mutation Carriers: Results from a Combined Analysis of 19 Studies

    PubMed Central

    Antoniou, Antonis C. ; Sinilnikova, Olga M. ; Simard, Jacques ; Léoné, Mélanie ; Dumont, Martine ; Neuhausen, Susan L. ; Struewing, Jeffery P. ; Stoppa-Lyonnet, Dominique ; Barjhoux, Laure ; Hughes, David J. ; Coupier, Isabelle ; Belotti, Muriel ; Lasset, Christine ; Bonadona, Valérie ; Bignon, Yves-Jean ; Rebbeck, Timothy R. ; Wagner, Theresa ; Lynch, Henry T. ; Domchek, Susan M. ; Nathanson, Katherine L. ; Garber, Judy E. ; Weitzel, Jeffrey ; Narod, Steven A. ; Tomlinson, Gail ; Olopade, Olufunmilayo I. ; Godwin, Andrew ; Isaacs, Claudine ; Jakubowska, Anna ; Lubinski, Jan ; Gronwald, Jacek ; Górski, Bohdan ; Byrski, Tomasz ; Huzarski, Tomasz ; Peock, Susan ; Cook, Margaret ; Baynes, Caroline ; Murray, Alexandra ; Rogers, Mark ; Daly, Peter A. ; Dorkins, Huw ; Schmutzler, Rita K. ; Versmold, Beatrix ; Engel, Christoph ; Meindl, Alfons ; Arnold, Norbert ; Niederacher, Dieter ; Deissler, Helmut ; Spurdle, Amanda B. ; Chen, Xiaoqing ; Waddell, Nicola ; Cloonan, Nicole ; Kirchhoff, Tomas ; Offit, Kenneth ; Friedman, Eitan ; Kaufmann, Bella ; Laitman, Yael ; Galore, Gilli ; Rennert, Gad ; Lejbkowicz, Flavio ; Raskin, Leon ; Andrulis, Irene L. ; Ilyushik, Eduard ; Ozcelik, Hilmi ; Devilee, Peter ; Vreeswijk, Maaike P. G. ; Greene, Mark H. ; Prindiville, Sheila A. ; Osorio, Ana ; Benítez, Javier ; Zikan, Michal ; Szabo, Csilla I. ; Kilpivaara, Outi ; Nevanlinna, Heli ; Hamann, Ute ; Durocher, Francine ; Arason, Adalgeir ; Couch, Fergus J. ; Easton, Douglas F. ; Chenevix-Trench, Georgia 

    2007-01-01

    RAD51 is an important component of double-stranded DNA–repair mechanisms that interacts with both BRCA1 and BRCA2. A single-nucleotide polymorphism (SNP) in the 5′ untranslated region (UTR) of RAD51, 135G→C, has been suggested as a possible modifier of breast cancer risk in BRCA1 and BRCA2 mutation carriers. We pooled genotype data for 8,512 female mutation carriers from 19 studies for the RAD51 135G→C SNP. We found evidence of an increased breast cancer risk in CC homozygotes (hazard ratio [HR] 1.92 [95% confidence interval {CI} 1.25–2.94) but not in heterozygotes (HR 0.95 [95% CI 0.83–1.07]; P=.002, by heterogeneity test with 2 degrees of freedom [df]). When BRCA1 and BRCA2 mutation carriers were analyzed separately, the increased risk was statistically significant only among BRCA2 mutation carriers, in whom we observed HRs of 1.17 (95% CI 0.91–1.51) among heterozygotes and 3.18 (95% CI 1.39–7.27) among rare homozygotes (P=.0007, by heterogeneity test with 2 df). In addition, we determined that the 135G→C variant affects RAD51 splicing within the 5′ UTR. Thus, 135G→C may modify the risk of breast cancer in BRCA2 mutation carriers by altering the expression of RAD51. RAD51 is the first gene to be reliably identified as a modifier of risk among BRCA1/2 mutation carriers. PMID:17999359

  16. ER stress suppresses DNA double-strand break repair and sensitizes tumor cells to ionizing radiation by stimulating proteasomal degradation of Rad51.

    PubMed

    Yamamori, Tohru; Meike, Shunsuke; Nagane, Masaki; Yasui, Hironobu; Inanami, Osamu

    2013-10-11

    In this study, we provide evidence that endoplasmic reticulum (ER) stress suppresses DNA double-strand break (DSB) repair and increases radiosensitivity of tumor cells by altering Rad51 levels. We show that the ER stress inducer tunicamycin stimulates selective degradation of Rad51 via the 26S proteasome, impairing DSB repair and enhancing radiosensitivity in human lung cancer A549 cells. We also found that glucose deprivation, which is a physiological inducer of ER stress, triggered similar events. These findings suggest that ER stress caused by the intratumoral environment influences tumor radiosensitivity, and that it has potential as a novel target to improve cancer radiotherapy.

  17. Xrcc3 induces cisplatin resistance by stimulation of Rad51-related recombinational repair, S-phase checkpoint activation, and reduced apoptosis.

    PubMed

    Xu, Zhi-Yuan; Loignon, Martin; Han, Fei-Yu; Panasci, Lawrence; Aloyz, Raquel

    2005-08-01

    Eukaryotic cells respond to DNA damage by activation of DNA repair, cell cycle arrest, and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signaling other cellular responses. The Rad51-guided homologous recombination repair system plays an important role in the recognition and repair of DNA interstrand crosslinks (ICLs), and cells deficient in this repair pathway become hypersensitive to ICL-inducing agents such as cisplatin and melphalan. We investigated the possible role of the Rad51-paralog protein Xrcc3 in drug resistance. Xrcc3 overexpression in MCF-7 cells resulted in 1) a 2- to 6-fold resistance to cisplatin/melphalan, 2) a 2-fold increase in drug-induced Rad51 foci, 3) an increased cisplatin-induced S-phase arrest, 4) decreased cisplatin-induced apoptosis, and 5) increased cisplatin-induced DNA synthesis arrest. Interestingly, Xrcc3 overexpression did not alter the doubling time or cell cycle progression in the absence of DNA damage. Furthermore, Xrcc3 overexpression is associated with increased Rad51C protein levels consistent with the known interaction of these two proteins. Our results demonstrate that Xrcc3 is an important factor in DNA cross-linking drug resistance in human tumor cells and suggest that the response of the homologous recombinational repair machinery and cell cycle checkpoints to DNA cross-linking agents is intertwined.

  18. Panobinostat enhances cytarabine and daunorubicin sensitivities in AML cells through suppressing the expression of BRCA1, CHK1, and Rad51.

    PubMed

    Xie, Chengzhi; Drenberg, Christina; Edwards, Holly; Caldwell, J Timothy; Chen, Wei; Inaba, Hiroto; Xu, Xuelian; Buck, Steven A; Taub, Jeffrey W; Baker, Sharyn D; Ge, Yubin

    2013-01-01

    Acute myeloid leukemia (AML) remains a challenging disease to treat and urgently requires new therapies to improve its treatment outcome. In this study, we investigated the molecular mechanisms underlying the cooperative antileukemic activities of panobinostat and cytarabine or daunorubicin (DNR) in AML cell lines and diagnostic blast samples in vitro and in vivo. Panobinostat suppressed expression of BRCA1, CHK1, and RAD51 in AML cells in a dose-dependent manner. Further, panobinostat significantly increased cytarabine- or DNR-induced DNA double-strand breaks and apoptosis, and abrogated S and/or G2/M cell cycle checkpoints. Analogous results were obtained by shRNA knockdown of BRCA1, CHK1, or RAD51. Cotreatment of NOD-SCID-IL2Rγ(null) mice bearing AML xenografts with panobinostat and cytarabine significantly increased survival compared to either cytarabine or panobinostat treatment alone. Additional studies revealed that panobinostat suppressed the expression of BRCA1, CHK1, and RAD51 through downregulation of E2F1 transcription factor. Our results establish a novel mechanism underlying the cooperative antileukemic activities of these drug combinations in which panobinostat suppresses expression of BRCA1, CHK1, and RAD51 to enhance cytarabine and daunorubicin sensitivities in AML cells.

  19. Association of RAD 51 135 G/C, 172 G/T and XRCC3 Thr241Met gene polymorphisms with increased risk of head and neck cancer.

    PubMed

    Kayani, Mahmood Akhtar; Khan, Sumeera; Baig, Ruqia Mehmood; Mahjabeen, Ishrat

    2014-01-01

    Homologous recombination repair (HRR) plays an important role in protection against carcinogenic factors. Genes regulating the HRR mechanisms may impair their functions and consequently result in increased cancer susceptibility. RAD 51 and XRCC3 are key regulators of the HRR pathway and genetic variability in these may contribute to the appearance and progression of various cancers including head and neck cancer (HNC). The aim of the present study was to compare the distribution of genotypes of RAD51 (135G/C, 172 G/T) and XRCC3 (Thr241Met) polymorphisms between HNC patients and controls. Each polymorphism was genotyped using the polymerase chain reaction-restriction fragment length polymerase (PCR-RFLP) technique in 200 pathologically confirmed HNC patients along with 150 blood samples from normal, disease free healthy individuals. We observed that homozygous variant CC genotype of RAD51 135G/C was associated with a 2.5 fold increased HNC risk (OR=2.5; 95%CI=0.69-9.53; p<0.02), while second polymorphism of RAD 51 172 G/T, heterozygous variant GT genotype was associated with a 1.68 fold (OR=1.68; 95%CI=1.08-2.61; p<0.02) elevation when compared with controls. In the case of the Thr241Met polymorphism of XRCC3, we observed a 16 fold (OR=16; 95% CI= 3.78-69.67; p<0.0002) increased HNC risk in patients compared to controls. These results further suggested that RAD51 (135G/C, 172 G/T) and XRCC3 (Thr241Met) polymorphisms may be effective biomarkers for genetic susceptibility to HNC. Larger studies are needed to confirm our findings and identify the underlying mechanisms.

  20. CHK1 and RAD51 activation after DNA damage is regulated via urokinase receptor/TLR4 signaling

    PubMed Central

    Narayanaswamy, Pavan B; Tkachuk, Sergey; Haller, Hermann; Dumler, Inna; Kiyan, Yulia

    2016-01-01

    Mechanisms of DNA damage and repair signaling are not completely understood that hinder the efficiency of cancer therapy. Urokinase-type plasminogen activator receptor (PLAUR) is highly expressed in most solid cancers and serves as a marker of poor prognosis. We show that PLAUR actively promotes DNA repair in cancer cells. On the contrary, downregulation of PLAUR expression results in delayed DNA repair. We found PLAUR to be essential for activation of Checkpoint kinase 1 (CHK1); maintenance of cell cycle arrest after DNA damage in a TP53-dependent manner; expression, nuclear import and recruitment to DNA-damage foci of RAD51 recombinase, the principal protein involved in the homologous recombination repair pathway. Underlying mechanism implies auto-/paracrine signaling of PLAUR/TLR4 receptor complex leading to activation of CHK1 and DNA repair. The signaling is induced by a danger molecule released by DNA-damaged cells and mediates, at least partially, activation of DNA-damage response. This study describes a new mechanism of DNA repair activation initiated by auto-/paracrine signaling of membrane receptors PLAUR/TLR4. It adds to the understanding of role of PLAUR in cancer and provides a rationale for therapeutic targeting of PLAUR/TLR4 interaction in TP53-positive cancers. PMID:27685627

  1. FANCJ uses its motor ATPase to destabilize protein-DNA complexes, unwind triplexes, and inhibit RAD51 strand exchange.

    PubMed

    Sommers, Joshua A; Rawtani, Nina; Gupta, Rigu; Bugreev, Dmitry V; Mazin, Alexander V; Cantor, Sharon B; Brosh, Robert M

    2009-03-20

    Mutations in the FANCJ helicase predispose individuals to breast cancer and are genetically linked to the Fanconi anemia (FA) complementation group J. FA is a chromosomal instability disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk. FANCJ has been proposed to function downstream of FANCD2 monoubiquitination, a critical event in the FA pathway. Evidence supports a role for FANCJ in a homologous recombination pathway of double strand break repair. In an effort to understand the molecular functions of FANCJ, we have investigated the ability of purified FANCJ recombinant protein to use its motor ATPase function for activities in addition to unwinding of conventional duplex DNA substrates. These efforts have led to the discovery that FANCJ ATP hydrolysis can be used to destabilize protein-DNA complexes and unwind triple helix alternate DNA structures. These novel catalytic functions of FANCJ may be important for its role in cellular DNA repair, recombination, or resolving DNA structural obstacles to replication. Consistent with this, we show that FANCJ can inhibit RAD51 strand exchange, an activity that is likely to be important for its role in controlling DNA repair through homologous recombination.

  2. Reduced FANCD2 influences spontaneous SCE and RAD51 foci formation in uveal melanoma and Fanconi anaemia.

    PubMed

    Gravells, P; Hoh, L; Solovieva, S; Patil, A; Dudziec, E; Rennie, I G; Sisley, K; Bryant, H E

    2013-11-14

    Uveal melanoma (UM) is unique among cancers in displaying reduced endogenous levels of sister chromatid exchange (SCE). Here we demonstrate that FANCD2 expression is reduced in UM and that ectopic expression of FANCD2 increased SCE. Similarly, FANCD2-deficient fibroblasts (PD20) derived from Fanconi anaemia patients displayed reduced spontaneous SCE formation relative to their FANCD2-complemented counterparts, suggesting that this observation is not specific to UM. In addition, spontaneous RAD51 foci were reduced in UM and PD20 cells compared with FANCD2-proficient cells. This is consistent with a model where spontaneous SCEs are the end product of endogenous recombination events and implicates FANCD2 in the promotion of recombination-mediated repair of endogenous DNA damage and in SCE formation during normal DNA replication. In both UM and PD20 cells, low SCE was reversed by inhibiting DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Finally, we demonstrate that both PD20 and UM are sensitive to acetaldehyde, supporting a role for FANCD2 in repair of lesions induced by such endogenous metabolites. Together, these data suggest FANCD2 may promote spontaneous SCE by influencing which double-strand break repair pathway predominates during normal S-phase progression.

  3. Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination.

    PubMed

    Prakash, Rohit; Satory, Dominik; Dray, Eloïse; Papusha, Almas; Scheller, Jürgen; Kramer, Wilfried; Krejci, Lumir; Klein, Hannah; Haber, James E; Sung, Patrick; Ira, Grzegorz

    2009-01-01

    Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks.

  4. Reduced FANCD2 influences spontaneous SCE and RAD51 foci formation in uveal melanoma and Fanconi anaemia

    PubMed Central

    Gravells, P; Hoh, L; Solovieva, S; Patil, A; Dudziec, E; Rennie, I G; Sisley, K; Bryant, H E

    2013-01-01

    Uveal melanoma (UM) is unique among cancers in displaying reduced endogenous levels of sister chromatid exchange (SCE). Here we demonstrate that FANCD2 expression is reduced in UM and that ectopic expression of FANCD2 increased SCE. Similarly, FANCD2-deficient fibroblasts (PD20) derived from Fanconi anaemia patients displayed reduced spontaneous SCE formation relative to their FANCD2-complemented counterparts, suggesting that this observation is not specific to UM. In addition, spontaneous RAD51 foci were reduced in UM and PD20 cells compared with FANCD2-proficient cells. This is consistent with a model where spontaneous SCEs are the end product of endogenous recombination events and implicates FANCD2 in the promotion of recombination-mediated repair of endogenous DNA damage and in SCE formation during normal DNA replication. In both UM and PD20 cells, low SCE was reversed by inhibiting DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Finally, we demonstrate that both PD20 and UM are sensitive to acetaldehyde, supporting a role for FANCD2 in repair of lesions induced by such endogenous metabolites. Together, these data suggest FANCD2 may promote spontaneous SCE by influencing which double-strand break repair pathway predominates during normal S-phase progression. PMID:23318456

  5. Dose-Response Curves of the FDXR and RAD51 Genes with 6 and 18 MV Beam Energies in Human Peripheral Blood Lymphocytes.

    PubMed

    Saberi, Alihossein; Khodamoradi, Ehsan; Tahmasebi Birgani, Mohammad Javad; Makvandi, Manoochehr; Noori, Bijan

    2016-11-01

    Rapid dose assessment using biological dosimetry methods is essential to increase the chance of survival of exposed individuals in radiation accidents. We compared the expression levels of the FDXR and RAD51 genes at 6 and 18 MV beam energies in human peripheral blood lymphocytes. The results of our study can be used to analyze radiation energy in biological dosimetry. For this in vitro experimental study, from 36 students in the medical physics and virology departments, seven voluntary, healthy, non-smoking male blood donors of Khuzestan ethnicity with no history of exposure to ionization radiation were selected using simple randomized sampling. Sixty-three peripheral blood samples were collected from the seven healthy donors. Human peripheral blood was then exposed to doses of 0, 0.2, 0.5, 2, and 4 Gy with 6 and 18 MV beam energies in a Linac Varian 2100C/D (Varian, USA) at Golestan hospital in Ahvaz, Iran. After RNA extraction and cDNA synthesis, the expression levels of FDXR and RAD51 were determined 24 hours post-irradiation using the gel-purified reverse transcription polymerase chain reaction (RT-PCR) technique and TaqMan strategy (by real-time PCR). The expression level of FDXR gene was significantly increased at doses of 2 Gy and 4 Gy in the 6 - 18 MV energy range (P < 0.001 and P < 0.008, respectively). The medians with interquartile ranges (IQRs) of the copy numbers of the FDXR gene at 2 Gy and 4 Gy doses under 6 and 18 MV beam energies were 2393.59 (1798.21, 2575.37) and 2983.00 (2199.48, 3643.82) and 3779.12 (3051.40, 5120.74) and 5051.26 (4704.83, 5859.17), respectively. However, RAD51 gene expression levels only showed a significant difference between samples at a dose of 2 Gy with 6 and 18 MV beam energies, respectively (P < 0.040). The medians with IQRs of the copy numbers of the RAD51 gene were 2092.77 (1535.78, 2705.61) and 3412.57 (2979.72, 4530.61) at beam energies of 6 and 18 MV, respectively. The data suggest that the expression analysis of

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

    PubMed

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

    2015-08-13

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

  7. Dose–Response Curves of the FDXR and RAD51 Genes with 6 and 18 MV Beam Energies in Human Peripheral Blood Lymphocytes

    PubMed Central

    Saberi, Alihossein; Khodamoradi, Ehsan; Tahmasebi Birgani, Mohammad Javad; Makvandi, Manoochehr; Noori, Bijan

    2016-01-01

    Background Rapid dose assessment using biological dosimetry methods is essential to increase the chance of survival of exposed individuals in radiation accidents. Objectives We compared the expression levels of the FDXR and RAD51 genes at 6 and 18 MV beam energies in human peripheral blood lymphocytes. The results of our study can be used to analyze radiation energy in biological dosimetry. Methods For this in vitro experimental study, from 36 students in the medical physics and virology departments, seven voluntary, healthy, non-smoking male blood donors of Khuzestan ethnicity with no history of exposure to ionization radiation were selected using simple randomized sampling. Sixty-three peripheral blood samples were collected from the seven healthy donors. Human peripheral blood was then exposed to doses of 0, 0.2, 0.5, 2, and 4 Gy with 6 and 18 MV beam energies in a Linac Varian 2100C/D (Varian, USA) at Golestan hospital in Ahvaz, Iran. After RNA extraction and cDNA synthesis, the expression levels of FDXR and RAD51 were determined 24 hours post-irradiation using the gel-purified reverse transcription polymerase chain reaction (RT-PCR) technique and TaqMan strategy (by real-time PCR). Results The expression level of FDXR gene was significantly increased at doses of 2 Gy and 4 Gy in the 6 - 18 MV energy range (P < 0.001 and P < 0.008, respectively). The medians with interquartile ranges (IQRs) of the copy numbers of the FDXR gene at 2 Gy and 4 Gy doses under 6 and 18 MV beam energies were 2393.59 (1798.21, 2575.37) and 2983.00 (2199.48, 3643.82) and 3779.12 (3051.40, 5120.74) and 5051.26 (4704.83, 5859.17), respectively. However, RAD51 gene expression levels only showed a significant difference between samples at a dose of 2 Gy with 6 and 18 MV beam energies, respectively (P < 0.040). The medians with IQRs of the copy numbers of the RAD51 gene were 2092.77 (1535.78, 2705.61) and 3412.57 (2979.72, 4530.61) at beam energies of 6 and 18 MV, respectively. Conclusions

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

    SciTech Connect

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

    2011-09-15

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

  9. DNA repair and cytotoxic drugs: the potential role of RAD51 in clinical outcome of non-small-cell lung cancer patients.

    PubMed

    Nogueira, Augusto; Assis, Joana; Catarino, Raquel; Medeiros, Rui

    2013-04-01

    Many of the cytotoxic drugs used in the treatment of non-small-cell lung carcinoma patients can interfere with DNA activity and the definition of an individual DNA repair profile could be a key strategy to achieve better response to chemotherapeutic treatment. Although DNA repair mechanisms are important factors in the prevention of carcinogenesis, these molecular pathways are also involved in therapy response. RAD51 is a crucial element in DNA repair by homologous recombination and has been shown to interfere with the prognosis of patients treated with chemoradiotherapy. There is increasing evidence that genetic polymorphisms in repair enzymes can influence DNA repair capacity and, consequently, affect chemotherapy efficacy. We conducted this review to show the possible influence of the RAD51 genetic variants in damage repair capacity and treatment response in non-small-cell lung carcinoma patients.

  10. Atmospheric-pressure plasma jet induces DNA double-strand breaks that require a Rad51-mediated homologous recombination for repair in Saccharomyces cerevisiae.

    PubMed

    Lee, Yoonna; Kim, Kangil; Kang, Kyu-Tae; Lee, Jong-Soo; Yang, Sang Sik; Chung, Woo-Hyun

    2014-10-15

    Non-thermal plasma generated under atmospheric pressure produces a mixture of chemically reactive molecules and has been developed for a number of biomedical applications. Recently, plasma jet has been proposed as novel cancer therapies based on the observation that free radicals generated by plasma jet induce mitochondria-mediated apoptotic cell death. We show here that air plasma jet induces DNA double-strand breaks (DSBs) in yeast chromosomes leading to genomic instability and loss of viability, which are alleviated by Rad51, the yeast homolog of Escherichiacoli RecA recombinase, through DNA damage repair by a homologous recombination (HR) process. Hypersensitivity of rad51 mutant to air plasma was not restored by antioxidant treatment unlike sod1 mutant that was highly sensitive to reactive oxygen species (ROS) challenge, suggesting that plasma jet induces DSB-mediated cell death independent of ROS generation. These results may provide a new insight into the mechanism of air plasma jet-induced cell death.

  11. Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1.

    PubMed

    Chang, Hao-Yen; Liao, Chia-Yu; Su, Guan-Chin; Lin, Sheng-Wei; Wang, Hong-Wei; Chi, Peter

    2015-08-07

    DMC1 and RAD51 are conserved recombinases that catalyze homologous recombination. DMC1 and RAD51 share similar properties in DNA binding, DNA-stimulated ATP hydrolysis, and catalysis of homologous DNA strand exchange. A large body of evidence indicates that attenuation of ATP hydrolysis leads to stabilization of the RAD51-ssDNA presynaptic filament and enhancement of DNA strand exchange. However, the functional relationship of ATPase activity, presynaptic filament stability, and DMC1-mediated homologous DNA strand exchange has remained largely unexplored. To address this important question, we have constructed several mutant variants of human DMC1 and characterized them biochemically to gain mechanistic insights. Two mutations, K132R and D223N, that change key residues in the Walker A and B nucleotide-binding motifs ablate ATP binding and render DMC1 inactive. On the other hand, the nucleotide-binding cap D317K mutant binds ATP normally but shows significantly attenuated ATPase activity and, accordingly, forms a highly stable presynaptic filament. Surprisingly, unlike RAD51, presynaptic filament stabilization achieved via ATP hydrolysis attenuation does not lead to any enhancement of DMC1-catalyzed homologous DNA pairing and strand exchange. This conclusion is further supported by examining wild-type DMC1 with non-hydrolyzable ATP analogues. Thus, our results reveal an important mechanistic difference between RAD51 and DMC1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway.

    PubMed

    Hirsch, Matthew L; Li, Chengwen; Bellon, Isabella; Yin, Chaoying; Chavala, Sai; Pryadkina, Marina; Richard, Isabelle; Samulski, Richard Jude

    2013-12-01

    A drawback of gene therapy using adeno-associated virus (AAV) is the DNA packaging restriction of the viral capsid (<4.7 kb). Recent observations demonstrate oversized AAV genome transduction through an unknown mechanism. Herein, AAV production using an oversized reporter (6.2 kb) resulted in chloroform and DNase-resistant particles harboring distinct "fragment" AAV (fAAV) genomes (5.0, 2.4, and 1.6 kb). Fractionation experiments determined that only the larger "fragments" mediated transduction in vitro, and relatively efficient transduction was also demonstrated in the muscle, the eye, and the liver. In contrast with concatemerization-dependent large-gene delivery by split AAV, fAAV transduction is independent of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in vitro and in vivo while disproportionately reliant on the DNA strand-annealing protein Rad51C. Importantly, fAAV's unique dependence on DNA repair proteins, compared with intact AAV, strongly suggests that the majority of oversized AAV transduction is mediated by fragmented genomes. Although fAAV transduction is less efficient than intact AAV, it is enhanced fourfold in muscle and sevenfold in the retina compared with split AAV transduction. Furthermore, fAAV carrying codon-optimized therapeutic dysferlin cDNA in a 7.5 kb expression cassette restored dysferlin levels in a dystrophic model. Collectively, oversized AAV genome transduction requires unique DNA repair pathways and offers an alternative, more efficient strategy for large-gene therapy.

