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Sample records for rad-51 filament disassembly

  1. Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent Plasmodium falciparum SSB as a Reporter for Single-Stranded DNA.

    PubMed

    Davenport, Eric Parker; Harris, Derek F; Origanti, Sofia; Antony, Edwin

    2016-01-01

    Single-stranded DNA binding (SSB) proteins coordinate DNA replication, repair, and recombination and are critical for maintaining genomic integrity. SSB binds to single-stranded DNA (ssDNA) rapidly and with very high affinity making it a useful molecular tool to detect free ssDNA in solution. We have labeled SSB from Plasmodium falciparum (Pf-SSB) with the MDCC (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)-carbonyl)coumarin) fluorophore which yields a four-fold increase in fluorescence upon binding to ssDNA. Pf-SSBMDCC binding to DNA is unaffected by NaCl or Mg2+ concentration and does not display salt-dependent changes in DNA binding modes or cooperative binding on long DNA substrates. These features are unique to Pf-SSB, making it an ideal tool to probe the presence of free ssDNA in any biochemical reaction. Using this Pf-SSBMDCC probe as a sensor for free ssDNA, we have investigated the clearing of preformed yeast Rad51 nucleoprotein filaments by the Srs2 helicase during HR. Our studies provide a rate for the disassembly of the Rad51 filament by full length Srs2 on long ssDNA substrates. Mutations in the conserved 2B domain in the homologous bacterial UvrD, Rep and PcrA helicases show an enhancement of DNA unwinding activity, but similar mutations in Srs2 do not affect its DNA unwinding or Rad51 clearing properties. These studies showcase the utility of the Pf-SSB probe in mechanistic investigation of enzymes that function in DNA metabolism. PMID:27416037

  2. Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent Plasmodium falciparum SSB as a Reporter for Single-Stranded DNA

    PubMed Central

    Davenport, Eric Parker; Harris, Derek F.; Origanti, Sofia

    2016-01-01

    Single-stranded DNA binding (SSB) proteins coordinate DNA replication, repair, and recombination and are critical for maintaining genomic integrity. SSB binds to single-stranded DNA (ssDNA) rapidly and with very high affinity making it a useful molecular tool to detect free ssDNA in solution. We have labeled SSB from Plasmodium falciparum (Pf-SSB) with the MDCC (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)-carbonyl)coumarin) fluorophore which yields a four-fold increase in fluorescence upon binding to ssDNA. Pf-SSBMDCC binding to DNA is unaffected by NaCl or Mg2+ concentration and does not display salt-dependent changes in DNA binding modes or cooperative binding on long DNA substrates. These features are unique to Pf-SSB, making it an ideal tool to probe the presence of free ssDNA in any biochemical reaction. Using this Pf-SSBMDCC probe as a sensor for free ssDNA, we have investigated the clearing of preformed yeast Rad51 nucleoprotein filaments by the Srs2 helicase during HR. Our studies provide a rate for the disassembly of the Rad51 filament by full length Srs2 on long ssDNA substrates. Mutations in the conserved 2B domain in the homologous bacterial UvrD, Rep and PcrA helicases show an enhancement of DNA unwinding activity, but similar mutations in Srs2 do not affect its DNA unwinding or Rad51 clearing properties. These studies showcase the utility of the Pf-SSB probe in mechanistic investigation of enzymes that function in DNA metabolism. PMID:27416037

  3. 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

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

    PubMed

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

    2016-08-23

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

  5. Without Binding ATP, Human Rad51 Does Not Form Helical Filaments on ssDNA.

    PubMed

    Schay, Gusztáv; Borka, Bálint; Kernya, Linda; Bulyáki, Éva; Kardos, József; Fekete, Melinda; Fidy, Judit

    2016-03-10

    Construction of the presynaptic filament (PSF) of proper helical structure by Rad51 recombinases is a prerequisite of the progress of homologous recombination repair. We studied the contribution of ATP-binding to this structure of wt human Rad51 (hRad51). We exploited the protein-dissociation effect of high hydrostatic pressure to determine the free energy of dissociation of the protomer interfaces in hRad51 oligomer states and used electron microscopy to obtain topological parameters. Without cofactors ATP and Ca(2+) and template DNA, hRad51 did not exist in monomer form, but it formed rodlike long filaments without helical order. ΔG(diss) indicated a strong inherent tendency of aggregation. Binding solely ssDNA left the filament unstructured with slightly increased ΔG(diss). Adding only ATP and Ca(2+) to the buffer disintegrated the self-associated rods into rings and short helices of further increased ΔG(diss). Rad51 binding to ssDNA only with ATP and Ca bound could lead to ordered helical filament formation of proper pitch size with interface contacts of K(d) ∼ 2 × 10(-11) M, indicating a structure of outstanding stability. ATP/Ca binding increased the ΔG(diss) of protomer contacts in the filament by 16 kJ/mol. The results emphasize that ATP-binding in the PSF of hRad51 has an essential, yet purely structural, role. PMID:26890079

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

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

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

    2010-09-03

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-10-01

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  12. 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.

  13. Regulation of Rad51 promoter.

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    Karlin, Justin; Fischhaber, Paula L.

    2013-01-01

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

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

    SciTech Connect

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

    2010-11-05

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

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

    PubMed Central

    Khade, Nilesh V.; Sugiyama, Tomohiko

    2016-01-01

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

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

    PubMed

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

    2015-08-18

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

  18. 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.

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

    PubMed

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

    2016-07-27

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

  20. 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.

  1. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2016-06-01

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

  6. RAD51B in Familial Breast Cancer

    PubMed Central

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

    2016-01-01

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

  7. RAD51B in Familial Breast Cancer.

    PubMed

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

    2016-01-01

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

  8. 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

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

    PubMed

    Polakova, Silvia; Molnarova, Lucia; Hyppa, Randy W; Benko, Zsigmond; Misova, Ivana; Schleiffer, Alexander; Smith, Gerald R; Gregan, Juraj

    2016-06-01

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

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

    PubMed

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

    2016-06-20

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

  11. 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

  12. Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans.

    PubMed

    McClendon, T Brooke; Sullivan, Meghan R; Bernstein, Kara A; Yanowitz, Judith L

    2016-05-01

    Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSB-inducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1 Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1 We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability. PMID:26936927

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

    PubMed

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

    2016-06-01

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

  14. 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.

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2016-05-01

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

  18. Rad51 supports triple negative breast cancer metastasis

    PubMed Central

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

    2014-01-01

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

  19. The recombination mediator RAD51D promotes geminiviral infection.

    PubMed

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

    2016-06-01

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

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

    PubMed

    Zhao, Weixing; Sung, Patrick

    2015-04-30

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

  1. 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

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

    PubMed

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

    2015-03-11

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

  3. 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.

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

    Ira, Grzegorz; Haber, James E.

    2002-01-01

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

  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. 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.

  8. GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

    PubMed Central

    Takizawa, Yoshimasa; Qing, Yong; Takaku, Motoki; Ishida, Takako; Morozumi, Yuichi; Tsujita, Takashi; Kogame, Toshiaki; Hirota, Kouji; Takahashi, Masayuki; Shibata, Takehiko; Kurumizaka, Hitoshi; Takeda, Shunichi

    2010-01-01

    RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51–DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator. PMID:20403813

  9. 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

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

    PubMed

    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. PMID:26652273

  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. Small-molecule inhibitors that target protein-protein interactions in the RAD51 family of recombinases.

    PubMed

    Scott, Duncan E; Coyne, Anthony G; Venkitaraman, Ashok; Blundell, Tom L; Abell, Chris; Hyvönen, Marko

    2015-02-01

    The development of small molecules that inhibit protein-protein interactions continues to be a challenge in chemical biology and drug discovery. Herein we report the development of indole-based fragments that bind in a shallow surface pocket of a humanised surrogate of RAD51. RAD51 is an ATP-dependent recombinase that plays a key role in the repair of double-strand DNA breaks. It both self-associates, forming filament structures with DNA, and interacts with the BRCA2 protein through a common "FxxA" tetrapeptide motif. We elaborated previously identified fragment hits that target the FxxA motif site and developed small-molecule inhibitors that are approximately 500-fold more potent than the initial fragments. The lead compounds were shown to compete with the BRCA2-derived Ac-FHTA-NH2 peptide and the self-association peptide of RAD51, but they had no effect on ATP binding. This study is the first reported elaboration of small-molecular-weight fragments against this challenging target. PMID:25470112

  13. Small-Molecule Inhibitors That Target Protein–Protein Interactions in the RAD51 Family of Recombinases

    PubMed Central

    Scott, Duncan E; Coyne, Anthony G; Venkitaraman, Ashok; Blundell, Tom L; Abell, Chris; Hyvönen, Marko

    2015-01-01

    The development of small molecules that inhibit protein–protein interactions continues to be a challenge in chemical biology and drug discovery. Herein we report the development of indole-based fragments that bind in a shallow surface pocket of a humanised surrogate of RAD51. RAD51 is an ATP-dependent recombinase that plays a key role in the repair of double-strand DNA breaks. It both self-associates, forming filament structures with DNA, and interacts with the BRCA2 protein through a common “FxxA” tetrapeptide motif. We elaborated previously identified fragment hits that target the FxxA motif site and developed small-molecule inhibitors that are approximately 500-fold more potent than the initial fragments. The lead compounds were shown to compete with the BRCA2-derived Ac-FHTA-NH2 peptide and the self-association peptide of RAD51, but they had no effect on ATP binding. This study is the first reported elaboration of small-molecular-weight fragments against this challenging target. PMID:25470112

  14. 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.

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

    PubMed

    Alexiadis, Vassilios; Kadonaga, James T

    2002-11-01

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

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

    PubMed

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

    2015-01-01

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

  17. 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

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

    PubMed

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

    2015-01-01

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

  19. 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.

  20. Cyclic hypoxia does not alter RAD51 expression or PARP inhibitor cell kill in tumor cells.

    PubMed

    Kumareswaran, Ramya; Chaudary, Naz; Jaluba, Karolina; Meng, Alice; Sykes, Jenna; Borhan, Asm; Hill, Richard P; Bristow, Robert G

    2015-09-01

    Solid tumors contain regions of chronic and cyclic hypoxia. Chronic hypoxia can downregulate RAD51 and sensitize cells to PARP inhibition. Herein, we show that RAD51 expression, cell survival and toxicity to PARP inhibition is not affected under cyclic hypoxic conditions. This suggests that PARP inhibition may be selectively toxic in tumor sub-regions associated with chronic hypoxia. PMID:25842967

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

    PubMed

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

    2016-08-01

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

  2. 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.

