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

PubMed Central

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

2010-01-01

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

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae.

PubMed

Saparbaev, M; Prakash, L; Prakash, S

1996-03-01

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3 delta mutation has an effect on recombination similar to that of the rad1 delta and rad10 delta mutations. The msh2 delta mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saparbaev, M.; Prakash, L.; Prakash, S.

1996-03-01

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3-A mutation has an effect on recombination similar to that of the rad1{Delta} and rad10{Delta} mutations. The msh2{Delta} mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integrationmore » of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAM-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process. 59 refs., 2 figs., 7 tabs.« less

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation.

PubMed

De Tullio, Luisina; Kaniecki, Kyle; Kwon, Youngho; Crickard, J Brooks; Sung, Patrick; Greene, Eric C

2017-10-17

Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3'→5' direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Corrupting the DNA damage response: a critical role for Rad52 in tumor cell survival.

PubMed

Lieberman, Rachel; You, Ming

2017-07-15

The DNA damage response enables cells to survive, maintain genome integrity, and to safeguard the transmission of high-fidelity genetic information. Upon sensing DNA damage, cells respond by activating this multi-faceted DNA damage response leading to restoration of the cell, senescence, programmed cell death, or genomic instability if the cell survives without proper repair. However, unlike normal cells, cancer cells maintain a marked level of genomic instability. Because of this enhanced propensity to accumulate DNA damage, tumor cells rely on homologous recombination repair as a means of protection from the lethal effect of both spontaneous and therapy-induced double-strand breaks (DSBs) in DNA. Thus, modulation of DNA repair pathways have important consequences for genomic instability within tumor cell biology and viability maintenance under high genotoxic stress. Efforts are underway to manipulate specific components of the DNA damage response in order to selectively induce tumor cell death by augmenting genomic instability past a viable threshold. New evidence suggests that RAD52, a component of the homologous recombination pathway, is important for the maintenance of tumor genome integrity. This review highlights recent reports indicating that reducing homologous recombination through inhibition of RAD52 may represent an important focus for cancer therapy and the specific efforts that are already demonstrating potential.

RAD51 interconnects between DNA replication, DNA repair and immunity.

PubMed

Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

2017-05-05

RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells.

PubMed

King, Harry O; Brend, Tim; Payne, Helen L; Wright, Alexander; Ward, Thomas A; Patel, Karan; Egnuni, Teklu; Stead, Lucy F; Patel, Anjana; Wurdak, Heiko; Short, Susan C

2017-01-10

Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

PubMed

Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

2013-12-01

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

Rad52 function prevents chromosome loss and truncation in Candida albicans

PubMed Central

Andaluz, E.; Bellido, A.; Gómez-Raja, J.; Selmecki, A.; Bouchonville, K.; Calderone, R.; Berman, J.; Larriba, G.

2013-01-01

Summary RAD52 is required for almost all recombination events in S. cerevisiae. We took advantage of the heterozygosity of HIS4 in the C. albicans SC5314 lineage to study the role of Rad52 in the genomic stability of this important fungal pathogen. The rate of loss of heterozygosity (LOH) at HIS4 in rad52-ΔΔ strains was ~10−3, at least 100-fold higher than in Rad52+ strains. LOH of whole chromosome 4 or truncation of the homologue that carries the functional HIS4 allele was detected in all 80 rad52-ΔΔ His auxotrophs (GLH –GL lab His−) obtained from six independent experiments. Isolates that had undergone whole chromosome LOH, presumably due to loss of chromosome, carried two copies of the remaining homolog. Isolates with truncations carried centric fragments of broken chromosomes healed by de novo telomere addition. GLH strains exhibited variable degrees of LOH across the genome, including two strains that became homozygous for all the heterozygous markers tested. In addition, GLH strains exhibited increased chromosomal instability (CIN), which was abolished by reintroduction of RAD52. CIN of GLH isolates is reminiscent of genomic alterations leading to cancer in human cells, and support the mutator hypothesis in which a mutator mutation or CIN phenotype facilitate more mutations/aneuploidies. PMID:21272099

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment

PubMed Central

Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

2013-01-01

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3′ endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes. PMID:24298055

Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

PubMed Central

Kramer, K M; Brock, J A; Bloom, K; Moore, J K; Haber, J E

1994-01-01

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells. PMID:8289808

SAW1 is required for SDSA double-strand break repair in S. cerevisiae.

PubMed

Diamante, Graciel; Phan, Claire; Celis, Angie S; Krueger, Jonas; Kelson, Eric P; Fischhaber, Paula L

2014-03-14

SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1-Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1-Rad10 to SDSA sites, possibly even binding as a protein-protein complex, but departs the repair site in advance of Rad1-Rad10. Copyright © 2014 Elsevier Inc. All rights reserved.

Transformation of Saccharomyces cerevisiae with UV-irradiated single-stranded plasmid.

PubMed

Zgaga, Z

1991-08-01

UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains. The low doses of UV used in this study (10-75 J/m2) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad+ strain, while they had no effect on transformation with double-stranded plasmids of comparable size. Neither the rev3 mutation, nor the rad18 or rad52 mutations influenced the efficiency of transformation with irradiated single-stranded plasmid. However, it was found to be decreased in the double rev3 rad52 mutant. Extracellular irradiation of plasmid that contains both URA3 and LEU2 genes (psLU) gave rise to up to 5% Leu- transformants among selected Ura+ ones in the repair-proficient strain. Induction of Leu- transformants was dose-dependent and only partially depressed in the rev3 mutant. These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged single-stranded DNA in yeast.

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair.

PubMed

Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

2016-10-01

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

PubMed Central

Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

2016-01-01

ABSTRACT USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1. PMID:27463890

Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI).

PubMed

Fukuda, Tomoyuki; Ohya, Yoshikazu

2006-02-01

During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.

Roles for the Rad27 Flap Endonuclease in Mitochondrial Mutagenesis and Double-Strand Break Repair in Saccharomyces cerevisiae.

PubMed

Nagarajan, Prabha; Prevost, Christopher T; Stein, Alexis; Kasimer, Rachel; Kalifa, Lidza; Sia, Elaine A

2017-06-01

The structure-specific nuclease, Rad27p/FEN1, plays a crucial role in DNA repair and replication mechanisms in the nucleus. Genetic assays using the rad27-∆ mutant have shown altered rates of DNA recombination, microsatellite instability, and point mutation in mitochondria. In this study, we examined the role of Rad27p in mitochondrial mutagenesis and double-strand break (DSB) repair in Saccharomyces cerevisiae Our findings show that Rad27p is essential for efficient mitochondrial DSB repair by a pathway that generates deletions at a region flanked by direct repeat sequences. Mutant analysis suggests that both exonuclease and endonuclease activities of Rad27p are required for its role in mitochondrial DSB repair. In addition, we found that the nuclease activities of Rad27p are required for the prevention of mitochondrial DNA (mtDNA) point mutations, and in the generation of spontaneous mtDNA rearrangements. Overall, our findings underscore the importance of Rad27p in the maintenance of mtDNA, and demonstrate that it participates in multiple DNA repair pathways in mitochondria, unlinked to nuclear phenotypes. Copyright © 2017 by the Genetics Society of America.

Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.

PubMed

Mbantenkhu, MacMillan; Wang, Xiaowen; Nardozzi, Jonathan D; Wilkens, Stephan; Hoffman, Elizabeth; Patel, Anamika; Cosgrove, Michael S; Chen, Xin Jie

2011-12-09

Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

DNA-repair scaffolds dampen checkpoint signalling by counteracting the adaptor Rad9.

PubMed

Ohouo, Patrice Y; Bastos de Oliveira, Francisco M; Liu, Yi; Ma, Chu Jian; Smolka, Marcus B

2013-01-03

In response to genotoxic stress, a transient arrest in cell-cycle progression enforced by the DNA-damage checkpoint (DDC) signalling pathway positively contributes to genome maintenance. Because hyperactivated DDC signalling can lead to a persistent and detrimental cell-cycle arrest, cells must tightly regulate the activity of the kinases involved in this pathway. Despite their importance, the mechanisms for monitoring and modulating DDC signalling are not fully understood. Here we show that the DNA-repair scaffolding proteins Slx4 and Rtt107 prevent the aberrant hyperactivation of DDC signalling by lesions that are generated during DNA replication in Saccharomyces cerevisiae. On replication stress, cells lacking Slx4 or Rtt107 show hyperactivation of the downstream DDC kinase Rad53, whereas activation of the upstream DDC kinase Mec1 remains normal. An Slx4-Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phosphorylated histone H2A, two positive regulators of Rad9-dependent Rad53 activation. A decrease in DDC signalling results from hypomorphic mutations in RAD53 and H2A and rescues the hypersensitivity to replication stress of cells lacking Slx4 or Rtt107. We propose that the Slx4-Rtt107 complex modulates Rad53 activation by a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings show that DDC signalling is monitored and modulated through the direct action of DNA-repair factors.

Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and spindle assembly checkpoint regulation

PubMed Central

Lim, Gyubum

2017-01-01

Rad52 is well known as a key factor in homologous recombination. Here, we report that Rad52 has functions unrelated to homologous recombination in Saccharomyces cerevisiae; it plays a role in the recruitment of Mps1 to the kinetochores and the maintenance of spindle assembly checkpoint (SAC) activity. Deletion of RAD52 causes various phenotypes related to the dysregulation of chromosome biorientation. Rad52 directly affects efficient operation of the SAC and accurate chromosome segregation. Remarkably, by using an in vitro kinase assay, we found that Rad52 is a substrate of Ipl1/Aurora and Mps1 in yeast and humans. Ipl1-dependent phosphorylation of Rad52 facilitates the kinetochore accumulation of Mps1, and Mps1-dependent phosphorylation of Rad52 is important for the accurate regulation of the SAC under spindle damage conditions. Taken together, our data provide detailed insights into the regulatory mechanism of chromosome biorientation by mitotic kinases. PMID:29078282

Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and spindle assembly checkpoint regulation.

PubMed

Lim, Gyubum; Huh, Won-Ki

2017-10-31

Rad52 is well known as a key factor in homologous recombination. Here, we report that Rad52 has functions unrelated to homologous recombination in Saccharomyces cerevisiae ; it plays a role in the recruitment of Mps1 to the kinetochores and the maintenance of spindle assembly checkpoint (SAC) activity. Deletion of RAD52 causes various phenotypes related to the dysregulation of chromosome biorientation. Rad52 directly affects efficient operation of the SAC and accurate chromosome segregation. Remarkably, by using an in vitro kinase assay, we found that Rad52 is a substrate of Ipl1/Aurora and Mps1 in yeast and humans. Ipl1-dependent phosphorylation of Rad52 facilitates the kinetochore accumulation of Mps1, and Mps1-dependent phosphorylation of Rad52 is important for the accurate regulation of the SAC under spindle damage conditions. Taken together, our data provide detailed insights into the regulatory mechanism of chromosome biorientation by mitotic kinases. Published under the PNAS license.

Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA.

PubMed

Guzder, S N; Sung, P; Prakash, L; Prakash, S

1998-11-20

Saccharomyces cerevisiae Rad4 and Rad23 proteins are required for the nucleotide excision repair of UV light-damaged DNA. Previous studies have indicated that these two DNA repair proteins are associated in a tight complex, which we refer to as nucleotide excision repair factor 2 (NEF2). In a reconstituted nucleotide excision repair reaction, incision of UV-damaged DNA is dependent on NEF2, indicating a role of NEF2 in an early step of the repair process. NEF2 does not, however, possess an enzymatic activity, and its function in the damage-specific incision reaction has not yet been defined. Here we use a DNA mobility shift assay to demonstrate that NEF2 binds specifically to UV-damaged DNA. Elimination of cyclobutane pyrimidine dimers from the UV-damaged DNA by enzymatic photoreactivation has little effect on the affinity of NEF2 for the DNA, suggesting that NEF2 recognizes the 6-(1, 2)-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4-(1H,3H)-pyrimidinedione photoproducts in the damaged DNA. These results highlight the intricacy of the DNA damage-demarcation reaction during nucleotide excision repair in eukaryotes.

Role of Rad23 and Dsk2 in Nucleotide Excision Repair and Spindle Pole Body Duplication

DTIC Science & Technology

2006-03-01

AD Award Number: W81XWH-05-1-0310 TITLE: Role of Rad23 and Dsk2 in Nucleotide Excision Repair and Spindle Pole Body Duplication PRINCIPAL...Feb 2006 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Role of Rad23 and Dsk2 in Nucleotide Excision Repair and Spindle Pole Body Duplication Sb. GRANT...Degradation, Cell Cycle, Spindle Pole Body 16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON OF ABSTRACT OF

Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation

PubMed Central

Dobson, Rachel; Stockdale, Christopher; Lapsley, Craig; Wilkes, Jonathan; McCulloch, Richard

2011-01-01

Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue. PMID:21615552

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

PubMed Central

Yang, Yang; Durando, Michael; Smith-Roe, Stephanie L.; Sproul, Chris; Greenwalt, Alicia M.; Kaufmann, William; Oh, Sehyun; Hendrickson, Eric A.; Vaziri, Cyrus

2013-01-01

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H2O2-induced DNA damage. UVC and H2O2 treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G0, G1 and S-phase. Rad18 was important for repressing H2O2-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G1, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H2O2-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H2O2-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G1. In contrast with G1-synchronized cultures, S-phase cells were H2O2-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G1 (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase. PMID:23295675

Coordination of Rad1-Rad10 interactions with Msh2-Msh3, Saw1 and RPA is essential for functional 3' non-homologous tail removal.

PubMed

Eichmiller, Robin; Medina-Rivera, Melisa; DeSanto, Rachel; Minca, Eugen; Kim, Christopher; Holland, Cory; Seol, Ja-Hwan; Schmit, Megan; Oramus, Diane; Smith, Jessica; Gallardo, Ignacio F; Finkelstein, Ilya J; Lee, Sang Eun; Surtees, Jennifer A

2018-06-01

Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.

The Exonuclease Homolog OsRAD1 Promotes Accurate Meiotic Double-Strand Break Repair by Suppressing Nonhomologous End Joining.

PubMed

Hu, Qing; Tang, Ding; Wang, Hongjun; Shen, Yi; Chen, Xiaojun; Ji, Jianhui; Du, Guijie; Li, Yafei; Cheng, Zhukuan

2016-10-01

During meiosis, programmed double-strand breaks (DSBs) are generated to initiate homologous recombination, which is crucial for faithful chromosome segregation. In yeast, Radiation sensitive1 (RAD1) acts together with Radiation sensitive9 (RAD9) and Hydroxyurea sensitive1 (HUS1) to facilitate meiotic recombination via cell-cycle checkpoint control. However, little is known about the meiotic functions of these proteins in higher eukaryotes. Here, we characterized a RAD1 homolog in rice (Oryza sativa) and obtained evidence that O. sativa RAD1 (OsRAD1) is important for meiotic DSB repair. Loss of OsRAD1 led to abnormal chromosome association and fragmentation upon completion of homologous pairing and synapsis. These aberrant chromosome associations were independent of OsDMC1. We found that classical nonhomologous end-joining mediated by Ku70 accounted for most of the ectopic associations in Osrad1 In addition, OsRAD1 interacts directly with OsHUS1 and OsRAD9, suggesting that these proteins act as a complex to promote DSB repair during rice meiosis. Together, these findings suggest that the 9-1-1 complex facilitates accurate meiotic recombination by suppressing nonhomologous end-joining during meiosis in rice. © 2016 American Society of Plant Biologists. All Rights Reserved.

RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, E.; Prakash, L.; Guzder, S.N.

1992-12-01

Xeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified. Homologs of human excision repair genes ERCC1, XPDC/ERCC2, and XPAC have been identified in the yeast Saccharomyces cerevisiae. Since no homolog of human XPBC/ERCC3 existed among the known yeast genes, we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog, RAD25 (SSL2), encodes a protein of 843 amino acids (M[sub r] 95,356). Themore » RAD25 (SSL2)- and XPCX-encoded proteins share 55% identical and 72% conserved amino acid residues, and the two proteins resemble one another in containing the conserved DNA helicase sequence motifs. A nonsense mutation at codon 799 that deletes the 45 C-terminal amino acid residues in RAD25 (SSL2) confers UV sensitivity. This mutation shows epistasis with genes in the excision repair group, whereas a synergistic increase in UN sensitivity occurs when it is combined with mutations in genes in other DNA repair pathways, indicating that RAD25 (SSL2) functions in excision repair but not in other repair pathways. We also show that RAD25 (SSL2) is an essential gene. A mutation of the Lys[sup 392] residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD 25 (SSL2) ATPase/DNA helicase activity in viability. 40 refs., 3 figs., 1 tab.« less

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

PubMed

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

2015-12-23

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

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

PubMed Central

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

2015-01-01

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-01-25

Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

PubMed

Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

2013-10-01

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23.

PubMed Central

Masutani, C; Sugasawa, K; Yanagisawa, J; Sonoyama, T; Ui, M; Enomoto, T; Takio, K; Tanaka, K; van der Spek, P J; Bootsma, D

1994-01-01

Complementation group C of xeroderma pigmentosum (XP) represents one of the most common forms of this cancer-prone DNA repair syndrome. The primary defect is located in the subpathway of the nucleotide excision repair system, dealing with the removal of lesions from the non-transcribing sequences ('genome-overall' repair). Here we report the purification to homogeneity and subsequent cDNA cloning of a repair complex by in vitro complementation of the XP-C defect in a cell-free repair system containing UV-damaged SV40 minichromosomes. The complex has a high affinity for ssDNA and consists of two tightly associated proteins of 125 and 58 kDa. The 125 kDa subunit is an N-terminally extended version of previously reported XPCC gene product which is thought to represent the human homologue of the Saccharomyces cerevisiae repair gene RAD4. The 58 kDa species turned out to be a human homologue of yeast RAD23. Unexpectedly, a second human counterpart of RAD23 was identified. All RAD23 derivatives share a ubiquitin-like N-terminus. The nature of the XP-C defect implies that the complex exerts a unique function in the genome-overall repair pathway which is important for prevention of skin cancer. Images PMID:8168482

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

NASA Astrophysics Data System (ADS)

Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; Park, Beomseok; Shim, Yoonjung; Kim, Youngchang; Liu, Lili; van Houten, Bennett; He, Chuan; Ansari, Anjum; Min, Jung-Hyun

2015-01-01

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

PubMed

Hengel, Sarah R; Spies, M Ashley; Spies, Maria

2017-09-21

To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiese, Claudia; Dray, Eloise; Groesser, Torsten

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 RAD51AP1more » 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.« less

p73 coordinates with Δ133p53 to promote DNA double-strand break repair.

PubMed

Gong, Hongjian; Zhang, Yuxi; Jiang, Kunpeng; Ye, Shengfan; Chen, Shuming; Zhang, Qinghe; Peng, Jinrong; Chen, Jun

2018-03-06

Tumour repressor p53 isoform Δ133p53 is a target gene of p53 and an antagonist of p53-mediated apoptotic activity. We recently demonstrated that Δ133p53 promotes DNA double-strand break (DSB) repair by upregulating transcription of the repair genes RAD51, LIG4 and RAD52 in a p53-independent manner. However, Δ133p53 lacks the transactivation domain of full-length p53, and the mechanism by which it exerts transcriptional activity independently of full-length p53 remains unclear. In this report, we describe the accumulation of high levels of both Δ133p53 and p73 (a p53 family member) at 24 h post γ-irradiation (hpi). Δ133p53 can form a complex with p73 upon γ-irradiation. The co-expression of Δ133p53 and p73, but not either protein alone, can significantly promote DNA DSB repair mechanisms, including homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). p73 and Δ133p53 act synergistically to promote the expression of RAD51, LIG4 and RAD52 by joining together to bind to region containing a Δ133p53-responsive element (RE) and a p73-RE in the promoters of all three repair genes. In addition to its accumulation at 24 hpi, p73 protein expression also peaks at 4 hpi. The depletion of p73 not only reduces early-stage apoptotic frequency (4-6 hpi), but also significantly increases later-stage DNA DSB accumulation (48 hpi), leading to cell cycle arrest in the G2 phase and, ultimately, cell senescence. In summary, the apoptotic regulator p73 also coordinates with Δ133p53 to promote DNA DSB repair, and the loss of function of p73 in DNA DSB repair may underlie spontaneous and carcinogen-induced tumorigenesis in p73 knockout mice.

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

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

PubMed

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

2005-07-06

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

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

DOE PAGES

Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; ...

2015-01-06

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivitymore » arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Lastly, kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.« less

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

PubMed Central

Paulovich, A G; Armour, C D; Hartwell, L H

1998-01-01

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication. PMID:9725831

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

PubMed

Paulovich, A G; Armour, C D; Hartwell, L H

1998-09-01

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication.

Temperate Phages Acquire DNA from Defective Prophages by Relaxed Homologous Recombination: The Role of Rad52-Like Recombinases

PubMed Central

De Paepe, Marianne; Hutinet, Geoffrey; Son, Olivier; Amarir-Bouhram, Jihane; Schbath, Sophie; Petit, Marie-Agnès

2014-01-01

Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like

A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein

PubMed Central

Ng, Jessica M.Y.; Vermeulen, Wim; van der Horst, Gijsbertus T.J.; Bergink, Steven; Sugasawa, Kaoru; Vrieling, Harry; Hoeijmakers, Jan H.J.

2003-01-01

Primary DNA damage sensing in mammalian global genome nucleotide excision repair (GG-NER) is performed by the xeroderma pigmentosum group C (XPC)/HR23B protein complex. HR23B and HR23A are human homologs of the yeast ubiquitin-domain repair factor RAD23, the function of which is unknown. Knockout mice revealed that mHR23A and mHR23B have a fully redundant role in NER, and a partially redundant function in embryonic development. Inactivation of both genes causes embryonic lethality, but appeared still compatible with cellular viability. Analysis of mHR23A/B double-mutant cells showed that HR23 proteins function in NER by governing XPC stability via partial protection against proteasomal degradation. Interestingly, NER-type DNA damage further stabilizes XPC and thereby enhances repair. These findings resolve the primary function of RAD23 in repair and reveal a novel DNA-damage-dependent regulation mechanism of DNA repair in eukaryotes, which may be part of a more global damage-response circuitry. PMID:12815074

RAD6 promotes DNA repair and stem cell signaling in ovarian cancer and is a promising therapeutic target to prevent and treat acquired chemoresistance.

PubMed

Somasagara, R R; Spencer, S M; Tripathi, K; Clark, D W; Mani, C; Madeira da Silva, L; Scalici, J; Kothayer, H; Westwell, A D; Rocconi, R P; Palle, K

2017-11-30

Ovarian cancer (OC) is the most deadly gynecological cancer and unlike most other neoplasms, survival rates for OC have not significantly improved in recent decades. We show that RAD6, an ubiquitin-conjugating enzyme, is significantly overexpressed in ovarian tumors and its expression increases in response to carboplatin chemotherapy. RAD6 expression correlated strongly with acquired chemoresistance and malignant behavior of OC cells, expression of stem cell genes and poor prognosis of OC patients, suggesting an important role for RAD6 in ovarian tumor progression. Upregulated RAD6 enhances DNA damage tolerance and repair efficiency of OC cells and promotes their survival. Increased RAD6 levels cause histone 2B ubiquitination-mediated epigenetic changes that stimulate transcription of stem cell genes, including ALDH1A1 and SOX2, leading to a cancer stem cell phenotype, which is implicated in disease recurrence and metastasis. Downregulation of RAD6 or its inhibition using a small molecule inhibitor attenuated DNA repair signaling and expression of cancer stem cells markers and sensitized chemoresistant OC cells to carboplatin. Together, these results suggest that RAD6 could be a therapeutic target to prevent and treat acquired chemoresistance and disease recurrence in OC and enhance the efficacy of standard chemotherapy.

The induction of rho- mutants by UV or gamma-rays is independent of the nuclear recombinational repair pathway in Saccharomyces cerevisiae.

PubMed

Heude, M

1988-09-01

In order to discover whether the nuclear recombinational repair pathway also acts on lesions induced in mitochondrial DNA (mtDNA), the possible role of the RAD50, -51, -52, -55 and -56 genes on the induction of rho- mutants by radiations was studied. Such induction appeared to be independent of this pathway. Nevertheless, an efficient induction of respiration-deficient mutants was observed in gamma-irradiated rad52 diploids. We demonstrate that these mutants do not result from a lack of mtDNA repair, but from chromosome losses induced by gamma-rays. Such an impairment of the respiratory ability of diploids by chromosome losses was effectively observed in the aneuploid progeny of unirradiated RAD+ cdc6 diploids incubated at the restrictive temperature.

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

PubMed

Browning, Cynthia L; Wise, John Pierce

2017-09-15

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

The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.

PubMed

Vialard, J E; Gilbert, C S; Green, C M; Lowndes, N F

1998-10-01

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

Characterization of an Escherichia coli mutant (radB101) sensitive to. gamma. and uv radiation, and methyl methanesulfonate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sargentini, N.J.; Smith, K.C.

1983-03-01

After N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis of Escherichia coli K-12 (xthA14), an X-ray-sensitive mutant was isolated. This sensitivity is due to a mutation, radB101, which is located at 56.5 min on the E.coli K-12 linkage map. The radB101 mutation sensitized wild-type cells to ..gamma.. and uv radiation, and to methyl methanesulfonate. When known DNA repair-deficient mutants were ranked for their ..gamma..-radiation sensitivity relative to their uv-radiation sensitivity, their order was (starting with the most selectively ..gamma..-radiation-sensitive strain): recB21, radB101, wild type, polA1, recF143, lexA101, recA56, uvrD3, and uvrA6. The radB mutant was normal for ..gamma..- and uv-radiation mutagenesis, it showed only a slightmore » enhancement of ..gamma..- and uv-radiation-induced DNA degradation, and it was approx. 60% deficient in recombination ability. The radB gene is suggested to play a role in the recA gene-dependent (Type III) repair of DNA single-strand breaks after ..gamma.. irradiation and in postreplication repair after uv irradiation for the following reasons: the radB strain was normal for the host-cell reactivation of ..gamma..- and uv-irradiated bacteriophage lambda; the radB mutation did not sensitize a recA strain, but did sensitize a polA strain to ..gamma.. and uv radiation; the radB mutation sensitized a uvrB strain to uv radiation.« less

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.

Single-Incision Laparoscopic Repair of Spigelian Hernia

PubMed Central

Tran, Kim; Zajkowska, Marta; Lam, Vincent; Hawthorne, Wayne J.

2015-01-01

Introduction: Spigelian hernias represent only 1% to 2% of all abdominal wall hernias. The treatment, however, remains controversial but depends on institutional expertise. This case series reports the first experience with single-incision laparoscopic totally extraperitoneal (SILTEP) repair of Spigelian hernias with telescopic extraperitoneal dissection in combination with inguinal hernia repair. Methods: From February 2013 to April 2014, all patients referred with inguinal or Spigelian hernias, without histories of extraperitoneal intervention, underwent SILTEP repair with telescopic extraperitoneal dissection. A single-port device, 5.5 mm/52 cm/30° angled laparoscope, and conventional straight dissecting instruments were used for all cases. Extraperitoneal dissection was performed under direct vision with preservation of preperitoneal fascia overlying retroperitoneal nerves. Inguinal herniorrhaphy was performed with lightweight mesh that covered low-lying Spigelian defects. High-lying Spigelian defects were repaired with additional mesh. Results: There were 131 patients with 186 (92 direct) inguinal hernias and 7 patients with 8 Spigelian hernias (6 incidental, including 1 bilateral and 2 preoperatively diagnosed), with a mean age of 51.3 years and a mean body mass index of 25.1 kg/m2. An additional piece of mesh was used for 3 hernias. All Spigelian hernias were associated with direct inguinal hernias, and 8 combined inguinal and Spigelian hernias were successfully repaired with SILTEP repair with telescopic extraperitoneal dissection as day cases. There were no clinical recurrences during a mean follow-up period of 6 months (range, 1–15 months). Conclusions: Combined Spigelian and inguinal hernias can be successfully treated with SILTEP herniorrhaphy with telescopic extraperitoneal dissection. The high incidence of Spigelian hernias associated with direct inguinal hernias suggests a high index of suspicion for Spigelian hernias during laparoscopic inguinal

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

PubMed Central

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

2015-01-01

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

Single-Port Laparoscopic Parastomal Hernia Repair with Modified Sugarbaker Technique

PubMed Central

Turingan, Isidro; Zajkowska, Marta; Tran, Kim

2014-01-01

Introduction: Laparoscopic parastomal hernia repair with modified Sugarbaker technique has become increasingly the operation of choice because of its low recurrence rates. This study aimed to assess feasibility, safety, and efficiency of performing the same operation with single-incision laparoscopic surgery. Materials and Methods: All patients referred from March 2010 to February 2013 were considered for single-port laparoscopic repair with modified Sugarbaker technique. A SILS port (Covidien, Norwalk, Connecticut, USA) was used together with conventional straight dissecting instruments and a 5.5- mm/52-cm/30° laparoscope. Important technical aspects include modified dissection techniques, namely, “inline” and “chopsticks” to overcome loss of triangulation, insertion of a urinary catheter into an ostomy for ostomy limb identification, safe adhesiolysis by avoiding electocautery, saline -jet dissection to demarcate tissue planes, dissection of an entire laparotomy scar to expose incidental incisional hernias, adequate mobilization of an ostomy limb for lateralization, and wide overlapping of defect with antiadhesive mesh. Results: Of 6 patients, 5 underwent single-port laparoscopic repair, and 1 (whose body mass index [BMI] of 39.4 kg/m2 did not permit SILS port placement) underwent multiport repair. Mean defect size was 10 cm, and mean mesh size was 660 cm2 with 4 patients having incidental incisional hernias repaired by the same mesh. Mean operation time was 270 minutes, and mean hospital stay was 4 days. Appliance malfunction ceased immediately, and pain associated with parastomal hernia disappeared. There was no recurrence with a follow-up of 2 to 36 months. Conclusion: Compared with multiport repair, single-port laparoscopic parastomal repair with modified Sugarbaker technique is safe and efficient, and it may eventually become the standard of care. PMID:24680140

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

2016-05-27

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.