  13. Assessment of HTLV-1 proviral load, LAT, BIM, c-FOS and RAD51 gene expression in adult T cell leukemia/lymphoma.

    PubMed

    Ramezani, Samaneh; Shirdel, Abbas; Rafatpanah, Houshang; Akbarin, Mohammad Mehdi; Tarokhian, Hanieh; Rahimi, Hossein; Bari, Alireza; Jahantigh, Hamid Reza; Rezaee, Seyed Abdolrahim

    2017-08-01

    Adult T cell leukemia/lymphoma (ATLL) is a life-threatening malignancy of HTLV-1 infected Th lymphocytes. In the present study host-virus interactions were investigated by assessment of HTLV-1 proviral load (PVL) and host gene expression. A cross-sectional study was carried out on 18 ATLL, 10 HAM/TSP patients and 18 HTLV-1 asymptomatic carriers (ACs). DNA and mRNA of the peripheral blood mononuclear cells were extracted for PVL and LAT, BIM, c-FOS and RAD51 gene expression measurement using qRT-PCR. The mean PVL in ATLL patients was 11,430 ± 3770 copies/10(4) which was statistically higher than ACs, 530 ± 119 copies/10(4), (p < 0.001). The expression of BIM, and c-FOS in ATLL patients were higher than HTLV-1 ACs; however, there were no statistically significant differences. The expression of RAD51 as an essential player on DNA repair showed around 160 times increase in ATLL group (166 ± 95) compared to ACs (1.04 ± 0.34) which is statistically significant (p < 0.001). Interestingly, there was a positive correlation between RAD51 expression and HTLV-PVL. The expression of LAT as a central adaptor in TCR signaling interestingly was around 36 times higher in ATLL group than ACs (ATLL; 41.33 ± 19.91 vs. ACs; 1.15 ± 0.22, p < 0.001). This finding showed that TCR signaling pathway mainly provides the growth factors for transformed cells. Furthermore, the overexpression of RAD51 which has been induced in HTLV-1 infected cells as a consequence of virus replication is not able to overcome the DNA damage toward cell transformation.

  14. The use of Olaparib (AZD2281) potentiates SN-38 cytotoxicity in colon cancer cells by indirect inhibition of Rad51-mediated repair of DNA double-strand breaks.

    PubMed

    Tahara, Makiko; Inoue, Takeshi; Sato, Futoshi; Miyakura, Yasuyuki; Horie, Hisanaga; Yasuda, Yoshikazu; Fujii, Hirofumi; Kotake, Kenjiro; Sugano, Kokichi

    2014-05-01

    Potent application of topoisomerase I inhibitor plus PARP inhibitor has been suggested to be an effective strategy for cancer therapy. Reportedly, mismatch repair (MMR)-deficient colon cancer cells are sensitive to topoisomerase I inhibitor, presumably due to microsatellite instability (MSI) of the MRE11 locus. We examined the synergy of SN-38, an active metabolite of irinotecan, in combination with the PARP inhibitor olaparib in colon cancer cells showing different MMR status, such as MSI or microsatellite stable (MSS) phenotype. Treatment with SN-38 and olaparib in combination almost halved the IC50 of SN-38 for a broad spectrum of colon cancer cells independent of the MMR status. Furthermore, olaparib potentiated S-phase-specific double-strand DNA breaks (DSB) induced by SN-38, which is followed by Rad51 recruitment. siRNA-mediated knockdown of Rad51, but not Mre11 or Rad50, increased the sensitivity to olaparib and/or SN-38 treatment in colon cancer cells. In vivo study using mouse xenograft demonstrated that olaparib was effective to potentiate the antitumor effect of irinotecan. In conclusion, olaparib shows a synergistic effect in colon cancer cells in combination with SN-38 or irinotecan, potentiated by the Rad51-mediated HR pathway, irrespective of the Mre11-mediated failure of the MRN complex. These results may contribute to future clinical trials using PARP inhibitor plus topoisomerase I inhibitor in combination. Furthermore, the synergistic effect comprising topoisomerase I-mediated DNA breakage-reunion reaction, PARP and Rad51-mediated HR pathway suggests the triple synthetic lethal pathways contribute to this event and are applicable as a potential target for future chemotherapy.

  15. Potential Role of Single Nucleotide Polymorphisms of XRCC1, XRCC3, and RAD51 in Predicting Acute Toxicity in Rectal Cancer Patients Treated With Preoperative Radiochemotherapy.

    PubMed

    Osti, Mattia F; Nicosia, Luca; Agolli, Linda; Gentile, Giovanna; Falco, Teresa; Bracci, Stefano; Di Nardo, Francesco; Minniti, Giuseppe; De Sanctis, Vitaliana; Valeriani, Maurizio; Maglio, Marianna; Borro, Marina; Simmaco, Maurizio; Enrici, Riccardo M

    2015-03-24

    To investigate the association between polymorphisms of DNA repair genes and xenobiotic with acute adverse effects in locally advanced rectal cancer patients treated with neoadjuvant radiochemotherapy. Sixty-seven patients were analyzed for the current study. Genotypes in DNA repair genes XRCC1 (G28152A), XRCC3 (A4541G), XRCC3 (C18067T), RAD51 (G315C), and GSTP1 (A313G) were determined by pyrosequencing technology. The observed grade ≥3 acute toxicity rates were 23.8%. Chemotherapy and radiotherapy were interrupted for 46 and 14 days, respectively, due to critical complications. Four patients were hospitalized, 6 patients had been admitted to the ER, and 5 patients received invasive procedures (2 bladder catheters, 2 blood transfusions, and 1 growth factor therapy).RAD51 correlated with acute severe gastrointestinal toxicity in heterozygosity (Aa) and homozygosity (AA) (P=0.036). Grade ≥3 abdominal/pelvis pain toxicity was higher in the Aa group (P=0.017) and in the Aa+AA group (P=0.027) compared with homozygous (aa) patients. Acute skin toxicity of any grade occurred in 55.6% of the mutated patients versus 22.8% in the wild-type group (P=0.04) for RAD51. XRCC1 correlated with skin toxicity of any grade in the Aa+AA group (P=0.03) and in the Aa group alone (P=0.044). Grade ≥3 urinary frequency/urgency was significantly higher in patients with AA (P=0.01), Aa (P=0.022), and Aa+AA (P=0.031) for XRCC3 compared with aa group. Our study suggested that RAD51, XRCC1, and XRCC3 polymorphisms may be predictive factors for radiation-induced acute toxicity in rectal cancer patients treated with preoperative combined therapy.

  16. Different mating-type-regulated genes affect the DNA repair defects of Saccharomyces RAD51, RAD52 and RAD55 mutants.

    PubMed

    Valencia-Burton, Maria; Oki, Masaya; Johnson, Jean; Seier, Tracey A; Kamakaka, Rohinton; Haber, James E

    2006-09-01

    Saccharomyces cerevisiae cells expressing both a- and alpha-mating-type (MAT) genes (termed mating-type heterozygosity) exhibit higher rates of spontaneous recombination and greater radiation resistance than cells expressing only MATa or MATalpha. MAT heterozygosity suppresses recombination defects of four mutations involved in homologous recombination: complete deletions of RAD55 or RAD57, an ATPase-defective Rad51 mutation (rad51-K191R), and a C-terminal truncation of Rad52, rad52-Delta327. We investigated the genetic basis of MAT-dependent suppression of these mutants by deleting genes whose expression is controlled by the Mata1-Matalpha2 repressor and scoring resistance to both campothecin (CPT) and phleomycin. Haploid rad55Delta strains became more damage resistant after deleting genes required for nonhomologous end-joining (NHEJ), a process that is repressed in MATa/MATalpha cells. Surprisingly, NHEJ mutations do not suppress CPT sensitivity of rad51-K191R or rad52-Delta327. However, rad51-K191R is uniquely suppressed by deleting the RME1 gene encoding a repressor of meiosis or its coregulator SIN4; this effect is independent of the meiosis-specific homolog, Dmc1. Sensitivity of rad52-Delta327 to CPT was unexpectedly increased by the MATa/MATalpha-repressed gene YGL193C, emphasizing the complex ways in which MAT regulates homologous recombination. The rad52-Delta327 mutation is suppressed by deleting the prolyl isomerase Fpr3, which is not MAT regulated. rad55Delta is also suppressed by deletion of PST2 and/or YBR052C (RFS1, rad55 suppressor), two members of a three-gene family of flavodoxin-fold proteins that associate in a nonrandom fashion with chromatin. All three recombination-defective mutations are made more sensitive by deletions of Rad6 and of the histone deacetylases Rpd3 and Ume6, although these mutations are not themselves CPT or phleomycin sensitive.

  17. Histone deacetylases 1 and 2 cooperate in regulating BRCA1, CHK1, and RAD51 expression in acute myeloid leukemia cells

    PubMed Central

    Edwards, Holly; Wang, Guan; Taub, Jeffrey W; Ge, Yubin

    2017-01-01

    Resistance to chemotherapy and a high relapse rate highlight the importance of finding new therapeutic options for the treatment of acute myeloid leukemia (AML). Histone deacetylase (HDAC) inhibitors (HDACIs) are a promising class of drugs for the treatment of AML. HDACIs have limited single-agent clinical activities, but when combined with conventional or investigational drugs they have demonstrated favorable outcomes. Previous studies have shown that decreasing expression of important DNA damage repair proteins enhances standard chemotherapy drugs. In our recent studies, the pan-HDACI panobinostat has been shown to enhance conventional chemotherapy drugs cytarabine and daunorubicin in AML cells by decreasing the expression of BRCA1, CHK1, and RAD51. In this study, we utilized class- and isoform-specific HDACIs and shRNA knockdown of individual HDACs to determine which HDACs are responsible for decreased expression of BRCA1, CHK1, and RAD51 following pan-HDACI treatment in AML cells. We found that inhibition of both HDAC1 and HDAC2 was necessary to decrease the expression of BRCA1, CHK1, and RAD51, enhance cytarabine- or daunorubicin-induced DNA damage and apoptosis, and abrogate cytarabine- or daunorubicin-induced cell cycle checkpoint activation in AML cells. These findings may aid in the development of rationally designed drug combinations for the treatment of AML. PMID:28030834

  18. Human DNA Helicase B Functions in Cellular Homologous Recombination and Stimulates Rad51-Mediated 5′-3′ Heteroduplex Extension In Vitro

    PubMed Central

    Liu, Hanjian; Yan, Peijun; Fanning, Ellen

    2015-01-01

    Homologous recombination is involved in the repair of DNA damage and collapsed replication fork, and is critical for the maintenance of genomic stability. Its process involves a network of proteins with different enzymatic activities. Human DNA helicase B (HDHB) is a robust 5′-3′ DNA helicase which accumulates on chromatin in cells exposed to DNA damage. HDHB facilitates cellular recovery from replication stress, but its role in DNA damage response remains unclear. Here we report that HDHB silencing results in reduced sister chromatid exchange, impaired homologous recombination repair, and delayed RPA late-stage foci formation induced by ionizing radiation. Ectopically expressed HDHB colocalizes with Rad51, Rad52, RPA, and ssDNA. In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5′-3′ direction. A helicase-defective mutant HDHB failed to promote this reaction. Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5′-3′ heteroduplex extension during Rad51-mediated strand exchange in vitro. PMID:25617833

  19. Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica.

    PubMed

    Charcas-Lopez, Ma del Socorro; Garcia-Morales, Lorena; Pezet-Valdez, Marisol; Lopez-Camarillo, Cesar; Zamorano-Carrillo, Absalom; Marchat, Laurence A

    2014-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan.

  20. Yap1 and Skn7 genetically interact with Rad51 in response to oxidative stress and DNA double-strand break in Saccharomyces cerevisiae.

    PubMed

    Yi, Dae Gwan; Kim, Myung Ju; Choi, Ji Eun; Lee, Jihyun; Jung, Joohee; Huh, Won-Ki; Chung, Woo-Hyun

    2016-12-01

    Reactive oxygen species (ROS)-mediated DNA adducts as well as DNA strand breaks are highly mutagenic leading to genomic instability and tumorigenesis. DNA damage repair pathways and oxidative stress response signaling have been proposed to be highly associated, but the underlying interaction remains unknown. In this study, we employed mutant strains lacking Rad51, the homolog of E. coli RecA recombinase, and Yap1 or Skn7, two major transcription factors responsive to ROS, to examine genetic interactions between double-strand break (DSB) repair proteins and cellular redox regulators in budding yeast Saccharomyces cerevisiae. Abnormal expression of YAP1 or SKN7 aggravated the mutation rate of rad51 mutants and their sensitivity to DSB- or ROS-generating reagents. Rad51 deficiency exacerbated genome instability in the presence of increased levels of ROS, and the accumulation of DSB lesions resulted in elevated intracellular ROS levels. Our findings suggest that evident crosstalk between DSB repair pathways and ROS signaling proteins contributes to cell survival and maintenance of genome integrity in response to genotoxic stress.

  1. RECK impedes DNA repair by inhibiting the erbB/JAB1/Rad51 signaling axis and enhances chemosensitivity of breast cancer cells

    PubMed Central

    Hong, Kun-Jing; Hsu, Ming-Chuan; Hung, Wen-Chun

    2015-01-01

    The reversion-inducing cysteine-rich protein with kazal motif (RECK) is an endogenous matrix metalloproteinase (MMP) inhibitor and a tumor suppressor. Its expression is dramatically down-regulated in human cancers. Our recent results suggest a novel MMP-independent anti-cancer activity of RECK by inhibiting the erbB signaling. Activation of the erbB signaling is associated with chemotherapeutic resistance, however, whether RECK could modulate drug sensitivity is still unknown. Here we demonstrated that expression of RECK induced the activation of ATM and ATR pathways, and the formation of γ-H2AX foci in breast cancer cells. RECK inhibited the erbB signaling and attenuated the expression of the downstream molecules Jun activation domain-binding protein 1 (JAB1) and the DNA repair protein RAD51 to impede DNA repair and to increase drug sensitivity. Treatment of epidermal growth factor or over-expression of HER-2 effectively reversed the inhibitory effect of RECK. In addition, ectopic expression of JAB1 counteracted RECK-induced RAD51 reduction and drug sensitization. Our results elucidate a novel function of RECK to modulate DNA damage response and drug resistance by inhibiting the erbB/Jab1/RAD51 signaling axis. Restoration of RECK expression in breast cancer cells may increase sensitivity to chemotherapeutic agents. PMID:26396917

  2. Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins.

    PubMed Central

    Hays, S L; Firmenich, A A; Berg, P

    1995-01-01

    The repair of DNA double-strand breaks in Saccharomyces cerevisiae requires genes of the RAD52 epistasis group, of which RAD55 and RAD57 are members. Here, we show that the x-ray sensitivity of rad55 and rad57 mutant strains is suppressible by overexpression of RAD51 or RAD52. Virtually complete suppression is provided by the simultaneous overexpression of RAD51 and RAD52. This suppression occurs at 23 degrees C, where these mutants are more sensitive to x-rays, as well as at 30 degrees C and 36 degrees C. In addition, a recombination defect of rad55 and rad57 mutants is similarly suppressed. Direct in vivo interactions between the Rad51 and Rad55 proteins, and between Rad55 and Rad57, have also been identified by using the two-hybrid system. These results indicate that these four proteins constitute part of a complex, a "recombinosome," to effect the recombinational repair of double-strand breaks. PMID:7624345

  3. Assessment of DNA binding to human Rad51 protein by using quartz crystal microbalance and atomic force microscopy: effects of ADP and BRC4-28 peptide inhibitor.

    PubMed

    Esnault, Charles; Renodon-Cornière, Axelle; Takahashi, Masayuki; Casse, Nathalie; Delorme, Nicolas; Louarn, Guy; Fleury, Fabrice; Pilard, Jean-François; Chénais, Benoît

    2014-12-01

    The interaction of human Rad51 protein (HsRad51) with single-stranded deoxyribonucleic acid (ssDNA) was investigated by using quartz crystal microbalance (QCM) monitoring and atomic force microscopy (AFM) visualization. Gold surfaces for QCM and AFM were modified by electrografting of the in situ generated aryldiazonium salt from the sulfanilic acid to obtain the organic layer Au-ArSO3 H. The Au-ArSO3 H layer was activated by using a solution of PCl5 in CH2 Cl2 to give a Au-ArSO2 Cl layer. The modified surface was then used to immobilize long ssDNA molecules. The results obtained showed that the presence of adenosine diphosphate promotes the protein autoassociation rather than nucleation around DNA. In addition, when the BRC4-28 peptide inhibitor was used, both QCM and AFM confirmed the inhibitory effect of BRC4-28 toward HsRad51 autoassociation. Altogether these results show the suitability of this modified surface to investigate the kinetics and structure of DNA-protein interactions and for the screening of inhibitors.

  4. Augmentation of Response to Chemotherapy by microRNA-506 Through Regulation of RAD51 in Serous Ovarian Cancers

    PubMed Central

    Liu, Guoyan; Yang, Da; Rupaimoole, Rajesha; Pecot, Chad V.; Sun, Yan; Mangala, Lingegowda S.; Li, Xia; Ji, Ping; Cogdell, David; Hu, Limei; Wang, Yingmei; Rodriguez-Aguayo, Cristian; Lopez-Berestein, Gabriel; Shmulevich, Ilya; De Cecco, Loris; Chen, Kexin; Mezzanzanica, Delia; Xue, Fengxia; Sood, Anil K.

    2015-01-01

    Background: Chemoresistance is a major challenge in cancer treatment. miR-506 is a potent inhibitor of the epithelial-to-mesenchymal transition (EMT), which is also associated with chemoresistance. We characterized the role of miR-506 in chemotherapy response in high-grade serous ovarian cancers. Methods: We used Kaplan-Meier and log-rank methods to analyze the relationship between miR-506 and progression-free and overall survival in The Cancer Genome Atlas (TCGA) (n = 468) and Bagnoli (n = 130) datasets, in vitro experiments to study whether miR-506 is associated with homologous recombination, and response to chemotherapy agents. We used an orthotopic ovarian cancer mouse model (n = 10 per group) to test the effect of miR-506 on cisplatin and PARP inhibitor sensitivity. All statistical tests were two-sided. Results: MiR-506 was associated with better response to therapy and longer progression-free and overall survival in two independent epithelial ovarian cancer patient cohorts (PFS: high vs low miR-506 expression; Bagnoli: hazard ratio [HR] = 3.06, 95% confidence interval [CI] = 1.90 to 4.70, P < .0001; TCGA: HR = 1.49, 95% CI = 1.00 to 2.25, P = 0.04). MiR-506 sensitized cells to DNA damage through directly targeting the double-strand DNA damage repair gene RAD51. Systemic delivery of miR-506 in 8–12 week old female athymic nude mice statistically significantly augmented the cisplatin and olaparib response (mean tumor weight ± SD, control miRNA plus cisplatin vs miR-506 plus cisplatin: 0.36±0.05g vs 0.07±0.02g, P < .001; control miRNA plus olaparib vs miR-506 plus olaparib: 0.32±0.13g vs 0.05±0.02g, P = .045, respectively), thus recapitulating the clinical observation. Conclusions: MiR-506 is a robust clinical marker for chemotherapy response and survival in serous ovarian cancers and has important therapeutic value in sensitizing cancer cells to chemotherapy. PMID:25995442

  5. The role of repair protein Rad51 in synergistic cytotoxicity and mutagenicity induced by epidermal growth factor receptor inhibitor (Gefitinib, Iressa{sup R}) and benzo[a]pyrene in human lung cancer

    SciTech Connect

    Ko, J.-C.; Hong, J.-H.; Wang, L.-H.; Lin, Y.-W.

    2008-05-01

    Rad51 protein is essential for homologous recombination repair of DNA damage, and is over-expressed in chemo- or radioresistant carcinomas. The polycyclic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) affects MAPKs transduction pathways. Gefitinib (Iressa{sup R}, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that blocks growth factor-mediated cell proliferation and ERK1/2 activation. We hypothesized that gefitinib enhances B[a]P-mediated cytotoxicity by decreasing ERK1/2 activation. Exposure of human lung cancer cells to gefitinib decreased B[a]P-elicited ERK1/2 activation and induced Rad51 protein expression. Gefitinib and B[a]P co-treatment decreased Rad51 protein stability by triggering degradation via a 26S proteasome-dependent pathway. Expression of constitutive active MKK1/2 vectors (MKK1/2-CA) rescues the decreased ERK1/2 activity, and restores Rad51 protein level and stability under gefitinib and B[a]P co-treatment. Gefitinib enhances B[a]P-induced growth inhibition, cytotoxicity and mutagenicity. Co-treatment with gefitinib and B[a]P can further inhibit cell growth significantly after depletion of endogenous Rad51 by siRad51 RNA transfection. Enhancement of ERK1/2 activation by MKK1-CA expression decrease B[a]P- and gefitinib-induced cytotoxicity, and B[a]P-induced mutagenicity. Rad51 protein protects lung cancer cells from synergistic cytotoxic and mutagenic effects induced by gefitinib and B[a]P. Suppression of Rad51 protein expression may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib.

  6. Gemcitabine sensitization by Chk1 inhibition correlates with inhibition of a Rad51 DNA damage response in pancreatic cancer cells1

    PubMed Central

    Parsels, Leslie A.; Morgan, Meredith A.; Tanska, Daria M.; Parsels, Joshua D.; Palmer, Brian D.; Booth, R. John; Denny, William A.; Canman, Christine E.; Kraker, Alan J.; Lawrence, Theodore S.; Maybaum, Jonathan

    2009-01-01

    The protein kinase Chk1 has been implicated as a key regulator of cell cycle progression and DNA repair and inhibitors of Chk1 (e.g., UCN-01, EXEL-9844) potentiate the cytotoxic actions of chemotherapeutic drugs in tumor cells. We have examined the ability of PD-321852, a small-molecule Chk1 inhibitor, to potentiate gemcitabine (Gem)-induced clonogenic death in a panel of pancreatic cancer cell lines, and evaluated the relationship between endpoints associated with Chk1 inhibition and chemosensitization. Gem chemosensitization by minimally-toxic concentrations of PD-321852 ranged from minimal (< 3 fold change in survival) in Panc1 cells, to over 30-fold in MiaPaCa2 cells. PD-321852 inhibited Chk1 in all cell lines, as evidenced by stabilization of Cdc25A, and, in combination with Gem, a synergistic loss of Chk1 protein was observed in the more sensitized cell lines. Gem chemosensitization, however, did not correlate with abrogation of the S/M or G2/M checkpoint; PD-321852 did not induce premature mitotic entry in Gem-treated BxPC3 or M-Panc96 cells, which were sensitized to Gem 6.2- and 4.6-fold, respectively. In the more sensitized cells lines, PD-321852 not only inhibited Gem-induced Rad51 focus formation and the recovery from Gem-induced replication stress, as evidenced by persistence of γ-H2AX, but also depleted these cells of Rad51 protein. Our data suggest the inhibition of this Chk1-mediated Rad51 response to Gem-induced replication stress is an important factor in determining Gem chemosensitization by Chk1 inhibition in pancreatic cancer cells. PMID:19139112

  7. Role of the insulin-like growth factor I/insulin receptor substrate 1 axis in Rad51 trafficking and DNA repair by homologous recombination.

    PubMed

    Trojanek, Joanna; Ho, Thu; Del Valle, Luis; Nowicki, Michal; Wang, Jin Ying; Lassak, Adam; Peruzzi, Francesca; Khalili, Kamel; Skorski, Tomasz; Reiss, Krzysztof

    2003-11-01

    The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome.

  8. 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

  9. The tumor suppressor PML specifically accumulates at RPA/Rad51-containing DNA damage repair foci but is nonessential for DNA damage-induced fibroblast senescence.

    PubMed

    Münch, Sandra; Weidtkamp-Peters, Stefanie; Klement, Karolin; Grigaravicius, Paulius; Monajembashi, Shamci; Salomoni, Paolo; Pandolfi, Pier Paolo; Weißhart, Klaus; Hemmerich, Peter

    2014-05-01

    The PML tumor suppressor has been functionally implicated in DNA damage response and cellular senescence. Direct evidence for such a role based on PML knockdown or knockout approaches is still lacking. We have therefore analyzed the irradiation-induced DNA damage response and cellular senescence in human and mouse fibroblasts lacking PML. Our data show that PML nuclear bodies (NBs) nonrandomly associate with persistent DNA damage foci in unperturbed human skin and in high-dose-irradiated cell culture systems. PML bodies do not associate with transient γH2AX foci after low-dose gamma irradiation. Superresolution microscopy reveals that all PML bodies within a nucleus are engaged at Rad51- and RPA-containing repair foci during ongoing DNA repair. The lack of PML (i) does not majorly affect the DNA damage response, (ii) does not alter the efficiency of senescence induction after DNA damage, and (iii) does not affect the proliferative potential of primary mouse embryonic fibroblasts during serial passaging. Thus, while PML NBs specifically accumulate at Rad51/RPA-containing lesions and senescence-derived persistent DNA damage foci, they are not essential for DNA damage-induced and replicative senescence of human and murine fibroblasts.

  10. Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase.

    PubMed

    Liberi, Giordano; Maffioletti, Giulio; Lucca, Chiara; Chiolo, Irene; Baryshnikova, Anastasia; Cotta-Ramusino, Cecilia; Lopes, Massimo; Pellicioli, Achille; Haber, James E; Foiani, Marco

    2005-02-01

    S-phase cells overcome chromosome lesions through replication-coupled recombination processes that seem to be assisted by recombination-dependent DNA structures and/or replication-related sister chromatid junctions. RecQ helicases, including yeast Sgs1 and human BLM, have been implicated in both replication and recombination and protect genome integrity by preventing unscheduled mitotic recombination events. We have studied the RecQ helicase-mediated mechanisms controlling genome stability by analyzing replication forks encountering a damaged template in sgs1 cells. We show that, in sgs1 mutants, recombination-dependent cruciform structures accumulate at damaged forks. Their accumulation requires Rad51 protein, is counteracted by Srs2 DNA helicase, and does not prevent fork movement. Sgs1, but not Srs2, promotes resolution of these recombination intermediates. A functional Rad53 checkpoint kinase that is known to protect the integrity of the sister chromatid junctions is required for the accumulation of recombination intermediates in sgs1 mutants. Finally, top3 and top3 sgs1 mutants accumulate the same structures as sgs1 cells. We suggest that, in sgs1 cells, the unscheduled accumulation of Rad51-dependent cruciform structures at damaged forks result from defective maturation of recombination-dependent intermediates that originate from the replication-related sister chromatid junctions. Our findings might contribute to explaining some of the recombination defects of BLM cells.