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

    PubMed

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

    2015-03-31

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

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  8. 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

  9. Germline mutations in RAD51D confer susceptibility to ovarian cancer.

    PubMed

    Loveday, Chey; Turnbull, Clare; Ramsay, Emma; Hughes, Deborah; Ruark, Elise; Frankum, Jessica R; Bowden, Georgina; Kalmyrzaev, Bolot; Warren-Perry, Margaret; Snape, Katie; Adlard, Julian W; Barwell, Julian; Berg, Jonathan; Brady, Angela F; Brewer, Carole; Brice, Glen; Chapman, Cyril; Cook, Jackie; Davidson, Rosemarie; Donaldson, Alan; Douglas, Fiona; Greenhalgh, Lynn; Henderson, Alex; Izatt, Louise; Kumar, Ajith; Lalloo, Fiona; Miedzybrodzka, Zosia; Morrison, Patrick J; Paterson, Joan; Porteous, Mary; Rogers, Mark T; Shanley, Susan; Walker, Lisa; Eccles, Diana; Evans, D Gareth; Renwick, Anthony; Seal, Sheila; Lord, Christopher J; Ashworth, Alan; Reis-Filho, Jorge S; Antoniou, Antonis C; Rahman, Nazneen

    2011-09-01

    Recently, RAD51C mutations were identified in families with breast and ovarian cancer. This observation prompted us to investigate the role of RAD51D in cancer susceptibility. We identified eight inactivating RAD51D mutations in unrelated individuals from 911 breast-ovarian cancer families compared with one inactivating mutation identified in 1,060 controls (P = 0.01). The association found here was principally with ovarian cancer, with three mutations identified in the 59 pedigrees with three or more individuals with ovarian cancer (P = 0.0005). The relative risk of ovarian cancer for RAD51D mutation carriers was estimated to be 6.30 (95% CI 2.86-13.85, P = 4.8 × 10(-6)). By contrast, we estimated the relative risk of breast cancer to be 1.32 (95% CI 0.59-2.96, P = 0.50). These data indicate that RAD51D mutation testing may have clinical utility in individuals with ovarian cancer and their families. Moreover, we show that cells deficient in RAD51D are sensitive to treatment with a PARP inhibitor, suggesting a possible therapeutic approach for cancers arising in RAD51D mutation carriers. PMID:21822267

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

    PubMed

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

    2016-05-26

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

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed

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

    2008-12-30

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

  13. ROLE OF THE HOMOLOGOUS RECOMBINATION GENES RAD51 and RAD59 IN THE RESISTANCE OF Candida albicans TO UV LIGHT, RADIOMIMETIC AND ANTI-TUMOR COMPOUNDS AND OXIDIZING AGENTS

    PubMed Central

    García-Prieto, Fátima; Gómez-Raja, Jonathan; Andaluz, Encarnación; Calderone, Richard; Larriba, Germán

    2010-01-01

    We have cloned and characterized the RAD51 and RAD59 orthologues of the pathogenic fungus Candida albicans. CaRad51 exhibited more than 50% identity with several other eukaryotes and the conserved the catalytic domain of a bacterial RecA. As compared to the parental strain, null strains of rad51 exhibited a filamentous morphology, had a decreased grow rate and exhibited a moderate sensitivity to UV light, oxidizing agents, and compounds that cause double-strand breaks (DSB), indicating a role in DNA repair. By comparison, the rad52 null had a higher percentage of filaments, a more severe growth defect and a greater sensitivity to DNA-damaging compounds. Null strains of rad59 showed a UV-sensitive phenotype but behaved similarly to the parental strain in the rest of the assays. As compared to S. cerevisiae, C. albicans was much more resistant to bleomycin and the same was true for their respective homologous recombination (HR) mutants. These results indicate that, as described in S. cerevisiae, RAD52 plays a more prominent role than RAD51 in the repair of DSBs in C. albicans and suggest the existence of at least two Rad52-dependent HR pathways, one dependent and one independent of Rad51. PMID:20206282

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

    PubMed

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

    2016-08-01

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

  15. 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

  16. Pseudorabies virus US3 leads to filamentous actin disassembly and contributes to viral genome delivery to the nucleus.

    PubMed

    Jacob, Thary; Van den Broeke, Céline; Grauwet, Korneel; Baert, Kim; Claessen, Christophe; De Pelsmaeker, Steffi; Van Waesberghe, Cliff; Favoreel, Herman W

    2015-06-12

    The conserved alphaherpesvirus US3 tegument protein induces rearrangements of the actin cytoskeleton, consisting of protrusion formation and stress fiber breakdown. Although US3 does not affect levels of total actin protein, it remains unclear whether US3 modulates the total levels of filamentous (F) actin. In this report, we show that the pseudorabies virus (PRV) US3 protein, via its kinase activity, leads to disassembly of F-actin in porcine ST cells. F-actin disassembly has been reported before to contribute to host cell entry of HIV. In line with this, in the current study, we report that US3 has a previously uncharacterized role in viral genome delivery to the nucleus, since quantitative polymerase chain reaction (qPCR) assays on nuclear fractions demonstrated a reduced nuclear delivery of US3null PRV compared to wild type PRV genomes. Treatment of cells with the actin depolymerizing drug cytochalasin D enhanced virus genome delivery to the nucleus, particularly of US3null PRV, supporting a role for F-actin disassembly during certain aspects of viral entry. In conclusion, the US3 kinase of PRV leads to F-actin depolymerization, and US3 and F-actin disassembly contribute to viral genome delivery to the nucleus. PMID:25869795

  17. 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.

  18. Targeting homologous recombination, new pre-clinical and clinical therapeutic combinations inhibiting RAD51.

    PubMed

    Ward, Ambber; Khanna, Kum Kum; Wiegmans, Adrian P

    2015-01-01

    The DNA damage response (DDR) is essential for maintaining genomic stability and cell survival. However, when tumour cells with deficiencies in HR are faced with radio- and chemotherapies they are forced to rely on error-prone, alternative repair pathways or aberrant HR for survival; threatening genome integrity and driving further mutation. Accurate therapeutic targeting of the key drivers of DNA repair can circumvent survival pathways and avoid aggressive therapy resistant mutants. Several studies have identified that stabilization of the cancer genome in HR deficient cells can be achieved by overexpression of the recombinase RAD51. Radio- and chemotherapeutic resistance is associated with overactive HR repair mechanisms. However no clinical trials have directly targeted RAD51, despite RAD51 displaying synergy in several drug screens against multiple cancer types. Currently synthetic lethality targeting the DDR pathways and HR deficiency has had clinical success with BRCA1 functional loss and PARP inhibition. In this review we suggest that clinical outcomes could be improved by additionally targeting RAD51. We examine the latest developments in directly and indirectly targeting RAD51. We scrutinize the potential treatment efficacy and future clinical applications of RAD51 inhibitors as single agents and in combination with other therapies and consider the best therapeutic options. PMID:25467108

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

    PubMed

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

    2015-01-01

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

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

    SciTech Connect

    Schild, David; Wiese, Claudia

    2009-10-15

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

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

    PubMed

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

    2005-07-01

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

  2. 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

  3. RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity.

    PubMed

    Yard, Brian D; Reilly, Nicole M; Bedenbaugh, Michael K; Pittman, Douglas L

    2016-06-01

    The RAD51 family is integral for homologous recombination (HR) mediated DNA repair and maintaining chromosome integrity. RAD51D, the fourth member of the family, is a known ovarian cancer susceptibility gene and required for the repair of interstrand crosslink DNA damage and preserving chromosomal stability. In this report, we describe the RNF138 E3 ubiquitin ligase that interacts with and ubiquitinates the RAD51D HR protein. RNF138 is a member of an E3 ligase family that contains an amino-terminal RING finger domain and a putative carboxyl-terminal ubiquitin interaction motif. In mammalian cells, depletion of RNF138 increased the stability of the RAD51D protein, suggesting that RNF138 governs ubiquitin-proteasome-mediated degradation of RAD51D. However, RNF138 depletion conferred sensitivity to DNA damaging agents, reduced RAD51 focus formation, and increased chromosomal instability. Site-specific mutagenesis of the RNF138 RING finger domain demonstrated that it was necessary for RAD51D ubiquitination. Presence of RNF138 also enhanced the interaction between RAD51D and a known interacting RAD51 family member XRCC2 in a yeast three-hybrid assay. Therefore, RNF138 is a newly identified regulatory component of the HR mediated DNA repair pathway that has implications toward understanding how ubiquitination modifies the functions of the RAD51 paralog protein complex. PMID:27161866

  4. 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

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

    PubMed

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

    2016-02-01

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

  6. Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular fluorescence complementation analysis

    SciTech Connect

    Min, Jaewon; Park, Pil-gu; Ko, Eunkyong; Choi, Eunhee; Lee, Hyunsook

    2007-11-03

    BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair.

  7. Rad51 promoter-targeted gene therapy is effective for in vivo visualization and treatment of cancer.

    PubMed

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

    2012-02-01

    Rad51 protein is overexpressed in a wide range of human cancers. Our previous in vitro studies demonstrated that a construct comprised Rad51 promoter driving expression of the diphtheria toxin A gene (pRad51-diphtheria toxin A (DTA)) destroys a variety of human cancer cell lines, with minimal to no toxicity to normal human cells. Here we delivered Rad51 promoter-based constructs in vivo using linear polyethylenimine nanoparticles, in vivo jetPEI, to visualize and treat tumors in mice with HeLa xenografts. For tumor detection, we used pRad51-Luc, a construct containing the firefly luciferase under the Rad51 promoter, administered by intraperitoneal (IP) injection. Tumors were detected with an in vivo bioluminescent camera. All mice with cancer displayed strong bioluminescence, while mice without cancer displayed no detectable bioluminescence. Treatment with pRad51-DTA/jetPEI decreased tumor mass of subcutaneous (SC) and IP tumors by sixfold and fourfold, respectively, along with the strong reduction of malignant ascites. Fifty percent of the mice with SC tumors were cancer-free after six pRad51-DTA/jetPEI injections, and for the mice with IP tumors, mean survival time increased by 90% compared to control mice. This study demonstrates the clinical potential of pRad51-based constructs delivered by nanoparticles for the diagnostics and treatment of a wide range of cancers. PMID:22008909

  8. Clinical and biological significance of RAD51 expression in breast cancer: a key DNA damage response protein.

    PubMed

    Alshareeda, Alaa Tarig; Negm, Ola H; Aleskandarany, Mohammed A; Green, Andrew R; Nolan, Christopher; TigHhe, Patrick J; Madhusudan, Srinivasan; Ellis, Ian O; Rakha, Emad A

    2016-08-01

    Impaired DNA damage response (DDR) may play a fundamental role in the pathogenesis of breast cancer (BC). RAD51 is a key player in DNA double-strand break repair. In this study, we aimed to assess the biological and clinical significance of RAD51 expression with relevance to different molecular classes of BC and patients' outcome. The expression of RAD51 was assessed immunohistochemically in a well-characterised annotated series (n = 1184) of early-stage invasive BC with long-term follow-up. A subset of cases of BC from patients with known BRCA1 germline mutations was included as a control group. The results were correlated with clinicopathological and molecular parameters and patients' outcome. RAD51 protein expression level was also assayed in a panel of cell lines using reverse phase protein array (RPPA). RAD51 was expressed in the nuclei (N) and cytoplasm (C) of malignant cells. Subcellular co-localisation phenotypes of RAD51 were significantly associated with clinicopathological features and patient outcome. Cytoplasmic expression (RAD51C(+)) and lack of nuclear expression (RAD51 N(-)) were associated with features of aggressive behaviour, including larger tumour size, high grade, lymph nodal metastasis, basal-like, and triple-negative phenotypes, together with aberrant expression of key DDR biomarkers including BRCA1. All BRCA1-mutated tumours had RAD51C(+)/N(-) phenotype. RPPA confirmed IHC results and showed differential expression of RAD51 in cell lines based on ER expression and BRCA1 status. RAD51 N(+) and RAD51C(+) tumours were associated with longer and shorter breast cancer-specific survival (BCSS), respectively. The RAD51 N(+) was an independent predictor of longer BCSS (P < 0.0001). Lack of RAD51 nuclear expression is associated with poor prognostic parameters and shorter survival in invasive BC patients. The significant associations between RAD51 subcellular localisation and clinicopathological features, molecular subtype and patients

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-03-31

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  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. Rad51 Expression in Nasopharyngeal Carcinoma and Its Association with Tumor Reduction: A Preliminary Study in Indonesia