Functional conservation of the yeast and Arabidopsis RAD54-like genes.

PubMed

Klutstein, Michael; Shaked, Hezi; Sherman, Amir; Avivi-Ragolsky, Naomi; Shema, Efrat; Zenvirth, Drora; Levy, Avraham A; Simchen, Giora

2008-04-01

The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance severalfold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54Delta mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-02-16

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

Rad59-Facilitated Acquisition of Y′ Elements by Short Telomeres Delays the Onset of Senescence

PubMed Central

Churikov, Dmitri; Charifi, Ferose; Simon, Marie-Noëlle; Géli, Vincent

2014-01-01

Telomerase-negative yeasts survive via one of the two Rad52-dependent recombination pathways, which have distinct genetic requirements. Although the telomere pattern of type I and type II survivors is well characterized, the mechanistic details of short telomere rearrangement into highly evolved pattern observed in survivors are still missing. Here, we analyze immediate events taking place at the abruptly shortened VII-L and native telomeres. We show that short telomeres engage in pairing with internal Rap1-bound TG1–3-like tracts present between subtelomeric X and Y′ elements, which is followed by BIR-mediated non-reciprocal translocation of Y′ element and terminal TG1–3 repeats from the donor end onto the shortened telomere. We found that choice of the Y′ donor was not random, since both engineered telomere VII-L and native VI-R acquired Y′ elements from partially overlapping sets of specific chromosome ends. Although short telomere repair was associated with transient delay in cell divisions, Y′ translocation on native telomeres did not require Mec1-dependent checkpoint. Furthermore, the homeologous pairing between the terminal TG1–3 repeats at VII-L and internal repeats on other chromosome ends was largely independent of Rad51, but instead it was facilitated by Rad59 that stimulates Rad52 strand annealing activity. Therefore, Y′ translocation events taking place during presenescence are genetically separable from Rad51-dependent Y′ amplification process that occurs later during type I survivor formation. We show that Rad59-facilitated Y′ translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation. PMID:25375789

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex

PubMed Central

Chakraborty, Sagnik; Steinbach, Peter J; Paul, Debamita; Mu, Hong; Broyde, Suse

2018-01-01

Abstract Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. PMID:29267981

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae.

PubMed Central

Soustelle, Christine; Vedel, Michèle; Kolodner, Richard; Nicolas, Alain

2002-01-01

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis. PMID:12072452

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

PubMed Central

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

2015-01-01

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

Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites

PubMed Central

Haas, Kalina T; Lee, MiYoung; Esposito, Alessandro; Venkitaraman, Ashok R

2018-01-01

Abstract RAD51 recombinase assembles on single-stranded (ss)DNA substrates exposed by DNA end-resection to initiate homologous recombination (HR), a process fundamental to genome integrity. RAD51 assembly has been characterized using purified proteins, but its ultrastructural topography in the cell nucleus is unexplored. Here, we combine cell genetics with single-molecule localization microscopy and a palette of bespoke analytical tools, to visualize molecular transactions during RAD51 assembly in the cellular milieu at resolutions approaching 30–40 nm. In several human cell types, RAD51 focalizes in clusters that progressively extend into long filaments, which abut—but do not overlap—with globular bundles of replication protein A (RPA). Extended filaments alter topographically over time, suggestive of succeeding steps in HR. In cells depleted of the tumor suppressor protein BRCA2, or overexpressing its RAD51-binding BRC repeats, RAD51 fails to assemble at damage sites, although RPA accumulates unhindered. By contrast, in cells lacking a BRCA2 carboxyl (C)-terminal region targeted by cancer-causing mutations, damage-induced RAD51 assemblies initiate but do not extend into filaments. We suggest a model wherein RAD51 assembly proceeds concurrently with end-resection at adjacent sites, via an initiation step dependent on the BRC repeats, followed by filament extension through the C-terminal region of BRCA2. PMID:29309696

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination

PubMed Central

Sugawara, Neal; Pâques, Frédéric; Colaiácovo, Mónica; Haber, James E.

1997-01-01

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3′ tail. PMID:9256462

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination.

PubMed

Sugawara, N; Pâques, F; Colaiácovo, M; Haber, J E

1997-08-19

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3' tail.

Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance.

PubMed

Khair, Lyne; Chang, Ya-Ting; Subramanian, Lakxmi; Russell, Paul; Nakamura, Toru M

2010-06-01

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.

RecA family proteins in archaea: RadA and its cousins.

PubMed

Haldenby, Sam; White, Malcolm F; Allers, Thorsten

2009-02-01

Recombinases of the RecA family are essential for homologous recombination and underpin genome stability, by promoting the repair of double-stranded DNA breaks and the rescue of collapsed DNA replication forks. Until now, our understanding of homologous recombination has relied on studies of bacterial and eukaryotic model organisms. Archaea provide new opportunities to study how recombination operates in a lineage distinct from bacteria and eukaryotes. In the present paper, we focus on RadA, the archaeal RecA family recombinase, and its homologues in archaea and other domains. On the basis of phylogenetic analysis, we propose that a family of archaeal proteins with a single RecA domain, which are currently annotated as KaiC, be renamed aRadC.

Enhancement of the RAD51 Recombinase Activity by the Tumor Suppressor PALB2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dray, Eloise; Etchin, Julia; Wiese, Claudia

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 RAD51more » 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.« less

Roles of the Checkpoint Sensor Clamp Rad9-Rad1-Hus1 (911)-Complex and the Clamp Loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe Telomere Maintenance

PubMed Central

Khair, Lyne; Chang, Ya-Ting; Subramanian, Lakxmi; Russell, Paul; Nakamura, Toru M.

2011-01-01

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1Δ cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1Δ cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes. PMID:20505337

Genotoxicity of diphenyl diselenide in bacteria and yeast.

PubMed

Rosa, Renato Moreira; Sulzbacher, Krisley; Picada, Jaqueline Nascimento; Roesler, Rafael; Saffi, Jenifer; Brendel, Martin; Henriques, João Antonio Pêgas

2004-10-10

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. This may increase the risk of human exposure to the chemical at the workplace. We have determined its mutagenic potential in the Salmonella/microsome assay and used the yeast Saccharomyces cerevisiae to assay for putative genotoxicity, recombinogenicity and to determine whether DNA damage produced by DPDS is repairable. Only in exponentially growing cultures was DPDS able to induce frameshift mutations in S. typhimurium and haploid yeast and to increase crossing over and gene conversion frequencies in diploid strains of S. cerevisiae. Thus, DPDS presents a behavior similar to that of an intercalating agent. Mutants defective in excision-resynthesis repair (rad3, rad1), in error-prone repair (rad6) and in recombinational repair (rad52) showed higher than WT-sensitivity to DPDS. It appears that this compound is capable of inducing single and/or double strand breaks in DNA. An epistatic interaction was shown between rad3-e5 and rad52-1 mutant alleles, indicating that excision-resynthesis and strand-break repair may possess common steps in the repair of DNA damage induced by DPDS. DPDS was able to enhance the mutagenesis induced by oxidative mutagens in bacteria. N-acetylcysteine, a glutathione biosynthesis precursor, prevented mutagenesis induced by DPDS in yeast. We have shown that DPDS is a weak mutagen which probably generates DNA strand breaks through both its intercalating action and pro-oxidant effect.

Resolving the Gordian Knot: Srs2 Strips Intermediates Formed during Homologous Recombination.

PubMed

Ghodke, Harshad; Lewis, Jacob S; van Oijen, Antoine M

2018-03-01

Cells use a suite of specialized enzymes to repair chromosomal double-strand breaks (DSBs). Two recent studies describe how single-molecule fluorescence imaging techniques are used in the direct visualization of some of the key molecular steps involved. De Tullio et al. and Kaniecki et al. watch individual Srs2 helicase molecules disrupt repair intermediates formed by RPA, Rad51, and Rad52 on DNA during homologous recombination. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interactions of RadB, a DNA repair protein in archaea, with DNA and ATP.

PubMed

Guy, Colin P; Haldenby, Sam; Brindley, Amanda; Walsh, David A; Briggs, Geoffrey S; Warren, Martin J; Allers, Thorsten; Bolt, Edward L

2006-04-21

The RecA family of recombinases (RecA, Rad51, RadA and UvsX) catalyse strand-exchange between homologous DNA molecules by utilising conserved DNA-binding modules and a common core ATPase domain. RadB was identified in archaea as a Rad51-like protein on the basis of conserved ATPase sequences. However, RadB does not catalyse strand exchange and does not turn over ATP efficiently. RadB does bind DNA, and here we report a triplet of residues (Lys-His-Arg) that is highly conserved at the RadB C terminus, and is crucial for DNA binding. This is consistent with the motif forming a "basic patch" of highly conserved residues identified in an atomic structure of RadB from Thermococcus kodakaraensis. As the triplet motif is conserved at the C terminus of XRCC2 also, a mammalian Rad51-paralogue, we present a phylogenetic analysis that clarifies the relationship between RadB, Rad51-paralogues and recombinases. We investigate interactions between RadB and ATP using genetics and biochemistry; ATP binding by RadB is needed to promote survival of Haloferax volcanii after UV irradiation, and ATP, but not other NTPs, induces pronounced conformational change in RadB. This is the first genetic analysis of radB, and establishes its importance for maintaining genome stability in archaea. ATP-induced conformational change in RadB may explain previous reports that RadB controls Holliday junction resolution by Hjc, depending on the presence or the absence of ATP.

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

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

PubMed

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

2016-11-01

The mechanisms cells use to maintain genetic fidelity via DNA repair and the accuracy of these processes have garnered interest from scientists engaged in basic research to clinicians seeking improved treatment for cancer patients. Despite the continued advances, many details of DNA repair are still incompletely understood. In addition, the inherent complexity of DNA repair processes, even at the most fundamental level, makes it a challenging topic. This primer is meant to assist both educators and students in using a recent paper, "Promotion of homologous recombination by SWS-1 in complex with RAD-51 paralogs in Caenorhabditis elegans," to understand mechanisms of DNA repair. The goals of this primer are to highlight and clarify several key techniques utilized, with special emphasis on the clustered, regularly interspaced, short palindromic repeats technique and the ways in which it has revolutionized genetics research, as well as to provide questions for deeper in-class discussion. Copyright © 2016 by the Genetics Society of America.

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

PubMed Central

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

2015-01-01

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

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-09-15

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1.

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed Central

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-01-01

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1. PMID:9722633

RNF168 forms a functional complex with RAD6 during the DNA damage response

PubMed Central

Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jennifer; Ma, Teng; Yu, Xiaochun

2013-01-01

Summary Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168–RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade. PMID:23525009

Prolonged Particulate Hexavalent Chromium Exposure Suppresses Homologous Recombination Repair in Human Lung Cells

PubMed Central

Browning, Cynthia L.; Qin, Qin; Kelly, Deborah F.; Prakash, Rohit; Vanoli, Fabio; Jasin, Maria

2016-01-01

Abstract Genomic instability is one of the primary models of carcinogenesis and a feature of almost all cancers. Homologous recombination (HR) repair protects against genomic instability by maintaining high genomic fidelity during the repair of DNA double strand breaks. The defining step of HR repair is the formation of the Rad51 nucleofilament, which facilitates the search for a homologous sequence and invasion of the template DNA strand. Particulate hexavalent chromium (Cr(VI)), a human lung carcinogen, induces DNA double strand breaks and chromosome instability. Since the loss of HR repair increases Cr(VI)-induced chromosome instability, we investigated the effect of extended Cr(VI) exposure on HR repair. We show acute (24 h) Cr(VI) exposure induces a normal HR repair response. In contrast, prolonged (120 h) exposure to particulate Cr(VI) inhibited HR repair and Rad51 nucleofilament formation. Prolonged Cr(VI) exposure had a profound effect on Rad51, evidenced by reduced protein levels and Rad51 mislocalization to the cytoplasm. The response of proteins involved in Rad51 nuclear import and nucleofilament formation displayed varying responses to prolonged Cr(VI) exposure. BRCA2 formed nuclear foci after prolonged Cr(VI) exposure, while Rad51C foci formation was suppressed. These results suggest that particulate Cr(VI), a major chemical carcinogen, inhibits HR repair by targeting Rad51, causing DNA double strand breaks to be repaired by a low fidelity, Rad51-independent repair pathway. These results further enhance our understanding of the underlying mechanism of Cr(VI)-induced chromosome instability and thus, carcinogenesis. PMID:27449664

Safety and Efficacy of Single Incision Laparoscopic Surgery for Total Extraperitoneal Inguinal Hernia Repair

PubMed Central

2011-01-01

Almost 20 years after the first laparoscopic inguinal hernia repair was performed, single incision laparoscopic surgery (SILS™) is set to revolutionize minimally invasive surgery. However, the loss of triangulation must be overcome before the technique can be popularized. This study reports the first 100 laparoscopic total extraperitoneal hernia repairs using a single incision. The study cohort comprised 68 patients with a mean age of 44 (range, 18 to 83): 36 unilateral and 32 bilateral hernias. Twelve patients also underwent umbilical hernia repair with the Ventralex patch requiring no additional incisions. A 2.5-cm to 3-cm crescentic incision within the confines of the umbilicus was performed. Standard dissecting instruments and 52-cm/5.5-mm/300 laparoscope were used. Operation times were 50 minutes for unilateral and 80 minutes for bilateral. There was one conversion to conventional 3-port laparoscopic repair and none to open surgery. Outpatient surgery was achieved in all (except one). Analgesic requirements were minimal: 8 Dextropropoxyphene tablets (range, 0 to 20). There were no intraoperative or postoperative complications with a high patient satisfaction score. Single-incision laparoscopic hernia repair is safe and efficient simply by modifying dissection techniques (so-called “inline” and “vertical”). Comparable success can be obtained while negating the risks of bowel and vascular injuries from sharp trocars and achieving improved cosmetic results. PMID:21902942

Single molecule techniques in DNA repair: A primer

PubMed Central

Hughes, Craig D.; Simons, Michelle; Mackenzie, Cassidy E.; Van Houten, Bennett; Kad, Neil M.

2016-01-01

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit – searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair. PMID:24819596

XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes.

PubMed Central

van der Spek, P J; Eker, A; Rademakers, S; Visser, C; Sugasawa, K; Masutani, C; Hanaoka, F; Bootsma, D; Hoeijmakers, J H

1996-01-01

The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER). The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al. (1994) EMBO J. 8, 1831-1843). Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC. In contrast, we cannot detect any bound HHR23A. Thus the HHR23 proteins may have an additional function independent of XPC. The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction. Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation. Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex. However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex. Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus. PMID:8692695

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

PubMed Central

Bolderson, Emma; Tomimatsu, Nozomi; Richard, Derek J.; Boucher, Didier; Kumar, Rakesh; Pandita, Tej K.; Burma, Sandeep; Khanna, Kum Kum

2010-01-01

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability. PMID:20019063

Relative contribution of homologous recombination and non-homologous end-joining to DNA double-strand break repair after oxidative stress in Saccharomyces cerevisiae.

PubMed

Letavayová, Lucia; Marková, Eva; Hermanská, Katarína; Vlcková, Viera; Vlasáková, Danusa; Chovanec, Miroslav; Brozmanová, Jela

2006-05-10

Oxidative damage to DNA seems to be an important factor in developing many human diseases including cancer. It involves base and sugar damage, base-free sites, DNA-protein cross-links and DNA single-strand (SSB) and double-strand (DSB) breaks. Oxidative DSB can be formed in various ways such as their direct induction by the drug or their generation either through attempted and aborted repair of primary DNA lesions or through DNA replication-dependent conversion of SSB. In general, two main pathways are responsible for repairing DSB, homologous recombination (HR) and non-homologous end-joining (NHEJ), with both of them being potential candidates for the repair of oxidative DSB. We have examined relative contribution of HR and NHEJ to cellular response after oxidative stress in Saccharomyces cerevisiae. Therefore, cell survival, mutagenesis and DSB induction and repair in the rad52, yku70 and rad52 yku70 mutants after hydrogen peroxide (H(2)O(2)), menadione (MD) or bleomycin (BLM) exposure were compared to those obtained for the corresponding wild type. We show that MD exposure does not lead to observable DSB induction in yeast, suggesting that the toxic effects of this agent are mediated by other types of DNA damage. Although H(2)O(2) treatment generates some DSB, their yield is relatively low and hence DSB may only partially be responsible for toxicity of H(2)O(2), particularly at high doses of the agent. On the other hand, the basis of the BLM toxicity resides primarily in DSB induction. Both HR and NHEJ act on BLM-induced DSB, although their relative participation in the process is not equal. Based on our results we suggest that the complexity and/or the quality of the BLM-induced DSB might represent an obstacle for the NHEJ pathway.

Jab1 Mediates Protein Degradation of Rad9/Rad1/Hus1 Checkpoint Complex

PubMed Central

Huang, Jin; Yuan, Honglin; Lu, Chongyuan; Liu, Ximeng; Cao, Xu; Wan, Mei

2009-01-01

Summary The Rad1-Rad9-Hus1 (9-1-1) complex serves a dual role as a DNA-damage sensor in checkpoint signaling and as a mediator in DNA repair pathway. However, the intercellular mechanisms that regulate 9-1-1 complex are poorly understood. Jab1, the fifth component of the COP9 signalosome complex, plays a central role in the degradation of multiple proteins and is emerging as an important regulator in cancer development. Here, we tested the hypothesis that Jab1 controls the protein stability of the 9-1-1 complex via the proteosome pathway. We provide evidence that Jab1 physically associates with the 9-1-1 complex. This association is mediated through direct interaction between Jab1 and Rad1, one of the subunits of 9-1-1 complex. Importantly, Jab1 causes the translocation of the 9-1-1 complex from the nucleus to the cytoplasm, mediating rapid degradation of the 9-1-1 complex via 26S proteasome. Furthermore, Jab1 significantly suppresses checkpoint signaling activation, DNA synthesis recovery from blockage and cell viability after replication stresses such as UV exposure, γ radiation and hydroxyurea treatment. These results suggest that Jab1 is an important regulator for 9-1-1 protein stability control in cells, which may provide novel information on the involvement of Jab1 in checkpoint and DNA repair signaling in response to DNA damage. PMID:17583730

Prolonged Particulate Hexavalent Chromium Exposure Suppresses Homologous Recombination Repair in Human Lung Cells.

PubMed

Browning, Cynthia L; Qin, Qin; Kelly, Deborah F; Prakash, Rohit; Vanoli, Fabio; Jasin, Maria; Wise, John Pierce

2016-09-01

Genomic instability is one of the primary models of carcinogenesis and a feature of almost all cancers. Homologous recombination (HR) repair protects against genomic instability by maintaining high genomic fidelity during the repair of DNA double strand breaks. The defining step of HR repair is the formation of the Rad51 nucleofilament, which facilitates the search for a homologous sequence and invasion of the template DNA strand. Particulate hexavalent chromium (Cr(VI)), a human lung carcinogen, induces DNA double strand breaks and chromosome instability. Since the loss of HR repair increases Cr(VI)-induced chromosome instability, we investigated the effect of extended Cr(VI) exposure on HR repair. We show acute (24 h) Cr(VI) exposure induces a normal HR repair response. In contrast, prolonged (120 h) exposure to particulate Cr(VI) inhibited HR repair and Rad51 nucleofilament formation. Prolonged Cr(VI) exposure had a profound effect on Rad51, evidenced by reduced protein levels and Rad51 mislocalization to the cytoplasm. The response of proteins involved in Rad51 nuclear import and nucleofilament formation displayed varying responses to prolonged Cr(VI) exposure. BRCA2 formed nuclear foci after prolonged Cr(VI) exposure, while Rad51C foci formation was suppressed. These results suggest that particulate Cr(VI), a major chemical carcinogen, inhibits HR repair by targeting Rad51, causing DNA double strand breaks to be repaired by a low fidelity, Rad51-independent repair pathway. These results further enhance our understanding of the underlying mechanism of Cr(VI)-induced chromosome instability and thus, carcinogenesis. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Domain mapping of the Rad51 paralog protein complexes

PubMed Central

Miller, Kristi A.; Sawicka, Dorota; Barsky, Daniel; Albala, Joanna S.

2004-01-01

The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B–Rad51C–Rad51D–Xrcc2 (BCDX2) and Rad51C–Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs. The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of Rad51B containing amino acid residues 1–75 interacts with the C-terminus and linker of Rad51C, residues 79–376, and this region of Rad51C also interacts with mRad51D and Xrcc3. We have also determined that the N-terminal domain of mRad51D, residues 4–77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77–328, binds Rad51C. By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes. PMID:14704354

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

PubMed

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

2016-07-01

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

Excision repair of UV radiation-induced DNA damage in Caenorhabditis elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartman, P.S.; Hevelone, J.; Dwarakanath, V.

1989-06-01

Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts (cyclobutane dimers and (6-4) photoproducts). Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). Thismore » correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.« less

Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

PubMed

Fornander, Louise H; Frykholm, Karolin; Reymer, Anna; Renodon-Cornière, Axelle; Takahashi, Masayuki; Nordén, Bengt

2012-06-01

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression.

PubMed

Lee, Shauna A; Roques, Céline; Magwood, Alissa C; Masson, Jean-Yves; Baker, Mark D

2009-02-01

The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.

Raman-Activated Droplet Sorting (RADS) for Label-Free High-Throughput Screening of Microalgal Single-Cells.

PubMed

Wang, Xixian; Ren, Lihui; Su, Yetian; Ji, Yuetong; Liu, Yaoping; Li, Chunyu; Li, Xunrong; Zhang, Yi; Wang, Wei; Hu, Qiang; Han, Danxiang; Xu, Jian; Ma, Bo

2017-11-21

Raman-activated cell sorting (RACS) has attracted increasing interest, yet throughput remains one major factor limiting its broader application. Here we present an integrated Raman-activated droplet sorting (RADS) microfluidic system for functional screening of live cells in a label-free and high-throughput manner, by employing AXT-synthetic industrial microalga Haematococcus pluvialis (H. pluvialis) as a model. Raman microspectroscopy analysis of individual cells is carried out prior to their microdroplet encapsulation, which is then directly coupled to DEP-based droplet sorting. To validate the system, H. pluvialis cells containing different levels of AXT were mixed and underwent RADS. Those AXT-hyperproducing cells were sorted with an accuracy of 98.3%, an enrichment ratio of eight folds, and a throughput of ∼260 cells/min. Of the RADS-sorted cells, 92.7% remained alive and able to proliferate, which is equivalent to the unsorted cells. Thus, the RADS achieves a much higher throughput than existing RACS systems, preserves the vitality of cells, and facilitates seamless coupling with downstream manipulations such as single-cell sequencing and cultivation.

SNPs in DNA repair or oxidative stress genes and late subcutaneous fibrosis in patients following single shot partial breast irradiation

PubMed Central

2012-01-01

Background The aim of this study was to evaluate the potential association between single nucleotide polymorphisms related response to radiotherapy injury, such as genes related to DNA repair or enzymes involved in anti-oxidative activities. The paper aims to identify marker genes able to predict an increased risk of late toxicity studying our group of patients who underwent a Single Shot 3D-CRT PBI (SSPBI) after BCS (breast conserving surgery). Methods A total of 57 breast cancer patients who underwent SSPBI were genotyped for SNPs (single nucleotide polymorphisms) in XRCC1, XRCC3, GST and RAD51 by Pyrosequencing technology. Univariate analysis (ORs and 95% CI) was performed to correlate SNPs with the risk of developing ≥ G2 fibrosis or fat necrosis. Results A higher significant risk of developing ≥ G2 fibrosis or fat necrosis in patients with: polymorphic variant GSTP1 (Ile105Val) (OR = 2.9; 95%CI, 0.88-10.14, p = 0.047). Conclusions The presence of some SNPs involved in DNA repair or response to oxidative stress seem to be able to predict late toxicity. Trial Registration ClinicalTrials.gov: NCT01316328 PMID:22272830

RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene.

PubMed

Aboussekhra, A; Chanet, R; Zgaga, Z; Cassier-Chauvat, C; Heude, M; Fabre, F

1989-09-25

A new type of radiation-sensitive mutant of S. cerevisiae is described. The recessive radH mutation sensitizes to the lethal effect of UV radiations haploids in the G1 but not in the G2 mitotic phase. Homozygous diploids are as sensitive as G1 haploids. The UV-induced mutagenesis is depressed, while the induction of gene conversion is increased. The mutation is believed to channel the repair of lesions engaged in the mutagenic pathway into a recombination process, successful if the events involve sister-chromatids but lethal if they involve homologous chromosomes. The sequence of the RADH gene reveals that it may code for a DNA helicase, with a Mr of 134 kDa. All the consensus domains of known DNA helicases are present. Besides these consensus regions, strong homologies with the Rep and UvrD helicases of E. coli were found. The RadH putative helicase appears to belong to the set of proteins involved in the error-prone repair mechanism, at least for UV-induced lesions, and could act in coordination with the Rev3 error-prone DNA polymerase.

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

PubMed Central

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

2015-01-01

The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs). Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments. PMID:26719980

The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae.

PubMed

Lettier, Gaëlle; Feng, Qi; de Mayolo, Adriana Antúnez; Erdeniz, Naz; Reid, Robert J D; Lisby, Michael; Mortensen, Uffe H; Rothstein, Rodney

2006-11-10

Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.

The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse

PubMed Central

Vasileva, Ana; Hopkins, Kevin M.; Wang, Xiangyuan; Weisbach, Melissa M.; Friedman, Richard A.; Wolgemuth, Debra J.; Lieberman, Howard B.

2013-01-01

Summary In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIβ-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I. PMID:23788429

Cyclic loading of rotator cuff reconstructions: single-row repair with modified suture configurations versus double-row repair.

PubMed

Lorbach, Olaf; Bachelier, Felix; Vees, Jochen; Kohn, Dieter; Pape, Dietrich

2008-08-01

Double-row repair is suggested to have superior biomechanical properties in rotator cuff reconstruction compared with single-row repair. However, double-row rotator cuff repair is frequently compared with simple suture repair and not with modified suture configurations. Single-row rotator cuff repairs with modified suture configurations have similar failure loads and gap formations as double-row reconstructions. Controlled laboratory study. We created 1 x 2-cm defects in 48 porcine infraspinatus tendons. Reconstructions were then performed with 4 single-row repairs and 2 double-row repairs. The single-row repairs included transosseous simple sutures; double-loaded corkscrew anchors in either a double mattress or modified Mason-Allen suture repair; and the Magnum Knotless Fixation Implant with an inclined mattress. Double-row repairs were either with Bio-Corkscrew FT using modified Mason-Allen stitches or a combination of Bio-Corkscrew FT and PushLock anchors using the SutureBridge Technique. During cyclic load (10 N to 60-200 N), gap formation was measured, and finally, ultimate load to failure and type of failure were recorded. Double-row double-corkscrew anchor fixation had the highest ultimate tensile strength (398 +/- 98 N) compared to simple sutures (105 +/- 21 N; P < .0001), single-row corkscrews using a modified Mason-Allen stitch (256 +/- 73 N; P = .003) or double mattress repair (290 +/- 56 N; P = .043), the Magnum Implant (163 +/- 13 N; P < .0001), and double-row repair with PushLock and Bio-Corkscrew FT anchors (163 +/- 59 N; P < .0001). Single-row double mattress repair was superior to transosseous sutures (P < .0001), the Magnum Implant (P = .009), and double-row repair with PushLock and Bio-Corkscrew FT anchors (P = .009). Lowest gap formation was found for double-row double-corkscrew repair (3.1 +/- 0.1 mm) compared to simple sutures (8.7 +/- 0.2 mm; P < .0001), the Magnum Implant (6.2 +/- 2.2 mm; P = .002), double-row repair with PushLock and Bio

Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae.

PubMed

Symington, L S; Kang, L E; Moreau, S

2000-12-01

A plasmid gap repair assay was used to assess the role of three known nucleases, Exo1, Mre11 and Rad1, in the processing of DNA ends and resolution of recombination intermediates during double-strand gap repair. In this assay, alterations in end processing or branch migration are reflected by the frequency of co-conversion of a chromosomal marker 200 bp from the gap. Gap repair associated with crossing over results in integration at the homologous chromosomal locus, whereas the plasmid remains episomal for non-crossover repair events. In mre11 strains, the frequency of gap repair was reduced 3- to 10-fold and conversion tracts were shorter than in the wild-type strain, consistent with a role for this nuclease in processing double-strand breaks. However, conversion tracts were longer in a strain containing the nuclease deficient allele, mre11-H125N, suggesting increased end processing by redundant nucleases. The frequency of gap repair was reduced 2-fold in rad1 mutants and crossing over was reduced, consistent with a role for Rad1 in cleaving recombination intermediates. The frequency of gap repair was increased in exo1 mutants with a significant increase in crossing over. In exo1 mre11 double mutants gap repair was reduced to below the mre11 single mutant level.

Meiotic Recombination Initiated by a Double-Strand Break in Rad50Δ Yeast Cells Otherwise Unable to Initiate Meiotic Recombination

PubMed Central

Malkova, A.; Ross, L.; Dawson, D.; Hoekstra, M. F.; Haber, J. E.

1996-01-01

Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast's 16 chromosomes. In Rad(+) strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad(+) and in rad50Δ cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50Δ cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50Δ diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination. PMID:8725223

Safety and Immunogenicity of a rAd35-EnvA Prototype HIV-1 Vaccine in Combination with rAd5-EnvA in Healthy Adults (VRC 012).