  11. Expression of PH Domain Leucine-rich Repeat Protein Phosphatase, Forkhead Homeobox Type O 3a and RAD51, and their Relationships with Clinicopathologic Features and Prognosis in Ovarian Serous Adenocarcinoma

    PubMed Central

    Zhang, Jun; Wang, Jun-Chao; Li, Yue-Hong; Wang, Rui-Xue; Fan, Xiao-Mei

    2017-01-01

    Background: Ovarian serous adenocarcinoma can be divided into low- and high-grade tumors, which exhibit substantial differences in pathogenesis, clinicopathology, and prognosis. This study aimed to investigate the differences in the PH domain leucine-rich repeat protein phosphatase (PHLPP), forkhead homeobox type O 3a (FoxO3a), and RAD51 protein expressions, and their associations with prognosis in patients with low- and high-grade ovarian serous adenocarcinomas. Methods: The PHLPP, FoxO3a, and RAD51 protein expressions were examined in 94 high- and 26 low-grade ovarian serous adenocarcinomas by immunohistochemistry. The differences in expression and their relationships with pathological features and prognosis were analyzed. Results: In high-grade serous adenocarcinomas, the positive rates of PHLPP and FoxO3a were 24.5% and 26.6%, while in low-grade tumors, they were 23.1% and 26.9%, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). The positive rates of RAD51 were 70.2% and 65.4% in high- and low-grade serous adenocarcinomas, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). Meanwhile, in high-grade tumors, Stage III/IV tumors and lymph node and omental metastases were significantly associated with lower PHLPP and FoxO3a and higher RAD51 expression. The 5-year survival rates of patients with PHLPP- and FoxO3a-positive high-grade tumors (43.5% and 36.0%) were significantly higher than in patients with PHLPP-negative tumors (5.6% and 7.2%, respectively; P < 0.05). Similarly, the 5-year survival rate of RAD51-positive patients (3.0%) was significantly lower than in negative patients (42.9%; P < 0.05). In low-grade tumors, the PHLPP, FoxO3a, and RAD51 expressions were not significantly correlated with lymph node metastasis, omental metastasis, Federation of Gynecology and Obstetrics stage, or prognosis. Conclusions: Abnormal PHLPP, FoxO3a, and RAD51 protein expressions may be involved in the development

  12. The fission yeast meiosis-specific Dmc1 recombinase mediates formation and branch migration of Holliday junctions by preferentially promoting strand exchange in a direction opposite to that of Rad51

    PubMed Central

    Murayama, Yasuto; Tsutsui, Yasuhiro; Iwasaki, Hiroshi

    2011-01-01

    Homologous recombination proceeds via the formation of several intermediates including Holliday junctions (HJs), which are important for creating crossover products. DNA strand exchange is a core reaction that produces these intermediates that is directly catalyzed by RecA family recombinases, of which there are two types in eukaryotes: universal Rad51 and meiosis-specific Dmc1. We demonstrated previously that Rad51 promotes four-strand exchange, mimicking the formation and branch migration of HJs. Here we show that Dmc1 from fission yeast has a similar activity, which requires ATP hydrolysis and is independent of an absolute requirement for the Swi5–Sfr1 complex. These features are critically different from three-strand exchange mediated by Dmc1, but similar to those of four-strand exchange mediated by Rad51, suggesting that strand exchange reactions between duplex–duplex and single-duplex DNAs are mechanistically different. Interestingly, despite similarities in protein structure and in reaction features, the preferential polarities of Dmc1 and Rad51 strand exchange are different (Dmc1 promotes exchange in the 5′-to-3′ direction and Rad51 promotes exchange in the 3′-to-5′ direction relative to the ssDNA region of the DNA substrate). The significance of the Dmc1 polarity is discussed within the context of the necessity for crossover production. PMID:21363965

  13. The fission yeast meiosis-specific Dmc1 recombinase mediates formation and branch migration of Holliday junctions by preferentially promoting strand exchange in a direction opposite to that of Rad51.

    PubMed

    Murayama, Yasuto; Tsutsui, Yasuhiro; Iwasaki, Hiroshi

    2011-03-01

    Homologous recombination proceeds via the formation of several intermediates including Holliday junctions (HJs), which are important for creating crossover products. DNA strand exchange is a core reaction that produces these intermediates that is directly catalyzed by RecA family recombinases, of which there are two types in eukaryotes: universal Rad51 and meiosis-specific Dmc1. We demonstrated previously that Rad51 promotes four-strand exchange, mimicking the formation and branch migration of HJs. Here we show that Dmc1 from fission yeast has a similar activity, which requires ATP hydrolysis and is independent of an absolute requirement for the Swi5-Sfr1 complex. These features are critically different from three-strand exchange mediated by Dmc1, but similar to those of four-strand exchange mediated by Rad51, suggesting that strand exchange reactions between duplex-duplex and single-duplex DNAs are mechanistically different. Interestingly, despite similarities in protein structure and in reaction features, the preferential polarities of Dmc1 and Rad51 strand exchange are different (Dmc1 promotes exchange in the 5'-to-3' direction and Rad51 promotes exchange in the 3'-to-5' direction relative to the ssDNA region of the DNA substrate). The significance of the Dmc1 polarity is discussed within the context of the necessity for crossover production.

  14. Brief Report: Identification of BACH2 and RAD51B as Rheumatoid Arthritis Susceptibility Loci in a Meta-Analysis of Genome-Wide Data

    PubMed Central

    McAllister, Kate; Yarwood, Annie; Bowes, John; Orozco, Gisela; Viatte, Sebastian; Diogo, Dorothée; Hocking, Lynne J; Steer, Sophia; Wordsworth, Paul; Wilson, A G; Morgan, Ann W; Kremer, Joel M; Pappas, Dimitrios; Gregersen, Peter; Klareskog, Lars; Plenge, Robert; Barton, Anne; Greenberg, Jeffrey; Worthington, Jane; Eyre, Stephen

    2013-01-01

    Objective A recent high-density fine-mapping (ImmunoChip) study of genetic associations in rheumatoid arthritis (RA) identified 14 risk loci with validated genome-wide significance, as well as a number of loci showing associations suggestive of significance (P = 5 × 10−5 < 5 × 10−8), but these have yet to be replicated. The aim of this study was to determine whether these potentially significant loci are involved in the pathogenesis of RA, and to explore whether any of the loci are associated with a specific RA serotype. Methods A total of 16 single-nucleotide polymorphisms (SNPs) were selected for genotyping and association analyses in 2 independent validation cohorts, comprising 6,106 RA cases and 4,290 controls. A meta-analysis of the data from the original ImmunoChip discovery cohort and from both validation cohorts was carried out, for a combined total of 17,581 RA cases and 20,160 controls. In addition, stratified analysis of patient subsets, defined according to their anti–cyclic citrullinated peptide (anti-CCP) antibody status, was performed. Results A significant association with RA risk (P < 0.05) was replicated for 6 of the SNPs assessed in the validation cohorts. All SNPs in the validation study had odds ratios (ORs) for RA susceptibility in the same direction as those in the ImmunoChip discovery study. One SNP, rs72928038, mapping to an intron of BACH2, achieved genome-wide significance in the meta-analysis (P = 1.2 × 10−8, OR 1.12), and a second SNP, rs911263, mapping to an intron of RAD51B, was significantly associated in the anti-CCP–positive RA subgroup (P = 4 × 10−8, OR 0.89), confirming that both are RA susceptibility loci. Conclusion This study provides robust evidence for an association of RA susceptibility with genes involved in B cell differentiation (BACH2) and DNA repair (RAD51B). The finding that the RAD51B gene exhibited different associations based on serologic subtype adds to the expanding knowledge base in defining

  15. 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.

  16. Acquisition of radioresistance in docetaxel-resistant human lung adenocarcinoma cells is linked with dysregulation of miR-451/c-Myc-survivin/rad-51 signaling

    PubMed Central

    Huang, Jia-Yuan; Zhang, Kai; Feng, Bing; Pan, Ban-Zhou; Chen, Jing; De, Wei; Chen, Long-Bang

    2014-01-01

    Chemoresistant tumors usually fail to respond to radiotherapy. However, the mechanisms involved in chemo- and radiotherapy cross resistance are not fully understood. Previously, we have identified microRNA (miR)-451 as a tumor suppressor in lung adenocarcinoma (LAD). However, whether miR-451 plays critical roles in chemo- and radiotherapy cross resistance in LAD is unclear. Here, we established two docetaxel-resistant LAD cell models (SPC-A1/DTX and H1299/DTX), and showed that miR-451 was significantly downregulated in docetaxel-resistant LAD cells. Gain - and loss - of - function assays indicated that re-expression of miR-451 could reverse radioresistance of docetaxel-resistant LAD cells both in vitro and in vivo through promoting apoptosis and DNA double-strand breaks (DSBs). The proto-oncogene c-Myc was identified as a direct target of miR-451, and re-expression of miR-451 inhibited survivin and rad-51 expression by reducing the amount of c-Myc protein binding to their promoters. Silencing of c-Myc could phenocopy the effects of miR-451 upregulation, and restoration of c-Myc could partially rescue the effect of miR-451 upregulation on radiosensitivity of docetaxel-resistant LAD cells. Therefore, dysregulation of miR-451/c-Myc-survivin/rad-51 signaling is responsible for radioresistance of docetaxel-resistant LAD cells, and targeting it will be a potential strategy for reversing chemo- and radiotherapy cross resistance of LAD patients. PMID:25026294

  17. Constitutive promoter methylation of BRCA1 and RAD51C in patients with familial ovarian cancer and early-onset sporadic breast cancer.

    PubMed

    Hansmann, Tamara; Pliushch, Galyna; Leubner, Monika; Kroll, Patricia; Endt, Daniela; Gehrig, Andrea; Preisler-Adams, Sabine; Wieacker, Peter; Haaf, Thomas

    2012-11-01

    Genetic defects in breast cancer (BC) susceptibility genes, most importantly BRCA1 and BRCA2, account for ~40% of hereditary BC and ovarian cancer (OC). Little is known about the contribution of constitutive (soma-wide) epimutations to the remaining cases. We developed bisulfite pyrosequencing assays to screen >600 affected BRCA1/BRCA2 mutation-negative patients from the German Consortium for Hereditary Breast and Ovarian Cancer for constitutive hypermethylation of ATM, BRCA1, BRCA2, RAD51C, PTEN and TP53 in blood cells. In a second step, patients with ≥6% promoter methylation were analyzed by bisulfite plasmid sequencing to demonstrate the presence of hypermethylated alleles (epimutations), indicative of epigenetic gene silencing. Altogether we identified nine (1.4%) patients with constitutive BRCA1 and three (0.5%) with RAD51C hypermethylation. Epimutations were found in both sporadic cases, in particular in 2 (5.5%) of 37 patients with early-onset BC, and familial cases, in particular 4 (10%) of 39 patients with OC. Hypermethylation was always confined to one of the two parental alleles in a subset (12-40%) of the analyzed cells. Because epimutations occurred in cell types from different embryonal layers, they most likely originated in single cells during early somatic development. We propose that analogous to germline genetic mutations constitutive epimutations may serve as the first hit of tumor development. Because the role of constitutive epimutations in cancer development is likely to be largely underestimated, future strategies for effective testing of susceptibility to BC and OC should include an epimutation screen.

  18. Common Breast Cancer Susceptibility Variants in LSP1 and RAD51L1 Are Associated with Mammographic Density Measures that Predict Breast Cancer Risk

    PubMed Central

    Vachon, Celine M.; Scott, Christopher G.; Fasching, Peter A.; Hall, Per; Tamimi, Rulla M.; Li, Jingmei; Stone, Jennifer; Apicella, Carmel; Odefrey, Fabrice; Gierach, Gretchen L.; Jud, Sebastian M.; Heusinger, Katharina; Beckmann, Matthias W.; Pollan, Marina; Fernández-Navarro, Pablo; González-Neira, Anna; Benítez, Javier; van Gils, Carla H.; Lokate, Mariëtte; Onland-Moret, N. Charlotte; Peeters, Petra H.M.; Brown, Judith; Leyland, Jean; Varghese, Jajini S.; Easton, Douglas F.; Thompson, Deborah J.; Luben, Robert N.; Warren, Ruth ML; Wareham, Nicholas J.; Loos, Ruth JF; Khaw, Kay-Tee; Ursin, Giske; Lee, Eunjung; Gayther, Simon A.; Ramus, Susan J.; Eeles, Rosalind A.; Leach, Martin O.; Kwan-Lim, Gek; Couch, Fergus J.; Giles, Graham G.; Baglietto, Laura; Krishnan, Kavitha; Southey, Melissa C.; Le Marchand, Loic; Kolonel, Laurence N.; Woolcott, Christy; Maskarinec, Gertraud; Haiman, Christopher A; Walker, Kate; Johnson, Nichola; McCormack, Valerie A.; Biong, Margarethe; Alnæs, Grethe I.G.; Gram, Inger Torhild; Kristensen, Vessela N.; Børresen-Dale, Anne-Lise; Lindström, Sara; Hankinson, Susan E.; Hunter, David J.; Andrulis, Irene L.; Knight, Julia A.; Boyd, Norman F.; Figueroa, Jonine D.; Lissowska, Jolanta; Wesolowska, Ewa; Peplonska, Beata; Bukowska, Agnieszka; Reszka, Edyta; Liu, JianJun; Eriksson, Louise; Czene, Kamila; Audley, Tina; Wu, Anna H.; Pankratz, V. Shane; Hopper, John L.; dos-Santos-Silva, Isabel

    2013-01-01

    Background Mammographic density adjusted for age and body mass index (BMI) is a heritable marker of breast cancer susceptibility. Little is known about the biological mechanisms underlying the association between mammographic density and breast cancer risk. We examined whether common low-penetrance breast cancer susceptibility variants contribute to inter-individual differences in mammographic density measures. Methods We established an international consortium (DENSNP) of 19 studies from 10 countries, comprising 16,895 Caucasian women, to conduct a pooled cross-sectional analysis of common breast cancer susceptibility variants in 14 independent loci and mammographic density measures. Dense and non-dense areas, and percent density, were measured using interactive-thresholding techniques. Mixed linear models were used to assess the association between genetic variants and the square roots of mammographic density measures adjusted for study, age, case status, body mass index (BMI) and menopausal status. Results Consistent with their breast cancer associations, the C-allele of rs3817198 in LSP1 was positively associated with both adjusted dense area (p=0.00005) and adjusted percent density (p=0.001) whereas the A-allele of rs10483813 in RAD51L1 was inversely associated with adjusted percent density (p=0.003), but not with adjusted dense area (p=0.07). Conclusion We identified two common breast cancer susceptibility variants associated with mammographic measures of radio-dense tissue in the breast gland. Impact We examined the association of 14 established breast cancer susceptibility loci with mammographic density phenotypes within a large genetic consortium and identified two breast cancer susceptibility variants, LSP1-rs3817198 and RAD51L1-rs10483813, associated with mammographic measures and in the same direction as the breast cancer association. PMID:22454379

  19. Characterization of recombinase DMC1B and its functional role as Rad51 in DNA damage repair in Giardia duodenalis trophozoites.

    PubMed

    Torres-Huerta, Ana Laura; Martínez-Miguel, Rosa María; Bazán-Tejeda, María Luisa; Bermúdez-Cruz, Rosa María

    2016-08-01

    Homologous recombination (HR) is a highly conserved pathway for the repair of chromosomes that harbor DNA double-stranded breaks (DSBs). The recombinase RAD51 plays a key role by catalyzing the pairing of homologous DNA molecules and the exchange of information between them. Two putative DMC1 homologs (DMC1A and DMC1B) have been identified in Giardia duodenalis. In terms of sequences, GdDMC1A and GdDMC1B bear all of the characteristic recombinase domains: DNA binding domains (helix-turn-helix motif, loops 1 and 2), an ATPcap and Walker A and B motifs associated with ATP binding and hydrolysis. Because GdDMC1B is expressed at the trophozoite stage and GdDMC1A is expressed in the cyst stage, we cloned the giardial dmc1B gene and expressed and purified its protein to determine its activities, including DNA binding, ATP hydrolysis, and DNA strand exchange. Our results revealed that it possessed these activities, and they were modulated by divalent metal ions in different manners. GdDMC1B expression at the protein and transcript levels, as well as its subcellular localization in trophozoites upon DNA damage, was assessed. We found a significant increase in GdDMC1B transcript and protein levels after ionizing radiation treatment. Additionally, GdDMC1B protein was mostly located in the nucleus of trophozoites after DNA damage. These results indicate that GdDMC1B is the recombinase responsible for DSBs repair in the trophozoite; therefore, a functional Rad51 role is proposed for GdDMC1B.

  20. RAD52 inactivation is synthetically lethal with deficiencies in BRCA1 and PALB2 in addition to BRCA2 through RAD51-mediated homologous recombination.

    PubMed

    Lok, B H; Carley, A C; Tchang, B; Powell, S N

    2013-07-25

    Synthetic lethality is an approach to study selective cell killing based on genotype. Previous work in our laboratory has shown that loss of RAD52 is synthetically lethal with BRCA2 deficiency, while exhibiting no impact on cell growth and viability in BRCA2-proficient cells. We now show that this same synthetically lethal relationship is evident in cells with deficiencies in BRCA1 or PALB2, which implicates BRCA1, PALB2 and BRCA2 in an epistatic relationship with one another. When RAD52 was depleted in BRCA1- or PALB2-deficient cells, a severe reduction in plating efficiency was observed, with many abortive attempts at cell division apparent in the double-depleted background. In contrast, when RAD52 was depleted in a BRCA1- or PALB2-wildtype background, a negligible decrease in colony survival was observed. The frequency of ionizing radiation-induced RAD51 foci formation and double-strand break-induced homologous recombination (HR) was decreased by 3- and 10-fold, respectively, when RAD52 was knocked down in BRCA1- or PALB2-depleted cells, with minimal effect in BRCA1- or PALB2-proficient cells. RAD52 function was independent of BRCA1 status, as evidenced by the lack of any defect in RAD52 foci formation in BRCA1-depleted cells. Collectively, these findings suggest that RAD52 is an alternative repair pathway of RAD51-mediated HR, and a target for therapy in cells deficient in the BRCA1-PALB2-BRCA2 repair pathway.

  1. The carboxyl-terminal of BRCA1 is required for subnuclear assembly of RAD51 after treatment with cisplatin but not ionizing radiation in human breast and ovarian cancer cells

    SciTech Connect

    Zhou Chenyi; Huang Peng; Liu Jinsong . E-mail: jliu@mdanderson.org

    2005-10-28

    BRCA1 plays an important role in maintaining genomic stability through its involvement in DNA repair. Although it is known that BRCA1 and RAD51 form distinct DNA repair subnuclear complexes, or foci, following environmental insults to the DNA, the role of BRCA1 in this process remains to be characterized. The purpose of the study was therefore to determine the role of BRCA1 in the formation of RAD51 foci following treatment with cisplatin and ionizing radiation. We found that although a functional BRCA1 is required for the subnuclear assembly of BRCA1 foci following treatment with either ionizing radiation or cisplatin, a functional BRCA1 is required for RAD51 foci to form following treatment with cisplatin but not with ionizing radiation. Similar results were obtained in SKOV-3 cells when the level of BRCA1 expression was knocked down by stable expression of a retrovirus-mediated small-interfering RNA against BRCA1. We also found that the carboxyl-terminal of BRCA1 contains uncharacterized phosphorylation sites that are responsive to cisplatin. The functional BRCA1 is also required for breast and ovarian cancer cells to mount resistance to cisplatin. These results suggest that the carboxyl-terminal of BRCA1 is required for the cisplatin-induced recruitment of RAD51 to the DNA-damage site, which may contribute to cisplatin resistance.

  2. Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta.

    PubMed

    Patil, Shrikant; Moeys, Sara; von Dassow, Peter; Huysman, Marie J J; Mapleson, Daniel; De Veylder, Lieven; Sanges, Remo; Vyverman, Wim; Montresor, Marina; Ferrante, Maria Immacolata

    2015-11-14

    Sexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase. The exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestral loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase. Our results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom.

  3. Modulation of Homology-Directed Repair in T98G Glioblastoma Cells Due to Interactions between Wildtype p53, Rad51 and HCMV IE1-72

    PubMed Central

    Kulkarni, Amit S.; Fortunato, Elizabeth A.

    2014-01-01

    Human cytomegalovirus (HCMV) is a ubiquitous pathogen capable of causing life threatening consequences in neonates and immune-compromised individuals. HCMV inflicts site-specific double strand breaks (DSBs) in the cellular genome. DNA damage infliction raises the corollary question of virus modulation of DNA repair. We recently reported HDR was stimulated in wt human foreskin fibroblasts (HFFs) during fully permissive infection or expression of the HCMV protein IE1-72 (IE72). These studies have been extended into semi-permissive T98G glioblastoma cells. T98Gs encode a mutant p53, which may contribute to their high baseline rate of HDR. We fully expected HCMV infection to increase HDR in T98Gs, similar to its effects in HFFs. Surprisingly in T98Gs HCMV infection, or sole expression of IE72, decreased HDR by two-fold. Transient expression of wt p53 in T98Gs also reduced HDR by two-fold. Dual transient expression of wt p53 and IE72 restored high baseline HDR levels. GST pulldown experiments revealed that both IE72 and wt p53 bound the important HDR protein, Rad51. We conclude that the expression of certain HCMV proteins can modulate HDR in an infected cell, dependent upon p53 status. We propose a model of the protein interactions explaining this behavior. PMID:24576846

  4. Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1 homolog, in Pyrococcus furiosus.

    PubMed

    Hayashi, I; Morikawa, K; Ishino, Y

    1999-12-15

    Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene and three other genes. Using a two-hybrid screening to examine the interactions of the proteins encoded by the operon, we identified a specific interaction between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein (RadB). To ensure the specific interaction between these two proteins, each gene in the operon was expressed in Escherichia coli or insect cells separately and the products were purified. The in vitro analyses using the purified proteins also showed the interaction between DP1 and RadB. The deletion mutant analysis of DP1 revealed that a region important for binding with RadB is located in the central part of the sequence (amino acid residues 206-498). This region has an overlap to the C-terminal half (amino acids 334-613), which is highly conserved among euryarchaeal DP1s and is essential for the activity of PolD. Our results suggest that, although RadB does not noticeably affect the primer extension ability of PolD in vitro, PolD may utilize the RadB protein in DNA synthesis under certain conditions.

  5. Sensitization of Tumor to {sup 212}Pb Radioimmunotherapy by Gemcitabine Involves Initial Abrogation of G2 Arrest and Blocked DNA Damage Repair by Interference With Rad51

    SciTech Connect

    Yong, Kwon Joong; Milenic, Diane E.; Baidoo, Kwamena E.; Brechbiel, Martin W.

    2013-03-15

    Purpose: To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using {sup 212}Pb-TCMC-trastuzumab together with gemcitabine for treatment of disseminated peritoneal cancers. Methods and Materials: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pretreated with gemcitabine, followed by {sup 212}Pb-TCMC-trastuzumab and compared with controls. Results: Treatment with {sup 212}Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase-arrested tumors induced by gemcitabine at earlier time points (6 to 24 hours). {sup 212}Pb-TCMC-trastuzumab after gemcitabine pretreatment abrogated G2/M arrest at the same time points, which may be associated with the inhibition of Chk1 phosphorylation and, in turn, cell cycle perturbation, resulting in apoptosis. {sup 212}Pb-TCMC-trastuzumab treatment after gemcitabine pretreatment caused depression of DNA synthesis, DNA double-strand breaks, accumulation of unrepaired DNA, and down-regulation of Rad51 protein, indicating that DNA damage repair was blocked. In addition, modification in the chromatin structure of p21 may be associated with transcriptionally repressed chromatin states, indicating that the open structure was delayed at earlier time points. Conclusion: These findings suggest that the cell-killing efficacy of {sup 212}Pb-TCMC-trastuzumab after gemcitabine pretreatment may be associated with abrogation of the G2/M checkpoint, inhibition of DNA damage repair, and chromatin remodeling.

  6. Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1 homolog, in Pyrococcus furiosus.

    PubMed Central

    Hayashi, I; Morikawa, K; Ishino, Y

    1999-01-01

    Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene and three other genes. Using a two-hybrid screening to examine the interactions of the proteins encoded by the operon, we identified a specific interaction between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein (RadB). To ensure the specific interaction between these two proteins, each gene in the operon was expressed in Escherichia coli or insect cells separately and the products were purified. The in vitro analyses using the purified proteins also showed the interaction between DP1 and RadB. The deletion mutant analysis of DP1 revealed that a region important for binding with RadB is located in the central part of the sequence (amino acid residues 206-498). This region has an overlap to the C-terminal half (amino acids 334-613), which is highly conserved among euryarchaeal DP1s and is essential for the activity of PolD. Our results suggest that, although RadB does not noticeably affect the primer extension ability of PolD in vitro, PolD may utilize the RadB protein in DNA synthesis under certain conditions. PMID:10572168

  7. A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair

    PubMed Central

    Herr, Patrick; Lundin, Cecilia; Evers, Bastiaan; Ebner, Daniel; Bauerschmidt, Christina; Kingham, Guy; Palmai-Pallag, Timea; Mortusewicz, Oliver; Frings, Oliver; Sonnhammer, Erik; Helleday, Thomas

    2015-01-01

    To identify new regulators of homologous recombination repair, we carried out a genome-wide short-interfering RNA screen combined with ionizing irradiation using RAD51 foci formation as readout. All candidates were confirmed by independent short-interfering RNAs and validated in secondary assays like recombination repair activity and RPA foci formation. Network analysis of the top modifiers identified gene clusters involved in recombination repair as well as components of the ribosome, the proteasome and the spliceosome, which are known to be required for effective DNA repair. We identified and characterized the RNA polymerase II-associated protein CDC73/Parafibromin as a new player in recombination repair and show that it is critical for genomic stability. CDC73 interacts with components of the SCF/Cullin and INO80/NuA4 chromatin-remodeling complexes to promote Histone ubiquitination. Our findings indicate that CDC73 is involved in local chromatin decondensation at sites of DNA damage to promote DNA repair. This function of CDC73 is related to but independent of its role in transcriptional elongation. PMID:27462432

  8. Germline Mutations in PALB2, BRCA1, and RAD51C, Which Regulate DNA Recombination Repair, in Patients With Gastric Cancer.