    PubMed Central

    Cahyanti, Dian; Rachmadi, Lisnawati; Wulani, Vally; Adham, Marlinda

    2016-01-01

    Background: Overexpression of Rad51 protein in many tumor cells has been proven to increase radioresistance and can be related to the resistance of chemosensitivity of tumor cells. This preliminary study was conducted to determine the relationship between the Rad51 expression level in nasopharyngeal carcinoma and the response of the treatment based on the measurement of the tumor reduction. Methods: Thirteen cases of the NPCs were analyzed. The expression levels of the Rad51 were examined from the pretreatment biopsies. Furthermore, tumor reductions were determined based on the change in sum longest diameter of the nasopharyngeal CT-scan before and after therapy. Results: The expression level of the Rad51 was associated with the reduction of tumor mass. The P value was 0.049 and the correlation coefficient was 0.479. Conclusion: The tumor cells Rad51 expression levels may affect the tumor reduction of NPC after the therapy. PMID:27499778

  15. Differential Requirements for RAD51 in Physcomitrella patens and Arabidopsis thaliana Development and DNA Damage Repair[W

    PubMed Central

    Markmann-Mulisch, Ulrich; Wendeler, Edelgard; Zobell, Oliver; Schween, Gabriele; Steinbiss, Hans-Henning; Reiss, Bernd

    2007-01-01

    RAD51, the eukaryotic homolog of the bacterial RecA recombinase, plays a central role in homologous recombination (HR) in yeast and animals. Loss of RAD51 function causes lethality in vertebrates but not in other animals or in the flowering plant Arabidopsis thaliana, suggesting that RAD51 is vital for highly developed organisms but not for others. Here, we found that loss of RAD51 function in the moss Physcomitrella patens, a plant of less complexity, caused a significant vegetative phenotype, indicating an important function for RAD51 in this organism. Moreover, loss of RAD51 caused marked hypersensitivity to the double-strand break-inducing agent bleomycin in P. patens but not in Arabidopsis. Therefore, HR is used for somatic DNA damage repair in P. patens but not in Arabidopsis. These data imply fundamental differences in the use of recombination pathways between plants. Moreover, these data demonstrate that the importance of RAD51 for viability is independent of taxonomic position or complexity of an organism. The involvement of HR in DNA damage repair in the slowly evolving species P. patens but not in fast-evolving Arabidopsis suggests that the choice of the recombination pathway is related to the speed of evolution in plants. PMID:17921313

  16. Further evidence for the contribution of the RAD51C gene in hereditary breast and ovarian cancer susceptibility.

    PubMed

    Vuorela, Mikko; Pylkäs, Katri; Hartikainen, Jaana M; Sundfeldt, Karin; Lindblom, Annika; von Wachenfeldt Wäppling, Anna; Haanpää, Maria; Puistola, Ulla; Rosengren, Annika; Anttila, Maarit; Kosma, Veli-Matti; Mannermaa, Arto; Winqvist, Robert

    2011-12-01

    RAD51C, a RAD51 paralogue involved in homologous recombination, is a recently established Fanconi anemia and breast cancer predisposing factor. In the initial report, RAD51C mutations were shown to confer a high risk for both breast and ovarian tumors, but most of the replication studies published so far have failed to identify any additional susceptibility alleles. Here, we report a full mutation screening of the RAD51C gene in 147 Finnish familial breast cancer cases and in 232 unselected ovarian cancer cases originating from Finland and Sweden. In addition, in order to resolve whether common RAD51C SNPs are risk factors for breast cancer, we genotyped five tagging single nucleotide polymorphisms, rs12946522, rs304270, rs304283, rs17222691, and rs28363312, all located within the gene, from 993 Finnish breast cancer cases and 871 controls for cancer associated variants. Whereas, none of the studied common SNPs associated with breast cancer susceptibility, mutation analysis revealed two clearly pathogenic alterations. RAD51C c.-13_14del27 was observed in one familial breast cancer case and c.774delT in one unselected ovarian cancer case, thus confirming that RAD51C mutations are implicated in breast and ovarian cancer predisposition, although their overall frequency seems to be low. Independent identification of the very recently reported RAD51C c.774delT mutation in yet another patient originating from Sweden suggests that it might be a recurrent mutation in that population and should be studied further. The reliable estimation of the clinical implications of carrying a defective RAD51C allele still requires the identification of additional mutation positive families. PMID:21750962

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

    PubMed

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

    2016-05-01

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

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

    PubMed

    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

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

    PubMed Central

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

    2013-01-01

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

  20. 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

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

    PubMed Central

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

    2015-01-01

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

  2. 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. PMID:25414342

  3. Structural determinants governing S100A4-induced isoform-selective disassembly of nonmuscle myosin II filaments.

    PubMed

    Kiss, Bence; Kalmár, Lajos; Nyitray, László; Pál, Gábor

    2016-06-01

    The Ca(2+) -binding protein S100A4 interacts with the C terminus of nonmuscle myosin IIA (NMIIA) causing filament disassembly, which is correlated with an increased metastatic potential of tumor cells. Despite high sequence similarity of the three NMII isoforms, S100A4 discriminates against binding to NMIIB. We searched for structural determinants of this selectivity. Based on paralog scanning using phage display, we identified a single position as major determinant of isoform selectivity. Reciprocal single amino acid replacements showed that at position 1907 (NMIIA numbering), the NMIIA/NMIIC-specific alanine provides about 60-fold higher affinity than the NMIIB-specific asparagine. The structural background of this can be explained in part by a communication between the two consecutive α-helical binding segments. This communication is completely abolished by the Ala-to-Asn substitution. Mutual swapping of the disordered tailpieces only slightly affects the affinity of the NMII chimeras. Interestingly, we found that the tailpiece and position 1907 act in a nonadditive fashion. Finally, we also found that the higher stability of the C-terminal coiled-coil region of NMIIB also discriminates against interaction with S100A4. Our results clearly show that the isoform-selective binding of S100A4 is determined at multiple levels in the structure of the three NMII isoforms and the corresponding functional elements of NMII act synergistically with one another resulting in a complex interaction network. The experimental and in silico results suggest two divergent evolutionary pathways: NMIIA and NMIIB evolved to possess S100A4-dependent and -independent regulations, respectively. PMID:27029887

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2008-11-01

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

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

    PubMed

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

    2016-05-15

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

  8. 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.

  9. RAD51 and Breast Cancer Susceptibility: No Evidence for Rare Variant Association in the Breast Cancer Family Registry Study

    PubMed Central

    Le Calvez-Kelm, Florence; Oliver, Javier; Damiola, Francesca; Forey, Nathalie; Robinot, Nivonirina; Durand, Geoffroy; Voegele, Catherine; Vallée, Maxime P.; Byrnes, Graham; Registry, Breast Cancer Family; Hopper, John L.; Southey, Melissa C.; Andrulis, Irene L.; John, Esther M.; Tavtigian, Sean V.; Lesueur, Fabienne

    2012-01-01

    Background Although inherited breast cancer has been associated with germline mutations in genes that are functionally involved in the DNA homologous recombination repair (HRR) pathway, including BRCA1, BRCA2, TP53, ATM, BRIP1, CHEK2 and PALB2, about 70% of breast cancer heritability remains unexplained. Because of their critical functions in maintaining genome integrity and already well-established associations with breast cancer susceptibility, it is likely that additional genes involved in the HRR pathway harbor sequence variants associated with increased risk of breast cancer. RAD51 plays a central biological function in DNA repair and despite the fact that rare, likely dysfunctional variants in three of its five paralogs, RAD51C, RAD51D, and XRCC2, have been associated with breast and/or ovarian cancer risk, no population-based case-control mutation screening data are available for the RAD51 gene. We thus postulated that RAD51 could harbor rare germline mutations that confer increased risk of breast cancer. Methodology/Principal Findings We screened the coding exons and proximal splice junction regions of the gene for germline sequence variation in 1,330 early-onset breast cancer cases and 1,123 controls from the Breast Cancer Family Registry, using the same population-based sampling and analytical strategy that we developed for assessment of rare sequence variants in ATM and CHEK2. In total, 12 distinct very rare or private variants were characterized in RAD51, with 10 cases (0.75%) and 9 controls (0.80%) carrying such a variant. Variants were either likely neutral missense substitutions (3), silent substitutions (4) or non-coding substitutions (5) that were predicted to have little effect on efficiency of the splicing machinery. Conclusion Altogether, our data suggest that RAD51 tolerates so little dysfunctional sequence variation that rare variants in the gene contribute little, if anything, to breast cancer susceptibility. PMID:23300655

  10. 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

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

    PubMed

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

    2015-03-01

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

  12. Gefitinib Synergizes with Irinotecan to Suppress Hepatocellular Carcinoma via Antagonizing Rad51-Mediated DNA-Repair.

    PubMed

    Shao, Jinjin; Xu, Zhifei; Peng, Xueming; Chen, Min; Zhu, Yuanrun; Xu, Li; Zhu, Hong; Yang, Bo; Luo, Peihua; He, Qiaojun

    2016-01-01

    Chemotherapy is the only choice for most of the advanced hepatocellular carcinoma (HCC) patients, while few agents were available, making it an urgent need to develop new chemotherapy strategies. A phase II clinical trial suggested that the efficacy of irinotecan in HCC was limited due to dose-dependent toxicities. Here, we found that gefitinib exhibited synergistic activity in combination with SN-38, an active metabolite of irinotecan, in HCC cell lines. And the enhanced apoptosis induced by gefitinib plus SN-38 was a result from caspase pathway activation. Mechanistically, gefitinib dramatically promoted the ubiquitin-proteasome-dependent degradation of Rad51 protein, suppressed the DNA repair, gave rise to more DNA damages, and ultimately resulted in the synergism of these two agents. In addition, the increased antitumor efficacy of gefitinib combined with irinotecan was further validated in a HepG2 xenograft mice model. Taken together, our data demonstrated for the first time that the combination of irinotecan and gefitinib showed potential benefit in HCC, which suggests that Rad51 is a promising target and provides a rationale for clinical trials investigating the efficacy of the combination of topoisomerase I inhibitors and gefitinib in HCC. PMID:26752698

  13. Gefitinib Synergizes with Irinotecan to Suppress Hepatocellular Carcinoma via Antagonizing Rad51-Mediated DNA-Repair

    PubMed Central

    Peng, Xueming; Chen, Min; Zhu, Yuanrun; Xu, Li; Zhu, Hong; Yang, Bo; Luo, Peihua; He, Qiaojun

    2016-01-01

    Chemotherapy is the only choice for most of the advanced hepatocellular carcinoma (HCC) patients, while few agents were available, making it an urgent need to develop new chemotherapy strategies. A phase II clinical trial suggested that the efficacy of irinotecan in HCC was limited due to dose-dependent toxicities. Here, we found that gefitinib exhibited synergistic activity in combination with SN-38, an active metabolite of irinotecan, in HCC cell lines. And the enhanced apoptosis induced by gefitinib plus SN-38 was a result from caspase pathway activation. Mechanistically, gefitinib dramatically promoted the ubiquitin–proteasome-dependent degradation of Rad51 protein, suppressed the DNA repair, gave rise to more DNA damages, and ultimately resulted in the synergism of these two agents. In addition, the increased antitumor efficacy of gefitinib combined with irinotecan was further validated in a HepG2 xenograft mice model. Taken together, our data demonstrated for the first time that the combination of irinotecan and gefitinib showed potential benefit in HCC, which suggests that Rad51 is a promising target and provides a rationale for clinical trials investigating the efficacy of the combination of topoisomerase I inhibitors and gefitinib in HCC. PMID:26752698

  14. 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.

  15. 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. PMID:26323318

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

    PubMed

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

    2010-12-28

    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

  17. 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.