PubMed

Crank, Michelle C; Wilson, Eleanor M P; Novik, Laura; Enama, Mary E; Hendel, Cynthia S; Gu, Wenjuan; Nason, Martha C; Bailer, Robert T; Nabel, Gary J; McDermott, Adrian B; Mascola, John R; Koup, Richard A; Ledgerwood, Julie E; Graham, Barney S

2016-01-01

VRC 012 was a Phase I study of a prototype recombinant adenoviral-vector serotype-35 (rAd35) HIV vaccine, the precursor to two recently published clinical trials, HVTN 077 and 083. On the basis of prior evaluation of multiclade rAd5 HIV vaccines, Envelope A (EnvA) was selected as the standard antigen for a series of prototype HIV vaccines to compare various vaccine platforms. In addition, prior studies of rAd5-vectored vaccines suggested pre-existing human immunity may be a confounding factor in vaccine efficacy. rAd35 is less seroprevalent across human populations and was chosen for testing alone and in combination with a rAd5-EnvA vaccine in the present two-part phase I study. First, five subjects each received a single injection of 109, 1010, or 1011 particle units (PU) of rAd35-EnvA in an open-label, dose-escalation study. Next, 20 Ad5/Ad35-seronegative subjects were randomized to blinded, heterologous prime-boost schedules combining rAd5-EnvA and rAd35-EnvA with a three month interval. rAd35-EnvA was given at 1010 or 1011 PU to ten subjects each; all rAd5-EnvA injections were 1010 PU. EnvA-specific immunogenicity was assessed four weeks post-injection. Solicited reactogenicity and clinical safety were followed after each injection. Vaccinations were well tolerated at all dosages. Antibody responses measured by ELISA were detected at 4 weeks in 30% and 50% of subjects after single doses of 1010 or 1011 PU rAd35, respectively, and in 89% after a single rAd5-EnvA 1010 PU injection. EnvA-specific IFN-γ ELISpot responses were detected at four weeks in 0%, 70%, and 50% of subjects after the respective rAd35-EnvA dosages compared to 89% of subjects after rAd5. T cell responses were higher after a single rAd5-EnvA 1010 PU injection than after a single rAd35-EnvA 1010 PU injection, and humoral responses were low after a single dose of either vector. Of those completing the vaccine schedule, 100% of rAd5-EnvA recipients and 90% of rAd35-EnvA recipients had both T cell

p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage.

PubMed

Gong, Lu; Gong, Hongjian; Pan, Xiao; Chang, Changqing; Ou, Zhao; Ye, Shengfan; Yin, Le; Yang, Lina; Tao, Ting; Zhang, Zhenhai; Liu, Cong; Lane, David P; Peng, Jinrong; Chen, Jun

2015-03-01

The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

PubMed

Liu, Mingming; Ba, Zhaoqing; Costa-Nunes, Pedro; Wei, Wei; Li, Lanxia; Kong, Fansi; Li, Yan; Chai, Jijie; Pontes, Olga; Qi, Yijun

2017-03-01

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair. © 2017 American Society of Plant Biologists. All rights reserved.

Homology-dependent repair is involved in 45S rDNA loss in plant CAF-1 mutants

PubMed Central

Muchová, Veronika; Amiard, Simon; Mozgová, Iva; Dvořáčková, Martina; Gallego, Maria E; White, Charles; Fajkus, Jiří

2015-01-01

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability. PMID:25359579

An Overview of the Molecular Mechanisms of Recombinational DNA Repair

PubMed Central

Kowalczykowski, Stephen C.

2015-01-01

Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution. PMID:26525148

Functional compartmentalization of Rad9 and Hus1 reveals diverse assembly of the 9‐1‐1 complex components during the DNA damage response in Leishmania

PubMed Central

Damasceno, Jeziel D.; Obonaga, Ricardo; Santos, Elaine V.; Scott, Alan; McCulloch, Richard

2016-01-01

Summary The Rad9‐Rad1‐Hus1 (9‐1‐1) complex is a key component in the coordination of DNA damage sensing, cell cycle progression and DNA repair pathways in eukaryotic cells. This PCNA‐related trimer is loaded onto RPA‐coated single stranded DNA and interacts with ATR kinase to mediate effective checkpoint signaling to halt the cell cycle and to promote DNA repair. Beyond these core activities, mounting evidence suggests that a broader range of functions can be provided by 9‐1‐1 structural diversification. The protozoan parasite Leishmania is an early‐branching eukaryote with a remarkably plastic genome, which hints at peculiar genome maintenance mechanisms. Here, we investigated the existence of homologs of the 9‐1‐1 complex subunits in L. major and found that LmRad9 and LmRad1 associate with chromatin in response to replication stress and form a complex in vivo with LmHus1. Similar to LmHus1, LmRad9 participates in telomere homeostasis and in the response to both replication stress and double strand breaks. However, LmRad9 and LmHus1‐deficient cells present markedly opposite phenotypes, which suggest their functional compartmentalization. We show that some of the cellular pool of LmRad9 forms an alternative complex and that some of LmHus1 exists as a monomer. We propose that the diverse assembly of the Leishmania 9‐1‐1 subunits mediates functional compartmentalization, which has a direct impact on the response to genotoxic stress. PMID:27301589

RAD18 mediates resistance to ionizing radiation in human glioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Chen; Wang, Hongwei; Cheng, Hongbin

Highlights: • RAD18 is an important mediator of the IR-induced resistance in glioma cell lines. • RAD18 overexpression confers resistance to IR-mediated apoptosis. • The elevated expression of RAD18 is associated with recurrent GBM who underwent IR therapy. - Abstract: Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). RAD18 a central regulator of translesion DNA synthesis (TLS), has been shown to play an important role in regulating genomic stability and DNA damage response. In the present study, we investigate the relationship between RAD18 and resistance to ionizing radiation (IR) and examined the expression levels of RAD18more » in primary and recurrent GBM specimens. Our results showed that RAD18 is an important mediator of the IR-induced resistance in GBM. The expression level of RAD18 in glioma cells correlates with their resistance to IR. Ectopic expression of RAD18 in RAD18-low A172 glioma cells confers significant resistance to IR treatment. Conversely, depletion of endogenous RAD18 in RAD18-high glioma cells sensitized these cells to IR treatment. Moreover, RAD18 overexpression confers resistance to IR-mediated apoptosis in RAD18-low A172 glioma cells, whereas cells deficient in RAD18 exhibit increased apoptosis induced by IR. Furthermore, knockdown of RAD18 in RAD18-high glioma cells disrupts HR-mediated repair, resulting in increased accumulation of DSB. In addition, clinical data indicated that RAD18 was significantly higher in recurrent GBM samples that were exposed to IR compared with the corresponding primary GBM samples. Collectively, our findings reveal that RAD18 may serve as a key mediator of the IR response and may function as a potential target for circumventing IR resistance in human GBM.« less

Protective role of RAD50 on chromatin bridges during abnormal cytokinesis.

PubMed

Schröder-Heurich, Bianca; Wieland, Britta; Lavin, Martin F; Schindler, Detlev; Dörk, Thilo

2014-03-01

Faithful chromosome segregation is required for preserving genomic integrity. Failure of this process may entail chromatin bridges preventing normal cytokinesis. To test whether RAD50, a protein normally involved in DNA double-strand break repair, is involved in abnormal cytokinesis and formation of chromatin bridges, we used immunocytochemical and protein interaction assays. RAD50 localizes to chromatin bridges during aberrant cytokinesis and subsequent stages of the cell cycle, either decorating the entire bridge or focally accumulating at the midbody zone. Ionizing radiation led to an ∼4-fold increase in the rate of chromatin bridges in an ataxia telangiectatica mutated (ATM)-dependent manner in human RAD50-proficient fibroblasts but not in RAD50-deficient cells. Cells with a RAD50-positive chromatin bridge were able to continue cell cycling and to progress through S phase (44%), whereas RAD50 knockdown caused a deficiency in chromatin bridges as well as an ∼4-fold prolonged duration of mitosis. RAD50 colocalized and directly interacted with Aurora B kinase and phospho-histone H3, and Aurora B kinase inhibition led to a deficiency in RAD50-positive bridges. Based on these observations, we propose that RAD50 is a crucial factor for the stabilization and shielding of chromatin bridges. Our study provides evidence for a hitherto unknown role of RAD50 in abnormal cytokinesis.

Microfluidic guillotine for single-cell wound repair studies

NASA Astrophysics Data System (ADS)

Blauch, Lucas R.; Gai, Ya; Khor, Jian Wei; Sood, Pranidhi; Marshall, Wallace F.; Tang, Sindy K. Y.

2017-07-01

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

PubMed

Botthof, Jan Gregor; Bielczyk-Maczyńska, Ewa; Ferreira, Lauren; Cvejic, Ana

2017-05-30

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

Human RAD50 makes a functional DNA-binding complex.

PubMed

Kinoshita, Eri; van Rossum-Fikkert, Sari; Sanchez, Humberto; Kertokalio, Aryandi; Wyman, Claire

2015-06-01

The MRE11-RAD50-NBS1 (MRN) complex has several distinct functions in DNA repair including important roles in both non-homologous end-joining (NHEJ) and homologous recombination (HR). The biochemical activities of MR(N) have been well characterized implying specific functional roles for the components. The arrangement of proteins in the complex implies interdependence of their biochemical activities making it difficult to separate specific functions. We obtained purified human RAD50 and observed that it binds ATP, undergoes ATP-dependent conformational changes as well as having ATPase activity. Scanning force microscopy analysis clearly showed that RAD50 binds DNA although not as oligomers. RAD50 alone was not functional in tethering DNA molecules. ATP increased formation of RAD50 multimers which were however globular lacking extended coiled coils, in contrast to the MR complex where ATP induced oligomers have obvious coiled coils protruding from a central domain. These results suggest that MRE11 is important in maintaining the structural arrangement of RAD50 in the protein complex and perhaps has a role in reinforcing proper alignment of the coiled coils in the ATP-bound state. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Cloning and characterization of p52, the fifth subunit of the core of the transcription/DNA repair factor TFIIH.

PubMed Central

Marinoni, J C; Roy, R; Vermeulen, W; Miniou, P; Lutz, Y; Weeda, G; Seroz, T; Gomez, D M; Hoeijmakers, J H; Egly, J M

1997-01-01

TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine genes encoding the subunits forming TFIIH have already been cloned. We report here the identification, cDNA cloning and gene structure of the 52 kDa polypeptide and its homology with the yeast counterpart TFB2. This protein, along with p89/XPB, p62, p44 and p34, forms the core of TFIIH. Moreover, using in vitro reconstituted transcription and nucleotide excision repair (NER) assays and microinjection experiments, we demonstrate that p52 is directly involved in both transcription and DNA repair mechanisms in vitro and in vivo. PMID:9118947

RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication.

PubMed

Ruff, Patrick; Donnianni, Roberto A; Glancy, Eleanor; Oh, Julyun; Symington, Lorraine S

2016-12-20

DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Single-Versus Double-Row Arthroscopic Rotator Cuff Repair in Massive Tears

PubMed Central

Wang, EnZhi; Wang, Liang; Gao, Peng; Li, ZhongJi; Zhou, Xiao; Wang, SongGang

2015-01-01

Background It is a challenge for orthopaedic surgeons to treat massive rotator cuff tears. The optimal management of massive rotator cuff tears remains controversial. Therefore, the goal of this study was to compare arthroscopic single- versus double-row rotator cuff repair with a larger sample size. Material/Methods Of the subjects with massive rotator cuff tears, 146 were treated using single-row repair, and 102 were treated using double-row repair. Pre- and postoperative functional outcomes and radiographic images were collected. The clinical outcomes were evaluated for a minimum of 2 years. Results No significant differences were shown between the groups in terms of functional outcomes. Regarding the integrity of the tendon, a lower rate of post-treatment retear was observed in patients who underwent double-row repair compared with single-row repair. Conclusions The results suggest that double-row repair is relatively superior in shoulder ROM and the strength of tendon compared with single-row repair. Future studies involving more patients in better-designed randomized controlled trials will be required. PMID:26017641

Protein dynamics during presynaptic complex assembly on individual ssDNA molecules

PubMed Central

Gibb, Bryan; Ye, Ling F.; Kwon, YoungHo; Niu, Hengyao; Sung, Patrick; Greene, Eric C.

2014-01-01

Homologous recombination is a conserved pathway for repairing double–stranded breaks, which are processed to yield single–stranded DNA overhangs that serve as platforms for presynaptic complex assembly. Here we use single–molecule imaging to reveal the interplay between Saccharomyce cerevisiae RPA, Rad52, and Rad51 during presynaptic complex assembly. We show that Rad52 binds RPA–ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 binding extends the ssDNA, and Rad52–RPA clusters remain interspersed along the presynaptic complex. These clusters promote additional binding of RPA and Rad52. Together, our work illustrates the spatial and temporal progression of RPA and Rad52 association with the presynaptic complex, and reveals a novel RPA–Rad52–Rad51–ssDNA intermediate, which has implications for understanding how the activities of Rad52 and RPA are coordinated with Rad51 during the later stages recombination. PMID:25195049

Corylin increases the sensitivity of hepatocellular carcinoma cells to chemotherapy through long noncoding RNA RAD51-AS1-mediated inhibition of DNA repair.

PubMed

Chen, Chin-Chuan; Chen, Chi-Yuan; Ueng, Shir-Hwa; Hsueh, Chuen; Yeh, Chau-Ting; Ho, Jar-Yi; Chou, Li-Fang; Wang, Tong-Hong

2018-05-10

Corylin, a biologically active agent extracted from Psoralea corylifolia L. (Fabaceae), promotes bone differentiation and inhibits inflammation. Currently, few reports have addressed the biological functions that are regulated by corylin, and to date, no studies have investigated its antitumor activity. In this study, we used cell functional assays to analyze the antitumor activity of corylin in hepatocellular carcinoma (HCC). Furthermore, whole-transcriptome assays were performed to identify the downstream genes that were regulated by corylin, and gain-of-function and loss-of-function experiments were conducted to examine the regulatory roles of the above genes. We found that corylin significantly inhibited the proliferation, migration, and invasion of HCC cells and increased the toxic effects of chemotherapeutic agents against HCC cells. These properties were due to the induction of a long noncoding RNA, RAD51-AS1, which bound to RAD51 mRNA, thereby inhibiting RAD51 protein expression, thus inhibiting the DNA damage repair ability of HCC cells. Animal experiments also showed that a combination treatment with corylin significantly increased the inhibitory effects of the chemotherapeutic agent etoposide (VP16) on tumor growth. These findings indicate that corylin has strong potential as an adjuvant drug in HCC treatment and that corylin can strengthen the therapeutic efficacy of chemotherapy and radiotherapy.

Evidence for conformational capture mechanism for damage recognition by NER protein XPC/Rad4.

NASA Astrophysics Data System (ADS)

Chakraborty, Sagnik; Steinbach, Peter J.; Paul, Debamita; Min, Jung-Hyun; Ansari, Anjum

Altered flexibility of damaged DNA sites is considered to play an important role in damage recognition by DNA repair proteins. Characterizing lesion-induced DNA dynamics has remained a challenge. We have combined ps-resolved fluorescence lifetime measurements with cytosine analog FRET pair uniquely sensitive to local unwinding/twisting to analyze DNA conformational distributions. This innovative approach maps out with unprecedented sensitivity the alternative conformations accessible to a series of DNA constructs containing 3-base-pair mismatch, suitable model lesions for the DNA repair protein xeroderma pigmentosum C (XPC) complex. XPC initiates eukaryotic nucleotide excision repair by recognizing various DNA lesions primarily through DNA deformability. Structural studies show that Rad4 (yeast ortholog of XPC) unwinds DNA at the lesion site and flips out two nucleotide pairs. Our results elucidate a broad range of conformations accessible to mismatched DNA even in the absence of the protein. Notably, the most severely distorted conformations share remarkable resemblance to the deformed conformation seen in the crystal structure of the Rad4-bound ``recognition'' complex supporting for the first time a possible ``conformational capture'' mechanism for damage recognition by XPC/Rad4. NSF Univ of Illinois-Chicago.

Interdependence of the rad50 hook and globular domain functions.

PubMed

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

2015-02-05

Rad50 contains a conserved Zn(2+) coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here, we focused on rad50 mutations flanking the Zn(2+)-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double-strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end joining, and DNA double-strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled-coil and globular ATPase domains, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions. Copyright © 2015 Elsevier Inc. All rights reserved.

DNA strand breaks signal the induction of DNA double-strand break repair in Saccharomyces cerevisiae.

PubMed

Singh, Rakesh Kumar; Krishna, Malini

2005-12-01

Genotoxic stress induces a checkpoint signaling cascade to generate a stress response. Saccharomyces cerevisiae shows an altered radiation response under different type of stress. Although the induction of repair has been implicated in enhanced survival after exposure to the challenging stress, the nature of the signal remains poorly understood. This study demonstrates that low doses of gamma radiation and bleomycin induce RAD52-dependent recombination repair pathway in the wild-type strain D-261. Prior exposure of cells to DNA-damaging agents (gamma radiation or bleomycin) equips them better for the subsequent damage caused by challenging doses. However, exposure to UV light, which does not cause strand breaks, was ineffective. This was confirmed by PFGE studies. This indicates that the strand breaks probably serve as the signal for induction of the recombination repair pathway while pyrimidine dimers do not. The nature of the induced repair was investigated by mutation scoring in special strain D-7, which showed that the induced repair is essentially error free.

Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation

PubMed Central

Koury, Emily; Harrell, Kailey

2018-01-01

Abstract Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence. PMID:29244155

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

PubMed Central

Szyjka, Shawn J.; Aparicio, Jennifer G.; Viggiani, Christopher J.; Knott, Simon; Xu, Weihong; Tavaré, Simon; Aparicio, Oscar M.

2008-01-01

Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2–Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Δ cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair. PMID:18628397

Double-row vs single-row rotator cuff repair: a review of the biomechanical evidence.

PubMed

Wall, Lindley B; Keener, Jay D; Brophy, Robert H

2009-01-01

A review of the current literature will show a difference between the biomechanical properties of double-row and single-row rotator cuff repairs. Rotator cuff tears commonly necessitate surgical repair; however, the optimal technique for repair continues to be investigated. Recently, double-row repairs have been considered an alternative to single-row repair, allowing a greater coverage area for healing and a possibly stronger repair. We reviewed the literature of all biomechanical studies comparing double-row vs single-row repair techniques. Inclusion criteria included studies using cadaveric, animal, or human models that directly compared double-row vs single-row repair techniques, written in the English language, and published in peer reviewed journals. Identified articles were reviewed to provide a comprehensive conclusion of the biomechanical strength and integrity of the repair techniques. Fifteen studies were identified and reviewed. Nine studies showed a statistically significant advantage to a double-row repair with regards to biomechanical strength, failure, and gap formation. Three studies produced results that did not show any statistical advantage. Five studies that directly compared footprint reconstruction all demonstrated that the double-row repair was superior to a single-row repair in restoring anatomy. The current literature reveals that the biomechanical properties of a double-row rotator cuff repair are superior to a single-row repair. Basic Science Study, SRH = Single vs. Double Row RCR.

Single-row versus double-row rotator cuff repair: techniques and outcomes.

PubMed

Dines, Joshua S; Bedi, Asheesh; ElAttrache, Neal S; Dines, David M

2010-02-01

Double-row rotator cuff repair techniques incorporate a medial and lateral row of suture anchors in the repair configuration. Biomechanical studies of double-row repair have shown increased load to failure, improved contact areas and pressures, and decreased gap formation at the healing enthesis, findings that have provided impetus for clinical studies comparing single-row with double-row repair. Clinical studies, however, have not yet demonstrated a substantial improvement over single-row repair with regard to either the degree of structural healing or functional outcomes. Although double-row repair may provide an improved mechanical environment for the healing enthesis, several confounding variables have complicated attempts to establish a definitive relationship with improved rates of healing. Appropriately powered rigorous level I studies that directly compare single-row with double-row techniques in matched tear patterns are necessary to further address these questions. These studies are needed to justify the potentially increased implant costs and surgical times associated with double-row rotator cuff repair.

Srs2 prevents Rad51 filament formation by repetitive motion on DNA.

PubMed

Qiu, Yupeng; Antony, Edwin; Doganay, Sultan; Koh, Hye Ran; Lohman, Timothy M; Myong, Sua

2013-01-01

Srs2 dismantles presynaptic Rad51 filaments and prevents its re-formation as an anti-recombinase. However, the molecular mechanism by which Srs2 accomplishes these tasks remains unclear. Here we report a single-molecule fluorescence study of the dynamics of Rad51 filament formation and its disruption by Srs2. Rad51 forms filaments on single-stranded DNA by sequential binding of primarily monomers and dimers in a 5'-3' direction. One Rad51 molecule binds to three nucleotides, and six monomers are required to achieve a stable nucleation cluster. Srs2 exhibits ATP-dependent repetitive motion on single-stranded DNA and this activity prevents re-formation of the Rad51 filament. The same activity of Srs2 cannot prevent RecA filament formation, indicating its specificity for Rad51. Srs2's DNA-unwinding activity is greatly suppressed when Rad51 filaments form on duplex DNA. Taken together, our results reveal an exquisite and highly specific mechanism by which Srs2 regulates the Rad51 filament formation.

Inducible DNA-repair systems in yeast: competition for lesions.

PubMed

Mitchel, R E; Morrison, D P

1987-03-01

DNA lesions may be recognized and repaired by more than one DNA-repair process. If two repair systems with different error frequencies have overlapping lesion specificity and one or both is inducible, the resulting variable competition for the lesions can change the biological consequences of these lesions. This concept was demonstrated by observing mutation in yeast cells (Saccharomyces cerevisiae) exposed to combinations of mutagens under conditions which influenced the induction of error-free recombinational repair or error-prone repair. Total mutation frequency was reduced in a manner proportional to the dose of 60Co-gamma- or 254 nm UV radiation delivered prior to or subsequent to an MNNG exposure. Suppression was greater per unit radiation dose in cells gamma-irradiated in O2 as compared to N2. A rad3 (excision-repair) mutant gave results similar to wild-type but mutation in a rad52 (rec-) mutant exposed to MNNG was not suppressed by radiation. Protein-synthesis inhibition with heat shock or cycloheximide indicated that it was the mutation due to MNNG and not that due to radiation which had changed. These results indicate that MNNG lesions are recognized by both the recombinational repair system and the inducible error-prone system, but that gamma-radiation induction of error-free recombinational repair resulted in increased competition for the lesions, thereby reducing mutation. Similarly, gamma-radiation exposure resulted in a radiation dose-dependent reduction in mutation due to MNU, EMS, ENU and 8-MOP + UVA, but no reduction in mutation due to MMS. These results suggest that the number of mutational MMS lesions recognizable by the recombinational repair system must be very small relative to those produced by the other agents. MNNG induction of the inducible error-prone systems however, did not alter mutation frequencies due to ENU or MMS exposure but, in contrast to radiation, increased the mutagenic effectiveness of EMS. These experiments demonstrate

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

PubMed

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

2017-02-01

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

Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henrique Barreta, Marcos; Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS; Garziera Gasperin, Bernardo

2012-10-01

This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes weremore » expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.« less

RAD21L, a novel cohesin subunit implicated in linking homologous chromosomes in mammalian meiosis.

PubMed

Lee, Jibak; Hirano, Tatsuya

2011-01-24

Cohesins are multi-subunit protein complexes that regulate sister chromatid cohesion during mitosis and meiosis. Here we identified a novel kleisin subunit of cohesins, RAD21L, which is conserved among vertebrates. In mice, RAD21L is expressed exclusively in early meiosis: it apparently replaces RAD21 in premeiotic S phase, becomes detectable on the axial elements in leptotene, and stays on the axial/lateral elements until mid pachytene. RAD21L then disappears, and is replaced with RAD21. This behavior of RAD21L is unique and distinct from that of REC8, another meiosis-specific kleisin subunit. Remarkably, the disappearance of RAD21L at mid pachytene correlates with the completion of DNA double-strand break repair and the formation of crossovers as judged by colabeling with molecular markers, γ-H2AX, MSH4, and MLH1. RAD21L associates with SMC3, STAG3, and either SMC1α or SMC1β. Our results suggest that cohesin complexes containing RAD21L may be involved in synapsis initiation and crossover recombination between homologous chromosomes.

Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation.

PubMed

Koury, Emily; Harrell, Kailey; Smolikove, Sarit

2018-01-25

Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Comparison between single-row and double-row rotator cuff repair: a biomechanical study.

PubMed

Milano, Giuseppe; Grasso, Andrea; Zarelli, Donatella; Deriu, Laura; Cillo, Mario; Fabbriciani, Carlo

2008-01-01

The aim of this study was to compare the mechanical behavior under cyclic loading test of single-row and double-row rotator cuff repair with suture anchors in an ex-vivo animal model. For the present study, 50 fresh porcine shoulders were used. On each shoulder, a crescent-shaped full-thickness tear of the infraspinatus was performed. Width of the tendon tear was 2 cm. The lesion was repaired using metal suture anchors. Shoulders were divided in four groups, according the type of repair: single-row tension-free repair (Group 1); single-row tension repair (Group 2); double-row tension-free repair (Group 3); double-row tension repair (Group 4); and a control group. Specimens were subjected to a cyclic loading test. Number of cycles at 5 mm of elongation and at failure, and total elongation were calculated. Single-row tension repair showed significantly poorest results for all the variables considered, when compared with the other groups. Regarding the mean number of cycles at 5 mm of elongation and at failure, there was a nonsignificant difference between Groups 3 and 4, and both of them were significantly greater than Group 1. For mean total elongation, the difference between Groups 1, 3, and 4 was not significant, but all of them were significantly lower than the control group. A single-row repair is particularly weak when performed under tension. Double-row repair is significantly more resistant to cyclic displacement than single-row repair in both tension-free and tension repair. Double-row repair technique can be primarily considered for large, unstable rotator cuff tears to improve mechanical strength of primary fixation of tendons to bone.

Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zafar, Faria; Seidler, Sara B.; Kronenberg, Amy

2010-06-29

To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, inmore » p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.« less

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

PubMed Central

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

2016-01-01

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

Biomechanical Comparison of Single- Versus Double-Row Capsulolabral Repair for Shoulder Instability: A Review.

PubMed

Yousif, Matthew John; Bicos, James

2017-12-01

The glenohumeral joint is the most commonly dislocated joint in the body. Failure rates of capsulolabral repair have been reported to be approximately 8%. Recent focus has been on restoration of the capsulolabral complex by a double-row capsulolabral repair technique in an effort to decrease redislocation rates after arthroscopic capsulolabral repair. To present a review of the biomechanical literature comparing single- versus double-row capsulolabral repairs and discuss the previous case series of double-row fixation. Narrative review. A simple review of the literature was performed by PubMed search. Only biomechanical studies comparing single- versus double-row capsulolabral repair were included for review. Only those case series and descriptive techniques with clinical results for double-row repair were included in the discussion. Biomechanical comparisons evaluating the native footprint of the labrum demonstrated significantly superior restoration of the footprint through double-row capsulolabral repair compared with single-row repair. Biomechanical comparisons of contact pressure at the repair interface, fracture displacement in bony Bankart lesion, load to failure, and decreased external rotation (suggestive of increased load to failure) were also significantly in favor of double- versus single-row repair. Recent descriptive techniques and case series of double-row fixation have demonstrated good clinical outcomes; however, no comparative clinical studies between single- and double-row repair have assessed functional outcomes. The superiority of double-row capsulolabral repair versus single-row repair remains uncertain because comparative studies assessing clinical outcomes have yet to be performed.

Lack of chemically induced mutation in repair-deficient mutants of yeast.

PubMed

Prakash, L

1974-12-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.

Biomechanical Comparison of Single- Versus Double-Row Capsulolabral Repair for Shoulder Instability: A Review

PubMed Central

Yousif, Matthew John; Bicos, James

2017-01-01

Background: The glenohumeral joint is the most commonly dislocated joint in the body. Failure rates of capsulolabral repair have been reported to be approximately 8%. Recent focus has been on restoration of the capsulolabral complex by a double-row capsulolabral repair technique in an effort to decrease redislocation rates after arthroscopic capsulolabral repair. Purpose: To present a review of the biomechanical literature comparing single- versus double-row capsulolabral repairs and discuss the previous case series of double-row fixation. Study Design: Narrative review. Methods: A simple review of the literature was performed by PubMed search. Only biomechanical studies comparing single- versus double-row capsulolabral repair were included for review. Only those case series and descriptive techniques with clinical results for double-row repair were included in the discussion. Results: Biomechanical comparisons evaluating the native footprint of the labrum demonstrated significantly superior restoration of the footprint through double-row capsulolabral repair compared with single-row repair. Biomechanical comparisons of contact pressure at the repair interface, fracture displacement in bony Bankart lesion, load to failure, and decreased external rotation (suggestive of increased load to failure) were also significantly in favor of double- versus single-row repair. Recent descriptive techniques and case series of double-row fixation have demonstrated good clinical outcomes; however, no comparative clinical studies between single- and double-row repair have assessed functional outcomes. Conclusion: The superiority of double-row capsulolabral repair versus single-row repair remains uncertain because comparative studies assessing clinical outcomes have yet to be performed. PMID:29230427

A biomechanical comparison of single and double-row fixation in arthroscopic rotator cuff repair.

PubMed

Smith, Christopher D; Alexander, Susan; Hill, Adam M; Huijsmans, Pol E; Bull, Anthony M J; Amis, Andrew A; De Beer, Joe F; Wallace, Andrew L

2006-11-01

The optimal method for arthroscopic rotator cuff repair is not yet known. The hypothesis of the present study was that a double-row repair would demonstrate superior static and cyclic mechanical behavior when compared with a single-row repair. The specific aims were to measure gap formation at the bone-tendon interface under static creep loading and the ultimate strength and mode of failure of both methods of repair under cyclic loading. A standardized tear of the supraspinatus tendon was created in sixteen fresh cadaveric shoulders. Arthroscopic rotator cuff repairs were performed with use of either a double-row technique (eight specimens) or a single-row technique (eight specimens) with nonabsorbable sutures that were double-loaded on a titanium suture anchor. The repairs were loaded statically for one hour, and the gap formation was measured. Cyclic loading to failure was then performed. Gap formation during static loading was significantly greater in the single-row group than in the double-row group (mean and standard deviation, 5.0 +/- 1.2 mm compared with 3.8 +/- 1.4 mm; p < 0.05). Under cyclic loading, the double-row repairs failed at a mean of 320 +/- 96.9 N whereas the single-row repairs failed at a mean of 224 +/- 147.9 N (p = 0.058). Three single-row repairs and three double-row repairs failed as a result of suture cut-through. Four single-row repairs and one double-row repair failed as a result of anchor or suture failure. The remaining five repairs did not fail, and a midsubstance tear of the tendon occurred. Although more technically demanding, the double-row technique demonstrates superior resistance to gap formation under static loading as compared with the single-row technique. A double-row reconstruction of the supraspinatus tendon insertion may provide a more reliable construct than a single-row repair and could be used as an alternative to open reconstruction for the treatment of isolated tears.