    PubMed

    Sahasrabudhe, Ruta; Lott, Paul; Bohorquez, Mabel; Toal, Ted; Estrada, Ana P; Suarez, John J; Brea-Fernández, Alejandro; Cameselle-Teijeiro, José; Pinto, Carla; Ramos, Irma; Mantilla, Alejandra; Prieto, Rodrigo; Corvalan, Alejandro; Norero, Enrique; Alvarez, Carolina; Tapia, Teresa; Carvallo, Pilar; Gonzalez, Luz M; Cock-Rada, Alicia; Solano, Angela; Neffa, Florencia; Della Valle, Adriana; Yau, Chris; Soares, Gabriela; Borowsky, Alexander; Hu, Nan; He, Li-Ji; Han, Xiao-You; Taylor, Philip R; Goldstein, Alisa M; Torres, Javier; Echeverry, Magdalena; Ruiz-Ponte, Clara; Teixeira, Manuel R; Carvajal-Carmona, Luis G

    2017-04-01

    Up to 10% of cases of gastric cancer are familial, but so far, only mutations in CDH1 have been associated with gastric cancer risk. To identify genetic variants that affect risk for gastric cancer, we collected blood samples from 28 patients with hereditary diffuse gastric cancer (HDGC) not associated with mutations in CDH1 and performed whole-exome sequence analysis. We then analyzed sequences of candidate genes in 333 independent HDGC and non-HDGC cases. We identified 11 cases with mutations in PALB2, BRCA1, or RAD51C genes, which regulate homologous DNA recombination. We found these mutations in 2 of 31 patients with HDGC (6.5%) and 9 of 331 patients with sporadic gastric cancer (2.8%). Most of these mutations had been previously associated with other types of tumors and partially co-segregated with gastric cancer in our study. Tumors that developed in patients with these mutations had a mutation signature associated with somatic homologous recombination deficiency. Our findings indicate that defects in homologous recombination increase risk for gastric cancer.

  9. Elevated Estrogen Receptor-α in VHL-Deficient Condition Induces Microtubule Organizing Center Amplification via Disruption of BRCA1/Rad51 Interaction

    PubMed Central

    Jung, Youn-Sang; Chun, Ho-Young; Yoon, Min-Ho; Park, Bum-Joon

    2014-01-01

    Since loss of VHL is frequently detected early phase genetic event in human renal cell carcinoma, pVHL is assumed to be indispensable for suppression of tumor initiation step. However, induction of HIF-1α, target of pVHL E3 ligase, is more adequate to angiogenesis step after tumor mass formation. Concerning this, it has been reported that pVHL is involved in centrosome location during metaphase and regulates ER-α signaling. Here, we provide the evidences that pVHL-mediated ER-α suppression is critical for microtubule organizing center (MTOC) maintaining and elevated ER-α promotes MTOC amplification through disruption of BRCA1-Rad51 interaction. In fact, numerous MTOC in VHL- or BRCA1-deficient cells are reduced by Fulvestrant, inhibitor of ER-α expression as well as antagonist. In addition, we reveal that activation of ER signaling can increase γ-tubulin, core factor of TuRC and render the resistance to Taxol. Thus, Fulvestrant but not Tamoxifen, antagonist against ER-α, can restore the Taxol sensitivity in VHL- or BRCA1-deficient cells. Our results suggest that pVHL-mediated ER-α suppression is important for regulation of MTOC as well as drug resistance. PMID:25499220

  10. Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes

    NASA Astrophysics Data System (ADS)

    Lee, Eon-Seok; Won, Yeo Jin; Kim, Byoung-Chul; Park, Daeui; Bae, Jin-Han; Park, Seong-Joon; Noh, Sung Jin; Kang, Yeong-Rok; Choi, Si Ho; Yoon, Je-Hyun; Heo, Kyu; Yang, Kwangmo; Son, Tae Gen

    2016-05-01

    Current evidence indicates that there is a relationship between microRNA (miRNA)-mediated gene silencing and low-dose irradiation (LDIR) responses. Here, alterations of miRNA expression in response to LDIR exposure in male BALB/c mice and three different types of hepatocytes were investigated. The miRNome of the LDIR-exposed mouse spleens (0.01 Gy, 6.5 mGy/h) was analyzed, and the expression of miRNA and mRNA was validated by qRT-PCR. Western blotting, chromatin immunoprecipitation (ChIP), and luciferase assays were also performed to evaluate the interaction between miRNAs and their target genes and to gain insight into the regulation of miRNA expression. The expression of miRNA-193b-3p was down-regulated in the mouse spleen and liver and in various hepatocytes (NCTC, Hepa, and HepG2 cell lines) in response to LDIR. The down-regulation of miR-193b-3p expression was caused by histone deacetylation on the miR-193b-3p promoter in the HepG2 cells irradiated with 0.01 Gy. However, the alteration of histone deacetylation and miR-193b-3p and Rad51 expression in response to LDIR was restored by pretreatment with N-acetyl-cyctein. In conclusion, we provide evidence that miRNA responses to LDIR include the modulation of cellular stress responses and repair mechanisms.

  11. Specific inhibition of Wee1 kinase and Rad51 recombinase: A strategy to enhance the sensitivity of leukemic T-cells to ionizing radiation-induced DNA double-strand breaks

    SciTech Connect

    Havelek, Radim; Cmielova, Jana; Kralovec, Karel; Bruckova, Lenka; Bilkova, Zuzana; Fousova, Ivana; Sinkorova, Zuzana; Vavrova, Jirina; Rezacova, Martina

    2014-10-24

    Highlights: • Pre-treatment with the inhibitors increased the sensitivity of Jurkat cells to irradiation. • Combining both inhibitors together resulted in a G2 cell cycle arrest abrogation in Jurkat. • Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24 h upon irradiation. • Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction in MOLT-4 cells. • When dosed together, the combination decreased MOLT-4 cell survival. - Abstract: Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a part of their anticancer treatment. The objectives of the current study were to investigate the effects of the small molecule inhibitors of Wee1 kinase II (681641) and Rad51 (RI-1) on cell cycle progression, DNA double-strand breaks repair and apoptosis following ionizing radiation exposure in human leukemic T-cells Jurkat and MOLT-4. Pre-treatment with the Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to irradiation, however combining both inhibitors together resulted in a further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24 h upon irradiation. MOLT-4 cells were less affected by inhibitors application prior to ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4 cells.

  12. Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta

    DOE PAGES

    Patil, Shrikant; Moeys, Sara; von Dassow, Peter; ...

    2015-11-14

    Sexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase. The exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestralmore » loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase. Lastly, our results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom.« less

  13. Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers

    PubMed Central

    Kueh, Hao Yuan; Charras, Guillaume T.; Mitchison, Timothy J.; Brieher, William M.

    2008-01-01

    Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315–324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs. PMID:18663144

  14. Force Generation by Microtubule Assembly/Disassembly in Mitosis and Related Movements

    PubMed Central

    Inoué, Shinya; Salmon, Edward D.

    1995-01-01

    In this article, we review the dynamic nature of the filaments (microtubules) that make up the labile fibers of the mitotic spindle and asters, we discuss the roles that assembly and disassembly of microtubules play in mitosis, and we consider how such assembling and disassembling polymer filaments can generate forces that are utilized by the living cell in mitosis and related movements. Images PMID:8590794

  15. Pit disassembly motion control

    SciTech Connect

    Christensen, L.; Pittman, P. C.

    2001-01-01

    A Department of Energy (DOE) Pit Disassembly and Conversion Facility (PDCF) is being designed for the Savannah River Site in South Carolina. The facility will recover plutonium from excess nuclear weapon pits defined in START II and START III treaties. The plutonium will be stored and used to produce mixed oxide reactor fuel at another new DOE facility. Because of radiation dose issues, much of the pit disassembly work and material transfer will be automated. Automated material handling systems will interface with disassembly lathes, conversion reactors that produce oxide for storage, robotic container welding stations, vault retrieval systems, and nondestructive assay (NDA) instrumentation. The goal is to use common motion control hardware for material transfer and possibly common motion controllers for the unique PDCF systems. The latter is complicated by the different directions manufactures are considering for distributed control, such as Firewire, SERCOS, etc., and by the unique control requirements of machines such as lathes compared to controls for an integrated NDA system. The current design approach is to standardize where possible, use network cables to replace wire bundles where possible, but to first select hardware and motion controllers that meet specific machine or process requirements.

  16. Community disassembly in ephemeral ecosystems.

    PubMed

    O'Neill, Brian J

    2016-12-01

    Community disassembly is the non-random process of progressive species declines and losses. This process is usually studied to determine how various forces extirpate species, such as catastrophic disturbance, species invasions, habitat fragmentation, or unnatural/anthropogenic stressors. However, in ephemeral ecosystems, community disassembly is a natural and repeatable process. While many ephemeral ecosystems are aquatic (vernal pools, playa lakes, rock pools, saline lakes, phytotelmata, etc.), some disassembly patterns are applicable to other ecosystem types, including terrestrial ecosystems. As ephemeral waterbodies near the end of their hydroperiod, certain aspects fundamentally change. These fundamental changes or mechanisms cause visible patterns of community disassembly. Decreasing habitat size eliminates microhabitats and increases encounter rates between organisms, possibly increasing predation and competition. A harshening habitat eliminates low-tolerance species, changes the proportions of specialists/generalists, and forces organisms to acclimate, emigrate, or die. Additionally, ultraviolet light affects more of the water column, eliminating unprotected species. Furthermore, the entire metacommunity is often in similar stages of disassembly and collapses. Many of these mechanisms drive disassembly of terrestrial ephemeral habitats, such as animal carcasses, dung pads, or fungal fruiting bodies. Organisms obligate to ephemeral habitats have evolved to optimize their life history for a rapid life cycle with specific adaptations for themselves or their offspring to survive through the inactive period of the ecosystem. While some disassembly may occur too fast for biotic interactions or compensatory dynamics to be important, organisms undergoing natural disassembly should "expect" it. Thus, predictions of disassembly based on internal or biotic patterns may be more common in natural disassembly scenarios than in human-induced disassembly of permanent

  17. Effect of species-specific differences in chromosome morphology on chromatin compaction and the frequency and distribution of RAD51 and MLH1 foci in two bovid species: cattle (Bos taurus) and the common eland (Taurotragus oryx).

    PubMed

    Sebestova, Hana; Vozdova, Miluse; Kubickova, Svatava; Cernohorska, Halina; Kotrba, Radim; Rubes, Jiri

    2016-03-01

    Meiotic recombination between homologous chromosomes is crucial for their correct segregation into gametes and for generating diversity. We compared the frequency and distribution of MLH1 foci and RAD51 foci, synaptonemal complex (SC) length and DNA loop size in two related Bovidae species that share chromosome arm homology but show an extreme difference in their diploid chromosome number: cattle (Bos taurus, 2n = 60) and the common eland (Taurotragus oryx, 2nmale = 31). Compared to cattle, significantly fewer MLH1 foci per cell were observed in the common eland, which can be attributed to the lower number of initial double-strand breaks (DSBs) detected as RAD51 foci in leptonema. Despite the significantly shorter total autosomal SC length and longer DNA loop size of the common eland bi-armed chromosomes compared to those of bovine acrocentrics, the overall crossover density in the common eland was still lower than in cattle, probably due to the reduction in the number of MLH1 foci in the proximal regions of the bi-armed chromosomes. The formation of centric fusions during karyotype evolution of the common eland accompanied by meiotic chromatin compaction has greater implications in the reduction in the number of DSBs in leptonema than in the decrease of MLH1 foci number in pachynema.

  18. Single Nucleotide Polymorphisms in Noncoding Regions of Rad51C Do Not Change the Risk of Unselected Breast Cancer but They Modulate the Level of Oxidative Stress and the DNA Damage Characteristics: A Case-Control Study

    PubMed Central

    Gresner, Peter; Gromadzinska, Jolanta; Jablonska, Ewa; Stepnik, Maciej; Zambrano Quispe, Oscar; Twardowska, Ewa; Wasowicz, Wojciech

    2014-01-01

    Deleterious and missense mutations of RAD51C have recently been suggested to modulate the individual susceptibility to hereditary breast and ovarian cancer and unselected ovarian cancer, but not unselected breast cancer (BrC). We enrolled 132 unselected BrC females and 189 cancer-free female subjects to investigate whether common single nucleotide polymorphisms (SNPs) in non-coding regions of RAD51C modulate the risk of BrC, and whether they affect the level of oxidative stress and the extent/characteristics of DNA damage. Neither SNPs nor reconstructed haplotypes were found to significantly affect the unselected BrC risk. Contrary to this, carriers of rs12946522, rs16943176, rs12946397 and rs17222691 rare-alleles were found to present significantly increased level of blood plasma TBARS compared to respective wild-type homozygotes (p<0.05). Furthermore, these carriers showed significantly decreased fraction of oxidatively generated DNA damage (34% of total damaged DNA) in favor of DNA strand breakage, with no effect on total DNA damage, unlike respective wild-types, among which more evenly distributed proportions between oxidatively damaged DNA (48% of total DNA damage) and DNA strand breakage was found (p<0.0005 for the difference). Such effects were found among both the BrC cases and healthy subjects, indicating that they cannot be assumed as causal factors contributing to BrC development. PMID:25343521

  19. AGC-2 Disassembly Report

    SciTech Connect

    William Windes

    2014-05-01

    The Next Generation Nuclear Plant (NGNP) Graphite Research and Development (R&D) Program is currently measuring irradiated material properties for predicting the behavior and operating performance of new nuclear graphite grades available for use within the cores of new very high temperature reactor designs. The Advanced Graphite Creep (AGC) experiment, consisting of six irradiation capsules, will generate irradiated graphite performance data for NGNP reactor operating conditions. The AGC experiment is designed to determine the changes to specific material properties such as thermal diffusivity, thermal expansion, elastic modulus, mechanical strength, irradiation induced dimensional change rate, and irradiation creep for a wide variety of nuclear grade graphite types over a range of high temperature, and moderate doses. A series of six capsules containing graphite test specimens will be used to expose graphite test samples to a dose range from 1 to 7 dpa at three different temperatures (600, 900, and 1200°C) as described in the Graphite Technology Development Plan. Since irradiation induced creep within graphite components is considered critical to determining the operational life of the graphite core, some of the samples will also be exposed to an applied load to determine the creep rate for each graphite type under both temperature and neutron flux. All six AGC capsules in the experiment will be irradiated in the Advanced Test Reactor (ATR). AGC-1 and AGC-2 will be irradiated in the south flux trap and AGC-3–AGC-6 will be irradiated in the east flux trap. The change in flux traps is due to NGNP irradiation priorities requiring the AGC experiment to be moved to accommodate Fuel irradiation experiments. After irradiation, all six AGC capsules will be cooled in the ATR Canal, sized for shipment, and shipped to the Materials and Fuels Complex (MFC) where the capsule will be disassembled in the Hot Fuel Examination Facility (HFEF). During disassembly, the metallic

  20. Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition.

    PubMed

    Morozov, V; Wawrousek, E F

    2008-03-01

    Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs alone, or in combination with other factors, to introduce a point mutation into the alphaB-crystallin gene in one-celled mouse embryos. We found that co-injection of ssDNA along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative nonhomologous end-joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been previously reported.

  1. Static Detection of Disassembly Errors

    SciTech Connect

    Krishnamoorthy, Nithya; Debray, Saumya; Fligg, Alan K

    2009-10-13

    Static disassembly is a crucial first step in reverse engineering executable files, and there is a consider- able body of work in reverse-engineering of binaries, as well as areas such as semantics-based security anal- ysis, that assumes that the input executable has been correctly disassembled. However, disassembly errors, e.g., arising from binary obfuscations, can render this assumption invalid. This work describes a machine- learning-based approach, using decision trees, for stat- ically identifying possible errors in a static disassem- bly; such potential errors may then be examined more closely, e.g., using dynamic analyses. Experimental re- sults using a variety of input executables indicate that our approach performs well, correctly identifying most disassembly errors with relatively few false positives.

  2. Partial disassembly of peroxisomes

    PubMed Central

    1985-01-01

    Rat liver peroxisomes were subjected to a variety of procedures intended to partially disassemble or damage them; the effects were analyzed by recentrifugation into sucrose gradients, enzyme analyses, electron microscopy, and SDS PAGE. Freezing and thawing or mild sonication released some matrix proteins and produced apparently intact peroxisomal "ghosts" with crystalloid cores and some fuzzy fibrillar content. Vigorous sonication broke open the peroxisomes but the membranes remained associated with cores and fibrillar and amorphous matrix material. The density of both ghosts and more severely damaged peroxisomes was approximately 1.23. Pyrophosphate (pH 9) treatment solubilized the fibrillar content, yielding ghosts that were empty except for cores. Some matrix proteins such as catalase and thiolase readily leak from peroxisomes. Other proteins were identified that remain in mechanically damaged peroxisomes but are neither core nor membrane proteins because they can be released by pyrophosphate treatment. These constitute a class of poorly soluble matrix proteins that appear to correspond to the fibrillar material observed morphologically. All of the peroxisomal beta-oxidation enzymes are located in the matrix, but they vary greatly in how easily they leak out. Palmitoyl coenzyme A synthetase is in the membrane, based on its co-distribution with the 22-kilodalton integral membrane polypeptide. PMID:2989301

  3. Associations of common variants at 1p11.2 and 14q24.1 (RAD51L1) with breast cancer risk and heterogeneity by tumor subtype: findings from the Breast Cancer Association Consortium.

    PubMed

    Figueroa, Jonine D; Garcia-Closas, Montserrat; Humphreys, Manjeet; Platte, Radka; Hopper, John L; Southey, Melissa C; Apicella, Carmel; Hammet, Fleur; Schmidt, Marjanka K; Broeks, Annegien; Tollenaar, Rob A E M; Van't Veer, Laura J; Fasching, Peter A; Beckmann, Matthias W; Ekici, Arif B; Strick, Reiner; Peto, Julian; dos Santos Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Sawyer, Elinor; Tomlinson, Ian; Kerin, Michael; Burwinkel, Barbara; Marme, Federik; Schneeweiss, Andreas; Sohn, Christof; Bojesen, Stig; Flyger, Henrik; Nordestgaard, Børge G; Benítez, Javier; Milne, Roger L; Ignacio Arias, Jose; Zamora, M Pilar; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Rahman, Nazneen; Turnbull, Clare; Seal, Sheila; Renwick, Anthony; Brauch, Hiltrud; Justenhoven, Christina; Brüning, Thomas; Chang-Claude, Jenny; Hein, Rebecca; Wang-Gohrke, Shan; Dörk, Thilo; Schürmann, Peter; Bremer, Michael; Hillemanns, Peter; Nevanlinna, Heli; Heikkinen, Tuomas; Aittomäki, Kristiina; Blomqvist, Carl; Bogdanova, Natalia; Antonenkova, Natalia; Rogov, Yuri I; Karstens, Johann Hinrich; Bermisheva, Marina; Prokofieva, Darya; Gantcev, Shamil Hanafievich; Khusnutdinova, Elza; Lindblom, Annika; Margolin, Sara; Chenevix-Trench, Georgia; Beesley, Jonathan; Chen, Xiaoqing; Mannermaa, Arto; Kosma, Veli-Matti; Soini, Ylermi; Kataja, Vesa; Lambrechts, Diether; Yesilyurt, Betül T; Chrisiaens, Marie-Rose; Peeters, Stephanie; Radice, Paolo; Peterlongo, Paolo; Manoukian, Siranoush; Barile, Monica; Couch, Fergus; Lee, Adam M; Diasio, Robert; Wang, Xianshu; Giles, Graham G; Severi, Gianluca; Baglietto, Laura; Maclean, Catriona; Offit, Ken; Robson, Mark; Joseph, Vijai; Gaudet, Mia; John, Esther M; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Andrulis, Irene; Knight, Julia A; Mulligan, Anna Marie; O'Malley, Frances P; Brinton, Louise A; Sherman, Mark E; Lissowska, Jolanta; Chanock, Stephen J; Hooning, Maartje; Martens, John W M; van den Ouweland, Ans M W; Collée, J Margriet; Hall, Per; Czene, Kamila; Cox, Angela; Brock, Ian W; Reed, Malcolm W R; Cross, Simon S; Pharoah, Paul; Dunning, Alison M; Kang, Daehee; Yoo, Keun-Young; Noh, Dong-Young; Ahn, Sei-Hyun; Jakubowska, Anna; Lubinski, Jan; Jaworska, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; McKay, James; Shen, Chen-Yang; Ding, Shian-ling; Hsu, Huan-Ming; Yu, Jyh-Cherng; Anton-Culver, Hoda; Ziogas, Argyrios; Ashworth, Alan; Swerdlow, Anthony; Jones, Michael; Orr, Nick; Trentham-Dietz, Amy; Egan, Kathleen; Newcomb, Polly; Titus-Ernstoff, Linda; Easton, Doug; Spurdle, Amanda B

    2011-12-01

    A genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) at 1p11.2 and 14q24.1 (RAD51L1) as breast cancer susceptibility loci. The initial GWAS suggested stronger effects for both loci for estrogen receptor (ER)-positive tumors. Using data from the Breast Cancer Association Consortium (BCAC), we sought to determine whether risks differ by ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), grade, node status, tumor size, and ductal or lobular morphology. We genotyped rs11249433 at 1p.11.2, and two highly correlated SNPs rs999737 and rs10483813 (r(2)= 0.98) at 14q24.1 (RAD51L1), for up to 46 036 invasive breast cancer cases and 46 930 controls from 39 studies. Analyses by tumor characteristics focused on subjects reporting to be white women of European ancestry and were based on 25 458 cases, of which 87% had ER data. The SNP at 1p11.2 showed significantly stronger associations with ER-positive tumors [per-allele odds ratio (OR) for ER-positive tumors was 1.13, 95% CI = 1.10-1.16 and, for ER-negative tumors, OR was 1.03, 95% CI = 0.98-1.07, case-only P-heterogeneity = 7.6 × 10(-5)]. The association with ER-positive tumors was stronger for tumors of lower grade (case-only P= 6.7 × 10(-3)) and lobular histology (case-only P= 0.01). SNPs at 14q24.1 were associated with risk for most tumor subtypes evaluated, including triple-negative breast cancers, which has not been described previously. Our results underscore the need for large pooling efforts with tumor pathology data to help refine risk estimates for SNP associations with susceptibility to different subtypes of breast cancer.

  4. RAD51 and XRCC3 polymorphisms: impact on the risk and treatment outcomes of de novo inv(16) or t(16;16)/CBFβ-MYH11(+) acute myeloid leukemia.

    PubMed

    Liu, Liang; Yang, Lin; Mi, Yingchang; Wang, Jianxiang; Li, Jianyong; Zhang, Yue; Ma, Xiaotang; Qin, Tiejun; Xu, Zefeng; Xiao, Zhijian

    2011-08-01

    DNA double-strand break repair via homologous recombination (HR) is essential in maintaining genetic integrity, and may modulate susceptibility to the development of acute myeloid leukemia (AML) and influence outcomes of AML. This study was designed to evaluate the effects of polymorphisms in HR repair genes RAD51 and XRCC3 on the risk and treatment outcomes of inv(16)/t(16;16)/CBFβ-MYH11(+) AML. The distribution of polymorphisms in RAD51-G135C and XRCC3-Thr241Met were studied by PCR-RFLP analysis in 625 cases of de novo AML, including 105 cases with inv(16)/t(16;16)/CBFβ-MYH11, 806 family controls and 704 volunteer controls. It was found that the XRCC3-241Met variant significantly increased the risk of the development of the AML with inv(16)/t(16;16) as compared with both the volunteer control (OR=7.22; 95% CI, 4.37-11.91) and the family control (OR=7.99; 95% CI, 5.03-12.69). A retrospective study conducted in 103 inv(16)/t(16;16) AML patients. In multivariate analysis for the potential prognostic factors, the XRCC3-241Met variant significantly reduced disease-free survival (DFS) in complete remission (CR) achieved patients (HR=2.34, 95% CI, 1.32-4.16). These data indicate that the XRCC3-241Met variant may not be only a susceptibility factor to the AML with inv(16)/t(16;16), but also an independent poor-prognostic factor for this AML subtype.

  5. Single-molecule studies of actin assembly and disassembly factors.

    PubMed

    Smith, Benjamin A; Gelles, Jeff; Goode, Bruce L

    2014-01-01

    The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks.

  6. Desmosome assembly and disassembly are membrane raft-dependent.

    PubMed

    Stahley, Sara N; Saito, Masataka; Faundez, Victor; Koval, Michael; Mattheyses, Alexa L; Kowalczyk, Andrew P

    2014-01-01

    Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV.

  7. Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

    PubMed Central

    Faundez, Victor; Koval, Michael; Mattheyses, Alexa L.; Kowalczyk, Andrew P.

    2014-01-01

    Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV. PMID:24498201

  8. Associations of common variants at 1p11.2 and 14q24.1 (RAD51L1) with breast cancer risk and heterogeneity by tumor subtype: findings from the Breast Cancer Association Consortium†

    PubMed Central

    Figueroa, Jonine D.; Garcia-Closas, Montserrat; Humphreys, Manjeet; Platte, Radka; Hopper, John L.; Southey, Melissa C.; Apicella, Carmel; Hammet, Fleur; Schmidt, Marjanka K.; Broeks, Annegien; Tollenaar, Rob A.E.M.; Van't Veer, Laura J.; Fasching, Peter A.; Beckmann, Matthias W.; Ekici, Arif B.; Strick, Reiner; Peto, Julian; dos Santos Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Sawyer, Elinor; Tomlinson, Ian; Kerin, Michael; Burwinkel, Barbara; Marme, Federik; Schneeweiss, Andreas; Sohn, Christof; Bojesen, Stig; Flyger, Henrik; Nordestgaard, Børge G.; Benítez, Javier; Milne, Roger L.; Ignacio Arias, Jose; Zamora, M. Pilar; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Rahman, Nazneen; Turnbull, Clare; Seal, Sheila; Renwick, Anthony; Brauch, Hiltrud; Justenhoven, Christina; Brüning, Thomas; Chang-Claude, Jenny; Hein, Rebecca; Wang-Gohrke, Shan; Dörk, Thilo; Schürmann, Peter; Bremer, Michael; Hillemanns, Peter; Nevanlinna, Heli; Heikkinen, Tuomas; Aittomäki, Kristiina; Blomqvist, Carl; Bogdanova, Natalia; Antonenkova, Natalia; Rogov, Yuri I.; Karstens, Johann Hinrich; Bermisheva, Marina; Prokofieva, Darya; Hanafievich Gantcev, Shamil; Khusnutdinova, Elza; Lindblom, Annika; Margolin, Sara; Chenevix-Trench, Georgia; Beesley, Jonathan; Chen, Xiaoqing; Mannermaa, Arto; Kosma, Veli-Matti; Soini, Ylermi; Kataja, Vesa; Lambrechts, Diether; Yesilyurt, Betül T.; Chrisiaens, Marie-Rose; Peeters, Stephanie; Radice, Paolo; Peterlongo, Paolo; Manoukian, Siranoush; Barile, Monica; Couch, Fergus; Lee, Adam M.; Diasio, Robert; Wang, Xianshu; Giles, Graham G.; Severi, Gianluca; Baglietto, Laura; Maclean, Catriona; Offit, Ken; Robson, Mark; Joseph, Vijai; Gaudet, Mia; John, Esther M.; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Andrulis, Irene; Knight, Julia A.; Marie Mulligan, Anna; O'Malley, Frances P.; Brinton, Louise A.; Sherman, Mark E.; Lissowska, Jolanta; Chanock, Stephen J.; Hooning, Maartje; Martens, John W.M.; van den Ouweland, Ans M.W.; Collée, J. Margriet; Hall, Per; Czene, Kamila; Cox, Angela; Brock, Ian W.; Reed, Malcolm W.R.; Cross, Simon S.; Pharoah, Paul; Dunning, Alison M.; Kang, Daehee; Yoo, Keun-Young; Noh, Dong-Young; Ahn, Sei-Hyun; Jakubowska, Anna; Lubinski, Jan; Jaworska, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; McKay, James; Shen, Chen-Yang; Ding, Shian-ling; Hsu, Huan-Ming; Yu, Jyh-Cherng; Anton-Culver, Hoda; Ziogas, Argyrios; Ashworth, Alan; Swerdlow, Anthony; Jones, Michael; Orr, Nick; Trentham-Dietz, Amy; Egan, Kathleen; Newcomb, Polly; Titus-Ernstoff, Linda; Easton, Doug; Spurdle, Amanda B.