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

    DOE PAGESBeta

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; Groesser, Torsten; Liang, Fengshan; Maranon, David G.; Leung, Stanley G.; Grundt, Kirsten; Dray, Eloïse; Idate, Rupa; et al

    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

  19. 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

  20. Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress.

    PubMed

    Chastain, Megan; Zhou, Qing; Shiva, Olga; Whitmore, Leanne; Jia, Pingping; Dai, Xueyu; Huang, Chenhui; Fadri-Moskwik, Maria; Ye, Ping; Chai, Weihang

    2016-08-01

    The telomeric CTC1/STN1/TEN1 (CST) complex has been implicated in promoting replication recovery under replication stress at genomic regions, yet its precise role is unclear. Here, we report that STN1 is enriched at GC-rich repetitive sequences genome-wide in response to hydroxyurea (HU)-induced replication stress. STN1 deficiency exacerbates the fragility of these sequences under replication stress, resulting in chromosome fragmentation. We find that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association of CST with RAD51 in an ATR-dependent manner. Strikingly, CST deficiency diminishes HU-induced RAD51 foci formation and reduces RAD51 recruitment to telomeres and non-telomeric GC-rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC-rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance. PMID:27487043

  1. 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

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

    PubMed Central

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

    2014-01-01

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

  3. p53 is involved in clearance of ionizing radiation-induced RAD51 foci in a human colon cancer cell line

    SciTech Connect

    Orre, Lukas M. . E-mail: Lukas.Orre@ki.se; Stenerloew, Bo; Dhar, Sumeer; Larsson, Rolf; Lewensohn, Rolf; Lehtioe, Janne

    2006-04-21

    We have investigated p53-related differences in cellular response to DNA damaging agents, focusing on p53s effects on RAD51 protein level and sub-cellular localization post exposure to ionizing radiation. In a human colon cancer cell line, HCT116 and its isogenic p53-/- subcell line we show here p53-independent RAD51 foci formation but interestingly the resolution of RAD51 foci showed clear p53 dependence. In p53 wt cells, but not in p53-/- cells, RAD51 protein level decreased 48 h post irradiation and fluorescence immunostaining showed resolution of RAD51 foci and relocalization of RAD51 to nucleoli at time points corresponding to the decrease in RAD51 protein level. Both cell lines rejoined DNA double strand breaks efficiently with similar kinetics and p53 status did not influence sensitivity to DNA damaging agents. We suggest that p53 has a role in RAD51 clearance post DSB repair and that nucleoli might be sites of RAD51 protein degradation.

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

    PubMed

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

    2013-01-01

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

  5. Polymorphism within the distal RAD51 gene promoter is associated with colorectal cancer in a Polish population

    PubMed Central

    Mucha, Bartosz; Kabzinski, Jacek; Dziki, Adam; Przybylowska-Sygut, Karolina; Sygut, Andrzej; Majsterek, Ireneusz; Dziki, Lukasz

    2015-01-01

    Background: colorectal cancer (CRC) is one of the most common cancers in developed countries. Annually, over one million of new cases in the world are recorded. Majority of CRCs occur sporadically with dominant phenotype of chromosomal instability (CIN). Permanent exposure to DNA damaging agents such as ionizing radiation result in DNA double-stranded breaks, which create favorable conditions for chromosomal aberration to arise. Homologous recombination repair (HRR) is the leading process engaged in maintaining of the genome integrity. RAD51 protein was recognized as crucial in HRR. Single nucleotide polymorphisms are the primary source of genetic variation which presence in the RAD51 promoter region can affect on its expression and consequently modulate HR efficiency. Objectives: The aim of this study was to analyze the distribution of genotypes and allele frequencies of -4791A/T and -4601A/G RAD51 gene polymorphisms, followed by an assessment of their relationship with the risk of CRC. Material and methods: The study included 115 patients with confirmed CRC. Control group was consisted of 118 cancer-free individuals with a negative family history. The genotypes were identified by PCR-RFLP method. Conclusion: This study revealed statistically significant association between appearance of G/A genotype in position -4601 of RAD51 gene and CRC risk. PMID:26617897

  6. Selective Chromatid Segregation Mechanism Invoked For the Human Congenital Mirror Hand Movement Disorder Development by RAD51 Mutations: A Hypothesis

    PubMed Central

    Klar, Amar J. S.

    2014-01-01

    The vertebrate body plan externally is largely symmetrical across the midline but internal organs develop asymmetrically. The biological basis of asymmetric organ development has been investigated extensively for years, although the proposed mechanisms remain controversial. By comparison, the biological origin of external organs symmetry has not been extensively investigated. Bimanual hand control is one such external organs symmetry allowing independent motor control movements of both hands to a person. This gap in our knowledge is illustrated by the recent reports of heterozygous rad51 mutations causing mysterious symptoms of congenital mirror hand movement disorder (MM) in humans with 50% penetrance by an unknown mechanism. The analysis of mutations that vary symmetry or asymmetry could be exploited to decipher the mechanisms of laterality development. Here I present a hypothesis for explaining 50% penetrance of the rad51 mutation. The MM's origin is explained with the Somatic Strand-specific Imprinting and selective sister chromatid Segregation (SSIS) hypothesis proposed originally as the mechanism of asymmetric cell division to promote visceral organs body plan laterality development in vertebrates. By hypothesis, random sister chromatid segregation in mitosis occurs for a specific chromosome due to rad51/RAD51 constitution causing MM disorder development in 50% of subjects. PMID:25210500

  7. 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

    2016-06-01

    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. PMID:26387543

  8. 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

  9. Selective chromatid segregation mechanism invoked for the human congenital mirror hand movement disorder development by RAD51 mutations: a hypothesis.

    PubMed

    Klar, Amar J S

    2014-01-01

    The vertebrate body plan externally is largely symmetrical across the midline but internal organs develop asymmetrically. The biological basis of asymmetric organ development has been investigated extensively for years, although the proposed mechanisms remain controversial. By comparison, the biological origin of external organs symmetry has not been extensively investigated. Bimanual hand control is one such external organs symmetry allowing independent motor control movements of both hands to a person. This gap in our knowledge is illustrated by the recent reports of heterozygous rad51 mutations causing mysterious symptoms of congenital mirror hand movement disorder (MM) in humans with 50% penetrance by an unknown mechanism. The analysis of mutations that vary symmetry or asymmetry could be exploited to decipher the mechanisms of laterality development. Here I present a hypothesis for explaining 50% penetrance of the rad51 mutation. The MM's origin is explained with the Somatic Strand-specific Imprinting and selective sister chromatid Segregation (SSIS) hypothesis proposed originally as the mechanism of asymmetric cell division to promote visceral organs body plan laterality development in vertebrates. By hypothesis, random sister chromatid segregation in mitosis occurs for a specific chromosome due to rad51/RAD51 constitution causing MM disorder development in 50% of subjects. PMID:25210500

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

    PubMed

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

    2015-12-01

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

  11. 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. PMID:21725594

  12. Dmc1 Functions in a Saccharomyces Cerevisiae Meiotic Pathway That Is Largely Independent of the Rad51 Pathway

    PubMed Central

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

    1997-01-01

    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. PMID:9335591

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

    PubMed Central

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

    2014-01-01

    AIM: To explore the efficacy of PCI-24781, a broad-spectrum, hydroxamic acid-derived histone deacetylase inhibitor, in the treatment of gastric cancer (GC). METHODS: With or without treatment of PCI-24781 and/or cis-diamminedichloroplatinum (CDDP), GC cell lines were subjected to functional analysis, including cell growth, apoptosis and clonogenic assays. Chromatin immunoprecipitation and luciferase reporter assays were used to determine the interacting molecules and the activity of the enzyme. An in vivo study was carried out in GC xenograft mice. Cell culture-based assays were represented as mean ± SD. ANOVA tests were used to assess differences across groups. All pairwise comparisons between tumor weights among treatment groups were made using the Tukey-Kramer method for multiple comparison adjustment to control experimental-wise type I error rates. Significance was set at P < 0.05. RESULTS: PCI-24781 significantly reduced the growth of the GC cells, enhanced cell apoptosis and suppressed clonogenicity, and these effects synergized with the effects of CDDP. PCI-24781 modulated the cell cycle and significantly reduced the expression of RAD51, which is related to homologous recombination. Depletion of RAD51 augmented the biological functions of PCI-24781, CDDP and the combination treatment, whereas overexpressing RAD51 had the opposite effects. Increased binding of the transcription suppressor E2F4 on the RAD51 promoter appeared to play a major role in these processes. Furthermore, significant suppression of tumor growth and weight in vivo was obtained following PCI-24781 treatment, which synergized with the anticancer effect of CDDP. CONCLUSION: These data suggest that RAD51 potentiates the synergistic effects of chemotherapy with PCI-24781 and CDDP on GC. PMID:25110436

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2015-01-01

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

  17. Interrogation of the Protein-Protein Interactions between Human BRCA2 BRC Repeats and RAD51 Reveals Atomistic Determinants of Affinity

    PubMed Central

    Cole, Daniel J.; Rajendra, Eeson; Roberts-Thomson, Meredith; Hardwick, Bryn; McKenzie, Grahame J.; Payne, Mike C.; Venkitaraman, Ashok R.; Skylaris, Chris-Kriton

    2011-01-01

    The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the

  18. Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.

    PubMed

    Cole, Daniel J; Rajendra, Eeson; Roberts-Thomson, Meredith; Hardwick, Bryn; McKenzie, Grahame J; Payne, Mike C; Venkitaraman, Ashok R; Skylaris, Chris-Kriton

    2011-07-01

    The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the

  19. Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase.

    PubMed

    Chanet, R; Heude, M; Adjiri, A; Maloisel, L; Fabre, F

    1996-09-01

    Suppressors of the methyl methanesulfonate sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase turned out to contain semidominant mutations in Rad5l, a homolog of the bacterial RecA protein. The nature of these mutations was determined by direct sequencing. The 26 mutations characterized were single base substitutions leading to amino acid replacements at 18 different sites. The great majority of these sites (75%) are conserved in the family of RecA-like proteins, and 10 of them affect sites corresponding to amino acids in RecA that are probably directly involved in ATP reactions, binding, and/or hydrolysis. Six mutations are in domains thought to be involved in interaction between monomers; they may also affect ATP reactions. By themselves, all the alleles confer a rad5l null phenotype. When heterozygous, however, they are, to varying degrees, negative semidominant for radiation sensitivity; presumably the mutant proteins are coassembled with wild-type Rad51 and poison the resulting nucleofilaments or recombination complexes. This negative effect is partially suppressed by an SRS2 deletion, which supports the hypothesis that Srs2 reverses recombination structures that contain either mutated proteins or numerous DNA lesions. PMID:8756636

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed Central

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

    2004-01-01

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

  2. 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

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

    PubMed

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

    2012-03-01

    Reproductive health of humans and animals exposed to daily irradiants from solar/cosmic particles remains largely understudied. We evaluated the sensitivities of bovine and mouse oocytes to bombardment by krypton-78 (1 Gy) or ultraviolet B (UV-B; 100 microjoules). Mouse oocytes responded to irradiation by undergoing massive activation of caspases, rapid loss of energy without cytochrome-c release, and subsequent necrotic death. In contrast, bovine oocytes became positive for annexin-V, exhibited cytochrome-c release, and displayed mild activation of caspases and downstream DNAses but with the absence of a complete cell death program; therefore, cytoplasmic fragmentation was never observed. However, massive cytoplasmic fragmentation and increased DNA damage were induced experimentally by both inhibiting RAD51 and increasing caspase 3 activity before irradiation. Microinjection of recombinant human RAD51 prior to irradiation markedly decreased both cytoplasmic fragmentation and DNA damage in both bovine and mouse oocytes. RAD51 response to damaged DNA occurred faster in bovine oocytes than in mouse oocytes. Therefore, we conclude that upon exposure to irradiation, bovine oocytes create a physiologically indeterminate state of partial cell death, attributed to rapid induction of DNA repair and low activation of caspases. The persistence of these damaged cells may represent an adaptive mechanism with potential implications for livestock productivity and long-term health risks associated with human activity in space. PMID:22190703