Incidence of retear with double-row versus single-row rotator cuff repair.

PubMed

Shen, Chong; Tang, Zhi-Hong; Hu, Jun-Zu; Zou, Guo-Yao; Xiao, Rong-Chi

2014-11-01

Rotator cuff tears have a high recurrence rate, even after arthroscopic rotator cuff repair. Although some biomechanical evidence suggests the superiority of the double-row vs the single-row technique, clinical findings regarding these methods have been controversial. The purpose of this study was to determine whether the double-row repair method results in a lower incidence of recurrent tearing compared with the single-row method. Electronic databases were systematically searched to identify reports of randomized, controlled trials (RCTs) comparing single-row with double-row rotator cuff repair. The primary outcome assessed was retear of the repaired cuff. Secondary outcome measures were the American Shoulder and Elbow Surgeons (ASES) shoulder score, the Constant shoulder score, and the University of California, Los Angeles (UCLA) score. Heterogeneity between the included studies was assessed. Six studies involving 428 patients were included in the review. Compared with single-row repair, double-row repair demonstrated a lower retear incidence (risk ratio [RR]=1.71 [95% confidence interval (CI), 1.18-2.49]; P=.005; I(2)=0%) and a reduced incidence of partial-thickness retears (RR=2.16 [95% CI, 1.26-3.71]; P=.005; I(2)=26%). Functional ASES, Constant, and UCLA scores showed no difference between single- and double-row cuff repairs. Use of the double-row technique decreased the incidence of retears, especially partial-thickness retears, compared with the single-row technique. The functional outcome was not significantly different between the 2 techniques. To improve the structural outcome of the repaired rotator cuff, surgeons should use the double-row technique. However, further long-term RCTs on this topic are needed. Copyright 2014, SLACK Incorporated.

RAD21L, a novel cohesin subunit implicated in linking homologous chromosomes in mammalian meiosis

PubMed Central

Lee, Jibak

2011-01-01

Cohesins are multi-subunit protein complexes that regulate sister chromatid cohesion during mitosis and meiosis. Here we identified a novel kleisin subunit of cohesins, RAD21L, which is conserved among vertebrates. In mice, RAD21L is expressed exclusively in early meiosis: it apparently replaces RAD21 in premeiotic S phase, becomes detectable on the axial elements in leptotene, and stays on the axial/lateral elements until mid pachytene. RAD21L then disappears, and is replaced with RAD21. This behavior of RAD21L is unique and distinct from that of REC8, another meiosis-specific kleisin subunit. Remarkably, the disappearance of RAD21L at mid pachytene correlates with the completion of DNA double-strand break repair and the formation of crossovers as judged by colabeling with molecular markers, γ-H2AX, MSH4, and MLH1. RAD21L associates with SMC3, STAG3, and either SMC1α or SMC1β. Our results suggest that cohesin complexes containing RAD21L may be involved in synapsis initiation and crossover recombination between homologous chromosomes. PMID:21242291

Single-Port Onlay Mesh Repair of Recurrent Inguinal Hernias after Failed Anterior and Laparoscopic Repairs

PubMed Central

Tran, Kim; Zajkowska, Marta; Lam, Vincent; Hawthorne, Wayne J.

2015-01-01

Background and Objectives: Despite the exponential increase in the use of laparoscopic inguinal herniorrhaphy, overall recurrence rates have remained unchanged. Therefore, a growing number of patients are presenting with recurrent hernias after conventional anterior and laparoscopic repairs have failed. This study reports our experience with single-incision laparoscopic (SIL) intraperitoneal onlay mesh (IPOM) repair of these hernias. Methods: Patients referred with two or more recurrences of inguinal hernia underwent SIL-IPOM from November 1, 2009, to June 24, 2014. A 2.5-cm infraumbilical incision was made, and an SIL port was placed intraperitoneally. Modified dissection techniques were used: chopstick and inline dissection, 5.5-mm/52-cm/30° angled laparoscope, and conventional straight dissecting instruments. The peritoneum was incised above the pubic symphysis, and dissection was continued laterally and proximally, raising the inferior flap below the previous extraperitoneal mesh while reducing any direct, indirect, femoral, or cord lipoma before placement of antiadhesive mesh, which was fixed to the pubic ramus, as well as superiorly, with nonabsorbable tacks before the inferior border was fixed with fibrin sealant. The inferior peritoneal flap was then tacked back onto the mesh. Results: Nine male patients underwent SIL-IPOM. Their mean age was 53 years and mean body mass index was 26.8 kg/m2. Mean mesh size was 275 cm2. Mean operation time was 125 minutes, with a hospital stay of 1 day. The umbilical scar length was 23 mm at the 6-week follow-up. There were no intra-/postoperative complications, port-site hernias, chronic groin pain, or recurrence of the hernia during a mean follow-up of 24 months. Conclusion: Inguinal hernias recurring after two or more failed conventional anterior and laparoscopic repairs can be safely and efficiently treated with SIL-IPOM. PMID:25848186

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

The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination.

PubMed

Eppink, Berina; Tafel, Agnieszka A; Hanada, Katsuhiro; van Drunen, Ellen; Hickson, Ian D; Essers, Jeroen; Kanaar, Roland

2011-11-10

Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS. Copyright © 2011 Elsevier B.V. All rights reserved.

Excess single-stranded DNA inhibits meiotic double-strand break repair.

PubMed

Johnson, Rebecca; Borde, Valérie; Neale, Matthew J; Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S H

2007-11-01

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant

Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair

PubMed Central

Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S. H

2007-01-01

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair

Dissecting transcription-coupled and global genomic repair in the chromatin of yeast GAL1-10 genes.

PubMed

Li, Shisheng; Smerdon, Michael J

2004-04-02

Transcription-coupled repair (TCR) and global genomic repair (GGR) of UV-induced cyclobutane pyrimidine dimers were investigated in the yeast GAL1-10 genes. Both Rpb9- and Rad26-mediated TCR are confined to the transcribed strands, initiating at upstream sites approximately 100 nucleotides from the upstream activating sequence shared by the two genes. However, TCR initiation sites do not correlate with either transcription start sites or TATA boxes. Rad16-mediated GGR tightly correlates with nucleosome positioning when the genes are repressed and are slow in the nucleosome core and fast in linker DNA. Induction of transcription enhanced GGR in nucleosome core DNA, especially in the nucleosomes around and upstream of the transcription start sites. Furthermore, when the genes were induced, GGR was slower in the transcribed regions than in the upstream regions. Finally, simultaneous deletion of RAD16, RAD26, and RPB9 resulted in no detectable repair in all sites along the region analyzed. Our results suggest that (a). TCR may be initiated by a transcription activator, presumably through the loading of RNA polymerase II, rather than by transcription initiation or elongation per se; (b). TCR and nucleosome disruption-enhanced GGR are the major causes of rapid repair in regions around and upstream of transcription start sites; (c). transcription machinery may hinder access of NER factors to a DNA lesion in the absence of a transcription-repair coupling factor; and (d). other than GGR mediated by Rad16 and TCR mediated by Rad26 and Rpb9, no other nucleotide excision repair pathway exists in these RNA polymerase II-transcribed genes.

Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance.

PubMed

Ghodke, Harshad; Ho, Han; van Oijen, Antoine M

2018-02-19

Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow individual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA-repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA-repair processes in prokaryotes. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

PubMed Central

Kadyk, L. C.; Hartwell, L. H.

1993-01-01

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination. PMID:8454200

Suture spanning augmentation of single-row rotator cuff repair: a biomechanical analysis.

PubMed

Early, Nicholas A; Elias, John J; Lippitt, Steven B; Filipkowski, Danielle E; Pedowitz, Robert A; Ciccone, William J

2017-02-01

This in vitro study evaluated the biomechanical benefit of adding spanning sutures to single-row rotator cuff repair. Mechanical testing was performed to evaluate 9 pairs of cadaveric shoulders with complete rotator cuff repairs, with a single-row technique used on one side and the suture spanning technique on the other. The spanning technique included sutures from 2 lateral anchors securing tendon near the musculotendinous junction, spanning the same anchor placement from single-row repair. The supraspinatus muscle was loaded to 100 N at 0.25 Hz for 100 cycles, followed by a ramp to failure. Markers and a video tracking system measured anterior and posterior gap formation across the repair at 25-cycle intervals. The force at which the stiffness decreased by 50% and 75% was determined. Data were compared using paired t-tests. One single-row repair failed at <25 cycles. Both anterior and posterior gap distances tended to be 1 to 2 mm larger for the single-row repairs than for the suture spanning technique. The difference was statistically significant at all cycles for the posterior gap formation (P ≤ .02). The trends were not significant for the anterior gap (P ≥ .13). The loads at which the stiffness decreased by 50% and 75% did not differ significantly between the 2 types of repair (P ≥ .10). The suture spanning technique primarily improved posterior gap formation. Decreased posterior gap formation could reduce failure rates for rotator cuff repair. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

Mechanism of homologous recombination and implications for aging-related deletions in mitochondrial DNA.

PubMed

Chen, Xin Jie

2013-09-01

Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells.

Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

PubMed Central

2013-01-01

SUMMARY Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells. PMID:24006472

Contributions of recombination and repair proteins to telomere maintenance in telomerase-positive and negative Ustilago maydis.

PubMed

Yu, Eun Young; Hsu, Min; Holloman, William K; Lue, Neal F

2018-01-01

Homologous recombination and repair factors are known to promote both telomere replication and recombination-based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance in Ustilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase-positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase-negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi-solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II-like as well as ALT-like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I-like telomere features. In addition, we observed direct physical interactions between Blm and two telomere-binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination. © 2017 John Wiley & Sons Ltd.

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

DOE PAGES

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

2015-08-31

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

Laser Cladding for Crack Repair of CMSX-4 Single-Crystalline Turbine Parts

NASA Astrophysics Data System (ADS)

Rottwinkel, Boris; Nölke, Christian; Kaierle, Stefan; Wesling, Volker

2017-03-01

The increase of the lifetime of modern single crystalline (SX) turbine blades is of high economic priority. The currently available repair methods using polycrystalline cladding of the damaged area do not address the issue of monocrystallinity and are restricted to few areas of the blade. The tip area of the blade is most prone to damage and undergoes the most wear, erosion and cracking during its lifetime. To repair such defects, the common procedure is to remove the whole tip with the damaged area and rebuild it by applying a polycrystalline solidification of the material. The repair of small cracks is conducted in the same way. To reduce repair cost, the investigation of a manufacturing process to repair these cracked areas while maintaining single-crystal solidification is of high interest as this does not diminish material properties and thereby its lifetime. To establish this single-crystal solidification, the realization of a directed temperature gradient is needed. The initial scope of this work is the computational prediction of the temperature field that arises and its verification during the process. The laser cladding process of CMSX-4 substrates was simulated and the necessary parameters calculated. These parameters were then applied to notched substrates and their microstructures analyzed. Starting with a simulation of the temperature field using ANSYS®, a process to repair parts of single crystalline nickel-based alloys was developed. It could be shown that damages to the tip area and cracks can be repaired by establishing a specific temperature gradient during the repair process in order to control the solidification process.

Single- and double-row repair for rotator cuff tears - biology and mechanics.

PubMed

Papalia, Rocco; Franceschi, Francesco; Vasta, Sebastiano; Zampogna, Biagio; Maffulli, Nicola; Denaro, Vincenzo

2012-01-01

We critically review the existing studies comparing the features of single- and double-row repair, and discuss suggestions about the surgical indications for the two repair techniques. All currently available studies comparing the biomechanical, clinical and the biological features of single and double row. Biomechanically, the double-row repair has greater performances in terms of higher initial fixation strength, greater footprint coverage, improved contact area and pressure, decreased gap formation, and higher load to failure. Results of clinical studies demonstrate no significantly better outcomes for double-row compared to single-row repair. Better results are achieved by double-row repair for larger lesions (tear size 2.5-3.5 cm). Considering the lack of statistically significant differences between the two techniques and that the double row is a high cost and a high surgical skill-dependent technique, we suggest using the double-row technique only in strictly selected patients. Copyright © 2012 S. Karger AG, Basel.

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

PubMed

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

2015-04-01

Giardia duodenalis is a well-known protozoan parasite of humans and other mammals. The repair of DNA double strand breaks (DSBs) is crucial for genomic stability and homologous recombination is one of the primary mechanisms used by cells to repair DNA. The Mre11 complex is comprised by Mre11, an endonuclease and 3'-5' exonuclease known to resect ends during homologous recombination, and Rad50, a member of the structural maintenance of chromosomes (SMC) family of ATPases. In this work we cloned, expressed and characterized the catalytic activities of the giardial Mre11 (GdMre11) and Rad50 (GdRad50) proteins. Our results show that while purified recombinant GdMre11 and GdRad50 proteins bind DNA, GdMre11 contains a 3'-5' exonuclease and purified recombinant GdRad50 has ATPase activity. The predicted structure for GdMre11 revealed a conserved Mn(2+) dependent binding pocket. We also explored the expression of giardial mre11 and rad50 genes after ionizing radiation, and our results indicate that both specific transcripts were increased after 1-2 h while their protein levels were found to be significantly increased 4 h after gamma radiation treatment. These proteins were localized in the nuclei before and after irradiation. The implication of these observations is discussed. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Mammalian DNA single-strand break repair: an X-ra(y)ted affair.

PubMed

Caldecott, K W

2001-05-01

The genetic stability of living cells is continuously threatened by the presence of endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of DNA single-strand breaks that arise in each cell, each day, both directly from disintegration of damaged sugars and indirectly from the excision repair of damaged bases. If un-repaired, single-strand breaks can be converted into double-strand breaks during DNA replication, potentially resulting in chromosomal rearrangement and genetic deletion. Consequently, cells have adopted multiple pathways to ensure the rapid and efficient removal of single-strand breaks. A general feature of these pathways appears to be the extensive employment of protein-protein interactions to stimulate both the individual component steps and the overall repair reaction. Our current understanding of DNA single-strand break repair is discussed, and testable models for the architectural coordination of this important process are presented. Copyright 2001 John Wiley & Sons, Inc.

Location of RAD51-like protein during meiotic prophase in Eimeria tenella.

PubMed

Del Cacho, Emilio; Gallego, Margarita; Pagés, Marc; Barbero, José Luís; Monteagudo, Luís; Sánchez-Acedo, Caridad

2011-05-31

This study focuses on reporting events in Eimeria tenella oocysts from early to late prophase I in terms of RAD51 protein in association with the synaptonemal complex formed between homologous chromosomes. The aim of the study was the sequential localization of RAD51 protein, which is involved in the repair of double-strand breaks (DSBs) on the eimerian chromosomes as they synapse and desynapse. Structural Maintenance of Chromosome protein SMC3, which plays a role in synaptonemal complex formation, was labeled to identify initiation and progress of chromosome synapsis and desynapsis in parallel with the appearance and disappearance of RAD51 foci. Antibodies directed against RAD51 and cohesin subunit SMC3 proteins were labeled with either fluorescence or colloidal gold to visualize RAD51 protein foci and synaptonemal complexes. RAD51 protein localization during prophase I was studied on meiotic chromosomes spreads obtained from oocysts at different points in time after the start of sporulation. The present findings showed that foci detected with the antibody directed against RAD51 protein first appeared at the pre-leptotene stage before homologous chromosomes began pairing. Subsequently, the foci were detected in association with the lateral elements at the precise sites where synapsis were in progress. These findings lead us to suggest that in E. tenella, homologous chromosome pairing was a DSB-dependent mechanism and reinforced the participation of RAD51 protein in meiotic homology search, alignment and pairing of chromosomes. Copyright © 2010 Elsevier B.V. All rights reserved.

Chromosomal localization of three repair genes: The xeroderma pigmentosum group C gene and two human homologs of yeast RAD23

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spek, P.J. van der; Smit, E.M.E.; Beverloo, H.B.

1994-10-01

The nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) is characterized by sun (UV) sensitivity, predisposition to skin cancer, and extensive genetic heterogeneity. Recently, we reported the cloning and analysis of three human NER genes, XPC, HHR23A, and HHR23B. The previously cloned XPC gene is involved in the common XP complementation group C, which is defective in excision repair of nontranscribed sequences in the genome. The XPC protein was found to be complexed with the product of HHR23B, one of the two human homologs of the Saccharomyes cerevisiae NER gene RAD23. Here we present the chromosomal localization by in situmore » hybridization using haptenized probes of all three genes. The HHR23A gene was assigned to chromosome 19p13.2. Interestingly, the HHR23B and XPC genes, the product of which forms a tight complex, were found to colocalize on band 3p25.1. Pulsed-field gel electrophoresis revealed that the HHR23B and XPC genes possibly share a MluI restriction fragment of about 625 kb. Potential involvement of the HHR23 genes in human genetic disorders is discussed. 53 refs., 4 figs., 2 tabs.« less

Rad-Hard/HI-REL FPGA

NASA Technical Reports Server (NTRS)

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

1997-01-01

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

Interrogation of ethnomedicinal plants for synthetic lethality effects in combination with deficiency in the DNA repair endonuclease RAD1 using a yeast cell-based assay.

PubMed

Aung, Hsu Mon; Huangteerakul, Chananya; Panvongsa, Wittaya; Jensen, Amornrat N; Chairoungdua, Arthit; Sukrong, Suchada; Jensen, Laran T

2018-09-15

Plant materials used in this study were selected based on the ethnobotanical literature. Plants have either been utilized by Thai practitioners as alternative treatments for cancer or identified to exhibit anti-cancer properties. To screen ethnomedicinal plants using a yeast cell-based assay for synthetic lethal interactions with cells deleted for RAD1, the yeast homologue of human ERCC4 (XPF) MATERIALS AND METHODS: Ethanolic extracts from thirty-two species of medicinal plants utilized in Thai traditional medicine were screened for synthetic lethal/sick interactions using a yeast cell-based assay. Cell growth was compared between the parental strain and rad1∆ yeast following exposure to select for specific toxicity of plant extracts. Candidate extracts were further examined for the mode of action using genetic and biochemical approaches. Screening a library of ethanolic extracts from medicinal plants identified Bacopa monnieri and Colubrina asiatica as having synthetic lethal effects in the rad1∆ cells but not the parental strain. Synthetic lethal effects for B. monneiri extracts were more apparent and this plant was examined further. Genetic analysis indicates that pro-oxidant activities and defective excision repair pathways do not significantly contribute to enhanced sensitivity to B. monneiri extracts. Exposure to B. monneiri extracts resulted in nuclear fragmentation and elevated levels of ethidium bromide staining in rad1∆ yeast suggesting promotion of an apoptosis-like event. Growth inhibition also observed in the human Caco-2 cell line suggesting the effects of B. monnieri extracts on both yeast and human cells may be similar. B. monneiri extracts may have utility in treatment of colorectal cancers that exhibit deficiency in ERCC4 (XPF). Copyright © 2018 Elsevier B.V. All rights reserved.

Disparate requirements for the Walker A and B ATPase motifs of human RAD51D in homologous recombination.

PubMed

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

2006-01-01

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 (ICLs). 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.

Rad51 Interacts with Non-structural 3 Protein of Hepatitis C Virus and Regulates Viral Production

PubMed Central

Son, Kidong; Nguyen, Tram T. T.; Choi, Jae-Woong; Pham, Long V.; Luong, Trang T. D.; Lim, Yun-Sook; Hwang, Soon B.

2017-01-01

Hepatitis C virus (HCV) is a leading cause of chronic liver disease affecting over 170 million people worldwide. Chronic infection with HCV progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma. HCV exploits host cellular factors for viral propagation. To investigate the cellular factors required for HCV propagation, we screened a siRNA library targeting human cell cycle genes using cell culture grown HCV-infected cells. In the present study, we selected and characterized a gene encoding Rad51. Rad51, a member of a conserved recombinase family, is an essential factor for homologous recombination and repair of double-strand DNA breaks. We demonstrated that siRNA-mediated knockdown of Rad51 significantly inhibited HCV propagation without affecting HCV RNA replication. Silencing of Rad51 impaired secretion of infectious HCV particles and thus intracellular viruses were accumulated. We showed that HCV NS3 specifically interacted with Rad51 and accumulated Rad51 in the cytosol. Furthermore, Rad51 was coprecipitated with NS3 and HCV RNA. By employing membrane flotation and protease protection assays, we also demonstrated that Rad51 was co-fractionated with HCV NS3 on the lipid raft. These data indicate that Rad51 may be a component of the HCV RNA replication complex. Collectively, these data suggest that HCV may exploit cellular Rad51 to promote viral propagation and thus Rad51 may be a potential therapeutic target for HCV. PMID:28729862

Atrioventricular valve repair in patients with single-ventricle physiology: mechanisms, techniques of repair, and clinical outcomes.

PubMed

Honjo, Osami; Mertens, Luc; Van Arsdell, Glen S

2011-01-01

Significant atrioventricular (AV) valve insufficiency in patient with single ventricle-physiology is strongly associated with poor survival. Herein we discuss the etiology and mechanism of development of significant AV valve insufficiency in patients with single-ventricle physiology, surgical indication and repair techniques, and clinical outcomes along with our 10-year surgical experience. Our recent clinical series and literature review indicate that it is of prime importance to appreciate the high incidence and clinical effect of the structural abnormalities of AV valve. Valve repair at stage II palliation may minimize the period of volume overload, thereby potentially preserving post-repair ventricular function. Since 85% of the AV valve insufficiency was associated with structural abnormalities, inspection of an AV valve that has more than mild to moderate insufficiency is recommended because they are not likely to be successfully treated with volume unloading surgery alone. Copyright © 2011 Elsevier Inc. All rights reserved.

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

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.

Repair of DNA Strand Breaks in a Minichromosome In Vivo: Kinetics, Modeling, and Effects of Inhibitors

PubMed Central

Kumala, Slawomir; Fujarewicz, Krzysztof; Jayaraju, Dheekollu; Rzeszowska-Wolny, Joanna; Hancock, Ronald

2013-01-01

To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ∼170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20–30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair. PMID:23382828

A comparative clinical evaluation of arthroscopic single-row versus double-row supraspinatus tendon repair.

PubMed

Buess, Eduard; Waibl, Bernhard; Vogel, Roger; Seidner, Robert

2009-10-01

Cadaveric studies and commercial pressure have initiated a strong trend towards double-row repair in arthroscopic cuff surgery. The objective of this study was to evaluate if the biomechanical advantages of a double-row supraspinatus tendon repair would result in superior clinical outcome and higher abduction strength. A retrospective study of two groups of 32 single-row and 33 double-row repairs of small to medium cuff tears was performed. The Simple Shoulder Test (SST) and a visual analog scale for pain were used to evaluate the outcome. The participation rate was 100%. A subset of patients was further investigated with the Constant Score (CS) including electronic strength measurement. The double-row repair patients had significantly more (p = 0.01) yes answers in the SST than the single-row group, and pain reduction was slightly better (p = 0.03). No difference was found for the relative CS (p = 0.86) and abduction strength (p = 0.74). Patient satisfaction was 100% for double-row and 97% for single-row repair. Single- and double-row repairs both achieved excellent clinical results. Evidence of superiority of double-row repair is still scarce and has to be balanced against the added complexity of the procedure and higher costs.

ROBOTIC ASSISTED SINGLE SITE FOR BILATERAL INGUINAL HERNIA REPAIR.

PubMed

Bosi, Henrique Rasia; Guimarães, José Ricardo; Cavazzola, Leandro Totti

2016-01-01

The inguinal hernia is one of the most frequent surgical diseases, being frequent procedure and surgeon´s everyday practice. To present technical details in making hernioplasty using robotic equipment on bilateral inguinal hernia repair with single port and preliminary results with the method. The bilateral inguinal hernia repair was performed by using the Single-Site(c) Da Vinci Surgical Access Platform to the abdominal cavity and the placement of clamps. This technique proved to be effective for inguinal hernia and have more aesthetic result when compared to other techniques. Inguinal hernia repair robot-assisted with single-trocar is feasible and effective. However, still has higher costs needing surgical team special training. A hérnia inguinal é uma das doenças cirúrgicas mais frequentes, tornando-a procedimento frequente e do cotidiano do cirurgião. Apresentar detalhes da técnica da hernioplastia inguinal bilateral robótica por single-site e resultados preliminares com o método. Foi realizada hernioplastia inguinal bilateral assistida por robô, utilizando-se da Vinci Single-Site(c) Surgical Platform para acesso a cavidade abdominal e colocação das pinças. Esta técnica demonstrou-se efetiva para correção da hérnia inguinal, além de apresentar melhor resultado estético quando comparado às outras técnicas. A hernioplastia inguinal assistida por robô com trocarte único é viável e eficaz. Contudo, ainda apresenta custos mais elevados e necessidade de treinamento especial por parte da equipe cirúrgica.

Human longevity and variation in GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidant pathway genes: cross sectional and longitudinal studies.

PubMed

Soerensen, Mette; Dato, Serena; Tan, Qihua; Thinggaard, Mikael; Kleindorp, Rabea; Beekman, Marian; Jacobsen, Rune; Suchiman, H Eka D; de Craen, Anton J M; Westendorp, Rudi G J; Schreiber, Stefan; Stevnsner, Tinna; Bohr, Vilhelm A; Slagboom, P Eline; Nebel, Almut; Vaupel, James W; Christensen, Kaare; McGue, Matt; Christiansen, Lene

2012-05-01

Here we explore association with human longevity of common genetic variation in three major candidate pathways: GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidants by investigating 1273 tagging SNPs in 148 genes composing these pathways. In a case-control study of 1089 oldest-old (age 92-93) and 736 middle-aged Danes we found 1 pro/antioxidant SNP (rs1002149 (GSR)), 5 GH/IGF-1/INS SNPs (rs1207362 (KL), rs2267723 (GHRHR), rs3842755 (INS), rs572169 (GHSR), rs9456497 (IGF2R)) and 5 DNA repair SNPs (rs11571461 (RAD52), rs13251813 (WRN), rs1805329 (RAD23B), rs2953983 (POLB), rs3211994 (NTLH1)) to be associated with longevity after correction for multiple testing. In a longitudinal study with 11 years of follow-up on survival in the oldest-old Danes we found 2 pro/antioxidant SNPs (rs10047589 (TNXRD1), rs207444 (XDH)), 1 GH/IGF-1/INS SNP (rs26802 (GHRL)) and 3 DNA repair SNPs (rs13320360 (MLH1), rs2509049 (H2AFX) and rs705649 (XRCC5)) to be associated with mortality in late life after correction for multiple testing. When examining the 11 SNPs from the case-control study in the longitudinal data, rs3842755 (INS), rs13251813 (WRN) and rs3211994 (NTHL1) demonstrated the same directions of effect (p<0.05), while rs9456497 (IGF2R) and rs1157146 (RAD52) showed non-significant tendencies, indicative of effects also in late life survival. In addition, rs207444 (XDH) presented the same direction of effect when inspecting the 6 SNPs from the longitudinal study in the case-control data, hence, suggesting an effect also in survival from middle age to old age. No formal replications were observed when investigating the 11 SNPs from the case-control study in 1613 oldest-old (age 95-110) and 1104 middle-aged Germans, although rs11571461 (RAD52) did show a supportive non-significant tendency (OR=1.162, 95% CI=0.927-1.457). The same was true for rs10047589 (TNXRD1) (HR=0.758, 95%CI=0.543-1.058) when examining the 6 SNPs from the longitudinal study in a Dutch

Chronic low-dose ultraviolet-induced mutagenesis in nucleotide excision repair-deficient cells.

PubMed

Haruta, Nami; Kubota, Yoshino; Hishida, Takashi

2012-09-01

UV radiation induces two major types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts, which are both primarily repaired by nucleotide excision repair (NER). Here, we investigated how chronic low-dose UV (CLUV)-induced mutagenesis occurs in rad14Δ NER-deficient yeast cells, which lack the yeast orthologue of human xeroderma pigmentosum A (XPA). The results show that rad14Δ cells have a marked increase in CLUV-induced mutations, most of which are C→T transitions in the template strand for transcription. Unexpectedly, many of the CLUV-induced C→T mutations in rad14Δ cells are dependent on translesion synthesis (TLS) DNA polymerase η, encoded by RAD30, despite its previously established role in error-free TLS. Furthermore, we demonstrate that deamination of cytosine-containing CPDs contributes to CLUV-induced mutagenesis. Taken together, these results uncover a novel role for Polη in the induction of C→T transitions through deamination of cytosine-containing CPDs in CLUV-exposed NER deficient cells. More generally, our data suggest that Polη can act as both an error-free and a mutagenic DNA polymerase, depending on whether the NER pathway is available to efficiently repair damaged templates.

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

PubMed Central

Kozmin, Stanislav G.; Jinks-Robertson, Sue

2013-01-01

Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps. PMID:23307894

DNA Repair: The Search for Homology.

PubMed

Haber, James E

2018-05-01

The repair of chromosomal double-strand breaks (DSBs) by homologous recombination is essential to maintain genome integrity. The key step in DSB repair is the RecA/Rad51-mediated process to match sequences at the broken end to homologous donor sequences that can be used as a template to repair the lesion. Here, in reviewing research about DSB repair, I consider the many factors that appear to play important roles in the successful search for homology by several homologous recombination mechanisms. See also the video abstract here: https://youtu.be/vm7-X5uIzS8. © 2018 WILEY Periodicals, Inc.

Restoration of shoulder motion using single- versus dual-nerve repair in obstetrical brachial plexus injury.