    2011-01-01

    A genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) at 1p11.2 and 14q24.1 (RAD51L1) as breast cancer susceptibility loci. The initial GWAS suggested stronger effects for both loci for estrogen receptor (ER)-positive tumors. Using data from the Breast Cancer Association Consortium (BCAC), we sought to determine whether risks differ by ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), grade, node status, tumor size, and ductal or lobular morphology. We genotyped rs11249433 at 1p.11.2, and two highly correlated SNPs rs999737 and rs10483813 (r2= 0.98) at 14q24.1 (RAD51L1), for up to 46 036 invasive breast cancer cases and 46 930 controls from 39 studies. Analyses by tumor characteristics focused on subjects reporting to be white women of European ancestry and were based on 25 458 cases, of which 87% had ER data. The SNP at 1p11.2 showed significantly stronger associations with ER-positive tumors [per-allele odds ratio (OR) for ER-positive tumors was 1.13, 95% CI = 1.10–1.16 and, for ER-negative tumors, OR was 1.03, 95% CI = 0.98–1.07, case-only P-heterogeneity = 7.6 × 10−5]. The association with ER-positive tumors was stronger for tumors of lower grade (case-only P= 6.7 × 10−3) and lobular histology (case-only P= 0.01). SNPs at 14q24.1 were associated with risk for most tumor subtypes evaluated, including triple-negative breast cancers, which has not been described previously. Our results underscore the need for large pooling efforts with tumor pathology data to help refine risk estimates for SNP associations with susceptibility to different subtypes of breast cancer. PMID:21852249

  9. [Mutagenicity, genotoxicity and gene expression of Rad51C, Xiap, P53 and Nrf2 induced by antimalarial extracts of plants collected from the middle Vaupés region, Colombia].

    PubMed

    Barbosa, Claudia Viviana; Muskus, Carlos Enrique; Orozco, Luz Yaneth; Pabón, Adriana

    2017-09-01

    Due to Plasmodium resistance to antimalarial drugs, it is important to find new therapeutic alternatives for malaria treatment and control. Based on the knowledge of Colombian indigenous communities, we collected extracts of plants with potential antimalarial effects from the middle Vaupés region. To evaluate the mutagenic and genotoxic effects, as well as the gene expression of Rad51C, Xiap, P53 and Nrf2 induced by four ethanolic extracts with antimalarial activity (R001, T002, T015 and T028). We evaluated four ethanolic extracts with antimalarial activity using the Ames test to assess mutagenicity, and the comet assay on HepG2 cells to determine the genotoxicicity. We also evaluated the expression of Rad51C, Xiap, P53 and Nrf2 from HepG2 cells stimulated with the four extracts. None of the four extracts was mutagenic in Salmonella typhimurium TA98 strain in the presence and absence of S9 metabolic activity. Extracts R001, T015 and T028 were weakly mutagenic on the TA100 strain in the presence of S9, with mutagenic indexes (MI) of 1.58, 1.53 and 1.61, respectively. The T015 strain showed the same behavior without S9 with an MI of 1.36. The results of the comet assay showed that the four extracts produced category 1 or 2 damage, with comets between 36.7 and 51.48 μm in length. However, the genetic damage index suggested that most of the cells were affected by the treatments. Regarding gene expression, extracts R001 and T028 induced an overexpression of genes Xiap and P53 with an 1.84 to 3.99 fold-change compared with untreated cells. These results revealed that the T002 extract was the safest as it had antimalarial activity and was not cytotoxic on HepG2 cells. Moreover, it was not mutagenic and it only produced category 1 damage on the DNA. Also, the extract did not induce a change in the expression of the tested genes.

  10. An integrated in silico approach to analyze the involvement of single amino acid polymorphisms in FANCD1/BRCA2-PALB2 and FANCD1/BRCA2-RAD51 complex.

    PubMed

    Doss, C George Priya; Nagasundaram, N

    2014-11-01

    Fanconi anemia (FA) is an autosomal recessive human disease characterized by genomic instability and a marked increase in cancer risk. The importance of FANCD1 gene is manifested by the fact that deleterious amino acid substitutions were found to confer susceptibility to hereditary breast and ovarian cancers. Attaining experimental knowledge about the possible disease-associated substitutions is laborious and time consuming. The recent introduction of genome variation analyzing in silico tools have the capability to identify the deleterious variants in an efficient manner. In this study, we conducted in silico variation analysis of deleterious non-synonymous SNPs at both functional and structural level in the breast cancer and FA susceptibility gene BRCA2/FANCD1. To identify and characterize deleterious mutations in this study, five in silico tools based on two different prediction methods namely pathogenicity prediction (SIFT, PolyPhen, and PANTHER), and protein stability prediction (I-Mutant 2.0 and MuStab) were analyzed. Based on the deleterious scores that overlap in these in silico approaches, and the availability of three-dimensional structures, structure analysis was carried out with the major mutations that occurred in the native protein coded by FANCD1/BRCA2 gene. In this work, we report the results of the first molecular dynamics (MD) simulation study performed to analyze the structural level changes in time scale level with respect to the native and mutated protein complexes (G25R, W31C, W31R in FANCD1/BRCA2-PALB2, and F1524V, V1532F in FANCD1/BRCA2-RAD51). Analysis of the MD trajectories indicated that predicted deleterious variants alter the structural behavior of BRCA2-PALB2 and BRCA2-RAD51 protein complexes. In addition, statistical analysis was employed to test the significance of these in silico tool predictions. Based on these predictions, we conclude that the identification of disease-related SNPs by in silico methods, in combination with MD

  11. Methylation of RAD51B, XRCC3 and other homologous recombination genes is associated with expression of immune checkpoints and an inflammatory signature in squamous cell carcinoma of the head and neck, lung and cervix

    PubMed Central

    Rieke, Damian T.; Ochsenreither, Sebastian; Klinghammer, Konrad; Seiwert, Tanguy Y.; Klauschen, Frederick; Tinhofer, Inge; Keilholz, Ulrich

    2016-01-01

    Immune checkpoints are emerging treatment targets, but mechanisms underlying checkpoint expression are poorly understood. Since alterations in DNA repair genes have been connected to the efficacy of checkpoint inhibitors, we investigated associations between methylation of DNA repair genes and CTLA4 and CD274 (PD-L1) expression. A list of DNA repair genes (179 genes) was selected from the literature, methylation status and expression of inflammation-associated genes (The Cancer Genome Atlas data) was correlated in head and neck squamous cell carcinoma (HNSCC), cervical and lung squamous cell carcinoma. A significant positive correlation of the methylation status of 15, 3 and 2 genes with checkpoint expression was identified, respectively. RAD51B methylation was identified in all cancer subtypes. In HNSCC and cervical cancer, there was significant enrichment for homologous recombination genes. Methylation of the candidate genes was also associated with expression of other checkpoints, ligands, MHC- and T-cell associated genes as well as an interferon-inflammatory immune gene signature, predictive for the efficacy of PD-1 inhibition in HNSCC. Homologous recombination deficiency might therefore be mediated by DNA repair gene hypermethylation and linked to an immune-evasive phenotype in SCC. The methylation status of these genes could represent a new predictive biomarker for immune checkpoint inhibition. PMID:27683114

  12. Homologous and homeologous intermolecular gene conversion are not differentially affected by mutations in the DNA damage or the mismatch repair genes RAD1, RAD50, RAD51, RAD52, RAD54, PMS1 and MSH2

    SciTech Connect

    Porter, G.; Westmoreland, J.; Priebe, S.

    1996-06-01

    Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs. homologous recombination differentially. Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants. Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations. DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five- to 10-fold reduction in homeologous relative to homologous recombination regardless of background. Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence. Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction. A comparison of Rad{sup +} vs. rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts. 67 refs., 5 figs., 4 tabs.

  13. Chinese Herbal Mixture, Tien-Hsien Liquid, Induces G2/M Cycle Arrest and Radiosensitivity in MCF-7 Human Breast Cancer Cells through Mechanisms Involving DNMT1 and Rad51 Downregulation

    PubMed Central

    Chow, Jyh-Ming; Yang, Chia-Ming; Kuo, Hui-Ching; Chang, Chia-Lun; Lee, Hsin-Lun; Lai, I-Chun; Chuang, Shuang-En

    2016-01-01

    The Chinese herbal mixture, Tien-Hsien Liquid (THL), has been proven to suppress the growth and invasiveness of cancer cells and is currently regarded as a complementary medicine for the treatment of cancer. Our previous study using acute promyelocytic leukemia cells uncovered its effect on the downregulation of DNA methyltransferase 1 (DNMT1) which is often overexpressed in cancer cells resulting in the repression of tumor suppressors via hypermethylation. Herein, we explored the effects of THL in MCF-7 breast cancer cells that also demonstrate elevated DNMT1. The results show that THL dose-dependently downregulated DNMT1 accompanied by the induction of tumor suppressors such as p21 and p15. THL arrested cell cycle in G2/M phase and decreased the protein levels of cyclin A, cyclin B1, phospho-pRb, and AKT. DNMT1 inhibition was previously reported to exert a radiosensitizing effect in cancer cells through the repression of DNA repair. We found that THL enhanced radiation-induced clonogenic cell death in MCF-7 cells and decreased the level of DNA double-strand break repair protein, Rad51. Our observations may be the result of DNMT1 downregulation. Due to the fact that DNMT1 inhibition is now a mainstream strategy for anticancer therapy, further clinical trials of THL to confirm its clinical efficacy are warranted. PMID:27525019

  14. Homologous and Homeologous Intermolecular Gene Conversion Are Not Differentially Affected by Mutations in the DNA Damage or the Mismatch Repair Genes Rad1, Rad50, Rad51, Rad52, Rad54, Pms1 and Msh2

    PubMed Central

    Porter, G.; Westmoreland, J.; Priebe, S.; Resnick, M. A.

    1996-01-01

    Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs. homologous recombination differentially. Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants. Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations. DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five- to 10-fold reduction in homeologous relative to homologous recombination regardless of background. Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence. Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction. A comparison of Rad(+) vs. rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts. PMID:8725224

  15. Single-molecule imaging of a three-component ordered actin disassembly mechanism

    PubMed Central

    Jansen, Silvia; Collins, Agnieszka; Chin, Samantha M.; Ydenberg, Casey A.; Gelles, Jeff; Goode, Bruce L.

    2015-01-01

    The mechanisms by which cells destabilize and rapidly disassemble filamentous actin networks have remained elusive; however, Coronin, Cofilin and AIP1 have been implicated in this process. Here using multi-wavelength single-molecule fluorescence imaging, we show that mammalian Cor1B, Cof1 and AIP1 work in concert through a temporally ordered pathway to induce highly efficient severing and disassembly of actin filaments. Cor1B binds to filaments first, and dramatically accelerates the subsequent binding of Cof1, leading to heavily decorated, stabilized filaments. Cof1 in turn recruits AIP1, which rapidly triggers severing and remains bound to the newly generated barbed ends. New growth at barbed ends generated by severing was blocked specifically in the presence of all three proteins. This activity enabled us to reconstitute and directly visualize single actin filaments being rapidly polymerized by formins at their barbed ends while simultanteously being stochastically severed and capped along their lengths, and disassembled from their pointed ends. PMID:25995115

  16. Simulation-based disassembly systems design

    NASA Astrophysics Data System (ADS)

    Ohlendorf, Martin; Herrmann, Christoph; Hesselbach, Juergen

    2004-02-01

    Recycling of Waste of Electrical and Electronic Equipment (WEEE) is a matter of actual concern, driven by economic, ecological and legislative reasons. Here, disassembly as the first step of the treatment process plays a key role. To achieve sustainable progress in WEEE disassembly, the key is not to limit analysis and planning to merely disassembly processes in a narrow sense, but to consider entire disassembly plants including additional aspects such as internal logistics, storage, sorting etc. as well. In this regard, the paper presents ways of designing, dimensioning, structuring and modeling different disassembly systems. Goal is to achieve efficient and economic disassembly systems that allow recycling processes complying with legal requirements. Moreover, advantages of applying simulation software tools that are widespread and successfully utilized in conventional industry sectors are addressed. They support systematic disassembly planning by means of simulation experiments including consecutive efficiency evaluation. Consequently, anticipatory recycling planning considering various scenarios is enabled and decisions about which types of disassembly systems evidence appropriateness for specific circumstances such as product spectrum, throughput, disassembly depth etc. is supported. Furthermore, integration of simulation based disassembly planning in a holistic concept with configuration of interfaces and data utilization including cost aspects is described.

  17. Choreography of importin-α/CAS complex assembly and disassembly at nuclear pores.

    PubMed

    Sun, Changxia; Fu, Guo; Ciziene, Danguole; Stewart, Murray; Musser, Siegfried M

    2013-04-23

    Nuclear pore complexes (NPCs) mediate the exchange of macromolecules between the cytoplasm and the nucleoplasm. Soluble nuclear transport receptors bind signal-dependent cargos to form transport complexes that diffuse through the NPC and are then disassembled. Although transport receptors enable the NPC's permeability barrier to be overcome, directionality is established by complex assembly and disassembly. Here, we delineate the choreography of importin-α/CAS complex assembly and disassembly in permeabilized cells, using single-molecule fluorescence resonance energy transfer and particle tracking. Monitoring interaction sequences in intact NPCs ensures spatiotemporal preservation of structures and interactions critical for activity in vivo. We show that key interactions between components are reversible, multiple outcomes are often possible, and the assembly and disassembly of complexes are precisely controlled to occur at the appropriate place and time. Importin-α mutants that impair interactions during nuclear import were used together with cytoplasmic Ran GTPase-activating factors to demonstrate that importin-α/CAS complexes form in the nuclear basket region, at the termination of protein import, and disassembly of importin-α/CAS complexes after export occurs in the cytoplasmic filament region of the NPC. Mathematical models derived from our data emphasize the intimate connection between transport and the coordinated assembly and disassembly of importin-α/CAS complexes for generating productive transport cycles.

  18. Choreography of importin-α/CAS complex assembly and disassembly at nuclear pores

    PubMed Central

    Sun, Changxia; Fu, Guo; Ciziene, Danguole; Stewart, Murray; Musser, Siegfried M.

    2013-01-01

    Nuclear pore complexes (NPCs) mediate the exchange of macromolecules between the cytoplasm and the nucleoplasm. Soluble nuclear transport receptors bind signal-dependent cargos to form transport complexes that diffuse through the NPC and are then disassembled. Although transport receptors enable the NPC's permeability barrier to be overcome, directionality is established by complex assembly and disassembly. Here, we delineate the choreography of importin-α/CAS complex assembly and disassembly in permeabilized cells, using single-molecule fluorescence resonance energy transfer and particle tracking. Monitoring interaction sequences in intact NPCs ensures spatiotemporal preservation of structures and interactions critical for activity in vivo. We show that key interactions between components are reversible, multiple outcomes are often possible, and the assembly and disassembly of complexes are precisely controlled to occur at the appropriate place and time. Importin-α mutants that impair interactions during nuclear import were used together with cytoplasmic Ran GTPase-activating factors to demonstrate that importin-α/CAS complexes form in the nuclear basket region, at the termination of protein import, and disassembly of importin-α/CAS complexes after export occurs in the cytoplasmic filament region of the NPC. Mathematical models derived from our data emphasize the intimate connection between transport and the coordinated assembly and disassembly of importin-α/CAS complexes for generating productive transport cycles. PMID:23569239

  19. Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta

    SciTech Connect

    Patil, Shrikant; Moeys, Sara; von Dassow, Peter; Huysman, Marie J. J.; Mapleson, Daniel; De Veylder, Lieven; Sanges, Remo; Vyverman, Wim; Montresor, Marina; Ferrante, Maria Immacolata

    2015-11-14

    Sexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase. The exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestral loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase. Lastly, our results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom.

  20. Mechanism of interaction of Dictyostelium severin with actin filaments

    PubMed Central

    1982-01-01

    Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+ -dependent manner, was purified 500-fold to greater than 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29 A and consists of a single polypeptide chain. It contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluoresence-labeled severin shows that this protein changes its conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 approximately equal to 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 approximately equal to 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin: severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo. PMID:6897549

  1. Quantitative Analysis of Actin Turnover in Listeria Comet Tails: Evidence for Catastrophic Filament Turnover

    PubMed Central

    Kueh, Hao Yuan; Brieher, William M.; Mitchison, Timothy J.

    2010-01-01

    Rapid assembly and disassembly (turnover) of actin filaments in cytoplasm drives cell motility and shape remodeling. While many biochemical processes that facilitate filament turnover are understood in isolation, it remains unclear how they work together to promote filament turnover in cells. Here, we studied cellular mechanisms of actin filament turnover by combining quantitative microscopy with mathematical modeling. Using live cell imaging, we found that actin polymer mass decay in Listeria comet tails is very well fit by a simple exponential. By analyzing candidate filament turnover pathways using stochastic modeling, we found that exponential polymer mass decay is consistent with either slow treadmilling, slow Arp2/3-dissociation, or catastrophic bursts of disassembly, but is inconsistent with acceleration of filament turnover by severing. Imaging of single filaments in Xenopus egg extract provided evidence that disassembly by bursting dominates isolated filament turnover in a cytoplasmic context. Taken together, our results point to a pathway where filaments grow transiently from barbed ends, rapidly terminate growth to enter a long-lived stable state, and then undergo a catastrophic burst of disassembly. By keeping filament lengths largely constant over time, such catastrophic filament turnover may enable cellular actin assemblies to maintain their mechanical integrity as they are turning over. PMID:20923649

  2. CALUTRON ASSEMBLING AND DISASSEMBLING MEANS

    DOEpatents

    Andrews, R.E.; Thornton, J.

    1959-01-27

    This patent relates to the assembling and disassembling of a calutron and, more specifically describes a calutron having the ion separating mechanism carried by a fuce plate removably secured to the tank. When it is desired to withdraw the ion separating mechanism from the tank, a motor is energized and a carriage attached through a bracket to the fuce plate is driven along a track. The face plate moves out from the tank in substantially a linear direction, preventing injury to the ion separating mechanism.

  3. Disassemblability modeling technology of configurable product based on disassembly constraint relation weighted design structure matrix(DSM)

    NASA Astrophysics Data System (ADS)

    Qiu, Lemiao; Liu, Xiaojian; Zhang, Shuyou; Sun, Liangfeng

    2014-05-01

    The current research of configurable product disassemblability focuses on disassemblability evaluation and disassembly sequence planning. Little work has been done on quantitative analysis of configurable product disassemblability. The disassemblability modeling technology for configurable product based on disassembly constraint relation weighted design structure matrix (DSM) is proposed. Major factors affecting the disassemblability of configurable product are analyzed, and the disassembling degrees between components in configurable product are obtained by calculating disassembly entropies such as joint type, joint quantity, disassembly path, disassembly accessibility and material compatibility. The disassembly constraint relation weighted DSM of configurable product is constructed and configuration modules are formed by matrix decomposition and tearing operations. The disassembly constraint relation in configuration modules is strong coupling, and the disassembly constraint relation between modules is weak coupling, and the disassemblability configuration model is constructed based on configuration module. Finally, taking a hydraulic forging press as an example, the decomposed weak coupling components are used as configuration modules alone, components with a strong coupling are aggregated into configuration modules, and the disassembly sequence of components inside configuration modules is optimized by tearing operation. A disassemblability configuration model of the hydraulic forging press is constructed. By researching the disassemblability modeling technology of product configuration design based on disassembly constraint relation weighted DSM, the disassembly property in maintenance, recycling and reuse of configurable product are optimized.

  4. Modeling operational behavior of a disassembly line

    NASA Astrophysics Data System (ADS)

    Kizilkaya, Elif A.; Gupta, Surendra M.

    2004-12-01

    In this paper we present a dynamic kanban (pull) system specifically developed for disassembly lines. This type of kanban system is much more complex than the traditional kanban system used in assembly lines. For instance, unlike the assembly line where the external demand occurs only at the last station, the demands in the disassembly case also occur at any of the intermittent stations. The reason is that as a product moves on the disassembly line, various parts are disassembled at every station and accumulated at that station. Therefore, there are as many demand sources as there are number of parts. We consider a case example involving the end-of-life products. Based on the precedence relationships and other criteria such as hazardous properties of the parts, we balance the disassembly line. The results of the disassembly line-balancing problem (DLBP) are used as input to the proposed dynamic kanban system for disassembly line (DKSDL). We compare the performance of the DKSDL to the modified kanban system for disassembly line (MKSDL), which was previously introduced by the authors. We show, via simulation, that the DKSDL is far superior to MKSDL considered.

  5. 19 CFR 181.132 - Disassembly.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 2 2010-04-01 2010-04-01 false Disassembly. 181.132 Section 181.132 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY (CONTINUED) NORTH AMERICAN FREE TRADE AGREEMENT Rules of Origin § 181.132 Disassembly. (a) Treated as...

  6. 19 CFR 181.132 - Disassembly.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 2 2011-04-01 2011-04-01 false Disassembly. 181.132 Section 181.132 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY (CONTINUED) NORTH AMERICAN FREE TRADE AGREEMENT Rules of Origin § 181.132 Disassembly. (a) Treated as...

  7. 19 CFR 181.132 - Disassembly.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 2 2012-04-01 2012-04-01 false Disassembly. 181.132 Section 181.132 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY (CONTINUED) NORTH AMERICAN FREE TRADE AGREEMENT Rules of Origin § 181.132 Disassembly. (a) Treated as...

  8. 19 CFR 181.132 - Disassembly.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 2 2014-04-01 2014-04-01 false Disassembly. 181.132 Section 181.132 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY (CONTINUED) NORTH AMERICAN FREE TRADE AGREEMENT Rules of Origin § 181.132 Disassembly. (a) Treated as...

  9. 19 CFR 181.132 - Disassembly.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 2 2013-04-01 2013-04-01 false Disassembly. 181.132 Section 181.132 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY (CONTINUED) NORTH AMERICAN FREE TRADE AGREEMENT Rules of Origin § 181.132 Disassembly. (a) Treated as...

  10. Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation

    PubMed Central

    Yamagishi, Yuka; Abe, Hiroshi

    2015-01-01

    We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation. PMID:26424802

  11. Disassembling Iron Availability to Phytoplankton

    PubMed Central

    Shaked, Yeala; Lis, Hagar

    2012-01-01

    The bioavailability of iron to microorganisms and its underlying mechanisms have far reaching repercussions to many natural systems and diverse fields of research, including ocean biogeochemistry, carbon cycling and climate, harmful algal blooms, soil and plant research, bioremediation, pathogenesis, and medicine. Within the framework of ocean sciences, short supply and restricted bioavailability of Fe to phytoplankton is thought to limit primary production and curtail atmospheric CO2 drawdown in vast ocean regions. Yet a clear-cut definition of bioavailability remains elusive, with elements of iron speciation and kinetics, phytoplankton physiology, light, temperature, and microbial interactions, to name a few, all intricately intertwined into this concept. Here, in a synthesis of published and new data, we attempt to disassemble the complex concept of iron bioavailability to phytoplankton by individually exploring some of its facets. We distinguish between the fundamentals of bioavailability – the acquisition of Fe-substrate by phytoplankton – and added levels of complexity involving interactions among organisms, iron, and ecosystem processes. We first examine how phytoplankton acquire free and organically bound iron, drawing attention to the pervasiveness of the reductive uptake pathway in both prokaryotic and eukaryotic autotrophs. Turning to acquisition rates, we propose to view the availability of various Fe-substrates to phytoplankton as a spectrum rather than an absolute “all or nothing.” We then demonstrate the use of uptake rate constants to make comparisons across different studies, organisms, Fe-compounds, and environments, and for gaging the contribution of various Fe-substrates to phytoplankton growth in situ. Last, we describe the influence of aquatic microorganisms on iron chemistry and fate by way of organic complexation and bio-mediated redox transformations and examine the bioavailability of these bio-modified Fe species. PMID:22529839

  12. Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo.

    PubMed

    Burgess, Rebecca C; Lisby, Michael; Altmannova, Veronika; Krejci, Lumir; Sung, Patrick; Rothstein, Rodney

    2009-06-15

    Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 "anti-recombinase" restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we cytologically characterize Srs2 function in vivo and describe a novel mechanism for regulating the initiation of HR. We find that Srs2 is recruited separately to replication and repair centers and identify the genetic requirements for recruitment. In the absence of Srs2 activity, Rad51 foci accumulate, and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2 removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR.

  13. TubZ filament assembly dynamics requires the flexible C-terminal tail

    PubMed Central

    Fuentes-Pérez, Maria E.; Núñez-Ramírez, Rafael; Martín-González, Alejandro; Juan-Rodríguez, David; Llorca, Oscar; Moreno-Herrero, Fernando; Oliva, Maria A.

    2017-01-01

    Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY. PMID:28230082

  14. TubZ filament assembly dynamics requires the flexible C-terminal tail.

    PubMed

    Fuentes-Pérez, Maria E; Núñez-Ramírez, Rafael; Martín-González, Alejandro; Juan-Rodríguez, David; Llorca, Oscar; Moreno-Herrero, Fernando; Oliva, Maria A

    2017-02-23

    Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY.