  4. 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. PMID:26820992

  5. Design, synthesis, and characterization of BRC4 mutants based on the crystal structure of BRC4-RAD51(191-220).

    PubMed

    Zhao, Dongxin; Lu, Kui

    2015-11-01

    Breast cancer susceptibility gene 2 (BRCA2)-a human tumor suppressor gene-is related to various malignancies such as breast and ovarian cancer. This gene can induce the key protein RAD51 recombinase, which is involved in homologous recombination with single-stranded DNA in the human body and can regulate RAD51 to complete the repair of damaged double-stranded DNA. Eight highly conserved BRC repeat motifs in BRCA2 protein serve as sites for the interaction between BRCA2 and RAD51. BRCA2 regulates RAD51 through these motifs. However, the mechanism of this interaction still requires further research. In this study, the BRC4 motif that demonstrated strong interaction with RAD51 was selected as template peptide. On the basis of known data regarding the crystal structure of the BRC4-RAD51(191-220) complex, a series of BRC4 mutants was designed using PyMOL software based on the sequence of BRC4, and polypeptides were synthesized by the Fmoc solid-phase method. After purification by reversed-phase high-performance liquid chromatography, the purity of the polypeptides reached >95 %. The primary determination of circular dichroism spectra showed that the polypeptides exhibited slight changes in secondary structure, which indicated that mutation on the non-conserved sites in BRC4 probably affected the interaction with BRC4. These findings will facilitate research on the interaction between targeting peptides and BRC4 mutants, as well the basic rules covering this interaction. PMID:26522863

  6. 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

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

    PubMed

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

    2014-10-01

    Replication inhibitors cause replication fork stalling and double-strand breaks (DSB) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitize cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here, we investigate why, paradoxically, mutations in HR genes protect cells from killing by gemcitabine. Using DNA replication and DNA damage assays in mammalian cells, we show that even short gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression, and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at gemcitabine-stalled forks are converted into DSBs and thus contribute to gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumors. PMID:25053826

  8. 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

  9. Genomic evolution in Barrett’s adenocarcinoma cells: critical roles of elevated hsRAD51, homologous recombination and Alu sequences in the genome

    PubMed Central

    Pal, J; Bertheau, R; Buon, L; Qazi, A; Batchu, RB; Bandyopadhyay, S; Ali-Fehmi, R; Beer, DG; Weaver, DW; Reis, RJ Shmookler; Goyal, RK; Huang, Q; Munshi, NC; Shammas, MA

    2012-01-01

    A prominent feature of most cancers including Barrett’s adenocarcinoma (BAC) is genetic instability, which is associated with development and progression of disease. In this study, we investigated the role of recombinase (hsRAD51), a key component of homologous recombination (HR)/repair, in evolving genomic changes and growth of BAC cells. We show that the expression of RAD51 is elevated in BAC cell lines and tissue specimens, relative to normal cells. HR activity is also elevated and significantly correlates with RAD51 expression in BAC cells. The suppression of RAD51 expression, by short hairpin RNA (shRNA) specifically targeting this gene, significantly prevented BAC cells from acquiring genomic changes to either copy number or heterozygosity (P<0.02) in several independent experiments employing single-nucleotide polymorphism arrays. The reduction in copy-number changes, following shRNA treatment, was confirmed by Comparative Genome Hybridization analyses of the same DNA samples. Moreover, the chromosomal distributions of mutations correlated strongly with frequencies and locations of Alu interspersed repetitive elements on individual chromosomes. We conclude that the hsRAD51 protein level is systematically elevated in BAC, contributes significantly to genomic evolution during serial propagation of these cells and correlates with disease progression. Alu sequences may serve as substrates for elevated HR during cell proliferation in vitro, as they have been reported to do during the evolution of species, and thus may provide additional targets for prevention or treatment of this disease. PMID:21423218

  10. Design of Potent Inhibitors of Human RAD51 Recombinase Based on BRC Motifs of BRCA2 Protein: Modeling and Experimental Validation of a Chimera Peptide

    PubMed Central

    2010-01-01

    We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51−ssDNA complex formation than the original peptide. PMID:20684611

  11. 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

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

    PubMed

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

    2009-08-01

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

  13. RAD51 G135C genetic polymorphism and their potential role in gastric cancer induced by Helicobacter pylori infection in Bhutan.

    PubMed

    Trang, T T H; Nagashima, H; Uchida, T; Mahachai, V; Vilaichone, R-K; Tshering, L; Binh, T T; Yamaoka, Y

    2016-01-01

    In order to evaluate the role of the RAD51 G135C genetic polymorphism on the risk of gastric cancer induced by Helicobacter pylori infection, we determined allele frequency and genotype distribution of this polymorphism in Bhutan--a population documented with high prevalence of gastric cancer and extremely high prevalence of H. pylori infection. The status of RAD51 G135C was examined by restriction fragment length polymorphism analysis of PCR amplified fragments and sequencing. Histological scores were evaluated according to the updated Sydney system. G135C carriers showed significantly higher scores for intestinal metaplasia in the antrum than G135G carriers [mean (median) 0·33 (0) vs. 0·08 (0), P = 0·008]. Higher scores for intestinal metaplasia of G135C carriers compared to those of G135G carriers were also observed in H. pylori-positive patients [0·3 (0) vs. 0·1 (0), P = 0·002] and H. pylori-positive patients with gastritis [0·4 (0) vs. 0·1 (0), P = 0·002] but were not found in H. pylori-negative patients. Our findings revealed that a combination of H. pylori infection and RAD51 G135C genotype of the host showed an increasing score for intestinal metaplasia. Therefore, RAD51 G135C might be the important predictor for gastric cancer of H. pylori-infected patients. PMID:26119522

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

    PubMed Central

    Nagaraj, V; Norris, D

    2000-01-01

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

  15. In Vivo Delivery of miR-34a Sensitizes Lung Tumors to Radiation Through RAD51 Regulation.

    PubMed

    Cortez, Maria Angelica; Valdecanas, David; Niknam, Sharareh; Peltier, Heidi J; Diao, Lixia; Giri, Uma; Komaki, Ritsuko; Calin, George A; Gomez, Daniel R; Chang, Joe Y; Heymach, John Victor; Bader, Andreas G; Welsh, James William

    2015-01-01

    MiR-34a, an important tumor-suppressing microRNA, is downregulated in several types of cancer; loss of its expression has been linked with unfavorable clinical outcomes in non-small-cell lung cancer (NSCLC), among others. MiR-34a represses several key oncogenic proteins, and a synthetic mimic of miR-34a is currently being tested in a cancer trial. However, little is known about the potential role of miR-34a in regulating DNA damage response and repair. Here, we demonstrate that miR-34a directly binds to the 3' untranslated region of RAD51 and regulates homologous recombination, inhibiting double-strand-break repair in NSCLC cells. We further demonstrate the therapeutic potential of miR-34a delivery in combination with radiotherapy in mouse models of lung cancer. Collectively, our results suggest that administration of miR-34a in combination with radiotherapy may represent a novel strategy for treating NSCLC. PMID:26670277

  16. 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

  17. 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

  18. 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

  19. Increased expression of SET domain-containing proteins and decreased expression of Rad51 in different classes of renal cell carcinoma.

    PubMed

    Liu, Si; Li, Yiyang; Xu, Hongmei; Wang, Kaichen; Li, Nan; Li, Jia; Sun, Tao; Xu, Ying

    2016-07-01

    In the present study, we aimed to examine whether SET domain-containing methyltransferases are up-regulated in different classes of renal cell carcinoma. We immunoblotted against SET domain and quantified the expression of these modular domains. Furthermore, we examined the expression of Rad51, the key protein that confers genomic stability. There was enhanced expression of SET domain-containing histone methyltransferases in whole lysates of all classes of renal carcinoma. In metastatic high grade clear cell carcinoma, this expression was more pronounced. Though we could not demonstrate direct correlation, we showed that epigenetic modification by methylation is associated with decreased genomic translation of Rad51. PMID:27170370

  20. A novel PLAG1-RAD51L1 gene fusion resulting from a t(8;14)(q12;q24) in a case of lipoblastoma.

    PubMed

    Deen, Mazin; Ebrahim, Salah; Schloff, Debbie; Mohamed, Anwar N

    2013-06-01

    Lipoblastomas are rare benign tumors that arise from embryonic adipose tissue and occur predominantly in the pediatric population. Here, we report a case of lipoblastoma in an 8-month-old boy. Surgical excision and subsequent histopathologic examination were consistent with features of lipoblastoma. Chromosome analysis of the tumor revealed a clonal unbalanced t(8;14) translocation. Genomic microarray analysis of the tumor delineated the exact breakpoints at 8q12.1 and 14q24.1, which involved the PLAG1 and RADA51L1 genes, respectively. Furthermore, fluorescence in situ hybridization demonstrated that the translocation fused the PLAG1-RAD51L1 genes. These results suggest that RAD51L1 is an alternative fusion partner gene for the PLAG1 gene in a lipoblastoma with an 8q12 rearrangement. PMID:23890983

  1. Oversized AAV Transductifon Is Mediated via a DNA-PKcs-independent, Rad51C-dependent Repair Pathway

    PubMed Central

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

    2013-01-01

    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. PMID:23939025

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

    PubMed

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

    2014-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan. PMID:24534563

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

    PubMed Central

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

    2014-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan. PMID:24534563

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

    PubMed

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

    2014-12-01

    The interaction of human Rad51 protein (HsRad51) with single-stranded deoxyribonucleic acid (ssDNA) was investigated by using quartz crystal microbalance (QCM) monitoring and atomic force microscopy (AFM) visualization. Gold surfaces for QCM and AFM were modified by electrografting of the in situ generated aryldiazonium salt from the sulfanilic acid to obtain the organic layer Au-ArSO3 H. The Au-ArSO3 H layer was activated by using a solution of PCl5 in CH2 Cl2 to give a Au-ArSO2 Cl layer. The modified surface was then used to immobilize long ssDNA molecules. The results obtained showed that the presence of adenosine diphosphate promotes the protein autoassociation rather than nucleation around DNA. In addition, when the BRC4-28 peptide inhibitor was used, both QCM and AFM confirmed the inhibitory effect of BRC4-28 toward HsRad51 autoassociation. Altogether these results show the suitability of this modified surface to investigate the kinetics and structure of DNA-protein interactions and for the screening of inhibitors. PMID:25208912

  5. Homologous Recombination Repair Signaling in Chemical Carcinogenesis: Prolonged Particulate Hexavalent Chromium Exposure Suppresses the Rad51 Response in Human Lung Cells

    PubMed Central

    Qin, Qin; Xie, Hong; Wise, Sandra S.; Browning, Cynthia L.; Thompson, Kelsey N.; Holmes, Amie L.; Wise, John Pierce

    2014-01-01

    The aim of this study was to focus on hexavalent chromium, [Cr(VI)], a chemical carcinogen and major public health concern, and consider its ability to impact DNA double strand break repair. We further focused on particulate Cr(VI), because it is the more potent carcinogenic form of Cr(VI). DNA double strand break repair serves to protect cells against the detrimental effects of DNA double strand breaks. For particulate Cr(VI), data show DNA double strand break repair must be overcome for neoplastic transformation to occur. Acute Cr(VI) exposures reveal a robust DNA double strand break repair response, however, longer exposures have not been considered. Using the comet assay, we found longer exposures to particulate zinc chromate induced concentration-dependent increases in DNA double strand breaks indicating breaks were occurring throughout the exposure time. Acute (24 h) exposure induced DNA double strand break repair signaling by inducing Mre11 foci formation, ATM phosphorylation and phosphorylated ATM foci formation, Rad51 protein levels and Rad51 foci formation. However, longer exposures reduced the Rad51 response. These data indicate a major chemical carcinogen can simultaneously induce DNA double strand breaks and alter their repair and describe a new and important aspect of the carcinogenic mechanism for Cr(VI). PMID:25173789

  6. 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

  7. 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

  8. 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.