PubMed

Azzi, Alain Joe; AlNaeem, Hassan; Aubin-Lemay, Camille; Kvann, Julie Chakriya; Alam, Peter; Retrouvey, Helene; Aldekhayel, Salah; Zadeh, Teanoosh

2018-05-01

OBJECTIVE The purpose of this study was to compare shoulder abduction and external rotation (ER) after single-nerve repair of the upper trunk alone versus dual-nerve repair of both the upper trunk and the suprascapular nerve. METHODS A retrospective chart review of a single surgeon's experience repairing obstetrical brachial plexus injuries between June 1995 and June 2015 was performed. Eight patients underwent repair of the upper trunk alone, and 10 patients underwent repair of the upper trunk and the suprascapular nerve. Shoulder abduction and ER ranges of motion (ROMs) (in degrees) were recorded preoperatively and postoperatively. Postoperative ROM and the difference in ROM gained after surgery were compared by independent t-test analysis. RESULTS The mean follow-up time was 161.4 weeks (range 62-514 weeks, SD 124.0 weeks). The mean patient age at the time of surgery was 31.3 weeks (range 19.9-47.0 weeks, SD 6.9 weeks). The mean postoperative shoulder abduction ROMs were 145.0° (range 85°-180°, SD 39.4°) after single-nerve repair and 134.0° (range 90°-180°, SD 30.3°) after dual-nerve repair (p = 0.51). The mean postoperative shoulder ER ROMs were 67.5° (range 10°-95°, SD 28.8°) after single-nerve repair and 72.0° (range 10°-95°, SD 31.3°) after dual-nerve repair (p = 0.76). CONCLUSIONS The authors found no difference in shoulder abduction and ER between patients who underwent single-nerve repair of the upper trunk alone and those who underwent dual-nerve repair of both the upper trunk and the suprascapular nerve.

Single-row versus double-row repair of the distal Achilles tendon: a biomechanical comparison.

PubMed

Pilson, Holly; Brown, Philip; Stitzel, Joel; Scott, Aaron

2012-01-01

Surgery for recalcitrant insertional Achilles tendinopathy often consists of partial or total release of the insertion site, debridement of the diseased portion of the tendon, calcaneal ostectomy, and reattachment of the Achilles to the calcaneus. Although single-row and double-row techniques exist for repair of the detached Achilles tendon, biomechanical data are lacking to support one technique over the other. Based on data extrapolated from the study of rotator cuff repairs, we hypothesized that a double-row construct would provide superior fixation strength over a single-row repair. Eighteen human cadaveric Achilles tendons (9 matched pairs) with attached calcanei were repaired with single-row or double-row techniques. Specimens were mounted in a servohydraulic materials testing machine, subjected to a preconditioning cycle, and loaded to failure. Failure was defined as suture breakage or pullout, midsubstance tendon rupture, or anchor pullout. Among the failures were 12 suture failures, 5 proximal-row anchor failures, and 1 distal-row anchor failure. No midsubstance tendon ruptures or testing apparatus failures were observed. There were no statistically significant differences in the peak load to failure between the single-row and double-row repairs (p = .46). Similarly, no significant differences were observed with regards to mean energy expenditure to failure (p = .069). The present study demonstrated no biomechanical advantages of the double-row repair over a single-row repair. Despite the lack of a clear biomechanical advantage, there may exist clinical advantages of a double-row repair, such as reduction in knot prominence and restoration of the Achilles footprint. Copyright © 2012 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

Biomechanical evaluation of a single-row versus double-row repair for complete subscapularis tears.

PubMed

Wellmann, Mathias; Wiebringhaus, Philipp; Lodde, Ina; Waizy, Hazibullah; Becher, Christoph; Raschke, Michael J; Petersen, Wolf

2009-12-01

The purpose of the study was to compare a single-row repair and a double-row repair technique for the specific characteristics of a complete subscapularis lesion. Ten pairs of human cadaveric shoulder human shoulder specimens were tested for stiffness and ultimate tensile strength of the intact tendons in a load to failure protocol. After a complete subscapularis tear was provoked, the specimens were assigned to two treatment groups: single-row repair (1) and a double-row repair using a "suture bridge" technique (2). After repair cyclic loading a subsequent load to failure protocol was performed to determine the ultimate tensile load, the stiffness and the elongation behaviour of the reconstructions. The intact subscapularis tendons had a mean stiffness of 115 N/mm and a mean ultimate load of 720 N. The predominant failure mode of the intact tendons was a tear at the humeral insertion site (65%). The double-row technique restored 48% of the ultimate load of the intact tendons (332 N), while the single-row technique revealed a significantly lower ultimate load of 244 N (P = 0.001). In terms of the stiffness, the double-row technique showed a mean stiffness of 81 N/mm which is significantly higher compared to the stiffness of the single-row repairs of 55 N/mm (P = 0.001). The double-row technique has been shown to be stronger and stiffer when compared to a conventional single-row repair. Therefore, this technique is recommended from a biomechanical point of view irrespectively if performed by an open or arthroscopic approach.

[Rotator cuff repair: single- vs double-row. Clinical and biomechanical results].

PubMed

Baums, M H; Kostuj, T; Klinger, H-M; Papalia, R

2016-02-01

The goal of rotator cuff repair is a high initial mechanical stability as a requirement for adequate biological recovery of the tendon-to-bone complex. Notwithstanding the significant increase in publications concerning the topic of rotator cuff repair, there are still controversies regarding surgical technique. The aim of this work is to present an overview of the recently published results of biomechanical and clinical studies on rotator cuff repair using single- and double-row techniques. The review is based on a selective literature research of PubMed, Embase, and the Cochrane Database on the subject of the clinical and biomechanical results of single- and double-row repair. In general, neither the biomechanical nor the clinical evidence can recommend the use of a double-row concept for the treatment for every rotator cuff tear. Only tears of more than 3 cm seem to benefit from better results on both imaging and in clinical outcome studies compared with the use of single-row techniques. Despite a significant increase in publications on the surgical treatment of rotator cuff tears in recent years, the clinical results were not significantly improved in the literature so far. Unique information and algorithms, from which the optimal treatment of this entity can be derived, are still inadequate. Because of the cost-effectiveness and the currently vague evidence, the double-row techniques cannot be generally recommended for the repair of all rotator cuff tears.

Single versus double-row repair of the rotator cuff: does double-row repair with improved anatomical and biomechanical characteristics lead to better clinical outcome?

PubMed

Pauly, Stephan; Gerhardt, Christian; Chen, Jianhai; Scheibel, Markus

2010-12-01

Several techniques for arthroscopic repair of rotator cuff defects have been introduced over the past years. Besides established techniques such as single-row repairs, new techniques such as double-row reconstructions have gained increasing interest. The present article therefore provides an overview of the currently available literature on both repair techniques with respect to several anatomical, biomechanical, clinical and structural endpoints. Systematic literature review of biomechanical, clinical and radiographic studies investigating or comparing single- and double-row techniques. These results were evaluated and compared to provide an overview on benefits and drawbacks of the respective repair type. Reconstructions of the tendon-to-bone unit for full-thickness tears in either single- or double-row technique differ with respect to several endpoints. Double-row repair techniques provide more anatomical reconstructions of the footprint and superior initial biomechanical characteristics when compared to single-row repair. With regard to clinical results, no significant differences were found while radiological data suggest a better structural tendon integrity following double-row fixation. Presently published clinical studies cannot emphasize a clearly superior technique at this time. Available biomechanical studies are in favour of double-row repair. Radiographic studies suggest a beneficial effect of double-row reconstruction on structural integrity of the reattached tendon or reduced recurrent defect rates, respectively.

TOPBP1Dpb11 plays a conserved role in homologous recombination DNA repair through the coordinated recruitment of 53BP1Rad9

PubMed Central

Sims, Jennie Rae; Freire, Raimundo

2017-01-01

Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1–TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair. PMID:28228534

Single-row versus double-row arthroscopic rotator cuff repair in small- to medium-sized tears.

PubMed

Aydin, Nuri; Kocaoglu, Baris; Guven, Osman

2010-07-01

Double-row rotator cuff repair leads to superior cuff integrity and clinical results compared with single-row repair. The study enrolled 68 patients with a full-thickness rotator cuff tear who were divided into 2 groups of 34 patients according to repair technique. The patients were followed-up for at least 2 years. The results were evaluated by Constant score. Despite the biomechanical studies and cadaver studies that proved the superiority of double-row fixation over single-row fixation, our clinical results show no difference in functional outcome between the two methods. It is evident that double-row repair is more technically demanding, expensive, and time-consuming than single-row repair, without providing a significant improvement in clinical results. Comparison between groups did not show significant differences. At the final follow-up, the Constant score was 82.2 in the single-row group and 78.8 in the double-row group. Functional outcome was improved in both groups after surgery, but the difference between the 2 groups was not significant. At long-term follow-up, arthroscopic rotator cuff repair with the double-row technique showed no significant difference in clinical outcome compared with single-row repair in small to medium tears. 2010 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.

Biomechanical comparison of a single-row versus double-row suture anchor technique for rotator cuff repair.

PubMed

Kim, David H; Elattrache, Neal S; Tibone, James E; Jun, Bong-Jae; DeLaMora, Sergai N; Kvitne, Ronald S; Lee, Thay Q

2006-03-01

Reestablishment of the native footprint during rotator cuff repair has been suggested as an important criterion for optimizing healing potential and fixation strength. A double-row rotator cuff footprint repair will demonstrate superior biomechanical properties compared with a single-row repair. Controlled laboratory study. In 9 matched pairs of fresh-frozen cadaveric shoulders, the supraspinatus tendon from 1 shoulder was repaired with a double-row suture anchor technique: 2 medial anchors with horizontal mattress sutures and 2 lateral anchors with simple sutures. The tendon from the contralateral shoulder was repaired using a single lateral row of 2 anchors with simple sutures. Each specimen underwent cyclic loading from 10 to 180 N for 200 cycles, followed by tensile testing to failure. Gap formation and strain over the footprint area were measured using a video digitizing system; stiffness and failure load were determined from testing machine data. Gap formation for the double-row repair was significantly smaller (P < .05) when compared with the single-row repair for the first cycle (1.67 +/- 0.75 mm vs 3.10 +/- 1.67 mm, respectively) and the last cycle (3.58 +/- 2.59 mm vs 7.64 +/- 3.74 mm, respectively). The initial strain over the footprint area for the double-row repair was nearly one third (P < .05) the strain of the single-row repair. Adding a medial row of anchors increased the stiffness of the repair by 46% and the ultimate failure load by 48% (P < .05). Footprint reconstruction of the rotator cuff using a double-row repair improved initial strength and stiffness and decreased gap formation and strain over the footprint when compared with a single-row repair. To achieve maximal initial fixation strength and minimal gap formation for rotator cuff repair, reconstructing the footprint attachment with 2 rows of suture anchors should be considered.

Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

2008-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

Pseudosynapsis and Decreased Stringency of Meiotic Repair Pathway Choice on the Hemizygous Sex Chromosome of Caenorhabditis elegans Males

PubMed Central

Checchi, Paula M.; Lawrence, Katherine S.; Van, Mike V.; Larson, Braden J.; Engebrecht, JoAnne

2014-01-01

During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes. PMID:24939994

Gene and pathway level analyses of germline DNA-repair gene variants and prostate cancer susceptibility using the iCOGS-genotyping array.

PubMed

Saunders, Edward J; Dadaev, Tokhir; Leongamornlert, Daniel A; Al Olama, Ali Amin; Benlloch, Sara; Giles, Graham G; Wiklund, Fredrik; Gronberg, Henrik; Haiman, Christopher A; Schleutker, Johanna; Nordestgaard, Borge G; Travis, Ruth C; Neal, David; Pasayan, Nora; Khaw, Kay-Tee; Stanford, Janet L; Blot, William J; Thibodeau, Stephen N; Maier, Christiane; Kibel, Adam S; Cybulski, Cezary; Cannon-Albright, Lisa; Brenner, Hermann; Park, Jong Y; Kaneva, Radka; Batra, Jyotsna; Teixeira, Manuel R; Pandha, Hardev; Govindasami, Koveela; Muir, Ken; Easton, Douglas F; Eeles, Rosalind A; Kote-Jarai, Zsofia

2016-04-12

Germline mutations within DNA-repair genes are implicated in susceptibility to multiple forms of cancer. For prostate cancer (PrCa), rare mutations in BRCA2 and BRCA1 give rise to moderately elevated risk, whereas two of B100 common, low-penetrance PrCa susceptibility variants identified so far by genome-wide association studies implicate RAD51B and RAD23B. Genotype data from the iCOGS array were imputed to the 1000 genomes phase 3 reference panel for 21 780 PrCa cases and 21 727 controls from the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) consortium. We subsequently performed single variant, gene and pathway-level analyses using 81 303 SNPs within 20 Kb of a panel of 179 DNA-repair genes. Single SNP analyses identified only the previously reported association with RAD51B. Gene-level analyses using the SKAT-C test from the SNP-set (Sequence) Kernel Association Test (SKAT) identified a significant association with PrCa for MSH5. Pathway-level analyses suggested a possible role for the translesion synthesis pathway in PrCa risk and Homologous recombination/Fanconi Anaemia pathway for PrCa aggressiveness, even though after adjustment for multiple testing these did not remain significant. MSH5 is a novel candidate gene warranting additional follow-up as a prospective PrCa-risk locus. MSH5 has previously been reported as a pleiotropic susceptibility locus for lung, colorectal and serous ovarian cancers.

Laparoendoscopic single-site extraperitoneal inguinal hernia repair: initial experience in 10 patients.

PubMed

Do, Minh; Liatsikos, Evangelos; Beatty, John; Haefner, Tim; Dunn, Ian; Kallidonis, Panagiotis; Stolzenburg, Jens-Uwe

2011-06-01

Recent technical advances and a trend toward laparoscopic single incision surgery have led us to explore the feasibility of laparoendoscopic single-site (LESS) hernia repair. We present our technique and initial experience with LESS extraperitoneal inguinal hernia repair in 10 consecutive men with unilateral inguinal hernias. Age range was 43.7 (28-64) years. Mean body mass index was 28 (range 24-30). Six were left inguinal hernias. There were six indirect and four direct hernias. Three patients had undergone previous open appendectomy. Incarcerated or bilateral hernias were excluded from our initial series. All cases were performed by three surgeons who were experienced in conventional totally extraperitoneal laparoscopic hernia repair as well as experienced in LESS. A literature review of current single-port inguinal hernia repair data is also presented. The mean operative time was 53 minutes (range 45-65  min). The average length of skin incision was 2.8  cm (range 2.3-3.2  cm). No drain was necessary in any of the patients, while no recordable bleeding was observed. There were no intraoperative or immediate postoperative complications. Hospitalization period was 2 days for all patients. After a limited follow-up of 1 month, there have been no recurrences and no complaints of testicular pain. The results of the current series compare favorably with those found in a literature review. LESS extraperitoneal inguinal hernia repair is both feasible and safe, although more technically demanding than its conventional laparoscopic counterpart. Although the cosmetic result with the former approach may prove superior, there are standing questions regarding the complications and long-term outcome. Randomized and if possible blinded trials that compare conventional and single-incision laparoscopic hernia repair may help to distinguish the most advantageous technique.

Biomechanical characteristics of single-row repair in comparison to double-row repair with consideration of the suture configuration and suture material.

PubMed

Baums, M H; Buchhorn, G H; Spahn, G; Poppendieck, B; Schultz, W; Klinger, H-M

2008-11-01

The aim of the study was to evaluate the time zero mechanical properties of single- versus double-row configuration for rotator cuff repair in an animal model with consideration of the stitch technique and suture material. Thirty-two fresh-frozen sheep shoulders were randomly assigned to four repair groups: suture anchor single-row repair coupled with (1) braided, nonabsorbable polyester suture sized USP No. 2 (SRAE) or (2) braided polyblend polyethylene suture sized No. 2 (SRAH). The double-row repair was coupled with (3) USP No. 2 (DRAE) or (4) braided polyblend polyethylene suture No. 2 (DRAH). Arthroscopic Mason-Allen stitches were used (single-row) and combined with medial horizontal mattress stitches (double-row). Shoulders were cyclically loaded from 10 to 180 N. Displacement to gap formation of 5- and 10-mm at the repair site, cycles to failure, and the mode of failure were determined. The ultimate tensile strength was verified in specimens that resisted to 3,000 cycles. DRAE and DRAH had a lower frequency of 5- (P = 0.135) and 10-mm gap formation (P = 0.135). All DRAE and DRAH resisted 3,000 cycles while only three SRAE and one SRAH resisted 3,000 cycles (P < 0.001). The ultimate tensile strength in double-row specimens was significantly higher than in others (P < 0.001). There was no significant variation in using different suture material (P > 0.05). Double-row suture anchor repair with arthroscopic Mason-Allen/medial mattress stitches provides initial strength superior to single-row repair with arthroscopic Mason-Allen stitches under isometric cyclic loading as well as under ultimate loading conditions. Our results support the concept of double-row fixation with arthroscopic Mason-Allen/medial mattress stitches in rotator cuff tears with improvement of initial fixation strength and ultimate tensile load. Use of new polyblend polyethylene suture material seems not to increase the initial biomechanical aspects of the repair construct.

Initial load-to-failure and failure analysis in single- and double-row repair techniques for rotator cuff repair.

PubMed

Baums, M H; Buchhorn, G H; Gilbert, F; Spahn, G; Schultz, W; Klinger, H-M

2010-09-01

This experimental study aimed to compare the load-to-failure rate and stiffness of single- versus double-row suture techniques for repairing rotator cuff lesions using two different suture materials. Additionally, the mode of failure of each repair was evaluated. In 32 sheep shoulders, a standardized tear of the infraspinatus tendon was created. Then, n = 8 specimen were randomized to four repair methods: (1) Double-row Anchor Ethibond coupled with polyester sutures, USP No. 2; (2) Double-Row Anchor HiFi with polyblend polyethylene sutures, USP No. 2; (3) Single-Row Anchor Ethibond coupled with braided polyester sutures, USP No. 2; and (4) Single-Row Anchor HiFi with braided polyblend polyethylene sutures, USP No. 2. Arthroscopic Mason-Allen stitches were placed (single-row) and combined with medial horizontal mattress stitches (double-row). All specimens were loaded to failure at a constant displacement rate on a material testing machine. Group 4 showed lowest load-to-failure result with 155.7 +/- 31.1 N compared to group 1 (293.4 +/- 16.1 N) and group 2 (397.7 +/- 7.4 N) (P < 0.001). Stiffness was highest in group 2 (162 +/- 7.3 N/mm) and lowest in group 4 (84.4 +/- 19.9 mm) (P < 0.001). In group 4, the main cause of failure was due to the suture cutting through the tendon (n = 6), a failure case observed in only n = 1 specimen in group 2 (P < 0.001). A double-row technique combined with arthroscopic Mason-Allen/horizontal mattress stitches provides high initial failure strength and may minimize the risk of the polyethylene sutures cutting through the tendon in rotator cuff repair when a single load force is used.

Mutational analysis of the human nucleotide excision repair gene ERCC1.

PubMed Central

Sijbers, A M; van der Spek, P J; Odijk, H; van den Berg, J; van Duin, M; Westerveld, A; Jaspers, N G; Bootsma, D; Hoeijmakers, J H

1996-01-01

The human DNA repair protein ERCC1 resides in a complex together with the ERCC4, ERCC11 and XP-F correcting activities, thought to perform the 5' strand incision during nucleotide excision repair (NER). Its yeast counterpart, RAD1-RAD10, has an additional engagement in a mitotic recombination pathway, probably required for repair of DNA cross-links. Mutational analysis revealed that the poorly conserved N-terminal 91 amino acids of ERCC1 are dispensable for both repair functions, in contrast to a deletion of only four residues from the C-terminus. A database search revealed a strongly conserved motif in this C-terminus sharing sequence homology with many DNA break processing proteins, indicating that this part is primarily required for the presumed structure-specific endonuclease activity of ERCC1. Most missense mutations in the central region give rise to an unstable protein (complex). Accordingly, we found that free ERCC1 is very rapidly degraded, suggesting that protein-protein interactions provide stability. Survival experiments show that the removal of cross-links requires less ERCC1 than UV repair. This suggests that the ERCC1-dependent step in cross-link repair occurs outside the context of NER and provides an explanation for the phenotype of the human repair syndrome xeroderma pigmentosum group F. PMID:8811092

The cost-effectiveness of single-row compared with double-row arthroscopic rotator cuff repair.

PubMed

Genuario, James W; Donegan, Ryan P; Hamman, Daniel; Bell, John-Erik; Boublik, Martin; Schlegel, Theodore; Tosteson, Anna N A

2012-08-01

Interest in double-row techniques for arthroscopic rotator cuff repair has increased over the last several years, presumably because of a combination of literature demonstrating superior biomechanical characteristics and recent improvements in instrumentation and technique. As a result of the increasing focus on value-based health-care delivery, orthopaedic surgeons must understand the cost implications of this practice. The purpose of this study was to examine the cost-effectiveness of double-row arthroscopic rotator cuff repair compared with traditional single-row repair. A decision-analytic model was constructed to assess the cost-effectiveness of double-row arthroscopic rotator cuff repair compared with single-row repair on the basis of the cost per quality-adjusted life year gained. Two cohorts of patients (one with a tear of <3 cm and the other with a tear of ≥3 cm) were evaluated. Probabilities for retear and persistent symptoms, health utilities for the particular health states, and the direct costs for rotator cuff repair were derived from the orthopaedic literature and institutional data. The incremental cost-effectiveness ratio for double-row compared with single-row arthroscopic rotator cuff repair was $571,500 for rotator cuff tears of <3 cm and $460,200 for rotator cuff tears of ≥3 cm. The rate of radiographic or symptomatic retear alone did not influence cost-effectiveness results. If the increase in the cost of double-row repair was less than $287 for small or moderate tears and less than $352 for large or massive tears compared with the cost of single-row repair, then double-row repair would represent a cost-effective surgical alternative. On the basis of currently available data, double-row rotator cuff repair is not cost-effective for any size rotator cuff tears. However, variability in the values for costs and probability of retear can have a profound effect on the results of the model and may create an environment in which double-row repair

Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance

PubMed Central

Jablonski, Angela M.; Lamitina, Todd; Liachko, Nicole F.; Sabatella, Mariangela; Lu, Jiayin; Zhang, Lei; Ostrow, Lyle W.; Gupta, Preetika; Wu, Chia-Yen; Doshi, Shachee; Mojsilovic-Petrovic, Jelena; Lans, Hannes; Wang, Jiou; Kraemer, Brian

2015-01-01

Misfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate in amyotrophic lateral sclerosis. Loss of RAD-23 suppresses the locomotor deficit of Caenorhabditis elegans engineered to express mutTDP-43 or mutSOD1 and also protects against aging and proteotoxic insults. Knockdown of RAD-23 is further neuroprotective against the toxicity of SOD1 and TDP-43 expression in mammalian neurons. Biochemical investigation indicates that RAD-23 modifies mutTDP-43 and mutSOD1 abundance, solubility, and turnover in association with altering the ubiquitination status of these substrates. In human amyotrophic lateral sclerosis spinal cord, we find that RAD-23 abundance is increased and RAD-23 is mislocalized within motor neurons. We propose a novel pathophysiological function for RAD-23 in the stabilization of mutated proteins that cause neurodegeneration. SIGNIFICANCE STATEMENT In this work, we identify RAD-23, a component of the protein homeostasis network and nucleotide excision repair pathway, as a modifier of the toxicity of two disease-causing, misfolding-prone proteins, SOD1 and TDP-43. Reducing the abundance of RAD-23 accelerates the degradation of mutant SOD1 and TDP-43 and reduces the cellular content of the toxic species. The existence of endogenous proteins that act as “anti-chaperones” uncovers new and general targets for therapeutic intervention. PMID:26490867

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway.

PubMed

Mu, Hong; Geacintov, Nicholas E; Min, Jung-Hyun; Zhang, Yingkai; Broyde, Suse

2017-06-19

The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N 2 -dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the "pre-flipped" base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [ Mu , H. , ( 2015 ) Biochemistry , 54 ( 34 ), 5263 - 7 ]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway

PubMed Central

2017-01-01

The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N2-dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the “pre-flipped” base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [MuH., (2015) Biochemistry, 54(34), 5263−726270861]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER. PMID:28460163

Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time.

PubMed

Kong, Muwen; Beckwitt, Emily C; Springall, Luke; Kad, Neil M; Van Houten, Bennett

2017-01-01

Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein-DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair. © 2017 Elsevier Inc. All rights reserved.

Biomechanical comparison of single-row arthroscopic rotator cuff repair technique versus transosseous repair technique.

PubMed

Tocci, Stephen L; Tashjian, Robert Z; Leventhal, Evan; Spenciner, David B; Green, Andrew; Fleming, Braden C

2008-01-01

This study determined the effect of tear size on gap formation of single-row simple-suture arthroscopic rotator cuff repair (ARCR) vs transosseous Mason-Allen suture open RCR (ORCR) in 13 pairs of human cadaveric shoulders. A massive tear was created in 6 pairs and a large tear in 7. Repairs were cyclically tested in low-load and high-load conditions, with no significant difference in gap formation. Under low-load, gapping was greater in massive tears. Under high-load, there was a trend toward increased gap with ARCR for large tears. All repairs of massive tears failed in high-load. Gapping was greater posteriorly in massive tears for both techniques. Gap formation of a modeled RCR depends upon the tear size. ARCR of larger tears may have higher failure rates than ORCR, and the posterior aspect appears to be the site of maximum gapping. Specific attention should be directed toward maximizing initial fixation of larger rotator cuff tears, especially at the posterior aspect.

Structural Basis for Eukaryotic Transcription-Coupled DNA Repair Initiation

PubMed Central

Xu, Jun; Lahiri, Indrajit; Wang, Wei; Wier, Adam; Cianfrocco, Michael A.; Chong, Jenny; Hare, Alissa A.; Dervan, Peter B.; DiMaio, Frank; Leschziner, Andres E.; Wang, Dong

2017-01-01

Eukaryotic transcription-coupled repair (TCR), or transcription-coupled nucleotide excision repair (TC-NER), is an important and well-conserved sub-pathway of nucleotide excision repair (NER) that preferentially removes DNA lesions from the template strand blocking RNA polymerase II (Pol II) translocation1,2. Cockayne syndrome group B protein in humans (CSB, or ERCC6), or its yeast orthologs (Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe), is among the first proteins to be recruited to the lesion-arrested Pol II during initiation of eukaryotic TCR1,3–10. Mutations in CSB are associated with Cockayne syndrome, an autosomal-recessive neurologic disorder characterized by progeriod features, growth failure, and photosensitivity1. The molecular mechanism of eukaryotic TCR initiation remains elusive, with several long-standing questions unanswered: How do cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II? How does CSB interact with the arrested Pol II complex? What is the role of CSB in TCR initiation? The lack of structures of CSB or the Pol II-CSB complex have hindered our ability to answer those questions. Here we report the first structure of S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy (cryo-EM). The structure reveals that Rad26 binds to the DNA upstream of Pol II where it dramatically alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes forward movement of Pol II and elucidate key roles for Rad26/CSB in both TCR and transcription elongation. PMID:29168508

Excellent healing rates and patient satisfaction after arthroscopic repair of medium to large rotator cuff tears with a single-row technique augmented with bone marrow vents.

PubMed

Dierckman, Brian D; Ni, Jake J; Karzel, Ronald P; Getelman, Mark H

2018-01-01

This study evaluated the repair integrity and patient clinical outcomes following arthroscopic rotator cuff repair of medium to large rotator cuff tears using a single-row technique consisting of medially based, triple-loaded anchors augmented with bone marrow vents in the rotator cuff footprint lateral to the repair. This is a retrospective study of 52 patients (53 shoulders) comprising 36 males and 16 females with a median age of 62 (range 44-82) with more than 24-month follow-up, tears between 2 and 4 cm in the anterior-posterior dimension and utilizing triple-loaded anchors. Mann-Whitney test compared Western Ontario Rotator Cuff (WORC) outcome scores between patients with healed and re-torn cuff repairs. Multivariate logistic regression analysed association of variables with healing status and WORC score. Cuff integrity was assessed on MRI, read by a musculoskeletal fellowship-trained radiologist. Magnetic resonance imaging (MRI) demonstrated an intact repair in 48 of 53 shoulders (91%). The overall median WORC score was 95.7 (range 27.6-100.0). A significant difference in WORC scores were seen between patients with healed repairs 96.7 (range 56.7-100.0) compared with a re-tear 64.6 (27.6-73.8), p < 0.00056. Arthroscopic repair of medium to large rotator cuff tears using a triple-loaded single-row repair augmented with bone marrow vents resulted in a 91% healing rate by MRI and excellent patient reported clinical outcomes comparable to similar reported results in the literature. IV.

Wound Repair: Toward Understanding and Integration of Single-Cell and Multicellular Wound Responses

PubMed Central

Sonnemann, Kevin J.; Bement, William M.

2016-01-01

The importance of wound healing to medicine and biology has long been evident, and consequently, wound healing has been the subject of intense investigation for many years. However, several relatively recent developments have added new impetus to wound repair research: the increasing application of model systems; the growing recognition that single cells have a robust, complex, and medically relevant wound healing response; and the emerging recognition that different modes of wound repair bear an uncanny resemblance to other basic biological processes such as morphogenesis and cytokinesis. In this review, each of these developments is described, and their significance for wound healing research is considered. In addition, overlapping mechanisms of single-cell and multicellular wound healing are highlighted, and it is argued that they are more similar than is often recognized. Based on this and other information, a simple model to explain the evolutionary relationships of cytokinesis, single-cell wound repair, multicellular wound repair, and developmental morphogenesis is proposed. Finally, a series of important, but as yet unanswered, questions is posed. PMID:21721944

Biomechanical Comparison of Standard and Linked Single-Row Rotator Cuff Repairs in a Human Cadaver Model.