  15. First insights into disassembled "evapotranspiration"

    NASA Astrophysics Data System (ADS)

    Chormański, Jarosław; Kleniewska, Małgorzata; Berezowski, Tomasz; Szporak-Wasilewska, Sylwia; Okruszko, Tomasz; Szatyłowicz, Jan; Batelaan, Okke

    2015-04-01

    In this work we present an initial data analysis obtained from a complex tool for measuring water fluxes in wetland ecosystems. The tool was designed to quantify processes related to interception storage on plants leafs. The measurements are conducted by combining readings from various instruments, including: eddy covariance tower (EC), field spectrometer, SapFlow system, rain gauges above and under canopy, soil moisture probes and other. The idea of this set-up is to provide continuous measurement of overall water flux from the ecosystem (EC tower), intercepted water volume and timing (field spectrometers), through-fall (rain gauges above and under canopy), transpiration (SapFlow), evaporation and soil moisture (soil moisture probes). Disassembling the water flux to the above components allows giving more insight to the interception related processes and differentiates them fromthe total evapotranspiration. The measurements are conducted in the Upper Biebrza Basin (NE Poland). The study area is part of the valley and is covered by peat soils (mainly peat moss with the exception of areas near the river) and receives no inundations waters of the Biebrza. The plant community of Agrostietum-Carici caninae has a dominant share here creating an up to 0.6 km wide belt along the river. The area is covered also by Caricion lasiocarpae as well as meadows and pastures Molinio-Arrhenatheretea, Phragmitetum communis. Sedges form a hummock pattern characteristic for the sedge communities in natural river valleys with wetland vegetation. The main result of the measurement set-up will be the analyzed characteristics and dynamics of interception storage for sedge ecosystems and a developed methodology for interception monitoring by use spectral reflectance technique. This will give a new insight to processes of evapotranspiration in wetlands and its component transpiration, evaporation from interception and evaporation from soil. Moreover, other important results of this project

  16. Generation of control sequences for a pilot-disassembly system

    NASA Astrophysics Data System (ADS)

    Seliger, Guenther; Kim, Hyung-Ju; Keil, Thomas

    2002-02-01

    Closing the product and material cycles has emerged as a paradigm for industry in the 21st century. Disassembly plays a key role in a life cycle economy since it enables the recovery of resources. A partly automated disassembly system should adapt to a large variety of products and different degrees of devaluation. Also the amounts of products to be disassembled can vary strongly. To cope with these demands an approach to generate on-line disassembly control sequences will be presented. In order to react on these demands the technological feasibility is considered within a procedure for the generation of disassembly control sequences. Procedures are designed to find available and technologically feasible disassembly processes. The control system is formed by modularised and parameterised control units in the cell level within the entire control architecture. In the first development stage product and process analyses at the sample product washing machine were executed. Furthermore a generalized disassembly process was defined. Afterwards these processes were structured in primary and secondary functions. In the second stage the disassembly control at the technological level was investigated. Factors were the availability of the disassembly tools and the technological feasibility of the disassembly processes within the disassembly system. Technical alternative disassembly processes are determined as a result of availability of the tools and technological feasibility of processes. The fourth phase was the concept for the generation of the disassembly control sequences. The approach will be proved in a prototypical disassembly system.

  17. Combinatorial optimization methods for disassembly line balancing

    NASA Astrophysics Data System (ADS)

    McGovern, Seamus M.; Gupta, Surendra M.

    2004-12-01

    Disassembly takes place in remanufacturing, recycling, and disposal with a line being the best choice for automation. The disassembly line balancing problem seeks a sequence which: minimizes workstations, ensures similar idle times, and is feasible. Finding the optimal balance is computationally intensive due to factorial growth. Combinatorial optimization methods hold promise for providing solutions to the disassembly line balancing problem, which is proven to belong to the class of NP-complete problems. Ant colony optimization, genetic algorithm, and H-K metaheuristics are presented and compared along with a greedy/hill-climbing heuristic hybrid. A numerical study is performed to illustrate the implementation and compare performance. Conclusions drawn include the consistent generation of optimal or near-optimal solutions, the ability to preserve precedence, the speed of the techniques, and their practicality due to ease of implementation.

  18. Electronic waste disassembly with industrial waste heat.

    PubMed

    Chen, Mengjun; Wang, Jianbo; Chen, Haiyian; Ogunseitan, Oladele A; Zhang, Mingxin; Zang, Hongbin; Hu, Jiukun

    2013-01-01

    Waste printed circuit boards (WPCBs) are resource-rich but hazardous, demanding innovative strategies for post-consumer collection, recycling, and mining for economically precious constituents. A novel technology for disassembling electronic components from WPCBs is proposed, using hot air to melt solders and to separate the components and base boards. An automatic heated-air disassembling equipment was designed to operate at a heating source temperature at a maximum of 260 °C and an inlet pressure of 0.5 MPa. A total of 13 individual WPCBs were subjected to disassembling tests at different preheat temperatures in increments of 20 °C between 80 and 160 °C, heating source temperatures ranging from 220 to 300 °C in increments of 20 °C, and incubation periods of 1, 2, 4, 6, or 8 min. For each experimental treatment, the disassembly efficiency was calculated as the ratio of electronic components released from the board to the total number of its original components. The optimal preheat temperature, heating source temperature, and incubation period to disassemble intact components were 120 °C, 260 °C, and 2 min, respectively. The disassembly rate of small surface mount components (side length ≤ 3 mm) was 40-50% lower than that of other surface mount components and pin through hole components. On the basis of these results, a reproducible and sustainable industrial ecological protocol using steam produced by industrial exhaust heat coupled to electronic-waste recycling is proposed, providing an efficient, promising, and green method for both electronic component recovery and industrial exhaust heat reutilization.

  19. Cofilin-2 controls actin filament length in muscle sarcomeres

    PubMed Central

    Kremneva, Elena; Makkonen, Maarit H.; Skwarek-Maruszewska, Aneta; Gateva, Gergana; Michelot, Alphee; Dominguez, Roberto; Lappalainen, Pekka

    2014-01-01

    SUMMARY ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of ‘aged’ ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an ‘actin nucleotide-state sensor’ among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties allowing it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies. PMID:25373779

  20. Filament Breakaway

    NASA Image and Video Library

    2017-04-18

    A dark, elongated filament rose up and broke to the lower left and out from the sun seen by NASA Solar Dynamics Observatory, Apr.9-10, 2017. Filaments are cooler clouds of plasma tethered above the sun surface by magnetic forces. They are notoriously unstable and tend not to last more than a few days before they collapse into the sun or break away into space. A video, taken in extreme ultraviolet light, covers about nine hours of activity. Videos are available at https://photojournal.jpl.nasa.gov/catalog/PIA21592

  1. Neutron tube disassembly glovebox operational readiness check. Revision A

    SciTech Connect

    Not Available

    1994-04-01

    The neutron tube disassembly glove box was designed and constructed at Martin Marietta Specialty Components, Inc. It is intended to replace the non-enclosed tube disassembly operation. A primary concern is the containment of tritium-laden particulate matter generated during the disassembly of neutron tubes and preparation of other tritium loaded samples. The goal of this project is to minimize the spread of tritium-laden contamination to the tube disassembly worker and the environment.

  2. Microtubules regulate disassembly of epithelial apical junctions

    PubMed Central

    Ivanov, Andrei I; McCall, Ingrid C; Babbin, Brian; Samarin, Stanislav N; Nusrat, Asma; Parkos, Charles A

    2006-01-01

    Background Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion. Results Calcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex. Conclusion Our data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins. PMID:16509970

  3. Multi-kanban mechanism for appliance disassembly

    NASA Astrophysics Data System (ADS)

    Udomsawat, Gun; Gupta, Surendra M.

    2005-11-01

    The use of household appliances continues to rise every year. A significant number of End-Of-Life (EOL) appliances are generated because of the introduction of newer models that are more attractive, efficient and affordable. Others are, of course, generated when they become non-functional. Many regulations encourage recycling of EOL appliances to reduce the amount of waste sent to landfills. In addition, EOL appliances offer the appliance manufacturing and remanufacturing industries a source of less expensive raw materials and components. For this reason product recovery has become a subject of interest during the past decade. In this paper, we study the disassembly line for appliance disassembly. We discuss and incorporate some of the complications that are inherent in disassembly line including product arrival, demand arrival, inventory fluctuation and production control mechanisms. We show how to overcome such complications by implementing a multi-kanban system in the appliance disassembly line setting. The multi-kanban system (MKS) relies on dynamic routing of kanbans according to the state of the system. We investigate the multi-kanban mechanism using simulation and explore the effect of product mix on performance of the traditional push system (TPS) and MKS in terms of controlling the system's inventory while attempting to achieve a decent customer service level.

  4. Myosin flares and actin leptomeres as myofibril assembly/disassembly intermediates in sonic muscle fibers.

    PubMed

    Nahirney, Patrick C; Fischman, Donald A; Wang, Kuan

    2006-04-01

    The sonic muscle of type 1 male midshipman fish produces loud and enduring mating calls. Each sonic muscle fiber contains a tubular contractile apparatus with radially arranged myofibrillar plates encased in a desmin-rich cytoskeleton that is anchored to broad Z bands (approximately 1.2 micro m wide). Immunomicroscopy has revealed patches of myosin-rich "flares" emanating from the contractile tubes into the peripheral sarcoplasm along the length of the fibers. These flares contain swirls of thick filaments devoid of associated thin filaments. In other regions of the sarcoplasm at the inner surface of the sarcolemma and near Z bands, abundant ladder-like leptomeres occur with rungs every 160 nm. Leptomeres consist of dense arrays of filaments (approximately 4 nm) with a structure that resembles myofibrillar Z band structure. We propose that flares and leptomeres are distinct filamentous arrays representing site-specific processing of myofibrillar components during the assembly and disassembly of the sarcomere. Recent reports that myosin assembles into filamentous aggregates before incorporating into the A band in the skeletal muscles of vertebrates and Caenorhabditis elegans suggest that sonic fibers utilize a similar pathway. Thus, sonic muscle fibers, with their tubular design and abundant sarcoplasmic space, may provide an attractive muscle model to identify myofibrillar intermediates by structural and molecular techniques.

  5. Filament winding

    NASA Astrophysics Data System (ADS)

    Shibley, A. M.

    The major aspects of filament winding are discussed, emphasizing basic reinforcement and matrix materials, winding procedures, process controls, and cured composite properties. Fiberglass (E-glass and S-glass strengths are 500,000 and 665,000 psi respectively) and polyester resins are the principal reinforcement constituent materials. Graphite and aramid reinforcements are being used more frequently, primarily for the more critical pressure vessels. Matrix systems are most commonly based on epoxy as it has superior mechanical properties, fatigue behavior, and heat resistance as compard with polyesters. A fiberglass overwrap of PVC pipe is an anticipated development in on-site winding and combination winding, and the compression molding of filament wound lay-ups will be investigated. The fabrication of weight-sensitive structural components may be achieved by using such moldings.

  6. LLNL Torso Phantom Assembly and Disassembly

    SciTech Connect

    Hickman, D P

    2005-10-05

    This document from the LLNL In Vivo measurement Facility archives provides important historical as well as current methods for the proper handling of the LLNL Torso Phantom. This document was written circa 1980 and is intended for use by in vivo measurement facilities that perform calibrations using the LLNL Torso Phantom. Proper care and use of the LLNL Torso Phantom will greatly extend the useful lifetime of the phantom. The assembly, and disassembly of the Realistic Phantom are simple tasks and are, for the most part, self evident. However, there are some considerations that will make these tasks easier and assure long life of the parts. The assembly process is presented in Figures 1 through 9 while disassembly suggestions are illustrated in Figures 10 and 11.

  7. Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly.

    PubMed

    Ayoub, Ahmed T; Klobukowski, Mariusz; Tuszynski, Jack A

    2015-06-01

    Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αβ-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder's axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap), lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability.

  8. Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly

    PubMed Central

    Ayoub, Ahmed T.; Klobukowski, Mariusz; Tuszynski, Jack A.

    2015-01-01

    Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αβ-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder's axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap), lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability. PMID:26030285

  9. Filament turbulence

    NASA Astrophysics Data System (ADS)

    Davidsen, Joern

    2010-03-01

    How much information do you need to distinguish between different mechanisms for spatiotemporal chaos in three-dimensions? In this talk, I will show that the observation of the dynamics on the surface of a medium can be sufficient. Studying mechanisms for filament turbulence in the context of reaction-diffusion media, we found numerically that two major classes of instabilities leave a very different signature on what can be observed on the surface of a three-dimensional medium. These results are of direct relevance in the context of ventricular fibrillation - a turbulent electrical wave activity that destroys the coherent contraction of the ventricular muscle and its main pumping function leading to sudden cardiac death. While it has been proposed that the three-dimensional structure of the heart plays an important role in this type of filament turbulence, only the surface of the heart is currently accessible to experimental observation preventing the study of the full dynamics. Our results suggest that such observations might be sufficient.

  10. Human cognitive analysis of disassembly planning using fuzzy petri nets

    NASA Astrophysics Data System (ADS)

    Tang, Ying; Turowski, Mark

    2004-12-01

    Disassembly, as the process of systematic removal of desirable constituent parts from an assembly, is of growing importance due to the increasing environmental and economic pressures. Designing and improving disassembly process towards a more economic manner is worthwhile investigating. This paper extends our previous work on human factors in disassembly. More specifically, a Fuzzy Petri Net (FPN) is proposed to mathematically model uncertainties in the disassembly process due to the variations in operator skill level and product condition. An adaptive fuzzy system is further developed for the decision-making in disassembly process planning (DPP), where a learning mechanism is engaged to monitor the quality of various parameter estimates and exploit the past "knowledge" regarding uncertainties. This idea is exemplified through the disassembly of a discarded product with three components.

  11. The bacterial actin MamK: in vitro assembly behavior and filament architecture.

    PubMed

    Ozyamak, Ertan; Kollman, Justin; Agard, David A; Komeili, Arash

    2013-02-08

    It is now recognized that actin-like proteins are widespread in bacteria and, in contrast to eukaryotic actins, are highly diverse in sequence and function. The bacterial actin, MamK, represents a clade, primarily found in magnetotactic bacteria, that is involved in the proper organization of subcellular organelles, termed magnetosomes. We have previously shown that MamK from Magnetospirillum magneticum AMB-1 (AMB-1) forms dynamic filaments in vivo. To gain further insights into the molecular mechanisms that underlie MamK dynamics and function, we have now studied the in vitro properties of MamK. We demonstrate that MamK is an ATPase that, in the presence of ATP, assembles rapidly into filaments that disassemble once ATP is depleted. The mutation of a conserved active site residue (E143A) abolishes ATPase activity of MamK but not its ability to form filaments. Filament disassembly depends on both ATPase activity and potassium levels, the latter of which results in the organization of MamK filaments into bundles. These data are consistent with observations indicating that accessory factors are required to promote filament disassembly and for spatial organization of filaments in vivo. We also used cryo-electron microscopy to obtain a high resolution structure of MamK filaments. MamK adopts a two-stranded helical filament architecture, but unlike eukaryotic actin and other actin-like filaments, subunits in MamK strands are unstaggered giving rise to a unique filament architecture. Beyond extending our knowledge of the properties and function of MamK in magnetotactic bacteria, this study emphasizes the functional and structural diversity of bacterial actins in general.

  12. Multikanban model for disassembly line with demand fluctuation

    NASA Astrophysics Data System (ADS)

    Udomsawat, Gun; Gupta, Surendra M.; Al-Turki, Yousef A. Y.

    2004-02-01

    In recent years, the continuous growth in consumer waste and dwindling natural resources has seriously threatened the environment. Realizing this, several countries have passed regulations that force manufacturers not only to manufacture environmentally conscious products, but also to take back their used products from consumers so that the components and materials recovered from the products may be reused and/or recycled. Disassembly plays an important role in product recovery. A disassembly line is perhaps the most suitable setting for disassembly of products in large quantities. Because a disassembly line has a tendency to generate excessive inventory, employing a kanban system can reduce the inventory level and let the system run more efficiently. A disassembly line is quite different from an assembly line. For example, not only can the demand arrive at the last station, it can also arrive at any of the other stations in the system. The demand for a component on the disassembly line could fluctuate widely. In fact, there are many other complicating matters that need to be considered to implement the concept of kanbans in such an environment. In this paper, we discuss the complications that are unique to a disassembly line. We discuss the complications in utilizing the conventional production control mechanisms in a disassembly line setting. We then show how to overcome them by implementing kanbans in a disassembly line setting with demand fluctuation and introduce the concept of multi-kanban mechanism. We demonstrate its effectiveness using a simulation model. An example is presented to illustrate the concept.

  13. A Heuristic for Disassembly Planning in Remanufacturing System

    PubMed Central

    2014-01-01

    This study aims to improve the efficiency of disassembly planning in remanufacturing environment. Even though disassembly processes are considered as the reverse of the corresponding assembly processes, under some technological and management constraints the feasible and efficient disassembly planning can be achieved by only well-designed algorithms. In this paper, we propose a heuristic for disassembly planning with the existence of disassembled part/subassembly demands. A mathematical model is formulated for solving this problem to determine the sequence and quantity of disassembly operations to minimize the disassembly costs under sequence-dependent setup and capacity constraints. The disassembly costs consist of the setup cost, part inventory holding cost, disassembly processing cost, and purchasing cost that resulted from unsatisfied demand. A simple but efficient heuristic algorithm is proposed to improve the quality of solution and computational efficiency. The main idea of heuristic is to divide the planning horizon into the smaller planning windows and improve the computational efficiency without much loss of solution quality. Performances of the heuristic are investigated through the computational experiments. PMID:24895679

  14. Disassembly sequencing problem: a case study of a cell phone

    NASA Astrophysics Data System (ADS)

    Gupta, Surendra M.; Erbis, Evren; McGovern, Seamus M.

    2004-12-01

    Selection of an optimal disassembly sequence is essential for the efficient processing of a product at the end of its life. Disassembly sequences are listings of disassembly actions (such as the separation of an assembly into two or more subassemblies, or removing one or more connections between components). Disassembly takes place in remanufacturing, recycling, and disposal with a disassembly line being the best choice for automation. In this paper, the disassembly sequencing problem is solved for a cell phone case on a disassembly line, seeking a sequence which is feasible, minimizes the number of workstations (and hence idle times), provides for early removal of high demand/value parts, provides the removal of parts that lead to the access of greatest number of still-installed parts, and early removal of hazardous parts as well as for the grouping of parts for removal having identical part removal directions. Since finding the optimal sequence is computationally intensive due to factorial growth, a heuristic method is used taking into account various disassembly-specific matters. Using the experimentally determined precedence relationships and task times of a real-world cell phone, a MATLAB program is designed and a sequencing solution is generated. Finally, Design for Disassembly (DFD) improvements are recommended with respect to environmentally conscious manufacturing.

  15. Postulated accident scenarios in weapons disassembly

    SciTech Connect

    Payne, S.S.

    1997-06-01

    A very brief summary of three postulated accident scenarios for weapons disassembly is provided in the paper. The first deals with a tetrahedral configuration of four generic pits; the second, an infinite planar array of generic pits with varying interstitial water density; and the third, a spherical shell with internal mass suspension in water varying the size and mass of the shell. Calculations were performed using the Monte Carlo Neutron Photon transport code MCNP4A. Preliminary calculations pointed to a need for higher resolution of small pit separation regimes and snapshots of hydrodynamic processes of water/plutonium mixtures.

  16. Presynaptic Filament Dynamics in Homologous Recombination and DNA Repair

    PubMed Central

    Liu, Jie; Ehmsen, Kirk T.; Heyer, Wolf-Dietrich; Morrical, Scott W.

    2014-01-01

    Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity. PMID:21599536

  17. Impact of different disassembly line balancing algorithms on the performance of dynamic kanban system for disassembly line

    NASA Astrophysics Data System (ADS)

    Kizilkaya, Elif A.; Gupta, Surendra M.

    2005-11-01

    In this paper, we compare the impact of different disassembly line balancing (DLB) algorithms on the performance of our recently introduced Dynamic Kanban System for Disassembly Line (DKSDL) to accommodate the vagaries of uncertainties associated with disassembly and remanufacturing processing. We consider a case study to illustrate the impact of various DLB algorithms on the DKSDL. The approach to the solution, scenario settings, results and the discussions of the results are included.

  18. High-speed depolymerization at actin filament ends jointly catalysed by Twinfilin and Srv2/CAP.

    PubMed

    Johnston, Adam B; Collins, Agnieszka; Goode, Bruce L

    2015-11-01

    Purified actin filaments depolymerize slowly, and cytosolic conditions strongly favour actin assembly over disassembly, which has left our understanding of how actin filaments are rapidly turned over in vivo incomplete. One mechanism for driving filament disassembly is severing by factors such as Cofilin. However, even after severing, pointed-end depolymerization remains slow and unable to fully account for observed rates of actin filament turnover in vivo. Here we describe a mechanism by which Twinfilin and Cyclase-associated protein work in concert to accelerate depolymerization of actin filaments by 3-fold and 17-fold at their barbed and pointed ends, respectively. This mechanism occurs even under assembly conditions, allowing reconstitution and direct visualization of individual filaments undergoing tunable, accelerated treadmilling. Further, we use specific mutations to demonstrate that this activity is critical for Twinfilin function in vivo. These findings fill a major gap in our knowledge of cellular disassembly mechanisms, and suggest that depolymerization and severing may be deployed separately or together to control the dynamics and architecture of distinct actin networks.

  19. Probabilistic Risk Assessment of disassembly procedures

    SciTech Connect

    O`Brien, D.A.; Bement, T.R.; Letellier, B.C.

    1993-10-01

    Probabilistic Risk (Safety) Assessment (PRA or PSA) is an analytic methodology for identifying the combination of events that, if they occur, lead to accidents. Accidents are defined as those events causing loss or injury to people, property, or the environment. PRA also provides a method for estimating the frequency of occurrence of each combination of events and the consequences of each accident. The Los Alamos effort for this study is summarized as follows: The focus of the Los Alamos study was on evaluating the risks specifically associated with disassembling a Los Alamos-designed device. The PRA for the disassembly operation included a detailed evaluation only for those potential accident sequences which could lead to significant off-site consequences and affect public health. The overall purpose of this study was to investigate the feasibility of a risk consequence goal for DOE operations. Often called a Level 3 PRA (or PSA), the methods are general and can with a little modification be applied to other procedures or processes.

  20. Asymmetric disassembly and robustness in declining networks

    PubMed Central

    Saavedra, Serguei; Reed-Tsochas, Felix; Uzzi, Brian

    2008-01-01

    Mechanisms that enable declining networks to avert structural collapse and performance degradation are not well understood. This knowledge gap reflects a shortage of data on declining networks and an emphasis on models of network growth. Analyzing >700,000 transactions between firms in the New York garment industry over 19 years, we tracked this network's decline and measured how its topology and global performance evolved. We find that favoring asymmetric (disassortative) links is key to preserving the topology and functionality of the declining network. Based on our findings, we tested a model of network decline that combines an asymmetric disassembly process for contraction with a preferential attachment process for regrowth. Our simulation results indicate that the model can explain robustness under decline even if the total population of nodes contracts by more than an order of magnitude, in line with our observations for the empirical network. These findings suggest that disassembly mechanisms are not simply assembly mechanisms in reverse and that our model is relevant to understanding the process of decline and collapse in a broad range of biological, technological, and financial networks. PMID:18936489

  1. Asymmetric disassembly and robustness in declining networks.

    PubMed

    Saavedra, Serguei; Reed-Tsochas, Felix; Uzzi, Brian

    2008-10-28

    Mechanisms that enable declining networks to avert structural collapse and performance degradation are not well understood. This knowledge gap reflects a shortage of data on declining networks and an emphasis on models of network growth. Analyzing >700,000 transactions between firms in the New York garment industry over 19 years, we tracked this network's decline and measured how its topology and global performance evolved. We find that favoring asymmetric (disassortative) links is key to preserving the topology and functionality of the declining network. Based on our findings, we tested a model of network decline that combines an asymmetric disassembly process for contraction with a preferential attachment process for regrowth. Our simulation results indicate that the model can explain robustness under decline even if the total population of nodes contracts by more than an order of magnitude, in line with our observations for the empirical network. These findings suggest that disassembly mechanisms are not simply assembly mechanisms in reverse and that our model is relevant to understanding the process of decline and collapse in a broad range of biological, technological, and financial networks.

  2. Filamentous Fungi.

    PubMed

    Powers-Fletcher, Margaret V; Kendall, Brian A; Griffin, Allen T; Hanson, Kimberly E

    2016-06-01

    Filamentous mycoses are often associated with significant morbidity and mortality. Prompt diagnosis and aggressive treatment are essential for good clinical outcomes in immunocompromised patients. The host immune response plays an essential role in determining the course of exposure to potential fungal pathogens. Depending on the effectiveness of immune response and the burden of organism exposure, fungi can either be cleared or infection can occur and progress to a potentially fatal invasive disease. Nonspecific cellular immunity (i.e., neutrophils, natural killer [NK] cells, and macrophages) combined with T-cell responses are the main immunologic mechanisms of protection. The most common potential mold pathogens include certain hyaline hyphomycetes, endemic fungi, the Mucorales, and some dematiaceous fungi. Laboratory diagnostics aimed at detecting and differentiating these organisms are crucial to helping clinicians make informed decisions about treatment. The purpose of this chapter is to provide an overview of the medically important fungal pathogens, as well as to discuss the patient characteristics, antifungal-therapy considerations, and laboratory tests used in current clinical practice for the immunocompromised host.

  3. Alignment Pins for Assembling and Disassembling Structures

    NASA Technical Reports Server (NTRS)

    Campbell, Oliver C.

    2008-01-01

    Simple, easy-to-use, highly effective tooling has been devised for maintaining alignment of bolt holes in mating structures during assembly and disassembly of the structures. The tooling was originally used during removal of a body flap from the space shuttle Atlantis, in which misalignments during removal of the last few bolts could cause the bolts to bind in their holes. By suitably modifying the dimensions of the tooling components, the basic design of the tooling can readily be adapted to other structures that must be maintained in alignment. The tooling includes tapered, internally threaded alignment pins designed to fit in the bolt holes in one of the mating structures, plus a draw bolt and a cup that are used to install or remove each alignment pin. In preparation for disassembly of two mating structures, external supports are provided to prevent unintended movement of the structures. During disassembly of the structures, as each bolt that joins the structures is removed, an alignment pin is installed in its place. Once all the bolts have been removed and replaced with pins, the pins maintain alignment as the structures are gently pushed or pulled apart on the supports. In assembling the two structures, one reverses the procedure described above: pins are installed in the bolt holes, the structures are pulled or pushed together on the supports, then the pins are removed and replaced with bolts. The figure depicts the tooling and its use. To install an alignment pin in a bolt hole in a structural panel, the tapered end of the pin is inserted from one side of the panel, the cup is placed over the pin on the opposite side of the panel, the draw bolt is inserted through the cup and threaded into the pin, the draw bolt is tightened to pull the pin until the pin is seated firmly in the hole, then the draw bolt and cup are removed, leaving the pin in place. To remove an alignment pin, the cup is placed over the pin on the first-mentioned side of the panel, the draw

  4. 2-opt heuristic for the disassembly line balancing problem

    NASA Astrophysics Data System (ADS)

    McGovern, Seamus M.; Gupta, Surendra M.