  9. RAD51 135G>C and TP53 Arg72Pro polymorphisms and susceptibility to breast cancer in Serbian women.

    PubMed

    Krivokuca, Ana M; Malisic, Emina J; Dobricic, Jelena D; Brotto, Ksenija V; Cavic, Milena R; Jankovic, Radmila N; Tomasevic, Zorica I; Brankovic-Magic, Mirjana V

    2014-06-01

    Breast cancer is a complex disease with both genetic and environmental factors involved in its etiology. An important role of polymorphisms in genes involved in DNA repair has been reported related to breast cancer risk. We conducted a case-control study in order to investigate the association of RAD51 135G>C and TP53 Arg72Pro polymorphisms with breast cancer in Serbian women.48 BRCA negative women with breast cancer and family history of breast/ovarian cancer (hereditary group), 107 women with breast cancer but without family history of the disease (sporadic group) and 114 healthy women without a history of the disease (control group) were included. Restriction fragment length polymorphism was used for genotyping. Genotype and allelic frequencies, the odds ratio (OR) and the 95 % confidence interval (CI) were calculated as an estimate of relative risk. The Hardy-Weinberg equilibrium was tested using χ(2) test. Significance was considered for p < 0.05. RAD51 135G>C showed statistically significant association of CC genotype and increased breast cancer risk (OR 10.28, 95 % CI 1.12-94.5) in hereditary group of patients compared to the control group. Regarding the TP53 Arg72Pro, we showed statistical significance for ProPro + ProArg comparing to ArgArg (OR 2.34, 95 %, CI 1.17-4.70) in hereditary compared to sporadic group. RAD51 135G>C contributes to hereditary breast cancer in Serbian population, with CC genotype as a risk factor. We also found that carriers of Pro allele of TP53 codon 72 is related to hereditary cancer comparing to sporadic one, which indicates it as a potential risk factor for hereditary form of disease. PMID:24114315

  10. Aberrant Double-Strand Break Repair Resulting in Half Crossovers in Mutants Defective for Rad51 or the DNA Polymerase δ Complex▿

    PubMed Central

    Smith, Catherine E.; Lam, Alicia F.; Symington, Lorraine S.

    2009-01-01

    Homologous recombination is an error-free mechanism for the repair of DNA double-strand breaks (DSBs). Most DSB repair events occur by gene conversion limiting loss of heterozygosity (LOH) for markers downstream of the site of repair and restricting deleterious chromosome rearrangements. DSBs with only one end available for repair undergo strand invasion into a homologous duplex DNA, followed by replication to the chromosome end (break-induced replication [BIR]), leading to LOH for all markers downstream of the site of strand invasion. Using a transformation-based assay system, we show that most of the apparent BIR events that arise in diploid Saccharomyces cerevisiae rad51Δ mutants are due to half crossovers instead of BIR. These events lead to extensive LOH because one arm of chromosome III is deleted. This outcome is also observed in pol32Δ and pol3-ct mutants, defective for components of the DNA polymerase δ (Pol δ) complex. The half crossovers formed in Pol δ complex mutants show evidence of limited homology-dependent DNA synthesis and are partially Mus81 dependent, suggesting that strand invasion occurs and the stalled intermediate is subsequently cleaved. In contrast to rad51Δ mutants, the Pol δ complex mutants are proficient for repair of a 238-bp gap by gene conversion. Thus, the BIR defect observed for rad51 mutants is due to strand invasion failure, whereas the Pol δ complex mutants are proficient for strand invasion but unable to complete extensive tracts of recombination-initiated DNA synthesis. PMID:19139272

  11. Increased γ-H2AX and Rad51 DNA Repair Biomarker Expression in Human Cell Lines Resistant to the Chemotherapeutic Agents Nitrogen Mustard and Cisplatin.

    PubMed

    Adam-Zahir, Sheba; Plowman, Piers N; Bourton, Emma C; Sharif, Fariha; Parris, Christopher N

    2014-01-01

    Chemotherapeutic anticancer drugs mediate cytotoxicity by a number of mechanisms. However, alkylating agents which induce DNA interstrand crosslinks (ICL) are amongst the most effective anticancer agents and often form the mainstay of many anticancer therapies. The effectiveness of these drugs can be limited by the development of drug resistance in cancer cells and many studies have demonstrated that alterations in DNA repair kinetics are responsible for drug resistance. In this study we developed two cell lines resistant to the alkylating agents nitrogen mustard (HN2) and cisplatin (Pt). To determine if drug resistance was associated with enhanced ICL DNA repair we used immunocytochemistry and imaging flow cytometry to quantitate the number of γ-H2AX and Rad51 foci in the nuclei of cells after drug exposure. γ-H2AX was used to evaluate DNA strand breaks caused by repair incision nucleases and Rad51 was used to measure the activity of homologous recombination in the repair of ICL. In the drug-resistant derivative cell lines there was overall a significant increase in the number and persistence of both γ-H2AX and Rad51 foci in the nuclei of cells over a 72-hour period, when compared to the non-resistant parental cell lines (ANOVA p < 0.0001). In a Pt-resistant ovarian cancer cell line (A2780cis(R)) a similar enhancement of DNA repair was observed when compared to the non-drug-resistant wild-type ovarian cancer cells (A2780) following exposure to HN2. Our data suggest that using DNA repair biomarkers to evaluate mechanisms of resistance in cancer cell lines and human tumours may be of experimental and clinical benefit. We concede, however, that examination of a larger population of cell lines and tumours is required to fully evaluate the validity of this approach. PMID:26138778

  12. 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

  13. RAD51 135G>C substitution increases breast cancer risk in an ethnic-specific manner: a meta-analysis on 21,236 cases and 19,407 controls.

    PubMed

    Sekhar, Deepa; Pooja, Singh; Kumar, Sandeep; Rajender, Singh

    2015-01-01

    RAD51 is a homolog of bacterial RecA protein, which plays an important role in preserving stability of the genome. RAD51 interacts with BRCA1 and BRCA2 for homologous recombination repair. A functional polymorphism (135G > C) in the RAD51 gene has been a subject of great interest, which is evidenced by at least 28 case-control studies and eight meta-analyses undertaken on this polymorphism till now. We undertook a meta-analysis on RAD51 135G > C data for 21,236 cases and 19,407 controls pooled from 28 studies on breast cancer in women. Pooled data analysis suggested a significant association of the substitution with breast cancer in the recessive model (GG + GC versus CC) and in the co-dominant models comparing GG versus CC and GC versus CC. Analysis of the results suggested that 'CC' genotype is a significant breast cancer risk factor in comparison to 'GG' and 'GC' genotypes. We also undertook pooled analyses on different ethnic groups and found that 'CC' was a strong risk factor in Caucasians, but not in East-Asians and populations of mixed ethnicity. In conclusion, the RAD51 135G > C substitution in the homozygous form (CC) increases the risk of breast cancer in an ethnic-specific manner. PMID:26108708

  14. CNS myelin wrapping is driven by actin disassembly.

    PubMed

    Zuchero, J Bradley; Fu, Meng-Meng; Sloan, Steven A; Ibrahim, Adiljan; Olson, Andrew; Zaremba, Anita; Dugas, Jason C; Wienbar, Sophia; Caprariello, Andrew V; Kantor, Christopher; Leonoudakis, Dmitri; Leonoudakus, Dmitri; Lariosa-Willingham, Karen; Kronenberg, Golo; Gertz, Karen; Soderling, Scott H; Miller, Robert H; Barres, Ben A

    2015-07-27

    Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins and rapid disassembly of the oligodendrocyte actin cytoskeleton and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together, these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility. PMID:26166300

  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. 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. PMID:27234615

  17. 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.

  18. Single Nucleotide Polymorphisms (SNPs) of RAD51-G172T and XRCC2-41657C/T Homologous Recombination Repair Genes and the Risk of Triple- Negative Breast Cancer in Polish Women.

    PubMed

    Michalska, Magdalena M; Samulak, Dariusz; Romanowicz, Hanna; Smolarz, Beata

    2015-09-01

    Double strand DNA breaks are the most dangerous DNA damage which, if non-repaired or misrepaired, may result in genomic instability, cancer transformation or cell death. RAD51 and XRCC2 encode proteins that are important for the repair of double-strand DNA breaks by homologous recombination. Therefore, genetic variability in these genes may contribute to the occurrence and progression of triple-negative breast cancer. The polymorphisms of the XRCC2 gene -41657C/T (rs718282) and of the RAD51 gene, -172G/T (rs1801321), were investigated by PCR-RFLP in 70 patients with triple-negative breast cancer and 70 age- and sex matched non-cancer controls. The obtained results demonstrated a significant positive association between the RAD51 T/T genotype and TNBC, with an adjusted odds ratio (OR) of 4.94 (p = 0.001). The homozygous T/T genotype was found in 60 % of TNBC cases and in 14 % of the used controls. Variant 172 T allele of RAD51 increased cancer risk (OR = 2.81 (1.72-4.58), p < .0001). No significant associations were observed between -41657C/T genotype of XRCC2 and the incidence of TNBC. There were no significant differences between the distribution of XRCC2 -41657C/T genotypes in the subgroups assigned to histological grades. The obtained results indicate that the polymorphism of RAD51, but not of XRCC2 gene, may be positively associated with the incidence of triple-negative breast carcinoma in the population of Polish women. PMID:25743260

  19. Three New Genetic Loci (R1210C in CFH, Variants in COL8A1 and RAD51B) Are Independently Related to Progression to Advanced Macular Degeneration

    PubMed Central

    Seddon, Johanna M.; Reynolds, Robyn; Yu, Yi; Rosner, Bernard

    2014-01-01

    Objectives To assess the independent impact of new genetic variants on conversion to advanced stages of AMD, controlling for established risk factors, and to determine the contribution of genes in predictive models. Methods In this prospective longitudinal study of 2765 individuals, 777 subjects progressed to neovascular disease (NV) or geographic atrophy (GA) in either eye over 12 years. Recently reported genetic loci were assessed for their independent effects on incident advanced AMD after controlling for 6 established loci in 5 genes, and demographic, behavioral, and macular characteristics. New variants which remained significantly related to progression were then added to a final multivariate model to assess their independent effects. The contribution of genes to risk models was assessed using reclassification tables by determining risk within cross-classified quintiles for alternative models. Results Three new genetic variants were significantly related to progression: rare variant R1210C in CFH (hazard ratio (HR) 2.5, 95% confidence interval [CI] 1.2–5.3, P = 0.01), and common variants in genes COL8A1 (HR 2.0, 95% CI 1.1–3.5, P = 0.02) and RAD51B (HR 0.8, 95% CI 0.60–0.97, P = 0.03). The area under the curve statistic (AUC) was significantly higher for the 9 gene model (.884) vs the 0 gene model (.873), P = .01. AUC’s for the 9 vs 6 gene models were not significantly different, but reclassification analyses indicated significant added information for more genes, with adjusted odds ratios (OR) for progression within 5 years per one quintile increase in risk score of 2.7, P<0.001 for the 9 vs 6 loci model, and OR 3.5, P<0.001 for the 9 vs. 0 gene model. Similar results were seen for NV and GA. Conclusions Rare variant CFH R1210C and common variants in COL8A1 and RAD51B plus six genes in previous models contribute additional predictive information for advanced AMD beyond macular and behavioral phenotypes. PMID:24498017

  20. Sensitization of Tumor to 212Pb-radioimmunotherapy by gemcitabine involves initial abrogation of G2 arrest and blocked DNA damage repair by interference with Rad51

    PubMed Central

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

    2012-01-01

    Purpose To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using 212Pb-TCMC-trastuzumab together with gemcitabine (Gem) for treatment of disseminated peritoneal cancers. Methods and Materials Mice bearing human colon cancer LS-174T i.p. xenografts were pre-treated with Gem, followed by 212Pb-TCMC-trastuzumab and compared to controls. Results Treatment with 212Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase arrested tumors induced by Gem at earlier time points (6 to 24 hours). 212Pb-TCMC-trastuzumab after Gem pre-treatment 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. 212Pb-TCMC-trastuzumab treatment post-Gem pre-treatment 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 the open structure was delayed at earlier time points. Conclusion These findings suggest that the cell killing efficacy of 212Pb-TCMC-trastuzumab following Gem pre-treatment may be associated with abrogation of G2/M checkpoint, inhibition of DNA damage repair, and chromatin remodeling. PMID:23200172

  1. 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

  2. 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.