PubMed

Meisel, Adam F; Henninger, Heath B; Barber, F Alan; Getelman, Mark H

2017-05-01

The purpose of this study was to evaluate the time zero cyclic and failure loading properties of a linked single-row rotator cuff repair compared with a standard simple suture single-row repair using triple-loaded suture anchors. Eighteen human cadaveric shoulders from 9 matched pairs were dissected, and full-thickness supraspinatus tears were created. The tendon cross-sectional area was recorded. In each pair, one side was repaired with a linked single-row construct and the other with a simple suture single-row construct, both using 2 triple-loaded suture anchors. After preloading, specimens were cycled to 1 MPa of effective stress at 1 Hz for 500 cycles, and gap formation was recorded with a digital video system. Samples were then loaded to failure, and modes of failure were recorded. There was no statistical difference in peak gap formation between the control and linked constructs (3.6 ± 0.9 mm and 3.6 ± 1.2 mm, respectively; P = .697). Both constructs averaged below a 5-mm cyclic failure threshold. There was no statistical difference in ultimate load to failure between the control and linked repair (511.1 ± 139.0 N and 561.2 ± 131.8 N, respectively; P = .164), and both groups reached failure at loads similar to previous studies. Constructs failed predominantly via tissue tearing parallel to the medial suture line. The linked repair performed similarly to the simple single-row repair. Both constructs demonstrated high ultimate load to failure and good resistance to gap formation with cyclic loading, validating the time zero strength of both constructs in a human cadaveric model. The linked repair provided equivalent resistance to gap formation and failure loads compared with simple suture single-row repairs with triple-loaded suture anchors. This suggests that the linked repair is a simplified rip-stop configuration using the existing suture that may perform similarly to current rotator cuff repair techniques. Copyright © 2016 Arthroscopy

Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism

PubMed Central

Laffitte, Marie-Claude N.; Leprohon, Philippe; Hainse, Maripier; Légaré, Danielle; Masson, Jean-Yves; Ouellette, Marc

2016-01-01

The parasite Leishmania often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in Leishmania would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate RAD50 null mutants in a wild-type background failed and we provide evidence that this gene is essential. Remarkably, inactivation of RAD50 was possible in a MRE11 null mutant that we had previously generated, providing good evidence that RAD50 may be dispensable in the absence of MRE11. Inactivation of the MRE11 and RAD50 genes led to a decreased frequency of homologous recombination and analysis of the null mutants by whole genome sequencing revealed several chromosomal translocations. Sequencing of the junction between translocated chromosomes highlighted microhomology sequences at the level of breakpoint regions. Sequencing data also showed a decreased coverage at subtelomeric locations in many chromosomes in the MRE11-/-RAD50-/- parasites. This study demonstrates an MRE11-independent microhomology-mediated end-joining mechanism and a prominent role for MRE11 and RAD50 in the maintenance of genomic integrity. Moreover, we suggest the possible involvement of RAD50 in subtelomeric regions stability. PMID:27314941

Single incision laparoscopic surgery (SILS) inguinal hernia repair - recent clinical experiences of this novel technique.

PubMed

Yussra, Y; Sutton, P A; Kosai, N R; Razman, J; Mishra, R K; Harunarashid, H; Das, S

2013-01-01

Inguinal hernia remains the most commonly encountered surgical problem. Various methods of repair have been described, and the most suitable one debated. Single port access (SPA) surgery is a rapidly evolving field, and has the advantage of affording 'scarless' surgery. Single incision laparoscopic surgery (SILS) for inguinal hernia repair is seen to be feasible in both total extraperitoneal (TEP) and transabdominal pre-peritoneal (TAPP) approaches. Data and peri-operative information on both of these however are limited. We aimed to review the clinical experience, feasibility and short term complications related to laparoscopic inguinal hernia repair via single port access. A literature search was performed using Google Scholar, Springerlink Library, Highwire Press, Surgical Endoscopy Journal, World Journal of Surgery and Medscape. The following search terms were used: laparoscopic hernia repair, TAPP, TEP, single incision laparoscopic surgery (SILS). Fourteen articles in English language related to SILS inguinal hernia repair were identified. Nine articles were related to TEP repair and the remaining 5 to TAPP. A total of 340 patients were reported within these studies: 294 patients having a TEP repair and 46 a TAPP. Only two cases of recurrence were reported. Various ports have been utilized, including the SILS port, Tri-Port and a custom- made port using conventional laparoscopic instruments. The duration of surgery was 40-100 minutes and the average length of hospital stay was one day. Early outcomes of this novel technique show it to be feasible, safe and with potentially better cosmetic outcome.

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. IV. Influence of DNA replication and excision repair on REV2 dependent UV-mutagenesis and repair.

PubMed

Siede, W; Eckardt, F

1986-01-01

A double mutant being thermoconditionally defective in mutation induction as well as in repair of pre-lethal UV-induced DNA damage (rev2ts) and deficient in excision repair (rad3-2) was studied in temperature-shift experiments. The influence of inhibitors of DNA replication (hydroxyurea, aphidicolin) was determined. Additionally, an analysis of the dose-response pattern of mutation induction ("mutation kinetics") at several ochre alleles was carried out. It was concluded that the UV-inducible REV2 dependent mutagenic repair process is not induced in excision-deficient cells. In excision-deficient cells, REV2 dependent mutation fixation is slow and mostly post-replicative though not dependent on DNA replication. The REV2 mediated mutagenic process could be separated from the repair function.

Dynamic binding of replication protein a is required for DNA repair

PubMed Central

Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

2016-01-01

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

Generation of DNA single-strand displacement by compromised nucleotide excision repair

PubMed Central

Godon, Camille; Mourgues, Sophie; Nonnekens, Julie; Mourcet, Amandine; Coin, Fréderic; Vermeulen, Wim; Mari, Pierre-Olivier; Giglia-Mari, Giuseppina

2012-01-01

Nucleotide excision repair (NER) is a precisely coordinated process essential to avoid DNA damage-induced cellular malfunction and mutagenesis. Here, we investigate the mechanistic details and effects of the NER machinery when it is compromised by a pathologically significant mutation in a subunit of the repair/transcription factor TFIIH, namely XPD. In contrast to previous studies, we find that no single- or double-strand DNA breaks are produced at early time points after UV irradiation of cells bearing a specific XPD mutation, despite the presence of a clear histone H2AX phosphorylation (γH2AX) signal in the UV-exposed areas. We show that the observed γH2AX signal can be explained by the presence of longer single-strand gaps possibly generated by strand displacement. Our in vivo measurements also indicate a strongly reduced TFIIH-XPG binding that could promote single-strand displacement at the site of UV lesions. This finding not only highlights the crucial role of XPG's interactions with TFIIH for proper NER, but also sheds new light on how a faulty DNA repair process can induce extreme genomic instability in human patients. PMID:22863773

Long-term Outcomes after Truncus Arteriosus Repair: A Single-center Experience for More than 40 Years.

PubMed

Asagai, Seiji; Inai, Kei; Shinohara, Tokuko; Tomimatsu, Hirofumi; Ishii, Tetsuko; Sugiyama, Hisashi; Park, In-Sam; Nagashima, Mitsugi; Nakanishi, Toshio

2016-12-01

This study aimed to analyze long-term survival and functional outcomes after truncus arteriosus repair in a single institution with more than 40 years of follow-up. Medical records were analyzed retrospectively in 52 patients who underwent the Rastelli procedure for truncus arteriosus repair between 1974 and 2002. Thirty-five patients survived the initial repair. The median age at the initial operation was 2.8 months (range, 0.1-123 months) and the body weight was 3.9 kg (range, 1.6 to 15.0 kg). The median age at follow-up was 23.6 years (range, 12.4 to 44.5 years). The median follow-up duration was 23.4 years (range, 12.3 to 40.7 years). The actuarial survival rate was 97% at 10 years and 93% at both 20 years and 40 years after the initial operation. At follow-up, most patients were in New York Heart Association (NYHA) functional classes I (73%) and II (24%). Thirty-six percent of patients had full-time jobs, 40% were students, and 21% were unemployed. Most patients (97%) had undergone conduit reoperations. Freedom from reoperation for right ventricular (RV) outflow and pulmonary artery (PA) stenosis was 59% at 5 years, 28% at 10 years, and 3% at 20 years after the initial operation. Freedom from catheter interventions for RV outflow and PA stenosis was 59% at 5 years, 47% at 10 years, and 38% at 20 years after the initial operation. Freedom from truncal valve replacement was 88% at 5 years, 85% at 10 years, and 70% at 20 years after the initial operation. In this single-center retrospective study, with long-term follow-up after repair of truncus arteriosus, long-term survival and functional outcomes were acceptable, despite the requirement for reoperation and multiple catheter interventions for RV outflow and PA stenosis in almost all patients, and the frequent requirement for late truncal valve operations. © 2016 The Authors. Congenital Heart Disease published by Wiley Periodicals, Inc.

Load response and gap formation in a single-row cruciate suture rotator cuff repair.

PubMed

Huntington, Lachlan; Richardson, Martin; Sobol, Tony; Caldow, Jonathon; Ackland, David C

2017-06-01

Double-row rotator cuff tendon repair techniques may provide superior contact area and strength compared with single-row repairs, but are associated with higher material expenses and prolonged operating time. The purpose of this study was to evaluate gap formation, ultimate tensile strength and stiffness of a single-row cruciate suture rotator cuff repair construct, and to compare these results with those of the Mason-Allen and SutureBridge repair constructs. Infraspinatus tendons from 24 spring lamb shoulders were harvested and allocated to cruciate suture, Mason-Allen and SutureBridge repair groups. Specimens were loaded cyclically between 10 and 62 N for 200 cycles, and gap formation simultaneously measured using a high-speed digital camera. Specimens were then loaded in uniaxial tension to failure, and construct stiffness and repair strength were evaluated. Gap formation in the cruciate suture repair was significantly lower than that of the Mason-Allen repair (mean difference = 0.6 mm, P = 0.009) and no different from that of the SutureBridge repair (P > 0.05). Both the cruciate suture repair (mean difference = 15.7 N/mm, P = 0.002) and SutureBridge repair (mean difference = 15.8 N/mm, P = 0.034) were significantly stiffer than that of the Mason-Allen repair; however, no significant differences in ultimate tensile strength between repair groups were discerned (P > 0.05). The cruciate suture repair construct, which may represent a simple and cost-effective alternative to double-row and double-row equivalent rotator cuff repairs, has comparable biomechanical strength and integrity with that of the SutureBridge repair, and may result in improved construct longevity and tendon healing compared with the Mason-Allen repair. © 2017 Royal Australasian College of Surgeons.

Performance and precision of double digestion RAD (ddRAD) genotyping in large multiplexed datasets of marine fish species.

PubMed

Maroso, F; Hillen, J E J; Pardo, B G; Gkagkavouzis, K; Coscia, I; Hermida, M; Franch, R; Hellemans, B; Van Houdt, J; Simionati, B; Taggart, J B; Nielsen, E E; Maes, G; Ciavaglia, S A; Webster, L M I; Volckaert, F A M; Martinez, P; Bargelloni, L; Ogden, R

2018-06-01

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in "non-model" species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 10 12 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies. Crown Copyright © 2018. Published by Elsevier B.V. All rights

Meta-analysis of Clinical and Radiographic Outcomes After Arthroscopic Single-Row Versus Double-Row Rotator Cuff Repair.

PubMed

Perser, Karen; Godfrey, David; Bisson, Leslie

2011-05-01

Double-row rotator cuff repair methods have improved biomechanical performance when compared with single-row repairs. To review clinical outcomes of single-row versus double-row rotator cuff repair with the hypothesis that double-row rotator cuff repair will result in better clinical and radiographic outcomes. Published literature from January 1980 to April 2010. Key terms included rotator cuff, prospective studies, outcomes, and suture techniques. The literature was systematically searched, and 5 level I and II studies were found comparing clinical outcomes of single-row and double-row rotator cuff repair. Coleman methodology scores were calculated for each article. Meta-analysis was performed, with treatment effect between single row and double row for clinical outcomes and with odds ratios for radiographic results. The sample size necessary to detect a given difference in clinical outcome between the 2 methods was calculated. Three level I studies had Coleman scores of 80, 74, and 81, and two level II studies had scores of 78 and 73. There were 156 patients with single-row repairs and 147 patients with double-row repairs, both with an average follow-up of 23 months (range, 12-40 months). Double-row repairs resulted in a greater treatment effect for each validated outcome measure in 4 studies, but the differences were not clinically or statistically significant (range, 0.4-2.2 points; 95% confidence interval, -0.19, 4.68 points). Double-row repairs had better radiographic results, but the differences were also not statistically significant (P = 0.13). Two studies had adequate power to detect a 10-point difference between repair methods using the Constant score, and 1 study had power to detect a 5-point difference using the UCLA (University of California, Los Angeles) score. Double-row rotator cuff repair does not show a statistically significant improvement in clinical outcome or radiographic healing with short-term follow-up.

Involvement of DNA-PK(sub cs) in DSB Repair Following Fe-56 Ion Irradiation

NASA Technical Reports Server (NTRS)

O'Neill, Peter; Harper, Jane; Anderson, Jennifer a.; Cucinnota, Francis A.

2007-01-01

When cells are exposed to radiation, cellular lesions are induced in the DNA including double strand breaks (DSBs), single strand breaks and clustered DNA damage, which if not repaired with high fidelity may lead to detrimental biological consequences. Complex DSBs are induced by ionizing radiation and characterized by the presence of base lesions close to the break termini. They are believed to be one of the major causes of the biological effects of IR. The complexity of DSBs increases with the ionization density of the radiation and these complex DSBs are distinct from the damage induced by sparsely ionizing gamma-radiation. It has been hypothesized that complex DSBs produced by heavy ions in space pose problems to the DNA repair machinery. We have used imm uno-cyto-chemical staining of phosphorylated histone H2AX (gamma-H2AX) foci, as a marker of DSBs. We have investigated the formation and loss of gamma-H2AX foci and RAD51 foci (a protein involved in the homologous recombination pathway) in mammalian cells induced by low fluences of low-LET gamma-radiation and high-LET Fe-56 ions (1GeV/n, 151 keV/micron LET). M059J and M059K cells, which are deficient and proficient in DNA-PK(sub cs) activity respectively, were used to examine the role of DNA-PK(sub cs), a key protein in the non-homologous end joining (NHEJ) pathway of DSB repair, along with HF19 human fibroblasts. Followi ng irradiation with Fe-56 ions the rate of repair was slower in M059J cells compared with that in M059K, indicating a role for DNA-PK(sub cs) in the repair of DSB induced by Fe-56 ions. However a small percentage of DSBs induced are rejoined within 5 h although many DSBs still persist up to 24 h. When RAD51 was examined in M059J/K cells, RAD51 foci are visible 24 hours after irradiation in approximately 40% of M059J cells compared with <5% of M059K cells indicating that persistent DSBs or those formed at stalled replication forks recruit RAD51 in DNA-PK(sub cs) deficient cells. Following 1 Gy

The association of folate pathway and DNA repair polymorphisms with susceptibility to childhood acute lymphoblastic leukemia.

PubMed

Goričar, Katja; Erčulj, Nina; Faganel Kotnik, Barbara; Debeljak, Maruša; Hovnik, Tinka; Jazbec, Janez; Dolžan, Vita

2015-05-15

Genetic factors may play an important role in susceptibility to childhood acute lymphoblastic leukemia (ALL). The aim of our study was to evaluate the associations of genetic polymorphisms in folate pathway and DNA repair genes with susceptibility to ALL. In total, 121 children with ALL and 184 unrelated healthy controls of Slovenian origin were genotyped for 14 polymorphisms in seven genes of folate pathway, base excision repair and homologous recombination repair (TYMS, MTHFR, OGG1, XRCC1, NBN, RAD51, and XRCC3). In addition, the exon 6 of NBN was screened for the presence of mutations using denaturing high performance liquid chromatography. Twelve polymorphisms were in Hardy-Weinberg equilibrium in controls and their genotype frequencies were in agreement with those reported in other Caucasian populations. Among the investigated polymorphisms and mutations, NBN Glu185Gln significantly decreased susceptibility to B-cell ALL (p=0.037), while TYMS 3R allele decreased susceptibility to T-cell ALL (p=0.011). Moreover, significantly decreased susceptibility to ALL was observed for MTHFR TA (p=0.030) and RAD51 GTT haplotypes (p=0.016). Susceptibility to ALL increased with the increasing number of risk alleles (ptrend=0.007). We also observed significant influence of hOGG-RAD51 and NBN-RAD51 interactions on susceptibility to ALL. Our results suggest that combination of several polymorphisms in DNA repair and folate pathways may significantly affect susceptibility to childhood ALL. Copyright © 2015 Elsevier B.V. All rights reserved.

Initial load-to-failure and failure analysis in single- and double-row repair techniques for rotator cuff repair

PubMed Central

Buchhorn, G. H.; Gilbert, F.; Spahn, G.; Schultz, W.; Klinger, H.-M.

2010-01-01

Aim This experimental study aimed to compare the load-to-failure rate and stiffness of single- versus double-row suture techniques for repairing rotator cuff lesions using two different suture materials. Additionally, the mode of failure of each repair was evaluated. Method In 32 sheep shoulders, a standardized tear of the infraspinatus tendon was created. Then, n = 8 specimen were randomized to four repair methods: (1) Double-row Anchor Ethibond® coupled with polyester sutures, USP No. 2; (2) Double-Row Anchor HiFi® with polyblend polyethylene sutures, USP No. 2; (3) Single-Row Anchor Ethibond® coupled with braided polyester sutures, USP No. 2; and (4) Single-Row Anchor HiFi® with braided polyblend polyethylene sutures, USP No. 2. Arthroscopic Mason–Allen stitches were placed (single-row) and combined with medial horizontal mattress stitches (double-row). All specimens were loaded to failure at a constant displacement rate on a material testing machine. Results Group 4 showed lowest load-to-failure result with 155.7 ± 31.1 N compared to group 1 (293.4 ± 16.1 N) and group 2 (397.7 ± 7.4 N) (P < 0.001). Stiffness was highest in group 2 (162 ± 7.3 N/mm) and lowest in group 4 (84.4 ± 19.9 mm) (P < 0.001). In group 4, the main cause of failure was due to the suture cutting through the tendon (n = 6), a failure case observed in only n = 1 specimen in group 2 (P < 0.001). Conclusions A double-row technique combined with arthroscopic Mason-Allen/horizontal mattress stitches provides high initial failure strength and may minimize the risk of the polyethylene sutures cutting through the tendon in rotator cuff repair when a single load force is used. PMID:20049605

Cellular redistribution of Rad51 in response to DNA damage: novel role for Rad51C.

PubMed

Gildemeister, Otto S; Sage, Jay M; Knight, Kendall L

2009-11-13

Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.

Expanded CAG/CTG Repeat DNA Induces a Checkpoint Response That Impacts Cell Proliferation in Saccharomyces cerevisiae

PubMed Central

Sundararajan, Rangapriya; Freudenreich, Catherine H.

2011-01-01

Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases. PMID:21437275

Rad-Release

ScienceCinema

None

2017-12-09

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

Does double-row rotator cuff repair improve functional outcome of patients compared with single-row technique? A systematic review.

PubMed

DeHaan, Alexander M; Axelrad, Thomas W; Kaye, Elizabeth; Silvestri, Lorenzo; Puskas, Brian; Foster, Timothy E

2012-05-01

The advantage of single-row versus double-row arthroscopic rotator cuff repair techniques has been a controversial issue in sports medicine and shoulder surgery. There is biomechanical evidence that double-row techniques are superior to single-row techniques; however, there is no clinical evidence that the double-row technique provides an improved functional outcome. When compared with single-row rotator cuff repair, double-row fixation, although biomechanically superior, has no clinical benefit with respect to retear rate or improved functional outcome. Systematic review. The authors reviewed prospective studies of level I or II clinical evidence that compared the efficacy of single- and double-row rotator cuff repairs. Functional outcome scores included the American Shoulder and Elbow Surgeons (ASES) shoulder scale, the Constant shoulder score, and the University of California, Los Angeles (UCLA) shoulder rating scale. Radiographic failures and complications were also analyzed. A test of heterogeneity for patient demographics was also performed to determine if there were differences in the patient profiles across the included studies. Seven studies fulfilled our inclusion criteria. The test of heterogeneity across these studies showed no differences. The functional ASES, Constant, and UCLA outcome scores revealed no difference between single- and double-row rotator cuff repairs. The total retear rate, which included both complete and partial retears, was 43.1% for the single-row repair and 27.2% for the double-row repair (P = .057), representing a trend toward higher failures in the single-row group. Through a comprehensive literature search and meta-analysis of current arthroscopic rotator cuff repairs, we found that the single-row repairs did not differ from the double-row repairs in functional outcome scores. The double-row repairs revealed a trend toward a lower radiographic proven retear rate, although the data did not reach statistical significance. There

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

PubMed

Inano, Shojiro; Sato, Koichi; Katsuki, Yoko; Kobayashi, Wataru; Tanaka, Hiroki; Nakajima, Kazuhiro; Nakada, Shinichiro; Miyoshi, Hiroyuki; Knies, Kerstin; Takaori-Kondo, Akifumi; Schindler, Detlev; Ishiai, Masamichi; Kurumizaka, Hitoshi; Takata, Minoru

2017-06-01

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

MSL-RAD Cruise Operations Concept

NASA Technical Reports Server (NTRS)

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

2012-01-01

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

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

PubMed Central

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

2015-01-01

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

Inhibition of RAD51 by siRNA and Resveratrol Sensitizes Cancer Stem Cells Derived from HeLa Cell Cultures to Apoptosis

PubMed Central

Ruíz, Graciela; Valencia-González, Heriberto A.; León-Galicia, Ismael; García-Villa, Enrique

2018-01-01

Cervical cancer is the second most frequent tumor type in women worldwide with cases developing clinical recurrence, metastasis, and chemoresistance. The cancer stem cells (CSC) may be implicated in tumor resistance to therapy. RESveratrol (RES), a natural compound, is an antioxidant with multiple beneficial activities. We previously determined that the expression of RAD51 is decreased by RES. The aim of our study was to examine molecular mechanism by which CSC from HeLa cultures exhibit chemoresistance. We hypothesized CSC repair more efficiently DNA breaks and that RAD51 plays an important role in this mechanism. We found that CSC, derived from cervical cancer cell lines, overexpress RAD51 and are less sensitive to Etoposide (VP16). We inhibited RAD51 in CSC-enriched cultures using RES or siRNA against RAD51 messenger RNA and observed a decrease in cell viability and induction of apoptosis when treated simultaneously with VP16. In addition, we found that inhibition of RAD51 expression using RES also sensitizes CSC to VP16 treatment. Our results suggest that resveratrol is effective to sensitize cervical CSC because of RAD51 inhibition, targeting high RAD51 expressing CD49f-positive cells, which supports the possible therapeutic application of RES as a novel agent to treat cancer. PMID:29681946

BI-RADS update.

PubMed

Mercado, Cecilia L

2014-05-01

The updated American College of Radiology (ACR) Breast Imaging Reporting and Data System (BI-RADS) has been newly released. This article summarizes the changes and updates that have been made to BI-RADS. The goal of the revised edition continues to be the same: to improve clarification in image interpretation, maintain reporting standardization, and simplify the monitoring of outcomes. The new BI-RADS also introduces new terminology to provide a more universal lexicon across all 3 imaging modalities. Copyright © 2014 Elsevier Inc. All rights reserved.

Differentiation of prostatitis and prostate cancer using the Prostate Imaging-Reporting and Data System (PI-RADS).

PubMed

Meier-Schroers, Michael; Kukuk, Guido; Wolter, Karsten; Decker, Georges; Fischer, Stefan; Marx, Christian; Traeber, Frank; Sprinkart, Alois Martin; Block, Wolfgang; Schild, Hans Heinz; Willinek, Winfried

2016-07-01

To determine if prostate cancer (PCa) and prostatitis can be differentiated by using PI-RADS. 3T MR images of 68 patients with 85 cancer suspicious lesions were analyzed. The findings were correlated with histopathology. T2w imaging (T2WI), diffusion weighted imaging (DWI), dynamic contrast enhancement (DCE), and MR-Spectroscopy (MRS) were acquired. Every lesion was given a single PI-RADS score for each parameter, as well as a sum score and a PI-RADS v2 score. Furthermore, T2-morphology, ADC-value, perfusion type, citrate/choline-level, and localization were evaluated. 44 of 85 lesions showed PCa (51.8%), 21 chronic prostatitis (24.7%), and 20 other benign tissue such as hyperplasia or fibromuscular tissue (23.5%). The single PI-RADS score for T2WI, DWI, DCE, as well as the aggregated score including and not including MRS, and the PI-RADS v2-score were all significantly higher for PCa than for prostatitis or other tissue (p<0.001). The single PI-RADS score for MRS and the PI-RADS sum score including MRS were significantly higher for prostatitis than for other tissue (p=0.029 and p=0.020), whereas the other parameters were not different. Prostatitis usually presented borderline pathological PI-RADS scores, showed restricted diffusion with ADC≥900mm(2)/s in 100% of cases, was more often indistinctly hypointense on T2WI (66.7%), and localized in the transitional zone (57.1%). An ADC≥900mm(2)/s achieved the highest predictive value for prostatitis (AUC=0.859). Prostatitis can be differentiated from PCa using PI-RADS, since all available parameters are more distinct in cases of cancer. However, there is significant overlap between prostatitis and other benign findings, thus PI-RADS is only suitable to a limited extent for the primary assessment of prostatitis. Restricted diffusion with ADC≥900mm(2)/s is believed to be a good indicator for prostatitis. MRS can help to distinguish between prostatitis and other tissue. Copyright © 2016 Elsevier Ireland Ltd. All

Molecular Pathways

PubMed Central

Lok, Benjamin H.; Powell, Simon N.

2012-01-01

The Rad52 protein was largely ignored in humans and other mammals when the mouse knockout revealed a largely “no-effect” phenotype. However, using synthetic lethal approaches to investigate context dependent function, new studies have shown that Rad52 plays a key survival role in cells lacking the function of the BRCA1-BRCA2 pathway of homologous recombination. Biochemical studies also showed significant differences between yeast and human Rad52, in which yeast Rad52 can promote strand invasion of RPA-coated single-stranded DNA in the presence of Rad51, but human Rad52 cannot. This results in the paradox of how is human Rad52 providing Rad51 function: presumably there is something missing in the biochemical assays that exists in-vivo, but the nature of this missing factor is currently unknown. Recent studies have suggested that Rad52 provides back-up Rad51 function for all members of the BRCA1-BRCA2 pathway, suggesting that Rad52 may be a target for therapy in BRCA pathway deficient cancers. Screening for ways to inhibit Rad52 would potentially provide a complementary strategy for targeting BRCA-deficient cancers in addition to PARP inhibitors. PMID:23071261

Single-Word Intelligibility in Speakers with Repaired Cleft Palate

ERIC Educational Resources Information Center

Whitehill, Tara; Chau, Cynthia

2004-01-01

Many speakers with repaired cleft palate have reduced intelligibility, but there are limitations with current procedures for assessing intelligibility. The aim of this study was to construct a single-word intelligibility test for speakers with cleft palate. The test used a multiple-choice identification format, and was based on phonetic contrasts…

Meta-analysis of Clinical and Radiographic Outcomes After Arthroscopic Single-Row Versus Double-Row Rotator Cuff Repair

PubMed Central

Perser, Karen; Godfrey, David; Bisson, Leslie

2011-01-01

Context: Double-row rotator cuff repair methods have improved biomechanical performance when compared with single-row repairs. Objective: To review clinical outcomes of single-row versus double-row rotator cuff repair with the hypothesis that double-row rotator cuff repair will result in better clinical and radiographic outcomes. Data Sources: Published literature from January 1980 to April 2010. Key terms included rotator cuff, prospective studies, outcomes, and suture techniques. Study Selection: The literature was systematically searched, and 5 level I and II studies were found comparing clinical outcomes of single-row and double-row rotator cuff repair. Coleman methodology scores were calculated for each article. Data Extraction: Meta-analysis was performed, with treatment effect between single row and double row for clinical outcomes and with odds ratios for radiographic results. The sample size necessary to detect a given difference in clinical outcome between the 2 methods was calculated. Results: Three level I studies had Coleman scores of 80, 74, and 81, and two level II studies had scores of 78 and 73. There were 156 patients with single-row repairs and 147 patients with double-row repairs, both with an average follow-up of 23 months (range, 12-40 months). Double-row repairs resulted in a greater treatment effect for each validated outcome measure in 4 studies, but the differences were not clinically or statistically significant (range, 0.4-2.2 points; 95% confidence interval, –0.19, 4.68 points). Double-row repairs had better radiographic results, but the differences were also not statistically significant (P = 0.13). Two studies had adequate power to detect a 10-point difference between repair methods using the Constant score, and 1 study had power to detect a 5-point difference using the UCLA (University of California, Los Angeles) score. Conclusions: Double-row rotator cuff repair does not show a statistically significant improvement in clinical

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens.

PubMed

Kamisugi, Yasuko; Schaefer, Didier G; Kozak, Jaroslav; Charlot, Florence; Vrielynck, Nathalie; Holá, Marcela; Angelis, Karel J; Cuming, Andrew C; Nogué, Fabien

2012-04-01

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

PubMed Central

Kamisugi, Yasuko; Schaefer, Didier G.; Kozak, Jaroslav; Charlot, Florence; Vrielynck, Nathalie; Holá, Marcela; Angelis, Karel J.; Cuming, Andrew C.; Nogué, Fabien

2012-01-01

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development. PMID:22210882

2015 RAD Fall Partner Meeting

EPA Pesticide Factsheets

This meeting covered the following discussion topics: 2014 RAD partner achievements and trends, national and international efforts to address HFCs, enhancing RAD partner recognition, and communicating the benefits of RAD.

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

PubMed Central

Sasatani, Megumi; Xu, Yanbin; Kawai, Hidehiko; Cao, Lili; Tateishi, Satoshi; Shimura, Tsutomu; Li, Jianxiang; Iizuka, Daisuke; Noda, Asao; Hamasaki, Kanya; Kusunoki, Yoichiro; Kamiya, Kenji

2015-01-01

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure. PMID:25675240

Identification of small molecule inhibitors of cytokinesis and single cell wound repair

PubMed Central

Clark, Andrew G.; Sider, Jenny R.; Verbrugghe, Koen; Fenteany, Gabriel; von Dassow, George; Bement, William M.