    2004-02-01

    Disassembly activities are an important part of product recovery operations. The disassembly line is the best choice for automated disassembly of returned products. However, finding the optimal balance for a disassembly line is computationally intensive with exhaustive search quickly becoming prohibitively large. In this paper, a greedy algorithm is presented for obtaining optimal or near-optimal solutions to the disassembly line-balancing problem. The greedy algorithm is a first-fit decreasing algorithm further enhanced to preserve precedence relationships. The algorithm seeks to minimize the number of workstations while addressing hazardous and high demand components. A two optimal algorithm is then developed to balance the part removal sequence and attempt to further reduce the total number of workstations. Examples are considered to illustrate the methodology. The conclusions drawn from the study include the consistent generation of optimal or near-optimal solutions, the ability to preserve precedence, the speed of the algorithms and their practicality due to the ease of implementation.

  5. Montmorillonite-induced Bacteriophage φ6 Disassembly

    NASA Astrophysics Data System (ADS)

    Trusiak, A.; Gottlieb, P.; Katz, A.; Alimova, A.; Steiner, J. C.; Block, K. A.

    2012-12-01

    It is estimated that there are 1031 virus particles on Earth making viruses an order of magnitude more prevalent in number than prokaryotes with the vast majority of viruses being bacteriophages. Clays are a major component of soils and aquatic sediments and can react with RNA, proteins and bacterial biofilms. The clays in soils serve as an important moderator between phage and their host bacteria, helping to preserve the evolutionary balance. Studies on the effects of clays on viral infectivity have given somewhat contradictory results; possibly a consequence of clay-virus interactions being dependent on the unique structure of particular viruses. In this work, the interaction between montmorillonite and the bacteriophage φ6 is investigated. φ6 is a member of the cystovirus family that infects Pseudomonas syringe, a common plant pathogen. As a member of the cystovirus family with an enveloped structure, φ6 serves as a model for reoviruses, a human pathogen. Experiments were conducted with φ6 suspended in dilute, purified homoionic commercial-grade montmorillonite over a range of virus:clay ratios. At a 1:100000 virus:clay ratio, the clay reduced viral infectivity by 99%. The minimum clay to virus ratio which results in a measurable reduction of P. syringae infection is 1:1. Electron microscopy demonstrates that mixed suspensions of smectite and virus co-aggregate to form flocs encompassing virions within the smectite. Both free viral particles as well as those imbedded in the flocs are seen in the micrographs to be missing the envelope- leaving only the nucleocapsid (NC) intact; indicating that smectite inactivates the virus by envelope disassembly. These results have strong implications in the evolution of both the φ6 virus and its P. syringae host cells. TEM of aggregate showing several disassembled NCs.

  6. Fulvic acid inhibits aggregation and promotes disassembly of tau fibrils associated with Alzheimer's disease.

    PubMed

    Cornejo, Alberto; Jiménez, José M; Caballero, Leonardo; Melo, Francisco; Maccioni, Ricardo B

    2011-01-01

    Alzheimer's disease is a neurodegenerative disorder involving extracellular plaques (amyloid-β) and intracellular tangles of tau protein. Recently, tangle formation has been identified as a major event involved in the neurodegenerative process, due to the conversion of either soluble peptides or oligomers into insoluble filaments. At present, the current therapeutic strategies are aimed at natural phytocomplexes and polyphenolics compounds able to either inhibit the formation of tau filaments or disaggregate them. However, only a few polyphenolic molecules have emerged to prevent tau aggregation, and natural drugs targeting tau have not been approved yet. Fulvic acid, a humic substance, has several nutraceutical properties with potential activity to protect cognitive impairment. In this work we provide evidence to show that the aggregation process of tau protein, forming paired helical filaments (PHFs) in vitro, is inhibited by fulvic acid affecting the length of fibrils and their morphology. In addition, we investigated whether fulvic acid is capable of disassembling preformed PHFs. We show that the fulvic acid is an active compound against preformed fibrils affecting the whole structure by diminishing length of PHFs and probably acting at the hydrophobic level, as we observed by atomic force techniques. Thus, fulvic acid is likely to provide new insights in the development of potential treatments for Alzheimer's disease using natural products.

  7. Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia.

    PubMed

    Vitriol, Eric A; Wise, Ariel L; Berginski, Mathew E; Bamburg, James R; Zheng, James Q

    2013-07-01

    Cofilin is a key regulator of the actin cytoskeleton. It can sever actin filaments, accelerate filament disassembly, act as a nucleation factor, recruit or antagonize other actin regulators, and control the pool of polymerization-competent actin monomers. In cells these actions have complex functional outputs. The timing and localization of cofilin activity are carefully regulated, and thus global, long-term perturbations may not be sufficient to probe its precise function. To better understand cofilin's spatiotemporal action in cells, we implemented chromophore-assisted laser inactivation (CALI) to instantly and specifically inactivate it. In addition to globally inhibiting actin turnover, CALI of cofilin generated several profound effects on the lamellipodia, including an increase of F-actin, a rearward expansion of the actin network, and a reduction in retrograde flow speed. These results support the hypothesis that the principal role of cofilin in lamellipodia at steady state is to break down F-actin, control filament turnover, and regulate the rate of retrograde flow.

  8. TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent

    PubMed Central

    West, Michele A.; Prescott, Alan R.; Chan, Kui Ming; Zhou, Zhongjun; Rose-John, Stefan; Scheller, Jürgen; Watts, Colin

    2008-01-01

    Toll-like receptor (TLR) signaling induces a rapid reorganization of the actin cytoskeleton in cultured mouse dendritic cells (DC), leading to enhanced antigen endocytosis and a concomitant loss of filamentous actin–rich podosomes. We show that as podosomes are lost, TLR signaling induces prominent focal contacts and a transient reduction in DC migratory capacity in vitro. We further show that podosomes in mouse DC are foci of pronounced gelatinase activity, dependent on the enzyme membrane type I matrix metalloprotease (MT1-MMP), and that DC transiently lose the ability to degrade the extracellular matrix after TLR signaling. Surprisingly, MMP inhibitors block TLR signaling–induced podosome disassembly, although stimulated endocytosis is unaffected, which demonstrates that the two phenomena are not obligatorily coupled. Podosome disassembly caused by TLR signaling occurs normally in DC lacking MT1-MMP, and instead requires the tumor necrosis factor α–converting enzyme ADAM17 (a disintegrin and metalloprotease 17), which demonstrates a novel role for this “sheddase” in regulating an actin-based structure. PMID:18762577

  9. Polar patterns of driven filaments.

    PubMed

    Schaller, Volker; Weber, Christoph; Semmrich, Christine; Frey, Erwin; Bausch, Andreas R

    2010-09-02

    The emergence of collective motion exhibited by systems ranging from flocks of animals to self-propelled microorganisms to the cytoskeleton is a ubiquitous and fascinating self-organization phenomenon. Similarities between these systems, such as the inherent polarity of the constituents, a density-dependent transition to ordered phases or the existence of very large density fluctuations, suggest universal principles underlying pattern formation. This idea is followed by theoretical models at all levels of description: micro- or mesoscopic models directly map local forces and interactions using only a few, preferably simple, interaction rules, and more macroscopic approaches in the hydrodynamic limit rely on the systems' generic symmetries. All these models characteristically have a broad parameter space with a manifold of possible patterns, most of which have not yet been experimentally verified. The complexity of interactions and the limited parameter control of existing experimental systems are major obstacles to our understanding of the underlying ordering principles. Here we demonstrate the emergence of collective motion in a high-density motility assay that consists of highly concentrated actin filaments propelled by immobilized molecular motors in a planar geometry. Above a critical density, the filaments self-organize to form coherently moving structures with persistent density modulations, such as clusters, swirls and interconnected bands. These polar nematic structures are long lived and can span length scales orders of magnitudes larger than their constituents. Our experimental approach, which offers control of all relevant system parameters, complemented by agent-based simulations, allows backtracking of the assembly and disassembly pathways to the underlying local interactions. We identify weak and local alignment interactions to be essential for the observed formation of patterns and their dynamics. The presented minimal polar-pattern-forming system

  10. 13. View of disassembled steam engine showing cylinder, piston rod, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. View of disassembled steam engine showing cylinder, piston rod, parallel motion links and steam chest. - Hacienda Azucarera La Esperanza, Steam Engine & Mill, 2.65 Mi. N of PR Rt. 2 Bridge over Manati River, Manati, Manati Municipio, PR

  11. 12. View of disassembled steam engine sitting in open shed ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. View of disassembled steam engine sitting in open shed showing base, columns and entablature. - Hacienda Azucarera La Esperanza, Steam Engine & Mill, 2.65 Mi. N of PR Rt. 2 Bridge over Manati River, Manati, Manati Municipio, PR

  12. 17. View of disassembled reduction gear parts including bull and ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    17. View of disassembled reduction gear parts including bull and intermediate gears and pedestal bearing. - Hacienda Azucarera La Esperanza, Steam Engine & Mill, 2.65 Mi. N of PR Rt. 2 Bridge over Manati River, Manati, Manati Municipio, PR

  13. New package for Belleville spring permits rate change, easy disassembly

    NASA Technical Reports Server (NTRS)

    Mac Glashan, W. F.

    1964-01-01

    A spring package, with grooves to hold the spring washers at the inner and outer edges, reduces hysteresis to a minimum. Three-segment retainers permit easy disassembly so that the spring rate can be changed.

  14. Actin network disassembly powers dissemination of Listeria monocytogenes

    PubMed Central

    Talman, Arthur M.; Chong, Ryan; Chia, Jonathan; Svitkina, Tatyana; Agaisse, Hervé

    2014-01-01

    ABSTRACT Several bacterial pathogens hijack the actin assembly machinery and display intracellular motility in the cytosol of infected cells. At the cell cortex, intracellular motility leads to bacterial dissemination through formation of plasma membrane protrusions that resolve into vacuoles in adjacent cells. Here, we uncover a crucial role for actin network disassembly in dissemination of Listeria monocytogenes. We found that defects in the disassembly machinery decreased the rate of actin tail turnover but did not affect the velocity of the bacteria in the cytosol. By contrast, defects in the disassembly machinery had a dramatic impact on bacterial dissemination. Our results suggest a model of L. monocytogenes dissemination in which the disassembly machinery, through local recycling of the actin network in protrusions, fuels continuous actin assembly at the bacterial pole and concurrently exhausts cytoskeleton components from the network distal to the bacterium, which enables membrane apposition and resolution of protrusions into vacuoles. PMID:24155331

  15. Disassembly line balancing with limited supply and subassembly availability

    NASA Astrophysics Data System (ADS)

    Altekin, Fatma Tevhide; Kandiller, Levent; Ozdemirel, Nur Evin

    2004-02-01

    Disassembly line balancing problem (DLBP) aims at finding a feasible assignment of disassembly tasks to workstations such that precedence relations among tasks are satisfied and some measure of effectiveness is optimized. We consider partial disassembly under limited supply of a single product as well as availability of its subassemblies. Hence, in satisfying the demand for revenue generating parts, both discarded products and available subassemblies can be utilized. We assume that part revenues and demand, task times and costs, inventory holding costs, and station opening costs are given. We propose two DLBP formulations. The first one maximizes the profit per disassembly cycle. The second formulation maximizes the profit over the whole planning horizon. Proposed formulations and computational results are presented.

  16. Systems impacts of spent fuel disassembly alternatives

    SciTech Connect

    Not Available

    1984-07-01

    Three studies were completed to evaluate four alternatives to the disposal of intact spent fuel assemblies in a geologic repository. A preferred spent fuel waste form for disposal was recommended on consideration of (1) package design and fuel/package interaction, (2) long-term, in-repository performance of the waste form, and (3) overall process performance and costs for packaging, handling, and emplacement. The four basic alternative waste forms considered were (1) end fitting removal, (2) fission gas venting, (3) disassembly and close packing, and (4) shearing/immobilization. None of the findings ruled out any alternative on the basis of waste package considerations or long-term performance of the waste form. The third alternative offers flexibility in loading that may prove attractive in the various geologic media under consideration, greatly reduces the number of packages, and has the lowest unit cost. These studies were completed in October, 1981. Since then Westinghouse Electric Corporation and the Office of Nuclear Waste Isolation have completed studies in related fields. This report is now being published to provide publicly the background material that is contained within. 47 references, 28 figures, 31 tables.

  17. Filamentation in Laser Wakefields

    NASA Astrophysics Data System (ADS)

    Los, Eva; Trines, Raoul; Silva, Luis; Bingham, Robert

    2016-10-01

    Laser filamentation instability is observed in plasma wakefields with sub-critical densities, and in high density inertial fusion plasmas. This leads to non-uniform acceleration or compression respectively. Here, we present simulation results on laser filamentation in plasma wakefields. The 2-D simulations are carried out using the particle-in-cell code Osiris. The filament intensity was found to increase exponentially before saturating. The maximum amplitude to which the highest intensity filament grew for a specific set of parameters was also recorded, and plotted against a corresponding parameter value. Clear, positively correlated linear trends were established between plasma density, transverse wavenumber k, laser pulse amplitude and maximum filament amplitude. Plasma density and maximum filament amplitude also showed a positive correlation, which saturated after a certain plasma density. Pulse duration and interaction length did not affect either filament intensity or transverse k value in a predictable manner. There was no discernible trend between pulse amplitude and filament width.

  18. Collaborative protein filaments.

    PubMed

    Ghosal, Debnath; Löwe, Jan

    2015-09-14

    It is now well established that prokaryotic cells assemble diverse proteins into dynamic cytoskeletal filaments that perform essential cellular functions. Although most of the filaments assemble on their own to form higher order structures, growing evidence suggests that there are a number of prokaryotic proteins that polymerise only in the presence of a matrix such as DNA, lipid membrane or even another filament. Matrix-assisted filament systems are frequently nucleotide dependent and cytomotive but rarely considered as part of the bacterial cytoskeleton. Here, we categorise this family of filament-forming systems as collaborative filaments and introduce a simple nomenclature. Collaborative filaments are frequent in both eukaryotes and prokaryotes and are involved in vital cellular processes including chromosome segregation, DNA repair and maintenance, gene silencing and cytokinesis to mention a few. In this review, we highlight common principles underlying collaborative filaments and correlate these with known functions.

  19. Filament Eruption Onset

    NASA Technical Reports Server (NTRS)

    Sterling, Alphonse C.; Moore, Ronald L.

    2011-01-01

    We have been investigating filament eruptions in recent years. Use filament eruptions as markers of the coronal field evolution. Data from SoHO, Yohkoh, TRACE, Hinode, and other sources. We and others have observed: (1)Filaments often show slow rise, followed by fast rise, (2) Brightenings, preflares, microflares during slow rise (3) Magnetic evolution in hours prior to eruption onset. We investigated What do Hinode and SDO show for filament eruptions?

  20. Filament depolymerization can explain chromosome pulling during bacterial mitosis.

    PubMed

    Banigan, Edward J; Gelbart, Michael A; Gitai, Zemer; Wingreen, Ned S; Liu, Andrea J

    2011-09-01

    Chromosome segregation is fundamental to all cells, but the force-generating mechanisms underlying chromosome translocation in bacteria remain mysterious. Caulobacter crescentus utilizes a depolymerization-driven process in which a ParA protein structure elongates from the new cell pole, binds to a ParB-decorated chromosome, and then retracts via disassembly, pulling the chromosome across the cell. This poses the question of how a depolymerizing structure can robustly pull the chromosome that disassembles it. We perform Brownian dynamics simulations with a simple, physically consistent model of the ParABS system. The simulations suggest that the mechanism of translocation is "self-diffusiophoretic": by disassembling ParA, ParB generates a ParA concentration gradient so that the ParA concentration is higher in front of the chromosome than behind it. Since the chromosome is attracted to ParA via ParB, it moves up the ParA gradient and across the cell. We find that translocation is most robust when ParB binds side-on to ParA filaments. In this case, robust translocation occurs over a wide parameter range and is controlled by a single dimensionless quantity: the product of the rate of ParA disassembly and a characteristic relaxation time of the chromosome. This time scale measures the time it takes for the chromosome to recover its average shape after it is has been pulled. Our results suggest explanations for observed phenomena such as segregation failure, filament-length-dependent translocation velocity, and chromosomal compaction.

  1. Analysis of Keratin Filament Assembly/Disassembly and Structure Following Modification by Sulfur Mustard Analogs

    DTIC Science & Technology

    2005-07-01

    epidermolysis bullosa simplex (EBS) occurs at the basal cell layer [4, 5] and usually involves mutations in keratins k5 or k14 [4] [6], the keratin...Bonifas, J.M., A.L. Rothman, and E.H. Epstein, Jr., Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities [see... epidermolysis bullosa simplex diseases. Journal of Cell Biology, 1991. 115(6): p. 1661-74. 3. Lane, E.B., et al., A mutation in the conserved helix termination

  2. Feisty filaments: actin dynamics in the red blood cell membrane skeleton

    PubMed Central

    Gokhin, David S.; Fowler, Velia M.

    2016-01-01

    STRUCTURED ABSTRACT Purpose of review The purpose of this review is to discuss recent advances and unsolved questions in our understanding of actin filament organization and dynamics in the red blood cell (RBC) membrane skeleton, a two-dimensional quasi-hexagonal network consisting of (α1β1)2-spectrin tetramers interconnecting short actin filament-based junctional complexes. Recent findings In contrast to the long-held view that RBC actin filaments are static structures that do not exchange subunits with the cytosol, RBC actin filaments are dynamic structures that undergo subunit exchange and turnover, as evidenced by monomer incorporation experiments with rhodamine-actin and filament disruption experiments with actin-targeting drugs. The malaria-causing parasite, Plasmodium falciparum, co-opts RBC actin dynamics to construct aberrantly branched actin filament networks. Even though RBC actin filaments are dynamic, RBC actin filament lengths are highly uniform (~37 nm). RBC actin filament lengths are thought to be stabilized by the capping proteins, tropomodulin-1 and αβ-adducin, as well as the side-binding protein tropomyosin, present in an equimolar combination of two isoforms, TM5b (Tpm1.9) and TM5NM1 (Tpm3.1). Summary New evidence indicates that RBC actin filaments are not simply passive cytolinkers, but rather dynamic structures whose assembly and disassembly play important roles in RBC membrane function. PMID:27055045

  3. Special issue on filamentation

    NASA Astrophysics Data System (ADS)

    Li, Ruxin; Milchberg, Howard; Mysyrowicz, André

    2014-05-01

    Journal of Physics B: Atomic, Molecular and Optical Physics is delighted to announce a forthcoming special issue on filamentation, to appear in the spring of 2015, and invites you to submit a paper. This special issue will attempt to give an overview of the present status of this field in order to create synergies and foster future developments. The issue is open to papers on the following issues: Theoretical advances on filamentation. Self-focusing and collapse. Filamentation in various media. Pulse self-compression and ultrafast processes in filaments. Molecular alignment and rotation. Filamentation tailoring. Interaction between filaments. Filament weather and pollution control. Filament induced condensation and precipitation. Terahertz science with filaments. Lasing in filaments. Filament induced molecular excitation and reaction. Electric discharge and plasma. Cross-disciplinary applications. Novel concepts related to these topics are particularly welcome. Please submit your article by 1 October 2014 (expected web publication: spring 2015) using our website http://mc04.manuscriptcentral.com/jphysb-iop. Submissions received after this date will be considered for the journal, but may not be included in the special issue. The issue will be edited by Ruxin Li, Howard Milchberg and André Mysyrowicz.

  4. Directed disassembly of an interfacial rubisco protein network.

    PubMed

    Onaizi, Sagheer A; Malcolm, Andrew S; He, Lizhong; Middelberg, Anton P J

    2007-05-22

    We present the first study of the directed disassembly of a protein network at the air-water interface by the synergistic action of a surfactant and an enzyme. We seek to understand the fundamentals of protein network disassembly by using rubisco adsorbed at the air-water interface as a model. We propose that rubisco adsorption at the air-water interface results in the formation of a fishnet-like network of interconnected protein molecules, capable of transmitting lateral force. The mechanical properties of the rubisco network during assembly and disassembly at the air-water interface were characterized by direct measurement of laterally transmitted force through the protein network using the Cambridge interfacial tensiometer. We have shown that, when used individually, either 2 ppm of the surfactant, sodium dodecyl benzyl sulfonate (SDOBS), or 2 ppm of the enzyme, subtilisin A (SA), were insufficient to completely disassemble the rubisco network within 1 h of treatment. However, a combination of 2 ppm SDOBS and 2 ppm SA led to almost complete disassembly within 1 h. Increasing the concentration of SA in the mixture from 2 to 10 ppm, while keeping the SDOBS concentration constant, significantly decreased the time required to completely disassemble the rubisco network. Furthermore, the initial rate of network disassembly using formulations containing SDOBS was surprisingly insensitive to this increase in SA concentration. This study gives insight into the role of lateral interactions between protein molecules at interfaces in stabilizing interfacial protein networks and shows that surfactant and enzyme working in combination proves more effective at disrupting and mobilizing the interfacial protein network than the action of either agent alone.

  5. High Speed Depolymerization at Actin Filament Ends Jointly Catalyzed by Twinfilin and Srv2/CAP

    PubMed Central

    Johnston, Adam B.; Collins, Agnieszka; Goode, Bruce L.

    2015-01-01

    Purified actin filaments depolymerize slowly, and cytosolic conditions strongly favor actin assembly over disassembly, which has left our understanding of how actin filaments are rapidly turned over in vivo incomplete 1,2. One mechanism for driving filament disassembly is severing by factors such as Cofilin. However, even after severing, pointed end depolymerization remains slow and unable to fully account for observed rates of actin filament turnover in vivo. Here we describe a mechanism by which Twinfilin and Cyclase-associated protein work in concert to accelerate depolymerization of actin filaments by 3-fold and 17-fold at their barbed and pointed ends, respectively. This mechanism occurs even under assembly conditions, allowing reconstitution and direct visualization of individual filaments undergoing tunable, accelerated treadmilling. Further, we use specific mutations to demonstrate that this activity is critical for Twinfilin function in vivo. These findings fill a major gap in our knowledge of mechanisms, and suggest that depolymerization and severing may be deployed separately or together to control the dynamics and architecture of distinct actin networks. PMID:26458246

  6. Anaphase A: Disassembling Microtubules Move Chromosomes toward Spindle Poles

    PubMed Central

    Asbury, Charles L.

    2017-01-01

    The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through loss of tubulin subunits from the kinetochore-attached plus ends. In addition, kinetochore-fiber disassembly in many cells occurs partly through ‘flux’, where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive these disassembly-coupled movements, are discussed. PMID:28218660

  7. A symbolic methodology to improve disassembly process design.

    PubMed

    Rios, Pedro; Blyler, Leslie; Tieman, Lisa; Stuart, Julie Ann; Grant, Ed

    2003-12-01

    Millions of end-of-life electronic components are retired annually due to the proliferation of new models and their rapid obsolescence. The recovery of resources such as plastics from these goods requires their disassembly. The time required for each disassembly and its associated cost is defined by the operator's familiarity with the product design and its complexity. Since model proliferation serves to complicate an operator's learning curve, it is worthwhile to investigate the benefits to be gained in a disassembly operator's preplanning process. Effective disassembly process design demands the application of green engineering principles, such as those developed by Anastas and Zimmerman (Environ. Sci. Technol. 2003, 37, 94A-101A), which include regard for product complexity, structural commonality, separation energy, material value, and waste prevention. This paper introduces the concept of design symbolsto help the operator more efficiently survey product complexity with respect to location and number of fasteners to remove a structure that is common to all electronics: the housing. With a sample of 71 different computers, printers, and monitors, we demonstrate that appropriate symbols reduce the total disassembly planning time by 13.2 min. Such an improvement could well make efficient the separation of plastic that would otherwise be destined for waste-to-energy or landfill. The symbolic methodology presented may also improve Design for Recycling and Design for Maintenance and Support.

  8. Large-Scale Patterns of Filament Channels and Filaments

    NASA Astrophysics Data System (ADS)

    Mackay, Duncan

    2016-07-01

    In this review the properties and large-scale patterns of filament channels and filaments will be considered. Initially, the global formation locations of filament channels and filaments are discussed, along with their hemispheric pattern. Next, observations of the formation of filament channels and filaments are described where two opposing views are considered. Finally, the wide range of models that have been constructed to consider the formation of filament channels and filaments over long time-scales are described, along with the origin of the hemispheric pattern of filaments.

  9. Cooperative Macromolecular Disassembly via the Heat Shock Chaperone Hsc70

    NASA Astrophysics Data System (ADS)

    Puchalla, Jason; Krantz, Kelly; Austin, Robert; Rye, Hays

    2008-03-01

    Many essential cellular functions depend on the assembly and disassembly of macromolecular complexes. A general class of protein known as molecular chaperones regulates several of these processes. How can complex protein structure be quickly and efficiently disassembled by the action of a small number of these proteins? One such example is that of clathrin: a ubiquitous coat protein that stabilizes vesicular trafficking by forming a scaffold onto the membrane surface. This scaffold must be removed before the vesicle can deliver its cargo. We report on the cooperative disassembly of yeast-derived GFP-labeled clathrin baskets via its interaction with Hsc70. We exploit the highest signal-to-noise light bursts from single fluorescent baskets transiting a confocal excitation spot to recursively determine the brightness and size distribution of the baskets during the uncoating process. This minimal uncoating system demonstrates the ability of a surprisingly simple protein system to facilitate rapid structural changes through cooperative action.

  10. Intermediate Filaments at the Junction of Mechanotransduction, Migration, and Development

    PubMed Central

    Sanghvi-Shah, Rucha; Weber, Gregory F.