  3. 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.

  4. 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 PAGESBeta

    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 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

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

    PubMed

    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

    2016-01-01

    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. PMID:27225532

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27225532

  7. 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.

  8. 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.

    PubMed

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

    2014-10-24

    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 24h 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. PMID:25285634

  9. Gelsolin mediates calcium-dependent disassembly of Listeria actin tails

    PubMed Central

    Larson, Laura; Arnaudeau, Serge; Gibson, Bruce; Li, Wei; Krause, Ryoko; Hao, Binghua; Bamburg, James R.; Lew, Daniel P.; Demaurex, Nicolas; Southwick, Frederick

    2005-01-01

    The role of intracellular Ca2+ in the regulation of actin filament assembly and disassembly has not been clearly defined. We show that reduction of intracellular free Ca2+ concentration ([Ca2+]i) to <40 nM in Listeria monocytogenes-infected, EGFP–actin-transfected Madin–Darby canine kidney cells results in a 3-fold lengthening of actin filament tails. This increase in tail length is the consequence of marked slowing of the actin filament disassembly rate, without a significant change in assembly rate. The Ca2+-sensitive actin-severing protein gelsolin concentrates in the Listeria rocket tails at normal resting [Ca2+]i and disassociates from the tails when [Ca2+]i is lowered. Reduction in [Ca2+]i also blocks the severing activity of gelsolin, but not actin-depolymerizing factor (ADF)/cofilin microinjected into Listeria-infected cells. In Xenopus extracts, Listeria tail lengths are also calcium-sensitive, markedly shortening on addition of calcium. Immunodepletion of gelsolin, but not Xenopus ADF/cofilin, eliminates calcium-sensitive actin-filament shortening. Listeria tail length is also calcium-insensitive in gelsolin-null mouse embryo fibroblasts. We conclude that gelsolin is the primary Ca2+-sensitive actin filament recycling protein in the cell and is capable of enhancing Listeria actin tail disassembly at normal resting [Ca2+]i (145 nM). These experiments illustrate the unique and complementary functions of gelsolin and ADF/cofilin in the recycling of actin filaments. PMID:15671163

  10. Mechanism of ciliary disassembly.

    PubMed

    Liang, Yinwen; Meng, Dan; Zhu, Bing; Pan, Junmin

    2016-05-01

    As motile organelles and sensors, cilia play pivotal roles in cell physiology, development and organ homeostasis. Ciliary defects are associated with a class of cilia-related diseases or developmental disorders, termed ciliopathies. Even though the presence of cilia is required for diverse functions, cilia can be removed through ciliary shortening or resorption that necessitates disassembly of the cilium, which occurs normally during cell cycle progression, cell differentiation and in response to cellular stress. The functional significance of ciliary resorption is highlighted in controlling the G1-S transition during cell cycle progression. Internal or external cues that trigger ciliary resorption initiate signaling cascades that regulate several downstream events including depolymerization of axonemal microtubules, dynamic changes in actin and the ciliary membrane, regulation of intraflagellar transport and posttranslational modifications of ciliary proteins. To ensure ciliary resorption, both the active disassembly of the cilium and the simultaneous inhibition of ciliary assembly must be coordinately regulated. PMID:26869233

  11. 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

    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

  12. 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. PMID:26194101

  13. Cilium assembly and disassembly.

    PubMed

    Sánchez, Irma; Dynlacht, Brian David

    2016-06-28

    The primary cilium is an antenna-like, immotile organelle present on most types of mammalian cells, which interprets extracellular signals that regulate growth and development. Although once considered a vestigial organelle, the primary cilium is now the focus of considerable interest. We now know that ciliary defects lead to a panoply of human diseases, termed ciliopathies, and the loss of this organelle may be an early signature event during oncogenic transformation. Ciliopathies include numerous seemingly unrelated developmental syndromes, with involvement of the retina, kidney, liver, pancreas, skeletal system and brain. Recent studies have begun to clarify the key mechanisms that link cilium assembly and disassembly to the cell cycle, and suggest new possibilities for therapeutic intervention. PMID:27350441

  14. 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. PMID:21852249

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. A multi-stage genome-wide association in breast cancer identifies two novel risk alleles at 1p11.2 and 14q24.1 (RAD51L1)

    PubMed Central

    Thomas, Gilles; Jacobs, Kevin B.; Kraft, Peter; Yeager, Meredith; Wacholder, Sholom; Cox, David G.; Hankinson, Susan E.; Hutchinson, Amy; Wang, Zhaoming; Yu, Kai; Chatterjee, Nilanjan; Garcia-Closas, Montserrat; Gonzalez-Bosquet, Jesus; Prokunina-Olsson, Ludmila; Orr, Nick; Willett, Walter C.; Colditz, Graham A.; Ziegler, Regina G.; Berg, Christine D.; Buys, Saundra S.; McCarty, Catherine A.; Feigelson, Heather Spencer; Calle, Eugenia E.; Thun, Michael J.; Diver, Ryan; Prentice, Ross; Jackson, Rebecca; Kooperberg, Charles; Chlebowski, Rowan; Lissowska, Jolanta; Peplonska, Beata; Brinton, Louise A.; Sigurdson, Alice; Doody, Michele; Bhatti, Parveen; Alexander, Bruce H.; Buring, Julie; Lee, I-Min; Vatten, Lars J; Hveem, Kristian; Kumle, Merethe; Hayes, Richard B.; Tucker, Margaret; Gerhard, Daniela S.; Fraumeni, Joseph F.; Hoover, Robert N.; Chanock, Stephen J; Hunter, David J.

    2010-01-01

    The Cancer Genetic Markers of Susceptibility (CGEMS) initiative has conducted a three-stage genome-wide association study (GWAS) of breast cancer in 9,770 cases and 10,799 controls. In Stage 1, we genotyped 528,173 single nucleotide polymorphisms (SNPs) in 1,145 cases of invasive breast cancer among postmenopausal white women, and 1,142 controls; in Stage 2, 24,909 SNPs with low p values observed in Stage 1 were analyzed in 4,547 cases and 4,434 controls. In Stage 3 we investigated 21 loci in 4,078 cases and 5,223 controls with low p values from Stage 1 and 2 combined. Two novel loci achieved genome-wide significance. A pericentromeric SNP on chromosome 1p11.2, rs11249433, (p=6.74 × 10-10 adjusted genotype test with 2 degrees of freedom) resides in a large block of linkage disequilibrium neighboring NOTCH2 and FCGR1B and is predominantly associated with estrogen receptor-positive breast cancer. A second SNP, rs999737 on chromosome 14q24.1 (p=1.74 × 10−7), localizes to RAD51L1, a gene in the homologous recombination DNA repair pathway, a prior candidate pathway for breast cancer susceptibility. We confirmed previously reported markers on chromosome 2q35, 5q11.2, 5p12, 8q24, 10q26, and 16q12.1. Our results underscore the importance of large-scale replication in the identification of low penetrance breast cancer alleles. PMID:19330030

  2. 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

  3. 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.

  4. Genetic algorithm for disassembly process planning

    NASA Astrophysics Data System (ADS)

    Kongar, Elif; Gupta, Surendra M.

    2002-02-01

    When a product reaches its end of life, there are several options available for processing it including reuse, remanufacturing, recycling, and disposing (the least desirable option). In almost all cases, a certain level of disassembly may be necessary. Thus, finding an optimal (or near optimal) disassembly sequence is crucial to increasing the efficiency of the process. Disassembly operations are labor intensive, can be costly, have unique characteristics and cannot be considered as reverse of assembly operations. Since the complexity of determining the best disassembly sequence increases with the increase in the number of parts of the product, it is extremely crucial that an efficient methodology for disassembly process planning be developed. In this paper, we present a genetic algorithm for disassembly process planning. A case example is considered to demonstrate the functionality of the algorithm.

  5. 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.

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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.

  11. 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

  12. Biocomputing based on particle disassembly

    NASA Astrophysics Data System (ADS)

    Nikitin, Maxim P.; Shipunova, Victoria O.; Deyev, Sergey M.; Nikitin, Petr I.

    2014-09-01

    Nanoparticles with biocomputing capabilities could potentially be used to create sophisticated robotic devices with a variety of biomedical applications, including intelligent sensors and theranostic agents. DNA/RNA-based computing techniques have already been developed that can offer a complete set of Boolean logic functions and have been used, for example, to analyse cells and deliver molecular payloads. However, the computing potential of particle-based systems remains relatively unexplored. Here, we show that almost any type of nanoparticle or microparticle can be transformed into autonomous biocomputing structures that are capable of implementing a functionally complete set of Boolean logic gates (YES, NOT, AND and OR) and binding to a target as result of a computation. The logic-gating functionality is incorporated into self-assembled particle/biomolecule interfaces (demonstrated here with proteins) and the logic gating is achieved through input-induced disassembly of the structures. To illustrate the capabilities of the approach, we show that the structures can be used for logic-gated cell targeting and advanced immunoassays.

  13. Biocomputing based on particle disassembly.

    PubMed

    Nikitin, Maxim P; Shipunova, Victoria O; Deyev, Sergey M; Nikitin, Petr I

    2014-09-01

    Nanoparticles with biocomputing capabilities could potentially be used to create sophisticated robotic devices with a variety of biomedical applications, including intelligent sensors and theranostic agents. DNA/RNA-based computing techniques have already been developed that can offer a complete set of Boolean logic functions and have been used, for example, to analyse cells and deliver molecular payloads. However, the computing potential of particle-based systems remains relatively unexplored. Here, we show that almost any type of nanoparticle or microparticle can be transformed into autonomous biocomputing structures that are capable of implementing a functionally complete set of Boolean logic gates (YES, NOT, AND and OR) and binding to a target as result of a computation. The logic-gating functionality is incorporated into self-assembled particle/biomolecule interfaces (demonstrated here with proteins) and the logic gating is achieved through input-induced disassembly of the structures. To illustrate the capabilities of the approach, we show that the structures can be used for logic-gated cell targeting and advanced immunoassays. PMID:25129073

  14. Chromatin remodeling by nucleosome disassembly in vitro.

    PubMed

    Lorch, Yahli; Maier-Davis, Barbara; Kornberg, Roger D

    2006-02-28

    The RSC chromatin-remodeling complex completely disassembles a nucleosome in the presence of the histone chaperone Nap1 and ATP. Disassembly occurs in a stepwise manner, with the removal of H2A/H2B dimers, followed by the rest of the histones and the release of naked DNA. RSC and related chromatin-remodeling complexes may be responsible for the removal of promoter nucleosomes during transcriptional activation in vivo. PMID:16492771

  15. Damage-Free Relief-Valve Disassembly

    NASA Technical Reports Server (NTRS)

    Haselmaier, H.