2013-01-01

Screening of small molecule libraries offers the potential to identify compounds that inhibit specific biological processes and, ultimately, to identify macromolecules that are important players in such processes. To date, however, most screens of small molecule libraries have focused on identification of compounds that inhibit known proteins or particular steps in a given process, and have emphasized automated primary screens. Here we have used “low tech” in vivo primary screens to identify small molecules that inhibit both cytokinesis and single cell wound repair, two complex cellular processes that possess many common features. The “diversity set”, an ordered array of 1990 compounds available from the National Cancer Institute, was screened in parallel to identify compounds that inhibit cytokinesis in D. excentricus (sand dollar) embryos and single cell wound repair in X. laevis (frog) oocytes. Two small molecules were thus identified: Sph1 and Sph2. Sph1 reduces Rho activation in wound repair and suppresses formation of the spindle midzone during cytokinesis. Sph2 also reduces Rho activation in wound repair and may inhibit cytokinesis by blocking membrane fusion. The results identify two small molecules of interest for analysis of wound repair and cytokinesis, reveal that these processes are more similar than often realized and reveal the potential power of low tech screens of small molecule libraries for analysis of complex cellular processes. PMID:23125193

Radioresistance of chordoma cells is associated with the ATM/ATR pathway, in which RAD51 serves as an important downstream effector.

PubMed

Zhang, Chao; Wang, Bing; Li, Lei; Li, Yawei; Li, Pengzhi; Lv, Guohua

2017-09-01

Surgery followed by radiotherapy is the standard treatment for chordomas, which are a rare but low-grade type of bone cancer arising from remnants of the embryonic notochord. However, disease recurrence following radiotherapy is common, most likely due to endogenous DNA repair mechanisms that promote cell survival upon radiation strikes. The ataxia telangiectasia mutated/ataxia telangiectasia mutated and Rad3 related (ATM/ATR)-mediated pathway has a critical role in DNA repair mechanisms; however, it has rarely been investigated in chordomas. In the present study, the expression of signal molecules related to the ATM/ATR pathway in chordoma tissues and adjacent normal tissues were initially examined using immunohistochemistry and western blot analysis. Chordoma U-CH1 and U-CH2 cells were subsequently used to investigate cell responses to ionizing radiation and the potential protective actions mediated by the ATM/ATR pathway. Phosphorylated (p)-ATM, p-ATR, γ-H2A histone family, member X (H2AX) and RAD51 were significantly upregulated in chordoma tissues relative to adjacent normal tissues (P<0.05). No significant reductions were observed in the viability of U-CH1 and U-CH2 cells following exposure to low-dose (1 and 2 Gy) radiation. Radiation (1 and 2 Gy) triggered a significant upregulation in p-ATM, γ-H2AX and RAD51 expression in U-CH1 cells (P<0.05), as well as a significant upregulation in p-ATM, p-ATR and RAD51 levels in U-CH2 cells (P<0.05). RAD51 knockdown increased the responses of both U-CH1 and U-CH2 cells to 1 Gy radiation, as evidenced by the significantly decreased cell viability and increased apoptosis rate (P<0.05). Collectively, the results of the present study indicated that radioresistance of chordoma cells is associated with the ATM/ATR pathway, in which RAD51 serves as an important downstream effector. Thus, RAD51 presents a promising therapeutic target for improving the outcome of radiotherapy treatment in chordomas.

Rad5, HLTF, and SHPRH: A Fresh View of an Old Story.

PubMed

Elserafy, Menattallah; Abugable, Arwa A; Atteya, Reham; El-Khamisy, Sherif F

2018-05-25

Not only have helicase-like transcription factor (HLTF) and SNF2 histone-linker PHD-finger RING-finger helicase (SHPRH) proved to be important players in post-replication repair like their yeast counterpart, Rad5, but they are also involved in multiple biological functions and are associated with several human disorders. We provide here an updated view of their functions, associated diseases, and potential therapeutic approaches. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Sufficient Amounts of Functional HOP2/MND1 Complex Promote Interhomolog DNA Repair but Are Dispensable for Intersister DNA Repair during Meiosis in Arabidopsis[W

PubMed Central

Uanschou, Clemens; Ronceret, Arnaud; Von Harder, Mona; De Muyt, Arnaud; Vezon, Daniel; Pereira, Lucie; Chelysheva, Liudmila; Kobayashi, Wataru; Kurumizaka, Hitoshi; Schlögelhofer, Peter; Grelon, Mathilde

2013-01-01

During meiosis, homologous recombination (HR) is essential to repair programmed DNA double-strand breaks (DSBs), and a dedicated protein machinery ensures that the homologous chromosome is favored over the nearby sister chromatid as a repair template. The HOMOLOGOUS-PAIRING PROTEIN2/MEIOTIC NUCLEAR DIVISION PROTEIN1 (HOP2/MND1) protein complex has been identified as a crucial factor of meiotic HR in Arabidopsis thaliana, since loss of either MND1 or HOP2 results in failure of DNA repair. We isolated two mutant alleles of HOP2 (hop2-2 and hop2-3) that retained the capacity to repair meiotic DSBs via the sister chromatid but failed to use the homologous chromosome. We show that in these alleles, the recombinases RADIATION SENSITIVE51 (RAD51) and DISRUPTED MEIOTIC cDNA1 (DMC1) are loaded, but only the intersister DNA repair pathway is activated. The hop2-2 phenotype is correlated with a decrease in HOP2/MND1 complex abundance. In hop2-3, a truncated HOP2 protein is produced that retains its ability to bind to DMC1 and DNA but forms less stable complexes with MND1 and fails to efficiently stimulate DMC1-driven D-loop formation. Genetic analyses demonstrated that in the absence of DMC1, HOP2/MND1 is dispensable for RAD51-mediated intersister DNA repair, while in the presence of DMC1, a minimal amount of functional HOP2/MND1 is essential to drive intersister DNA repair. PMID:24363313

RadTown USA: Basic Information

MedlinePlus

... Burbs Countryside Waterfront Downtown Did you know? RadTown Games Educational Materials Bring RadTown into the classroom with ... Waterfront Downtown Educational Materials RadTown A to Z Games Link to Us Glossary Subscribe to Listserv News ...

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.

Broad ligament hernia successfully repaired by single-incision laparoscopy: A case report.

PubMed

Takeyama, Hiroshi; Kogita, Yuya; Nishigaki, Takahiko; Yamashita, Masafumi; Aikawa, Eriko; Hoshi, Minako; Taniguchi, Hirokazu; Maruyama, Yasuki; Nakajima, Kazuhiro; Yamamoto, Yoshimitsu; Adachi, Kazushige; Yamamoto, Hitoshi; Ikeda, Kimimasa; Kurokawa, Eiji

2017-11-08

A 52-year-old woman with a history of two parturitions presented with lower abdominal pain. Multi-detector CT of the abdomen showed discontinuity of the sigmoid colon near the broad ligament on the left side. We assigned a provisional diagnosis of an internal hernia progressing through a defect in the broad ligament. SILS revealed a total broad ligament defect on the left side but no signs of ischemic, necrotic bowel. We successfully repaired the broad ligament defect with suturing. At the 2-month follow-up, the patient remained well with no signs of recurrence. This case appears to be the first report of a broad ligament hernia successfully diagnosed and repaired by SILS. © 2017 Japan Society for Endoscopic Surgery, Asia Endosurgery Task Force and John Wiley & Sons Australia, Ltd.

RAD50 germline mutations are associated with poor survival in BRCA1/2-negative breast cancer patients.

PubMed

Fan, Cong; Zhang, Juan; Ouyang, Tao; Li, Jinfeng; Wang, Tianfeng; Fan, Zhaoqing; Fan, Tie; Lin, Benyao; Xie, Yuntao

2018-05-04

RAD50 is a highly conserved DNA double-strand break (DSB) repair gene. However, the associations between RAD50 germline mutations and the survival and risk of breast cancer have not been fully elucidated. Here, we aimed to investigate the clinical impact of RAD50 germline mutations in a large cohort of unselected breast cancer patients. In this study, RAD50 germline mutations were determined using next-generation sequencing in 7657 consecutive unselected breast cancer patients without BRCA1/2 mutations. We also screened for RAD50 recurrent mutations (L719fs, K994fs, and H1269fs) in 5000 healthy controls using Sanger sequencing. We found that 26 out of 7657 (0.34%) patients had RAD50 pathogenic mutations, and 16 patients carried one of the three recurrent mutations (L719fs, n=6 cases; K994fs, n=5 cases; and H1269fs, n=5 cases); the recurrent mutation rate was 0.21%. The frequency of the three recurrent mutations in the 5000 healthy controls was 0.18% (9/5000). These mutations did not confer an increased risk of breast cancer in the studied patients [odds ratios (OR), 1.16; 95% confidence interval (CI), 0.51-2.63; P = 0.72]. Nevertheless, multivariate analysis revealed that RAD50 pathogenic mutations were an independent unfavourable predictor of recurrence-free survival (RFS) [adjusted hazard ratio (HR) 2.66; 95% CI, 1.18-5.98; P=0.018] and disease-specific survival (DSS) (adjusted HR 4.36; 95% CI, 1.58-12.03; P=0.004) in the entire study cohort. Our study suggested that RAD50 germline mutations are not associated with an increased risk of breast cancer, but patients with RAD50 germline mutations have unfavourable survival compared with patients without these mutations. This article is protected by copyright. All rights reserved. © 2018 UICC.

Clinical outcomes of arthroscopic single and double row repair in full thickness rotator cuff tears.

PubMed

Ji, Jong-Hun; Shafi, Mohamed; Kim, Weon-Yoo; Kim, Young-Yul

2010-07-01

There has been a recent interest in the double row repair method for arthroscopic rotator cuff repair following favourable biomechanical results reported by some studies. The purpose of this study was to compare the clinical results of arthroscopic single row and double row repair methods in the full-thickness rotator cuff tears. 22 patients of arthroscopic single row repair (group I) and 25 patients who underwent double row repair (group II) from March 2003 to March 2005 were retrospectively evaluated and compared for the clinical outcomes. The mean age was 58 years and 56 years respectively for group I and II. The average follow-up in the two groups was 24 months. The evaluation was done by using the University of California Los Angeles (UCLA) rating scale and the shoulder index of the American Shoulder and Elbow Surgeons (ASES). In Group I, the mean ASES score increased from 30.48 to 87.40 and the mean ASES score increased from 32.00 to 91.45 in the Group II. The mean UCLA score increased from the preoperative 12.23 to 30.82 in Group I and from 12.20 to 32.40 in Group II. Each method has shown no statistical clinical differences between two methods, but based on the sub scores of UCLA score, the double row repair method yields better results for the strength, and it gives more satisfaction to the patients than the single row repair method. Comparing the two methods, double row repair group showed better clinical results in recovering strength and gave more satisfaction to the patients but no statistical clinical difference was found between 2 methods.

Indications for an inducible component of error-prone DNA repair in yeast.

PubMed

Siede, W; Eckardt, F

1984-01-01

In a thermoconditional mutant of mutagenic DNA repair (rev 2ts = rad 5-8) of Saccharomyces cerevisiae recovery of survival and mutation frequencies can be monitored by incubating UV-irradiated cells in growth medium at a permissive temperature (23 degrees C) before plating and a shift to restrictive temperature (36 degrees C). Inhibition of protein synthesis with cycloheximide during incubation at permissive conditions blocks this REV 2 dependent recovery process in stationary phase rev 2ts cells, whereas it can be reduced but not totally abolished in exponentially growing cells. These results indicate a strict dependence on post-irradiation protein synthesis in stationary phase cells and argue for a considerable constitutive level and only limited inducibility in logarithmic phase cells. The UV inducibility of the REV 2 coded function in stationary phase cells could be confirmed by analysis of the dose-response pattern of the his 5-2 reversion: in stationary phase rev 2ts cells, the quadratic component of the biphasic linear-quadratic induction kinetics found at 23 degrees C, which is interpreted as the consequence of induction of mutagenic repair, is eliminated at 36 degrees C.

Rad50S alleles of the Mre11 complex: questions answered and questions raised.

PubMed

Usui, Takehiko; Petrini, John H J; Morales, Monica

2006-08-15

We find that Rad50S mutations in yeast and mammals exhibit constitutive PIKK (PI3-kinase like kinase)-dependent signaling [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-4354.]. The signaling depends on Mre11 complex functions, consistent with its role as a DNA damage sensor. Rad50S is distinct from hypomorphic mutations of Mre11 and Nbs1 in mammals [M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-3054.; J.P. Carney, R.S. Maser, H. Olivares, E.M. Davis, Le M. Beau, J.R. Yates, III, L. Hays, W.F. Morgan, J.H. Petrini, The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93 (1998) 477-486.; G.S. Stewart, R.S. Maser, T. Stankovic, D.A. Bressan, M.I. Kaplan, N.G. Jaspers, A. Raams, P.J. Byrd, J.H. Petrini, A.M. Taylor, The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell 99 (1999) 577-587.; B.R. Williams, O.K. Mirzoeva, W.F. Morgan, J. Lin, W. Dunnick, J.H. Petrini, A murine model of nijmegen breakage syndrome. Curr. Biol. 12 (2002) 648-653.; J.W. Theunissen, M.I. Kaplan, P.A. Hunt, B.R. Williams, D.O. Ferguson, F.W. Alt, J.H. Petrini, Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice. Mol. Cell 12 (2003) 1511-1523.] and the Mre11 complex deficiency in yeast [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response

Single-stage repair of rectoperineal and rectovestibular fistulae can be safely delayed beyond the neonatal period.

PubMed

Short, Scott S; Bucher, Brian T; Barnhart, Douglas C; Van Der Watt, Nadia; Zobell, Sarah; Allen, Ashley; Rollins, Michael D

2018-02-12

We sought to examine the short-term outcomes following single-stage repair of rectoperineal and rectovestibular fistulae in infants and identify risk factors for wound complication. Patients with a rectoperineal or rectovestibular fistula treated with a single-stage repair beyond the neonatal period (>30days of age) at a pediatric colorectal center (2011-2016) were reviewed. 36 patients with a rectoperineal and 7 patients with a rectovestibular fistula were repaired using the Posterior Sagittal Anorectoplasty (PSARP) approach. Median follow-up was 31months. The median age and weight at the time of repair were 166days and 6.5kg. Four patients (11%) suffered a wound complication (3 rectoperineal, 1 rectovestibular). Two required a diverting colostomy to allow wound healing. Two patients suffered skin separation managed with local wound care. All 4 patients experienced satisfactory wound healing without anoplasty stricture. Two different patients developed a stricture of the neo-anus. Age and weight at time of repair, gender, and presence of a genitourinary anomaly were not associated with wound complications. Delayed single-stage repair of rectoperineal and rectovestibular fistulae can be performed safely in infants beyond the newborn period. With attentive treatment, satisfactory healing can be anticipated if a wound complication is encountered. Retrospective Comparative Study, Level III. Copyright © 2018. Published by Elsevier Inc.

A tetrad of bicuspid aortic valve association: A single-stage repair

PubMed Central

Barik, Ramachandra; Patnaik, A. N.; Mishra, Ramesh C.; Kumari, N. Rama; Gulati, A. S.

2012-01-01

We report a 27 years old male who presented with a combination of both congenital and acquired cardiac defects. This syndrome complex includes congenital bicuspid aortic valve, Seller's grade II aortic regurgitation, juxta- subclavian coarctation, stenosis of ostium of left subclavian artery and ruptured sinus of Valsalva aneurysm without any evidence of infective endocarditis. This type of constellation is extremely rare. Neither coarctation of aorta with left subclavian artery stenosis nor the rupture of sinus Valsalva had a favorable pathology for percutaneus intervention. Taking account into morbidity associated with repeated surgery and anesthesia patient underwent a single stage surgical repair of both the defects by two surgical incisions. The approaches include median sternotomy for rupture of sinus of Valsalva and lateral thoracotomy for coarctation with left subclavian artery stenosis. The surgery was uneventful. After three months follow up echocardiography showed mild residual gradient across the repaired coarctation segment, mild aortic regurgitation and no residual left to right shunt. This patient is under follow up. This is an extremely rare case of single stage successful repair of coarctation and rupture of sinus of Valsalva associated with congenital bicuspid aortic valve. PMID:22629035

A tetrad of bicuspid aortic valve association: A single-stage repair.

PubMed

Barik, Ramachandra; Patnaik, A N; Mishra, Ramesh C; Kumari, N Rama; Gulati, A S

2012-04-01

We report a 27 years old male who presented with a combination of both congenital and acquired cardiac defects. This syndrome complex includes congenital bicuspid aortic valve, Seller's grade II aortic regurgitation, juxta- subclavian coarctation, stenosis of ostium of left subclavian artery and ruptured sinus of Valsalva aneurysm without any evidence of infective endocarditis. This type of constellation is extremely rare. Neither coarctation of aorta with left subclavian artery stenosis nor the rupture of sinus Valsalva had a favorable pathology for percutaneus intervention. Taking account into morbidity associated with repeated surgery and anesthesia patient underwent a single stage surgical repair of both the defects by two surgical incisions. The approaches include median sternotomy for rupture of sinus of Valsalva and lateral thoracotomy for coarctation with left subclavian artery stenosis. The surgery was uneventful. After three months follow up echocardiography showed mild residual gradient across the repaired coarctation segment, mild aortic regurgitation and no residual left to right shunt. This patient is under follow up. This is an extremely rare case of single stage successful repair of coarctation and rupture of sinus of Valsalva associated with congenital bicuspid aortic valve.

Biomechanical evaluation of arthroscopic rotator cuff repairs: double-row compared with single-row fixation.

PubMed

Ma, C Benjamin; Comerford, Lyn; Wilson, Joseph; Puttlitz, Christian M

2006-02-01

Recent studies have shown that arthroscopic rotator cuff repairs can have higher rates of failure than do open repairs. Current methods of rotator cuff repair have been limited to single-row fixation of simple and horizontal stitches, which is very different from open repairs. The objective of this study was to compare the initial cyclic loading and load-to-failure properties of double-row fixation with those of three commonly used single-row techniques. Ten paired human supraspinatus tendons were split in half, yielding four tendons per cadaver. The bone mineral content at the greater tuberosity was assessed. Four stitch configurations (two-simple, massive cuff, arthroscopic Mason-Allen, and double-row fixation) were randomized and tested on each set of tendons. Specimens were cyclically loaded between 5 and 100 N at 0.25 Hz for fifty cycles and then loaded to failure under displacement control at 1 mm/sec. Conditioning elongation, peak-to-peak elongation, ultimate tensile load, and stiffness were measured with use of a three-dimensional tracking system and compared, and the failure type (suture or anchor pull-out) was recorded. No significant differences were found among the stitches with respect to conditioning elongation. The mean peak-to-peak elongation (and standard error of the mean) was significantly lower for the massive cuff (1.1 +/- 0.1 mm) and double-row stitches (1.1 +/- 0.1 mm) than for the arthroscopic Mason-Allen stitch (1.5 +/- 0.2 mm) (p < 0.05). The ultimate tensile load was significantly higher for double-row fixation (287 +/- 24 N) than for all of the single-row fixations (p < 0.05). Additionally, the massive cuff stitch (250 +/- 21 N) was found to have a significantly higher ultimate tensile load than the two-simple (191 +/- 18 N) and arthroscopic Mason-Allen (212 +/- 21 N) stitches (p < 0.05). No significant differences in stiffness were found among the stitches. Failure mechanisms were similar for all stitches. Rotator cuff repairs in the

RAD-ADAPT: Software for modelling clonogenic assay data in radiation biology.

PubMed

Zhang, Yaping; Hu, Kaiqiang; Beumer, Jan H; Bakkenist, Christopher J; D'Argenio, David Z

2017-04-01

We present a comprehensive software program, RAD-ADAPT, for the quantitative analysis of clonogenic assays in radiation biology. Two commonly used models for clonogenic assay analysis, the linear-quadratic model and single-hit multi-target model, are included in the software. RAD-ADAPT uses maximum likelihood estimation method to obtain parameter estimates with the assumption that cell colony count data follow a Poisson distribution. The program has an intuitive interface, generates model prediction plots, tabulates model parameter estimates, and allows automatic statistical comparison of parameters between different groups. The RAD-ADAPT interface is written using the statistical software R and the underlying computations are accomplished by the ADAPT software system for pharmacokinetic/pharmacodynamic systems analysis. The use of RAD-ADAPT is demonstrated using an example that examines the impact of pharmacologic ATM and ATR kinase inhibition on human lung cancer cell line A549 after ionizing radiation. Copyright © 2017 Elsevier B.V. All rights reserved.

Polymorphisms in DNA repair genes increase the risk for type 2 diabetes mellitus and hypertension.

PubMed

Das, Sambuddha; Purkayastha, Sukanya; Roy, Hirakjyoti; Sinha, Anima; Choudhury, Yashmin

2018-06-09

We investigated the effect of polymorphisms in four DNA repair genes, viz. RAD18 Arg302Gln (G>A) (rs373572), XPD Asp312Asn (G>A) (rs1799793), APE1 Asp148Glu (T>G) (rs3136820), and OGG1 Ser326Cys (C>G) (rs1052133) on the risk for type 2 diabetes mellitus (T2DM) and hypertension (HT) in association with smoking, tobacco chewing, and alcohol consumption in a population from Northeast India. The study subjects were comprised of 70 patients suffering from both T2DM and HT and 83 healthy controls. Genotyping was performed using ARMS-PCR for XPD Asp312Asn (G>A) and PCR-CTPP for RAD18 Arg302Gln (G>A), APE1 Asp148Glu (T>G) and OGG1 Ser326Cys (C>G). The RAD18 Gln/Gln genotype was found to significantly increase the risk for T2DM and HT by 30 fold. Significant high risk was observed for individuals with XPD Asn/Asn-RAD18 Arg/Gln genotypes. Smoking was found to be the single most important independent risk factor for T2DM and HT. This study concludes that RAD18 Arg302Gln and XPD Asp312Asn polymorphisms might increase the risk for T2DM and HT in association with smoking, tobacco chewing, and/or alcohol consumption, while APE1 Asp148Glu (T>G) and OGG1 Ser326Cys (C>G) polymorphisms do not contribute to such risk.

Clinical outcomes of arthroscopic single and double row repair in full thickness rotator cuff tears

PubMed Central

Ji, Jong-Hun; Shafi, Mohamed; Kim, Weon-Yoo; Kim, Young-Yul

2010-01-01

Background: There has been a recent interest in the double row repair method for arthroscopic rotator cuff repair following favourable biomechanical results reported by some studies. The purpose of this study was to compare the clinical results of arthroscopic single row and double row repair methods in the full-thickness rotator cuff tears. Materials and Methods: 22 patients of arthroscopic single row repair (group I) and 25 patients who underwent double row repair (group II) from March 2003 to March 2005 were retrospectively evaluated and compared for the clinical outcomes. The mean age was 58 years and 56 years respectively for group I and II. The average follow-up in the two groups was 24 months. The evaluation was done by using the University of California Los Angeles (UCLA) rating scale and the shoulder index of the American Shoulder and Elbow Surgeons (ASES). Results: In Group I, the mean ASES score increased from 30.48 to 87.40 and the mean ASES score increased from 32.00 to 91.45 in the Group II. The mean UCLA score increased from the preoperative 12.23 to 30.82 in Group I and from 12.20 to 32.40 in Group II. Each method has shown no statistical clinical differences between two methods, but based on the sub scores of UCLA score, the double row repair method yields better results for the strength, and it gives more satisfaction to the patients than the single row repair method. Conclusions: Comparing the two methods, double row repair group showed better clinical results in recovering strength and gave more satisfaction to the patients but no statistical clinical difference was found between 2 methods. PMID:20697485

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

DOE PAGES

Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.; ...

2016-07-15

Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here,more » we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.« less

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.

Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here,more » we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.« less

Outcomes of single-row and double-row arthroscopic rotator cuff repair: a systematic review.

PubMed

Saridakis, Paul; Jones, Grant

2010-03-01

Arthroscopic rotator cuff repair is a common procedure that is gaining wide acceptance among orthopaedic surgeons because it is less invasive than open repair techniques. However, there is little consensus on whether to employ single-row or double-row fixation. The purpose of the present study was to systematically review the English-language literature to see if there is a difference between single-row and double-row fixation techniques in terms of clinical outcomes and radiographic healing. PubMed, the Cochrane Central Register of Controlled Trials, and EMBASE were reviewed with the terms "arthroscopic rotator cuff," "single row repair," and "double row repair." The inclusion criteria were a level of evidence of III (or better), an in vivo human clinical study on arthroscopic rotator cuff repair, and direct comparison of single-row and double-row fixation. Excluded were technique reports, review articles, biomechanical studies, and studies with no direct comparison of arthroscopic rotator cuff repair techniques. On the basis of these criteria, ten articles were found, and a review of the full-text articles identified six articles for final review. Data regarding demographic characteristics, rotator cuff pathology, surgical techniques, biases, sample sizes, postoperative rehabilitation regimens, American Shoulder and Elbow Surgeons scores, University of California at Los Angeles scores, Constant scores, and the prevalence of recurrent defects noted on radiographic studies were extracted. Confidence intervals were then calculated for the American Shoulder and Elbow Surgeons, University of California at Los Angeles, and Constant scores. Quality appraisal was performed by the two authors to identify biases. There was no significant difference between the single-row and double-row groups within each study in terms of postoperative clinical outcomes. However, one study divided each of the groups into patients with small-to-medium tears (< 3 cm in length) and those with

Proteolytic activities in yeast after UV irradiation. II. Variation in proteinase levels in mutants blocked in DNA-repair pathways.

PubMed

Schwencke, J; Moustacchi, E

1982-01-01

When the levels of three common yeast proteinases in exponentially growing cells of mutants blocked in different repair pathways are compared to that of isogenic wild-type cells, it can be seen that the level of proteinase B is enhanced in the mutants whereas the levels of leucin aminopeptidase (Leu.AP) and lysine aminopeptidase (Lys.AP) are similar in all strains. As in its corresponding wild type, the level of proteinase B activity is further enhanced after UV-irradiation in a mutant blocked in excision-repair (rad1-3). In contrast, following the same treatment the level of proteinase B remains almost constant in a mutant blocked in a general error-prone repair system (rad6-1) and in a mutant defective in a more specific mutagenic repair pathway (pso2-1). Cycloheximide, an inhibitor of protein synthesis, blocks the post-UV enhancement in proteinase B activity observed in rad1-3 indicating that, as in the wild-type cells, an inducible process is involved. The levels of Lys.AP and Leu.AP are, respectively, either unaffected or only moderately increased following UV-treatment of the repair defective mutants, as in wild-type strains. It is obvious that the induction of protease B activity following UV-treatment in Saccharomyces cannot be equated to the induction of the recA protein in Escherichia coli. However the correlation found between the block in mutagenic repair and the lack of UV-induction of protease B activity leads to questions on the possible role of certain protease activities in mutagenic repair in eucaryotic cells.

Single-stage soft tissue reconstruction and orbital fracture repair for complex facial injuries.

PubMed

Wu, Peng Sen; Matoo, Reshvin; Sun, Hong; Song, Li Yuan; Kikkawa, Don O; Lu, Wei

2017-02-01

Orbital fractures with open periorbital wounds cause significant morbidity. Timing of debridement with fracture repair and soft tissue reconstruction is controversial. This study focuses on the efficacy of early single-stage repair in combined bony and soft tissue injuries. Retrospective review. Twenty-three patients with combined open soft tissue wounds and orbital fractures were studied for single-stage orbital reconstruction and periorbital soft tissue repair. Inclusion criteria were open soft tissue wounds with clinical and radiographic evidence of orbital fractures and repair performed within 48 h after injury. Surgical complications and reconstructive outcomes were assessed over 6 months. The main outcome measures were enophthalmos, pre- and post-CT imaging of orbits, scar evaluation, presence of diplopia, and eyelid position. Enophthalmos was corrected in 16/19 cases and improved in 3/19 cases. 3D reconstruction of CT images showed markedly improved orbital alignment with objective measurements of the optic foramen to cornea distance (mm) in reconstructed orbits relative to intact orbits of 0.66, 95% confidence interval [CI] (lower 0.33, upper 0.99) mm. The mean baseline of Stony Brook Scar Evaluation Scale was 0.6, 95%CI (0.30-0.92), and for 6 months, the mean score was 3.4, 95%CI (3.05-3.73). Residual diplopia in secondary gazes was present in two patients; one patient had ectropion. Complications included one case of local wound infection. An early single-stage repair of combined soft tissue and orbital fractures yields satisfactory functional and aesthetic outcomes. Complications are low and likely related to trauma severity. Copyright © 2016 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Long-Term Outcome of Laparoscopic Totally Extraperitoneal Repair of Bilateral Inguinal Hernias with a Large Single Mesh.

PubMed

Issa, Nidal; Ohana, Gil; Bachar, Gil Nissim; Powsner, Eldad

2016-02-01

A totally extraperitoneal (TEP) approach is currently the technique of choice for the laparoscopic repair of bilateral inguinal hernias in our institution. Most other surgeons use two meshes for the TEP repair, one for each side. We prefer a large single mesh when possible since it allows for easier correct placement of the mesh in one stage. We compared our long-term results of both techniques in terms of late complications and recurrence rates. This study retrospectively evaluated the medical records of 108 patients who underwent bilateral laparoscopic TEP repair in our institution between January 2002 and December 2003. Excluded were patients who had a conversion to a transabdominal preperitoneal or open approach. A total of 73 (67 %) patients fulfilled study entrance criteria and were enrolled: 39 had undergone single mesh repair and 34 had undergone double mesh repair. There were no significant group differences in demographics, operating time, postoperative morbidity, or hospital stay. Likewise, after a median follow-up of 102 months (range 94–115 months), there were no significant group differences between the single and double mesh groups in persistent pain (5.8 vs 2.5 %, respectively; p = 0.476) and recurrence (7.6 vs 8.8 %, respectively; p = 0.55). The use of a large single mesh is an effective and safe alternative technique for TEP repair of bilateral inguinal hernias, and is technically easy to perform.