    2017-01-01

    Mechanically induced signal transduction has an essential role in development. Cells actively transduce and respond to mechanical signals and their internal architecture must manage the associated forces while also being dynamically responsive. With unique assembly-disassembly dynamics and physical properties, cytoplasmic intermediate filaments play an important role in regulating cell shape and mechanical integrity. While this function has been recognized and appreciated for more than 30 years, continually emerging data also demonstrate important roles of intermediate filaments in cell signal transduction. In this review, with a particular focus on keratins and vimentin, the relationship between the physical state of intermediate filaments and their role in mechanotransduction signaling is illustrated through a survey of current literature. Association with adhesion receptors such as cadherins and integrins provides a critical interface through which intermediate filaments are exposed to forces from a cell's environment. As a consequence, these cytoskeletal networks are posttranslationally modified, remodeled and reorganized with direct impacts on local signal transduction events and cell migratory behaviors important to development. We propose that intermediate filaments provide an opportune platform for cells to both cope with mechanical forces and modulate signal transduction. PMID:28959689

  11. Intermediate Filaments at the Junction of Mechanotransduction, Migration, and Development.

    PubMed

    Sanghvi-Shah, Rucha; Weber, Gregory F

    2017-01-01

    Mechanically induced signal transduction has an essential role in development. Cells actively transduce and respond to mechanical signals and their internal architecture must manage the associated forces while also being dynamically responsive. With unique assembly-disassembly dynamics and physical properties, cytoplasmic intermediate filaments play an important role in regulating cell shape and mechanical integrity. While this function has been recognized and appreciated for more than 30 years, continually emerging data also demonstrate important roles of intermediate filaments in cell signal transduction. In this review, with a particular focus on keratins and vimentin, the relationship between the physical state of intermediate filaments and their role in mechanotransduction signaling is illustrated through a survey of current literature. Association with adhesion receptors such as cadherins and integrins provides a critical interface through which intermediate filaments are exposed to forces from a cell's environment. As a consequence, these cytoskeletal networks are posttranslationally modified, remodeled and reorganized with direct impacts on local signal transduction events and cell migratory behaviors important to development. We propose that intermediate filaments provide an opportune platform for cells to both cope with mechanical forces and modulate signal transduction.

  12. Long-range self-organization of cytoskeletal myosin II filament stacks.

    PubMed

    Hu, Shiqiong; Dasbiswas, Kinjal; Guo, Zhenhuan; Tee, Yee-Han; Thiagarajan, Visalatchi; Hersen, Pascal; Chew, Teng-Leong; Safran, Samuel A; Zaidel-Bar, Ronen; Bershadsky, Alexander D

    2017-02-01

    Although myosin II filaments are known to exist in non-muscle cells, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their 'registered' alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

  13. Capillarity-induced disassembly of virions in carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Fan, Xiaobin; Barclay, J. Elaine; Peng, Wenchao; Li, Yang; Li, Xianyu; Zhang, Guoliang; Evans, David J.; Zhang, Fengbao

    2008-04-01

    Studying the transport and fate of viruses through nanochannels is of great importance. By using the nanochannel of a carbon nanotube (CNT) as an ideal model, we evaluated the possibility of capillarity-induced viral transport through a closely fitting nanochannel and explored the mechanisms involved. It is shown both experimentally and theoretically that Cowpea mosaic virus can enter CNTs by capillarity. However, when introduced into a nanotube the protein capsid may disassemble. During the initial capillary filling stage, anomalous needle-shaped high pressure exists in the centre of the nanotube's entrance. This high pressure, combining with the significant negative pressure within the nanotube, may account for the disassembly of the virions.

  14. Planning assembly/disassembly operations for space telerobotics

    NASA Technical Reports Server (NTRS)

    Sanderson, Arthur C.; Homem De Mello, Luiz

    1987-01-01

    Space telerobotic systems will perform complex tasks of assembly, disassembly, and repair of space-based equipment. Planning such tasks requires reasoning about the functional, physical, and geometrical properties of the equipment, as well as a representation of the characteristics and capabilities of the manipulators and sensors available for the task. The And/Or graph is a useful approach to representation of feasible assembly/disassembly sequences and provides the basis for search among alternative strategies. The paper describes the use of parts entropy measures as evaluation criteria for search in the And/Or graph space. This approach leads to candidate task plans which minimize the complexity of intermediate geometrical states.

  15. Coordinated process of polarized growth in filamentous fungi.

    PubMed

    Takeshita, Norio

    2016-09-01

    Filamentous fungi are extremely polarized organisms, exhibiting continuous growth at their hyphal tips. The hyphal form is related to their pathogenicity in animals and plants, and their high secretion ability for biotechnology. Polarized growth requires a sequential supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeleton. Therefore, the arrangement of the cytoskeleton is a crucial step to establish and maintain the cell polarity. This review summarizes recent findings unraveling the mechanism of polarized growth with special emphasis on the role of actin and microtubule cytoskeleton and polarity marker proteins. Rapid insertions of membranes via highly active exocytosis at hyphal tips could quickly dilute the accumulated polarity marker proteins. Recent findings by a super-resolution microscopy indicate that filamentous fungal cells maintain their polarity at the tips by repeating transient assembly and disassembly of polarity sites.

  16. Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy

    PubMed Central

    Cohen, Shenhav; Zhai, Bo; Gygi, Steven P.

    2012-01-01

    During muscle atrophy, myofibrillar proteins are degraded in an ordered process in which MuRF1 catalyzes ubiquitylation of thick filament components (Cohen et al. 2009. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200901052). Here, we show that another ubiquitin ligase, Trim32, ubiquitylates thin filament (actin, tropomyosin, troponins) and Z-band (α-actinin) components and promotes their degradation. Down-regulation of Trim32 during fasting reduced fiber atrophy and the rapid loss of thin filaments. Desmin filaments were proposed to maintain the integrity of thin filaments. Accordingly, we find that the rapid destruction of thin filament proteins upon fasting was accompanied by increased phosphorylation of desmin filaments, which promoted desmin ubiquitylation by Trim32 and degradation. Reducing Trim32 levels prevented the loss of both desmin and thin filament proteins. Furthermore, overexpression of an inhibitor of desmin polymerization induced disassembly of desmin filaments and destruction of thin filament components. Thus, during fasting, desmin phosphorylation increases and enhances Trim32-mediated degradation of the desmin cytoskeleton, which appears to facilitate the breakdown of Z-bands and thin filaments. PMID:22908310

  17. Tungsten Filament Fire

    ERIC Educational Resources Information Center

    Ruiz, Michael J.; Perkins, James

    2016-01-01

    We safely remove the outer glass bulb from an incandescent lamp and burn up the tungsten filament after the glass is removed. This demonstration dramatically illustrates the necessity of a vacuum or inert gas for the environment surrounding the tungsten filament inside the bulb. Our approach has added historical importance since the incandescent…

  18. Tungsten filament fire

    NASA Astrophysics Data System (ADS)

    Ruiz, Michael J.; Perkins, James

    2016-05-01

    We safely remove the outer glass bulb from an incandescent lamp and burn up the tungsten filament after the glass is removed. This demonstration dramatically illustrates the necessity of a vacuum or inert gas for the environment surrounding the tungsten filament inside the bulb. Our approach has added historical importance since the incandescent light bulb is being replaced by compact fluorescent and LED lamps.

  19. Tungsten Filament Fire

    ERIC Educational Resources Information Center

    Ruiz, Michael J.; Perkins, James

    2016-01-01

    We safely remove the outer glass bulb from an incandescent lamp and burn up the tungsten filament after the glass is removed. This demonstration dramatically illustrates the necessity of a vacuum or inert gas for the environment surrounding the tungsten filament inside the bulb. Our approach has added historical importance since the incandescent…

  20. Teaching Assembly for Disassembly; An Under-Graduate Module Experience

    ERIC Educational Resources Information Center

    Alexandri, Eleftheria

    2014-01-01

    This paper is about the experience of teaching Assembly for Disassembly to fourth year architect students within the module of sustainable design. When designing a sustainable building one should take into consideration the fact that the building is going to be demolished in some years; thus the materials should be assembled in such a way so that…

  1. Binary Disassembly Block Coverage by Symbolic Execution vs. Recursive Descent

    DTIC Science & Technology

    2012-03-01

    19 2.2.2 Defensive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.2.3 Understanding... 20 2.2.4 Future-Proofing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.3 Issues with Disassembly and Symbolic...Execution . . . . . . . . . . . . . . 20 2.3.1 Data Type Inference . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.3.1.1 Variable Recovery

  2. CPAP promotes timely cilium disassembly to maintain neural progenitor pool.

    PubMed

    Gabriel, Elke; Wason, Arpit; Ramani, Anand; Gooi, Li Ming; Keller, Patrick; Pozniakovsky, Andrei; Poser, Ina; Noack, Florian; Telugu, Narasimha Swamy; Calegari, Federico; Šarić, Tomo; Hescheler, Jürgen; Hyman, Anthony A; Gottardo, Marco; Callaini, Giuliano; Alkuraya, Fowzan Sami; Gopalakrishnan, Jay

    2016-04-15

    A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms ofNPCs maintenance remain unclear. Here, we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, andOFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  3. Motor-dependent microtubule disassembly driven by tubulin tyrosination

    PubMed Central

    Wagenbach, Michael; Lafanechère, Laurence; Brocard, Jacques; Moore, Ayana T.; Kozielski, Frank; Job, Didier; Wordeman, Linda

    2009-01-01

    In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of α-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)–inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation. PMID:19564401

  4. Assembly via disassembly: A case in machine perceptual development

    NASA Technical Reports Server (NTRS)

    Bajcsy, Ruzena K.; Tsikos, Constantine J.

    1989-01-01

    First results in the effort of learning about representations of objects is presented. The questions attempted to be answered are: What is innate and what must be derived from the environment. The problem is casted in the framework of disassembly of an object into two parts.

  5. Multi-kanban mechanism for personal computer disassembly

    NASA Astrophysics Data System (ADS)

    Udomsawat, Gun; Gupta, Surendra M.; Kamarthi, Sagar V.

    2004-12-01

    The use of personal computers (PCs) continues to increase every year. According to a 1999 figure, 50 percent of all US households owned PCs, a figure that continues to rise every year. With continuous development of sophisticated software, PCs are becoming increasingly powerful. In addition, the price of a PC continues to steadily decline. Furthermore, the typical life of a PC in the workplace is approximately two to three years while in the home it is three to five years. As these PCs become obsolete, they are replaced and the old PCs are disposed of. It is estimated that between 14 and 20 million PCs are retired annually in the US. While 20 to 30% of the units may be resold, the others are discarded. These discards represent a significant potential source of lead for the waste stream. In some communities, waste cathode ray tubes (CRTs) represent the second largest source of lead in the waste stream after vehicular lead acid batteries. PCs are, therefore, not suitable for dumping in landfills. Besides, several components of a PC can be reused and then there are other valuable materials that can also be harvested. And with the advent of product stewardship, product recovery is the best solution for manufacturers. Disassembly line is perhaps the most suitable set up for disassembling PCs. However, planning and scheduling of disassembly on a disassembly line is complicated. In this paper, we discuss some of the complications including product arrival, demand arrival, inventory fluctuation and production control mechanisms. We then show how to overcome them by implementing a multi-kanban mechanism in the PC disassembly line setting. The multi-kanban mechanism relies on dynamic routing of kanbans according to the state of the system. We investigate the multi-kanban mechanism using simulation and demonstrate that this mechanism is superior to the traditional push system in terms of controlling the system"s inventory while maintaining a decent customer service level.

  6. Criticality safety evaluation for disassembly basin sand filter

    SciTech Connect

    Rosser, M.A.

    1994-05-19

    As a result of the Reactor Division`s disassembly basin cleanup program, it has been determined that fissile-isotopes are present in the sludge that has accumulated at the bottom of the disassembly basins. Good criticality safety practices require that the potential for obtaining a critical configuration with this fissile material be evaluated. As part of this process, the disassembly basin sand filter system has been identified as a potential area of concern. Because disassembly basin water flows through the sand filter, it is conceivable that fissile material, from the basin, could accumulate in the sand filter. Previous calculations have indicated that the mass of some fissile isotopes in the basin sludge exceeds subcritical mass limits. This report documents the criticality safety evaluation that was performed to address the possibility of forming a critical configuration within the sand filter. This evaluation is applicable to K and L Areas, since the fissile masses listed in Table 1 are bounding for both areas. Applicability to P Area will be examined following the completion of sludge sample analyses for that Area. Although it is conceivable that fissile material could accumulate in the sand filter, because of the required fissile mass and necessary critical geometries it is highly unlikely that a critical configuration could be assembled. The mass of fissile material required for criticality, for present and anticipated sand filter geometries and operational characteristics, is much greater than that available in the sludge, as indicated by sludge sample analyses. In short, there is no identified mechanism by which a critical configuration could be assembled in the disassembly basin sand filter.

  7. Foraging traits modulate stingless bee community disassembly under forest loss.

    PubMed

    Lichtenberg, Elinor M; Mendenhall, Chase D; Brosi, Berry

    2017-08-20

    1. Anthropogenic land use change is an important driver of impacts to biological communities and the ecosystem services they provide. Pollination is one ecosystem service that may be threatened by community disassembly. Relatively little is known about changes in bee community composition in the tropics, where pollination limitation is most severe and land use change is rapid. Understanding how anthropogenic changes alter community composition and functioning has been hampered by high variability in responses of individual species. Trait-based approaches, however, are emerging as a potential method for understanding responses of ecologically-similar species to global change. 2. We studied how communities of tropical, eusocial stingless bees (Apidae: Meliponini) disassemble when forest is lost. These bees are vital tropical pollinators that exhibit high trait diversity, but are under considerable threat from human activities. 3. We compared functional traits of stingless bee species found in pastures surrounded by differing amounts forest in an extensively deforested landscape in southern Costa Rica. 4. Our results suggest that foraging traits modulate competitive interactions that underlie community disassembly patterns. In contrast to both theoretical predictions and temperate bee communities, we found that stingless bee species with the widest diet breadths were less likely to persist in sites with less forest. These wide-diet-breadth species also tend to be solitary foragers, and are competitively subordinate to group-foraging stingless bee species. Thus, displacement by dominant, group-foraging species may make subordinate species more dependent on the larger or more diversified resource pool that natural habitats offer. We also found that traits that may reduce reliance on trees - nesting in the ground or inside nests of other species - correlated with persistence in highly deforested landscapes. 5. The functional trait perspective we employed enabled

  8. Snake Filament Eruption

    NASA Image and Video Library

    A very long solar filament that had been snaking around the Sun erupted on Dec. 6, 2010 with a flourish. NASA's Solar Dynamics Observatory (SDO) caught the action in dramatic detail in extreme ultr...

  9. Fundamentals of Filament Interaction

    DTIC Science & Technology

    2017-05-19

    Report) 4.1.2 Single filament propagation through aerosol clouds . The work reported the final year on this topic refers principally to studies that...examine the propagation of filaments through clouds . With the support of a DURIP from AFOSR, we have designed, constructed, assembled and tested a...5 m long cloud chamber capable of creating clouds of aerosol of given diameter and density, with control over temperature and background pressure

  10. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin.

    PubMed

    Chaudhry, Faisal; Breitsprecher, Dennis; Little, Kristin; Sharov, Grigory; Sokolova, Olga; Goode, Bruce L

    2013-01-01

    Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

  11. CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor

    PubMed Central

    Zhao, Shuangshuang; Jiang, Yuxiang; Zhao, Yang; Huang, Shanjin; Yuan, Ming; Zhao, Yanxiu; Guo, Yan

    2016-01-01

    The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA. Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF. PMID:27268429

  12. Status of the pit disassembly and conversion facility

    NASA Astrophysics Data System (ADS)

    Wood, Warren T.; Christensen, Lowell T.

    2000-07-01

    A planned new facility, the Pit Disassembly and Conversion Facility (PDCF), will be used to disassemble the nation's inventory of surplus nuclear weapons pits and convert the plutonium from those pits into a form suitable for storage, international inspection, and final disposition. Sized to handle 35 metric tons of plutonium from pits and other sources over its 10-year operating life, the PDCF will apply the Advanced Recovery and Integrated Extraction System (ARIES) technology. ARIES process technology has been developed at Los Alamos National Laboratory (LANL) and Lawrence Livermore National Laboratory (LLNL), and an integrated system is being demonstrated at LANL. The Los Alamos National Laboratory is the lead for technical design oversight of the PDCF. Technical data gained from the ARIES demonstrations is integral for the proper design of the PDCF.

  13. On the optimal design of the disassembly and recovery processes.

    PubMed

    Xanthopoulos, A; Iakovou, E

    2009-05-01

    This paper tackles the problem of the optimal design of the recovery processes of the end-of-life (EOL) electric and electronic products, with a special focus on the disassembly issues. The objective is to recover as much ecological and economic value as possible, and to reduce the overall produced quantities of waste. In this context, a medium-range tactical problem is defined and a novel two-phased algorithm is presented for a remanufacturing-driven reverse supply chain. In the first phase, we propose a multicriteria/goal-programming analysis for the identification and the optimal selection of the most 'desirable' subassemblies and components to be disassembled for recovery, from a set of different types of EOL products. In the second phase, a multi-product, multi-period mixed-integer linear programming (MILP) model is presented, which addresses the optimization of the recovery processes, while taking into account explicitly the lead times of the disassembly and recovery processes. Moreover, a simulation-based solution approach is proposed for capturing the uncertainties in reverse logistics. The overall approach leads to an easy-to-use methodology that could support effectively middle level management decisions. Finally, the applicability of the developed methodology is illustrated by its application on a specific case study.

  14. Disassembly of the cystovirus ϕ6 envelope by montmorillonite clay.

    PubMed

    Block, Karin A; Trusiak, Adrianna; Katz, Al; Gottlieb, Paul; Alimova, Alexandra; Wei, Hui; Morales, Jorge; Rice, William J; Steiner, Jeffrey C

    2014-02-01

    Prior studies of clay-virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. We hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. The bacteriophage Cystoviridae species φ6 used in this study is a good model for enveloped pathogens. The interaction between φ6 and montmorillonite (MMT) clay (the primary component of bentonite) is explored by transmission electron microscopy. The analyses show that MMT-φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between MMT platelet layers or attached to platelet edges. The virions swell and undergo disassembly resulting in partial or total envelope loss. Edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. The nucleocapsid (NCs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced NCs which exhibit no distortion. This visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. The MMT-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments.

  15. Disassembly of the cystovirus ϕ6 envelope by montmorillonite clay

    PubMed Central

    Block, Karin A; Trusiak, Adrianna; Katz, Al; Gottlieb, Paul; Alimova, Alexandra; Wei, Hui; Morales, Jorge; Rice, William J; Steiner, Jeffrey C

    2014-01-01

    Prior studies of clay–virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. We hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. The bacteriophage Cystoviridae species φ6 used in this study is a good model for enveloped pathogens. The interaction between φ6 and montmorillonite (MMT) clay (the primary component of bentonite) is explored by transmission electron microscopy. The analyses show that MMT–φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between MMT platelet layers or attached to platelet edges. The virions swell and undergo disassembly resulting in partial or total envelope loss. Edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. The nucleocapsid (NCs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced NCs which exhibit no distortion. This visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. The MMT-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments. PMID:24357622

  16. Interactome disassembly during apoptosis occurs independent of caspase cleavage.

    PubMed

    Scott, Nichollas E; Rogers, Lindsay D; Prudova, Anna; Brown, Nat F; Fortelny, Nikolaus; Overall, Christopher M; Foster, Leonard J

    2017-01-12

    Protein-protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas-mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome-wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases.

  17. On the optimal design of the disassembly and recovery processes

    SciTech Connect

    Xanthopoulos, A.; Iakovou, E.

    2009-05-15

    This paper tackles the problem of the optimal design of the recovery processes of the end-of-life (EOL) electric and electronic products, with a special focus on the disassembly issues. The objective is to recover as much ecological and economic value as possible, and to reduce the overall produced quantities of waste. In this context, a medium-range tactical problem is defined and a novel two-phased algorithm is presented for a remanufacturing-driven reverse supply chain. In the first phase, we propose a multicriteria/goal-programming analysis for the identification and the optimal selection of the most 'desirable' subassemblies and components to be disassembled for recovery, from a set of different types of EOL products. In the second phase, a multi-product, multi-period mixed-integer linear programming (MILP) model is presented, which addresses the optimization of the recovery processes, while taking into account explicitly the lead times of the disassembly and recovery processes. Moreover, a simulation-based solution approach is proposed for capturing the uncertainties in reverse logistics. The overall approach leads to an easy-to-use methodology that could support effectively middle level management decisions. Finally, the applicability of the developed methodology is illustrated by its application on a specific case study.

  18. (HFR-B1 experiment reporting and capsule disassembly)

    SciTech Connect

    Myers, B.F.

    1991-02-22

    The traveler visited the Joint Research Centre (JRC), Petten, The Netherlands, the Forschungszentrum GmbH (KFA), Juelich, Germany; and the Zentralinstitut fuer Kernforschung (ZfK), Rossendorf, Germany, during the period January 28 through February 9. At JRC, the analysis of the experiment HFR-B1 was discussed; a new schedule for issuance of the final data report was established. Other discussions at JRC concerned the capabilities of Petten to conduct two reactor experiments being proposed under the US/FRG cooperative program and the initial results of a proof test of Germany fuel spheres. At KFA, the main emphasis was on the disassembly of capsules 2 and 3 of the HFR-B1 experiment and agreement on the examinations and tests to be conducted with the disassembled components. The disassembly of capsule 3 was observed. Extensive discussions were conducted on the work, both experimental and analytical, being conducted in the Institut fuer Sicherheitsforschung und Reaktor Technologie. A major portion of the experimental work is being conducted at ZfK and a visit to this laboratory, sponosored by the KFA, was made on February 6 and 7. Cooperation with the US on the experimental and analytical work in the safety area was strongly emphasized. 1 tab.

  19. Filament-Filament Switching Can Be Regulated by Separation Between Filaments Together with Cargo Motor Number

    PubMed Central

    Erickson, Robert P.

    2013-01-01

    How intracellular transport controls the probability that cargos switch at intersections between filaments is not well understood. In one hypothesis some motors on the cargo attach to one filament while others attach to the intersecting filament, and the ensuing tug-of-war determines which filament is chosen. We investigate this hypothesis using 3D computer simulations, and discover that switching at intersections increases with the number of motors on the cargo, but is not strongly dependent on motor number when the filaments touch. Thus, simply controlling the number of active motors on the cargo cannot account for in vivo observations that found reduced switching with increasing motor number, suggesting additional mechanisms of regulation. We use simulations to show that one possible way to regulate switching is by simultaneously adjusting the separation between planes containing the crossing filaments and the total number of active motors on the cargo. Heretofore, the effect of filament-filament separation on switching has been unexplored. We find that the switching probability decreases with increasing filament separation. This effect is particularly strong for cargos with only a modest number of motors. As the filament separation increases past the maximum head-to-head distance of the motor, individual motors walking along a filament will be unable to reach the intersecting filament. Thus, any switching requires that other motors on the cargo attach to the intersecting filament and haul the cargo along it, while motor(s) engaged on the original filament detach. Further, if the filament separation is large enough, the cargo can have difficulty proceeding along the initial filament because the engaged motors can walk underneath the intersecting filament, but the cargo itself cannot fit between the filaments. Thus, the cargo either detaches entirely from the original filament, or must dip to the side of the initial filament and then pass below the crossing

  20. Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics.

    PubMed

    Appaduray, Mark A; Masedunskas, Andrius; Bryce, Nicole S; Lucas, Christine A; Warren, Sean C; Timpson, Paul; Stear, Jeffrey H; Gunning, Peter W; Hardeman, Edna C

    2016-01-01

    The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.

  1. Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics

    PubMed Central

    Appaduray, Mark A.; Masedunskas, Andrius; Lucas, Christine A.; Warren, Sean C.; Timpson, Paul; Stear, Jeffrey H.

    2016-01-01

    The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin. PMID:27977753

  2. SAVANNAH RIVER SITE R REACTOR DISASSEMBLY BASIN IN SITU DECOMMISSIONING

    SciTech Connect

    Langton, C.; Blankenship, J.; Griffin, W.; Serrato, M.

    2009-12-03

    The US DOE concept for facility in-situ decommissioning (ISD) is to physically stabilize and isolate in tact, structurally sound facilities that are no longer needed for their original purpose of, i.e., generating (reactor facilities), processing(isotope separation facilities) or storing radioactive materials. The 105-R Disassembly Basin is the first SRS reactor facility to undergo the in-situ decommissioning (ISD) process. This ISD process complies with the105-R Disassembly Basin project strategy as outlined in the Engineering Evaluation/Cost Analysis for the Grouting of the R-Reactor Disassembly Basin at the Savannah River Site and includes: (1) Managing residual water by solidification in-place or evaporation at another facility; (2) Filling the below grade portion of the basin with cementitious materials to physically stabilize the basin and prevent collapse of the final cap - Sludge and debris in the bottom few feet of the basin will be encapsulated between the basin floor and overlying fill material to isolate if from the environment; (3) Demolishing the above grade portion of the structure and relocating the resulting debris to another location or disposing of the debris in-place; and (4) Capping the basin area with a concrete slab which is part of an engineered cap to prevent inadvertent intrusion. The estimated total grout volume to fill the 105-R Reactor Disassembly Basin is 24,424 cubic meters or 31,945 cubic yards. Portland cement-based structural fill materials were design and tested for the reactor ISD project and a placement strategy for stabilizing the basin was developed. Based on structural engineering analyses and work flow considerations, the recommended maximum lift height is 5 feet with 24 hours between lifts. Pertinent data and information related to the SRS 105-R-Reactor Disassembly Basin in-situ decommissioning include: regulatory documentation, residual water management, area preparation activities, technology needs, fill material designs

  3. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins.

    PubMed

    Blanchoin, L; Amann, K J; Higgs, H N; Marchand, J B; Kaiser, D A; Pollard, T D

    2000-04-27

    Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.

  4. Characterization of HI Filaments

    NASA Astrophysics Data System (ADS)

    Lubar, Emily; Verschuur, Gerrit L.

    2017-01-01

    We characterized the properties of dramatic interstellar HI filaments to learn more about the dynamics and structure of such features. Using Gauss fitting software, we searched the Effelsburg-Bonn HI Survey data for indications of a simple twisting (toroidal) motion across these filaments. Instead, we found that the structure was more complicated than expected. Apparent angular widths of several filaments were measured using the Galactic Arecibo L-band Feed Array HI (GALFA-HI), Bonn, and Leident/Argentine/Bonn (LAB) surveys. Based on filament widths and other parameters, we conclude that magnetism is the dominant force opposing internal motion and maintaining the structure of these filaments. The apparent width as a function of beam width closely follows a relationship reported in 1993 for HI featu