    1986-01-01

    Tool safely disassembles relief valves without damage to sensitive parts. Relief-valve disassembly tool used to extract valve nozzle from its housing. Holding device on tool grops nozzle. When user strikes hammer against impact disk, holding device pulls nozzle from press fit. Previously, nozzle dislodged by striking spindle above it, but practice often damaged retaining screw. New tool removes nozzle directly. With minor modifications, tool adapted to valves from different manufacturers.

  16. Filament disappearances

    NASA Technical Reports Server (NTRS)

    Wagner, William J.

    1986-01-01

    The phenomenon of the sudden filament disappearance (Disparition Brusque) is a familiar one to observers at H alpha telescopes. Nevertherless, the importance in Disparition Brusques (DB) continues to grow for several reasons which are cited in the discussion. It is reported that there seems to be more interest on building and maintain filaments than in destroying them. As a consequence, this sub-group is smaller than most of the others. All the same, progress in this area of filament disapperences seems steady and assured. The importance and interest in DBs is discussed and future directions are indicated.

  17. Helical filaments

    NASA Astrophysics Data System (ADS)

    Barbieri, Nicholas; Hosseinimakarem, Zahra; Lim, Khan; Durand, Magali; Baudelet, Matthieu; Johnson, Eric; Richardson, Martin

    2014-06-01

    The shaping of laser-induced filamenting plasma channels into helical structures by guiding the process with a non-diffracting beam is demonstrated. This was achieved using a Bessel beam superposition to control the phase of an ultrafast laser beam possessing intensities sufficient to induce Kerr effect driven non-linear self-focusing. Several experimental methods were used to characterize the resulting beams and confirm the observed structures are laser air filaments.

  18. 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

  19. 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

  20. 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

  1. D-amino acids trigger biofilm disassembly.

    PubMed

    Kolodkin-Gal, Ilana; Romero, Diego; Cao, Shugeng; Clardy, Jon; Kolter, Roberto; Losick, Richard

    2010-04-30

    Bacteria form communities known as biofilms, which disassemble over time. In our studies outlined here, we found that, before biofilm disassembly, Bacillus subtilis produced a factor that prevented biofilm formation and could break down existing biofilms. The factor was shown to be a mixture of D-leucine, D-methionine, D-tyrosine, and D-tryptophan that could act at nanomolar concentrations. D-amino acid treatment caused the release of amyloid fibers that linked cells in the biofilm together. Mutants able to form biofilms in the presence of D-amino acids contained alterations in a protein (YqxM) required for the formation and anchoring of the fibers to the cell. D-amino acids also prevented biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa. D-amino acids are produced by many bacteria and, thus, may be a widespread signal for biofilm disassembly. PMID:20431016

  2. 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.

  3. 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. PMID:24073987

  4. Biophysical characterization of cofilin-induced extension-torsion coupling in actin filaments.

    PubMed

    Kim, Jae In; Kwon, Junpyo; Baek, Inchul; Na, Sungsoo

    2016-06-14

    Cofilin makes the actin filament flexible and thermally unstable by disassembling the filament and inducing bending and torsional compliance. Actin monomers bound to cofilin are able to chemically and mechanically interact in response to external forces. In this study, we performed two molecular dynamics tensile tests for actin and cofilactin filaments under identical conditions. Surprisingly, cofilactin filaments were found to be twisted, generating shear stress caused by torsion. Additionally, analysis by plane stress assumption indicated that the extension-torsion coupling effect increases the amount of principal stress by 10%. Using elasticity and solid mechanics theories, our study elucidates the role of cofilin in the disassembly of actin filaments under tensile forces. PMID:27143106

  5. Myelination: actin disassembly leads the way

    PubMed Central

    Samanta, Jayshree; Salzer, James L.

    2016-01-01

    The mechanisms that drive the spiral wrapping of the myelin sheath around axons are poorly understood. Two papers in this issue of Developmental Cell demonstrate that actin disassembly, rather than actin assembly, predominates during oligodendrocyte maturation and is critical for the genesis of the central myelin sheath. PMID:26218317

  6. 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.

  7. Automatized disassembly of electrical industrial motors

    NASA Astrophysics Data System (ADS)

    Karlsson, Bjoern; Fugger, Erwin

    1998-10-01

    Since February 1996 a large-scale European project called REMPRODUSE-Cu has been in progress. Its main objective is to provide a comprehensive approach to overcome the problems found when electromechanical systems reach the end of their useful life. How these problems could be overcome by a smarter recycling system and a smarter product design is in this project exemplified for electric motors. Today small electric motors when worn out are put in a shredder, due to problems with the disassembly. To be able to perform the disassembly in a proper way measurement and sensing as well as industrial robots will play an important part. In this paper a robotized work station for end-of-life treatment of industrial motors is presented. There are two main steps in the work. The first step is an inspection where the functionality of the motor is checked and the second step is robotized automatic disassembly for motors that can not be reused. This paper deals mainly with the second step. The robotized disassembly station consists of two industrial robots with appliances.

  8. 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.

  9. Augmented stress fiber arrays after cytopharmacologic disassembly of microtubules

    SciTech Connect

    Godman, G.C.; Tannenbaum, J.; Brett, J.B.

    1986-03-01

    Disruption of microtubules (mt) of bovine aortic endothelial (BAE) cells, and normal and transformed fibroblasts, by exposure to 2.5 ..mu..M colchicine; 12 ..mu..M vinblastine; or 1 ..mu..M nocodazole, for 5 or 20 hrs results in aggregation of vimentin-intermediate filament (IF) and the development of markedly augmented stress fiber (SF) arrays. After disassembly of mt, confluent BAE, with circumferential marginal microfilament bands and few central SF, develop dense ribbon-like SF arrays, and spontaneously transformed fibroblasts (tHmf-e), which before treatment are apolar or epithelioid and have few or no SF, acquire extensive organized SF arrays. The axially oriented SF span the entire cell length and terminate in vinculin-containing adhesion plaques, polarizing these cells. The visible increase in SF associated actin is not accompanied by an increase either in actin synthesis (determined from electropherograms after pulse labeling with (/sup 35/S)methionine), or content (DNAse I assay for total cell actin). The reorganization of actin into SF and the development of vinculin adhesion plaques is independent of protein synthesis and occurs in the presence of cycloheximide (10 ..mu..g/ml). These results suggest a role for mt and IF in the regulation of the organizational state of the actin-based cytoskeleton.

  10. 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

  11. Actin cross-link assembly and disassembly mechanics for alpha-Actinin and fascin.

    PubMed

    Courson, David S; Rock, Ronald S

    2010-08-20

    Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and alpha-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. alpha-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo. PMID:20551315

  12. Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites

    PubMed Central

    Haase, Silvia; Zimmermann, Dennis; Olshina, Maya A.; Wilkinson, Mark; Fisher, Fabio; Tan, Yan Hong; Stewart, Rebecca J.; Tonkin, Christopher J.; Wong, Wilson; Kovar, David R.; Baum, Jake

    2015-01-01

    Proteins of the actin-depolymerizing factor (ADF)/cofilin family have been shown to be crucial for the motility and survival of apicomplexan parasites. However, the mechanisms by which ADF proteins fulfill their function remain poorly understood. In this study, we investigate the comparative activities of ADF proteins from Toxoplasma gondii and Plasmodium falciparum, the human malaria parasite, using a conditional T. gondii ADF-knockout line complemented with ADF variants from either species. We show that P. falciparum ADF1 can fully restore native TgADF activity, demonstrating functional conservation between parasites. Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development. In contrast, organelle segregation was severely impaired when complementing with a TgADF mutant lacking the corresponding residue (Lys-68). Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports. These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin. PMID:26157165

  13. Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites.

    PubMed

    Haase, Silvia; Zimmermann, Dennis; Olshina, Maya A; Wilkinson, Mark; Fisher, Fabio; Tan, Yan Hong; Stewart, Rebecca J; Tonkin, Christopher J; Wong, Wilson; Kovar, David R; Baum, Jake

    2015-09-01

    Proteins of the actin-depolymerizing factor (ADF)/cofilin family have been shown to be crucial for the motility and survival of apicomplexan parasites. However, the mechanisms by which ADF proteins fulfill their function remain poorly understood. In this study, we investigate the comparative activities of ADF proteins from Toxoplasma gondii and Plasmodium falciparum, the human malaria parasite, using a conditional T. gondii ADF-knockout line complemented with ADF variants from either species. We show that P. falciparum ADF1 can fully restore native TgADF activity, demonstrating functional conservation between parasites. Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development. In contrast, organelle segregation was severely impaired when complementing with a TgADF mutant lacking the corresponding residue (Lys-68). Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports. These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin. PMID:26157165

  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. 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.

  16. 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

  17. A heuristic for disassembly planning in remanufacturing system.

    PubMed

    Sung, Jinmo; Jeong, Bongju

    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

  18. 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.

  19. 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.

  20. 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. PMID:27337469

  1. 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.

  2. 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

  3. Disassembly and Sanitization of Classified Matter

    SciTech Connect

    Stockham, Dwight J.; Saad, Max P.

    2008-01-15

    The Disassembly Sanitization Operation (DSO) process was implemented to support weapon disassembly and disposition by using recycling and waste minimization measures. This process was initiated by treaty agreements and reconfigurations within both the DOD and DOE Complexes. The DOE is faced with disassembling and disposing of a huge inventory of retired weapons, components, training equipment, spare parts, weapon maintenance equipment, and associated material. In addition, regulations have caused a dramatic increase in the need for information required to support the handling and disposition of these parts and materials. In the past, huge inventories of classified weapon components were required to have long-term storage at Sandia and at many other locations throughout the DoE Complex. These materials are placed in onsite storage unit due to classification issues and they may also contain radiological and/or hazardous components. Since no disposal options exist for this material, the only choice was long-term storage. Long-term storage is costly and somewhat problematic, requiring a secured storage area, monitoring, auditing, and presenting the potential for loss or theft of the material. Overall recycling rates for materials sent through the DSO process have enabled 70 to 80% of these components to be recycled. These components are made of high quality materials and once this material has been sanitized, the demand for the component metals for recycling efforts is very high. The DSO process for NGPF, classified components established the credibility of this technique for addressing the long-term storage requirements of the classified weapons component inventory. The success of this application has generated interest from other Sandia organizations and other locations throughout the complex. Other organizations are requesting the help of the DSO team and the DSO is responding to these requests by expanding its scope to include Work-for- Other projects. For example

  4. 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.

  5. 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

  6. Filaments from L5

    NASA Technical Reports Server (NTRS)

    Sterling, Alphonse C.

    2011-01-01

    We've been investigating filament eruptions in recent years. Why do eruptions occur? Basic mechanism is magnetic, and can often include coronal mass ejections (CMEs), flares, and filament eruptions. Use filament eruptions as markers of the more-general eruption. From our studies, we can identify directions for future work to help predict when eruptions might occur.

  7. 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.

  8. 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

  9. 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

  10. Actin network disassembly powers dissemination of Listeria monocytogenes.

    PubMed

    Talman, Arthur M; Chong, Ryan; Chia, Jonathan; Svitkina, Tatyana; Agaisse, Hervé

    2014-01-01

    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