Single-row versus double-row capsulolabral repair: a comparative evaluation of contact pressure and surface area in the capsulolabral complex-glenoid bone interface.

PubMed

Kim, Doo-Sup; Yoon, Yeo-Seung; Chung, Hoi-Jeong

2011-07-01

Despite the attention that has been paid to restoration of the capsulolabral complex anatomic insertion onto the glenoid, studies comparing the pressurized contact area and mean interface pressure at the anatomic insertion site between a single-row repair and a double-row labral repair have been uncommon. The purpose of our study was to compare the mean interface pressure and pressurized contact area at the anatomic insertion site of the capsulolabral complex between a single-row repair and a double-row repair technique. Controlled laboratory study. Thirty fresh-frozen cadaveric shoulders (mean age, 61 ± 8 years; range, 48-71 years) were used for this study. Two types of repair were performed on each specimen: (1) a single-row repair and (2) a double-row repair. Using pressure-sensitive films, we examined the interface contact area and contact pressure. The mean interface pressure was greater for the double-row repair technique (0.29 ± 0.04 MPa) when compared with the single-row repair technique (0.21 ± 0.03 MPa) (P = .003). The mean pressurized contact area was also significantly greater for the double-row repair technique (211.8 ± 18.6 mm(2), 78.4% footprint) compared with the single-row repair technique (106.4 ± 16.8 mm(2), 39.4% footprint) (P = .001). The double-row repair has significantly greater mean interface pressure and pressurized contact area at the insertion site of the capsulolabral complex than the single-row repair. The double-row repair may be advantageous compared with the single-row repair in restoring the native footprint area of the capsulolabral complex.

Cdc13 prevents telomere uncapping and Rad50-dependent homologous recombination

PubMed Central

Grandin, Nathalie; Damon, Christelle; Charbonneau, Michel

2001-01-01

Cdc13 performs an essential function in telomere end protection in budding yeast. Here, we analyze the consequences on telomere dynamics of cdc13-induced telomeric DNA damage in proliferating cells. Checkpoint-deficient cdc13-1 cells accumulated DNA damage and eventually senesced. However, these telomerase-proficient cells could survive by using homologous recombination but, contrary to telomerase-deficient cells, did so without prior telomere shortening. Strikingly, homologous recombination in cdc13-1 mec3, as well as in telomerase-deficient cdc13-1 cells, which were Rad52- and Rad50-dependent but Rad51-independent, exclusively amplified the TG1–3 repeats. This argues that not only short telomeres are substrates for type II recombination. The Cdc13-1 mutant protein harbored a defect in its association with Stn1 and Ten1 but also an additional, unknown, defect that could not be cured by expressing a Cdc13-1– Ten1–Stn1 fusion. We propose that Cdc13 prevents telomere uncapping and inhibits recombination between telomeric sequences through a pathway distinct from and complementary to that used by telomerase. PMID:11689452

Summary of Meta-Analyses Dealing with Single-Row versus Double-Row Repair Techniques for Rotator Cuff Tears

PubMed Central

Spiegl, U.J.; Euler, S.A.; Millett, P.J.; Hepp, P.

2016-01-01

Background: Several meta-analyses of randomized clinical trials have been performed to analyze whether double-row (DR) rotator cuff repair (RCR) provides superior clinical outcomes and structural healing compared to single-row (SR) repair. The purpose of this study was to sum up the results of meta-analysis comparing SR and DR repair with respect on clinical outcomes and re-tear rates. Methods: A literature search was undertaken to identify all meta-analyses dealing with randomized controlled trials comparing clinical und structural outcomes after SR versus DR RCR. Results: Eight meta-analyses met the eligibility criteria: two including Level I studies only, five including both Level I and Level II studies, and one including additional Level III studies. Four meta-analyses found no differences between SR and DR RCR for patient outcomes, whereas four favored DR RCR for tears greater than 3 cm. Two meta-analyses found no structural healing differences between SR and DR RCR, whereas six found DR repair to be superior for tears greater than 3 cm tears. Conclusion: No clinical differences are seen between single-row and double-row repair for small and medium rotator cuff tears after a short-term follow-up period with a higher re-tear rate following single-row repairs. There seems to be a trend to superior results with double-row repair in large to massive tear sizes. PMID:27708735

Postoperative urinary retention after inguinal hernia repair: a single institution experience.

PubMed

Blair, A B; Dwarakanath, A; Mehta, A; Liang, H; Hui, X; Wyman, C; Ouanes, J P P; Nguyen, H T

2017-12-01

Inguinal hernia repair is a common general surgery procedure with low morbidity. However, postoperative urinary retention (PUR) occurs in up to 22% of patients, resulting in further extraneous treatments.This single institution series investigates whether patient comorbidities, surgical approaches, and anesthesia methods are associated with developing PUR after inguinal hernia repairs. This is a single institution retrospective review of inguinal hernia from 2012 to 2015. PUR was defined as patients without a postoperative urinary catheter who subsequently required bladder decompression due to an inability to void. Univariate and multivariate logistic regressions were performed to quantify the associations between patient, surgical, and anesthetic factors with PUR. Stratification analysis was conducted at age of 50 years. 445 patients were included (42.9% laparoscopic and 57.1% open). Overall rate of PUR was 11.2% (12% laparoscopic, 10.6% open, and p = 0.64). In univariate analysis, PUR was significantly associated with patient age >50 and history of benign prostatic hyperplasia (BPH). Risk stratification for age >50 revealed in this cohort a 2.49 times increased PUR risk with lack of intraoperative bladder decompression (p = 0.013). At our institution, we found that patient age, history of BPH, and bilateral repair were associated with PUR after inguinal hernia repair. No association was found with PUR and laparoscopic vs open approach. Older males may be at higher risk without intraoperative bladder decompression, and therefore, catheter placement should be considered in this population, regardless of surgical approach.

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells.

PubMed

Holton, Nathaniel W; Andrews, Joel F; Gassman, Natalie R

2017-09-05

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells.

PubMed

Delabaere, Laetitia; Ertl, Henry A; Massey, Dashiell J; Hofley, Carolyn M; Sohail, Faraz; Bienenstock, Elisa J; Sebastian, Hans; Chiolo, Irene; LaRocque, Jeannine R

2017-04-01

Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

[Dot1 and Set2 Histone Methylases Control the Spontaneous and UV-Induced Mutagenesis Levels in the Saccharomyces cerevisiae Yeasts].

PubMed

Kozhina, T N; Evstiukhina, T A; Peshekhonov, V T; Chernenkov, A Yu; Korolev, V G

2016-03-01

In the Saccharomyces cerevisiae yeasts, the DOT1 gene product provides methylation of lysine 79 (K79) of hi- stone H3 and the SET2 gene product provides the methylation of lysine 36 (K36) of the same histone. We determined that the dot1 and set2 mutants suppress the UV-induced mutagenesis to an equally high degree. The dot1 mutation demonstrated statistically higher sensitivity to the low doses of MMC than the wild type strain. The analysis of the interaction between the dot1 and rad52 mutations revealed a considerable level of spontaneous cell death in the double dot1 rad52 mutant. We observed strong suppression of the gamma-in- duced mutagenesis in the set2 mutant. We determined that the dot1 and set2 mutations decrease the sponta- neous mutagenesis rate in both single and d ouble mutants. The epistatic interaction between the dot1 and set2 mutations and almost similar sensitivity of the corresponding mutants to the different types of DNA damage allow one to conclude that both genes are involved in the control of the same DNA repair pathways, the ho- mologous-recombination-based and the postreplicative DNA repair.

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

Rare adipose disorders (RADs) masquerading as obesity

PubMed Central

Herbst, Karen L

2012-01-01

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

Single-Incision Laparoscopic Intraperitoneal Onlay Mesh Repair for the Treatment of Multiple Recurrent Inguinal Hernias

PubMed Central

Tran, Kim; Zajkowska, Marta; Lam, Vincent; Hawthorne, Wayne

2014-01-01

Introduction: Despite an exponential rise in laparoscopic surgery for inguinal herniorrhaphy, overall recurrence rates have remained unchanged. Therefore, an increasing number of patients present with recurrent hernias after having failed anterior and laparoscopic repairs. This study reports our experience with single-incision laparoscopic (SIL) intraperitoneal onlay mesh (IPOM) repair for these hernias. Materials and methods: All patients referred with multiply recurrent inguinal hernias underwent SIL-IPOM from November 1 2009 to October 30 2013. A 2.5-cm infraumbilical incision was made and a SIL surgical port was placed intraperitoneally. Modified dissection techniques, namely, “chopsticks” and “inline” dissection, 5.5 mm/52 cm/30° angled laparoscope and conventional straight dissecting instruments were used. The peritoneum was incised above the symphysis pubis and dissection continued laterally and proximally raising an inferior flap, below a previous extraperitoneal mesh, while reducing any direct/indirect/femoral/cord lipoma before placement of antiadhesive mesh that was fixed into the pubic ramus as well as superiorly with nonabsorbable tacks before fixing its inferior border with fibrin sealant. The inferior peritoneal flap was then tacked back onto the mesh. Results: There were 9 male patients who underwent SIL-IPOM. Mean age was 55 years old and mean body mass index was 26.8 kg/m2. Mean mesh size was 275 cm2. Mean operation time was 125 minutes with hospital stay of 1 day and umbilical scar length of 21 mm at 4 weeks' follow-up. There were no intraoperative/postoperative complications, port-site hernias, chronic groin pain, or recurrence with mean follow-up of 20 months. Conclusions: Multiply recurrent inguinal hernias after failed conventional anterior and laparoscopic repairs can be treated safely and efficiently with SIL-IPOM. PMID:25392643

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

PubMed Central

Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

2011-01-01

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

PubMed Central

Malivert, Laurent; McIlwraith, Michael J.; Pape, Tillman; West, Stephen C.; Zhang, Xiaodong

2014-01-01

Mutations in BRCA2 increase susceptibility to breast, ovarian and prostate cancers. The product of human BRCA2, BRCA2 protein, plays a key role in the repair of DNA double strand breaks and interstrand crosslinks by RAD51-mediated homologous recombination. Here, we present a biochemical and structural characterization of full length (3,418 amino acid) BRCA2, alone and in complex with RAD51. We show that BRCA2 facilitates nucleation of RAD51 filaments at multiple sites on single-stranded DNA. Three-dimensional electron microscopy reconstructions revealed that BRCA2 exists as a dimer and that two oppositely-oriented sets of RAD51 molecules bind the dimer. Single stranded DNA binds along the long axis of BRCA2, such that only one set of RAD51 monomers can form a productive complex with DNA and establish filament formation. Our data define the molecular mechanism by which this tumor suppressor facilitates RAD51-mediated homologous recombinational repair. PMID:25282148

Biomechanical Comparison of Arthroscopic Single- and Double-Row Repair Techniques for Acute Bony Bankart Lesions.

PubMed

Spiegl, Ulrich J; Smith, Sean D; Todd, Jocelyn N; Coatney, Garrett A; Wijdicks, Coen A; Millett, Peter J

2014-08-01

Single- and double-row arthroscopic reconstruction techniques for acute bony Bankart lesions have been described in the literature. The double-row fixation technique would provide superior reduction and stability of a simulated bony Bankart lesion at time zero in a cadaveric model compared with the single-row technique. Controlled laboratory study. Testing was performed on 14 matched pairs of glenoids with simulated bony Bankart fractures with a defect width of 25% of the glenoid diameter. Half of the fractures were repaired with a double-row technique, while the contralateral glenoids were repaired with a single-row technique. The quality of fracture reduction was measured with a coordinate measuring machine. To determine the biomechanical stability of the repairs, specimens were preconditioned with 10 sinusoidal cycles between 5 and 25 N at 0.1 Hz and then pulled to failure in the anteromedial direction at a rate of 5 mm/min. Loads at 1 mm and 2 mm of fracture displacement were determined. The double-row technique required significantly higher forces to achieve fracture displacements of 1 mm (mean, 60.6 N; range, 39.0-93.3 N; P = .001) and 2 mm (mean, 94.4 N; range, 43.4-151.2 N; P = .004) than the single-row technique (1 mm: mean, 30.2 N; range, 14.0-54.1 N and 2 mm: mean, 63.7 N; range, 26.6-118.8 N). Significantly reduced fracture displacement was seen after double-row repair for both the unloaded condition (mean, 1.1 mm; range, 0.3-2.4 mm; P = .005) and in response to a 10-N anterior force applied to the defect (mean, 1.6 mm; range, 0.5-2.7 mm; P = .001) compared with single-row repair (unloaded: mean, 2.1 mm; range, 1.3-3.4 mm and loaded: mean, 3.4 mm; range, 1.9-4.7 mm). The double-row fixation technique resulted in improved fracture reduction and superior stability at time zero in this cadaveric model. This information may influence the surgical technique used to treat large osseous Bankart fractures and the postoperative rehabilitation protocols

Single-row modified mason-allen versus double-row arthroscopic rotator cuff repair: a biomechanical and surface area comparison.

PubMed

Nelson, Cory O; Sileo, Michael J; Grossman, Mark G; Serra-Hsu, Frederick

2008-08-01

The purpose of this study was to compare the time-zero biomechanical strength and the surface area of repair between a single-row modified Mason-Allen rotator cuff repair and a double-row arthroscopic repair. Six matched pairs of sheep infraspinatus tendons were repaired by both techniques. Pressure-sensitive film was used to measure the surface area of repair for each configuration. Specimens were biomechanically tested with cyclic loading from 20 N to 30 N for 20 cycles and were loaded to failure at a rate of 1 mm/s. Failure was defined at 5 mm of gap formation. Double-row suture anchor fixation restored a mean surface area of 258.23 +/- 69.7 mm(2) versus 148.08 +/- 75.5 mm(2) for single-row fixation, a 74% increase (P = .025). Both repairs had statistically similar time-zero biomechanics. There was no statistical difference in peak-to-peak displacement or elongation during cyclic loading. Single-row fixation showed a higher mean load to failure (110.26 +/- 26.4 N) than double-row fixation (108.93 +/- 21.8 N). This was not statistically significant (P = .932). All specimens failed at the suture-tendon interface. Double-row suture anchor fixation restores a greater percentage of the anatomic footprint when compared with a single-row Mason-Allen technique. The time-zero biomechanical strength was not significantly different between the 2 study groups. This study suggests that the 2 factors are independent of each other. Surface area and biomechanical strength of fixation are 2 independent factors in the outcome of rotator cuff repair. Maximizing both factors may increase the likelihood of complete tendon-bone healing and ultimately improve clinical outcomes. For smaller tears, a single-row modified Mason-Allen suture technique may provide sufficient strength, but for large amenable tears, a double row can provide both strength and increased surface area for healing.

Triple-Loaded Suture Anchors Versus a Knotless Rip Stop Construct in a Single-Row Rotator Cuff Repair Model.

PubMed

Noyes, Matthew P; Lederman, Evan; Adams, Christopher R; Denard, Patrick J

2018-05-01

To compare the biomechanical properties of single-row repair with triple-loaded (TL) anchor repair versus a knotless rip stop (KRS) repair in a rotator cuff repair model. Rotator cuff tears were created in 8 cadaveric matched-pair specimens and repaired with a TL anchor or KRS construct. In the TL construct, anchors were placed in the greater tuberosity and then all suture limbs were passed through the rotator cuff as simple sutures and tied. In the KRS construct, a 2-mm suture tape was passed through the tendon in an inverted mattress fashion, and a free suture was passed medial to the suture tape to create a rip-stop. Then, the suture tape and free suture were secured with knotless anchors. Displacement was observed with video tracking after cyclic loading, and specimens were loaded to failure. The mean load to failure was 438 ± 59 N in TL anchor repairs compared with 457 ± 110 N in KRS repairs (P = .582). The mean displacement with cyclic loading was 3.8 ± 1.6 mm in TL anchor repairs versus 4.3 ± 1.8 mm in the KRS group (P = .297). Mode of failure was consistent in both groups, with 6 of 8 failures in the TL anchor group and 7 of 8 failures in KRS group occurring from anchor pullout. There is no statistical difference in load to failure and cyclic loading between TL anchor and KRS single-row repair techniques. KRS repair technique may be an alternative method of repairing full-thickness supraspinatus tendon tears with a single-row construct. Copyright © 2018 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells.

PubMed

Nojima, Kuniharu; Hochegger, Helfrid; Saberi, Alihossein; Fukushima, Toru; Kikuchi, Koji; Yoshimura, Michio; Orelli, Brian J; Bishop, Douglas K; Hirano, Seiki; Ohzeki, Mioko; Ishiai, Masamichi; Yamamoto, Kazuhiko; Takata, Minoru; Arakawa, Hiroshi; Buerstedde, Jean-Marie; Yamazoe, Mitsuyoshi; Kawamoto, Takuo; Araki, Kasumi; Takahashi, Jun A; Hashimoto, Nobuo; Takeda, Shunichi; Sonoda, Eiichiro

2005-12-15

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replication-dependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase Polzeta, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy.

Immunochip analysis identifies association of the RAD50/IL13 region with human longevity.

PubMed

Flachsbart, Friederike; Ellinghaus, David; Gentschew, Liljana; Heinsen, Femke-Anouska; Caliebe, Amke; Christiansen, Lene; Nygaard, Marianne; Christensen, Kaare; Blanché, Hélène; Deleuze, Jean-François; Derbois, Céline; Galan, Pilar; Büning, Carsten; Brand, Stephan; Peters, Anette; Strauch, Konstantin; Müller-Nurasyid, Martina; Hoffmann, Per; Nöthen, Markus M; Lieb, Wolfgang; Franke, Andre; Schreiber, Stefan; Nebel, Almut

2016-06-01

Human longevity is characterized by a remarkable lack of confirmed genetic associations. Here, we report on the identification of a novel locus for longevity in the RAD50/IL13 region on chromosome 5q31.1 using a combined European sample of 3208 long-lived individuals (LLI) and 8919 younger controls. First, we performed a large-scale association study on 1458 German LLI (mean age 99.0 years) and 6368 controls (mean age 57.2 years) by targeting known immune-associated loci covered by the Immunochip. The analysis of 142 136 autosomal single nucleotide polymorphisms (SNPs) revealed an Immunochip-wide significant signal (PI mmunochip  = 7.01 × 10(-9) ) for the SNP rs2075650 in the TOMM40/APOE region, which has been previously described in the context of human longevity. To identify novel susceptibility loci, we selected 15 markers with PI mmunochip  < 5 × 10(-4) for replication in two samples from France (1257 LLI, mean age 102.4 years; 1811 controls, mean age 49.1 years) and Denmark (493 LLI, mean age 96.2 years; 740 controls, mean age 63.1 years). The association at SNP rs2706372 replicated in the French study collection and showed a similar trend in the Danish participants and was also significant in a meta-analysis of the combined French and Danish data after adjusting for multiple testing. In a meta-analysis of all three samples, rs2706372 reached a P-value of PI mmunochip+Repl  = 5.42 × 10(-7) (OR = 1.20; 95% CI = 1.12-1.28). SNP rs2706372 is located in the extended RAD50/IL13 region. RAD50 seems a plausible longevity candidate due to its involvement in DNA repair and inflammation. Further studies are needed to identify the functional variant(s) that predispose(s) to a long and healthy life. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

RadShield: semiautomated shielding design using a floor plan driven graphical user interface

PubMed Central

Wu, Dee H.; Yang, Kai; Rutel, Isaac B.

2016-01-01

The purpose of this study was to introduce and describe the development of RadShield, a Java‐based graphical user interface (GUI), which provides a base design that uniquely performs thorough, spatially distributed calculations at many points and reports the maximum air‐kerma rate and barrier thickness for each barrier pursuant to NCRP Report 147 methodology. Semiautomated shielding design calculations are validated by two approaches: a geometry‐based approach and a manual approach. A series of geometry‐based equations were derived giving the maximum air‐kerma rate magnitude and location through a first derivative root finding approach. The second approach consisted of comparing RadShield results with those found by manual shielding design by an American Board of Radiology (ABR)‐certified medical physicist for two clinical room situations: two adjacent catheterization labs, and a radiographic and fluoroscopic (R&F) exam room. RadShield's efficacy in finding the maximum air‐kerma rate was compared against the geometry‐based approach and the overall shielding recommendations by RadShield were compared against the medical physicist's shielding results. Percentage errors between the geometry‐based approach and RadShield's approach in finding the magnitude and location of the maximum air‐kerma rate was within 0.00124% and 14 mm. RadShield's barrier thickness calculations were found to be within 0.156 mm lead (Pb) and 0.150 mm lead (Pb) for the adjacent catheterization labs and R&F room examples, respectively. However, within the R&F room example, differences in locating the most sensitive calculation point on the floor plan for one of the barriers was not considered in the medical physicist's calculation and was revealed by the RadShield calculations. RadShield is shown to accurately find the maximum values of air‐kerma rate and barrier thickness using NCRP Report 147 methodology. Visual inspection alone of the 2D X‐ray exam distribution by a

Evaluating the performance of PI-RADS v2 in the non-academic setting.

PubMed

Jordan, Eric J; Fiske, Charles; Zagoria, Ronald J; Westphalen, Antonio C

2017-11-01

To evaluate the utility of PI-RADS v2 to diagnose clinically significant prostate cancer (CS-PCa) with magnetic resonance ultrasound (MR/US) fusion-guided prostate biopsies in the non-academic setting. Retrospective analysis of men whom underwent prostate multiparametric MRI and subsequent MR/US fusion biopsies at a single non-academic center from 11/2014 to 3/2016. Prostate MRIs were performed on a 3-Tesla scanner with a surface body coil. The Prostate Imaging Reporting and Data System (PI-RADS) v2 scoring algorithm was utilized and MR/US fusion biopsies were performed in selected cases. Mixed effect logistic regression analyses and receiver-operating characteristic (ROC) curves were performed on PI-RADS v2 alone and combined with PSA density (PSAD) to predict CS-PCa. 170 patients underwent prostate MRI with 282 PI-RADS lesions. MR/US fusion diagnosed 71 CS-PCa, 33 Gleason score 3+3, and 168 negative. PI-RADS v2 score is a statistically significant predictor of CS-PCa (P < 0.001). For each one-point increase in the overall PI-RADS v2 score, the odds of having CS-PCa increases by 4.2 (95% CI 2.2-8.3). The area under the ROC curve for PI-RADS v2 is 0.69 (95% CI 0.63-0.76) and for PI-RADS v2 + PSAD is 0.76 (95% CI 0.69-0.82), statistically higher than PI-RADS v2 alone (P < 0.001). The rate of CS-PCa was about twice higher in men with high PSAD (≥0.15) compared to men with low PSAD (<0.15) when a PI-RADS 4 or 5 lesion was detected (P = 0.005). PI-RADS v2 is a strong predictor of CS-PCa in the non-academic setting and can be further strengthened when utilized with PSA density.

Single-Stage Primary Cleft Lip and Palate Repair: A Review of the Literature.

PubMed

Kantar, Rami S; Rifkin, William J; Cammarata, Michael J; Maliha, Samantha G; Diaz-Siso, J Rodrigo; Farber, Scott J; Flores, Roberto L

2018-06-26

Single-stage cleft lip and palate repair is a debated surgical approach. While some studies have described favorable outcomes, concerns include the effect on craniomaxillofacial growth and increased risk of complications. To this date, there has not been a comprehensive appraisal of available data following combined cleft lip and palate repair. An extensive literature review was performed to identify all relevant articles. The level of evidence of these articles was graded according to the Oxford Centre for Evidence-Based Medicine Levels of Evidence Scale. A total of 22 relevant articles were identified, all of which were retrospective in nature. Patient age at the time of surgery ranged from 1 month to 10 years, the longest duration of follow-up was 18 years, and the largest study included 106 patients. Review of the literature shows that overall surgical outcomes following combined cleft lip and palate repair are encouraging. An increased rate of postoperative fistulas with associated speech abnormalities in some studies is noteworthy. Importantly, there is no evidence to suggest an impact on craniomaxillofacial growth, and psychosocial outcomes and parental satisfaction seem to be improved with single-stage surgery as compared with the staged approach. Our review shows overall favorable outcomes associated with combined cleft lip and palate repair. The limited follow-up time or nature of evaluated outcomes in some studies may underrepresent the true rate of adverse events, and highlights the need for additional long-term studies with standardized follow-up. To our knowledge, our review is the first to evaluate existing data regarding outcomes following combined cleft lip and palate repair.

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. ©2016 American Association for Cancer Research.

Structural basis for the initiation of eukaryotic transcription-coupled DNA repair.

PubMed

Xu, Jun; Lahiri, Indrajit; Wang, Wei; Wier, Adam; Cianfrocco, Michael A; Chong, Jenny; Hare, Alissa A; Dervan, Peter B; DiMaio, Frank; Leschziner, Andres E; Wang, Dong

2017-11-30

Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II-CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.

Triple-Loaded Single-Row Versus Suture-Bridge Double-Row Rotator Cuff Tendon Repair With Platelet-Rich Plasma Fibrin Membrane: A Randomized Controlled Trial.

PubMed

Barber, F Alan

2016-05-01

To compare the structural healing and clinical outcomes of triple-loaded single-row with suture-bridging double-row repairs of full-thickness rotator cuff tendons when both repair constructs are augmented with platelet-rich plasma fibrin membrane. A prospective, randomized, consecutive series of patients diagnosed with full-thickness rotator cuff tears no greater than 3 cm in anteroposterior length were treated with a triple-loaded single-row (20) or suture-bridging double-row (20) repair augmented with platelet-rich plasma fibrin membrane. The primary outcome measure was cuff integrity by magnetic resonance imaging (MRI) at 12 months postoperatively. Secondary clinical outcome measures were American Shoulder and Elbow Surgeons, Rowe, Simple Shoulder Test, Constant, and Single Assessment Numeric Evaluation scores. The mean MRI interval was 12.6 months (range, 12-17 months). A total of 3 of 20 single-row repairs and 3 of 20 double-row repairs (15%) had tears at follow-up MRI. The single-row group had re-tears in 1 single tendon repair and 2 double tendon repairs. All 3 tears failed at the original attachment site (Cho type 1). In the double-row group, re-tears were found in 3 double tendon repairs. All 3 tears failed medial to the medial row near the musculotendinous junction (Cho type 2). All clinical outcome measures were significantly improved from the preoperative level (P < .0001), but there was no statistical difference between groups postoperatively. There is no MRI difference in rotator cuff tendon re-tear rate at 12 months postsurgery between a triple-loaded single-row repair or a suture-bridging double-row repair when both are augmented with platelet-rich plasma fibrin membrane. No difference could be demonstrated between these repairs on clinical outcome scores. I, Prospective randomized study. Copyright © 2016 Arthroscopy Association of North America. All rights reserved.

Proto-jet configurations in RADs orbiting a Kerr SMBH: symmetries and limiting surfaces

NASA Astrophysics Data System (ADS)

Pugliese, D.; Stuchlík, Z.

2018-05-01

Ringed accretion disks (RADs) are agglomerations of perfect-fluid tori orbiting around a single central attractor that could arise during complex matter inflows in active galactic nuclei. We focus our analysis to axi-symmetric accretion tori orbiting in the equatorial plane of a supermassive Kerr black hole; equilibrium configurations, possible instabilities, and evolutionary sequences of RADs were discussed in our previous works. In the present work we discuss special instabilities related to open equipotential surfaces governing the material funnels emerging at various regions of the RADs, being located between two or more individual toroidal configurations of the agglomerate. These open structures could be associated to proto-jets. Boundary limiting surfaces are highlighted, connecting the emergency of the jet-like instabilities with the black hole dimensionless spin. These instabilities are observationally significant for active galactic nuclei, being related to outflows of matter in jets emerging from more than one torus of RADs orbiting around supermassive black holes.

A critical re-assessment of DNA repair gene promoter methylation in non-small cell lung carcinoma

PubMed Central

Do, Hongdo; Wong, Nicholas C.; Murone, Carmel; John, Thomas; Solomon, Benjamin; Mitchell, Paul L.; Dobrovic, Alexander

2014-01-01

DNA repair genes that have been inactivated by promoter methylation offer potential therapeutic targets either by targeting the specific repair deficiency, or by synthetic lethal approaches. This study evaluated promoter methylation status for eight selected DNA repair genes (ATM, BRCA1, ERCC1, MGMT, MLH1, NEIL1, RAD23B and XPC) in 56 non-small cell lung cancer (NSCLC) tumours and 11 lung cell lines using the methylation-sensitive high resolution melting (MS-HRM) methodology. Frequent methylation in NEIL1 (42%) and infrequent methylation in ERCC1 (2%) and RAD23B (2%) are reported for the first time in NSCLC. MGMT methylation was detected in 13% of the NSCLCs. Contrary to previous studies, methylation was not detected in ATM, BRCA1, MLH1 and XPC. Data from The Cancer Genome Atlas (TCGA) was consistent with these findings. The study emphasises the importance of using appropriate methodology for accurate assessment of promoter methylation. PMID:24569633

Laparoendoscopic single-site repair of bladder rupture using a home-made single-port device: initial experience of treatment for a traumatic intraperitoneal bladder rupture.

PubMed

Lee, Joo Yong; Kang, Dong Hyuk; Lee, Seung Wook

2012-06-01

We report our initial experience with a laparoendoscopic single-site (LESS) repair of a bladder rupture using a home-made single-port device. A 37-year-old man presented to the emergency department with complaints of voiding difficulty and gross hematuria after blunt trauma. Cystography and computed tomography revealed an intraperitoneal bladder rupture. The patient underwent LESS repair of a bladder rupture using the Alexis wound retractor, which was inserted through the umbilical incision. A home-made single-port device was made by fixing 6½ surgical gloves to the outer rim of the retractor and securing the glove finger to the end of 3 trocars with a tie. Using the flexible laparoscopic instruments and rigid instruments, LESS surgery was performed using a procedure similar to conventional laparoscopic surgery. The patient did not have any voiding problem after removal of the urethral Foley catheter on the 10th postoperative day. To our knowledge, this is the first published report of LESS repair of a traumatic bladder rupture using a home-made single-port device in the literature.

Simultaneous ATM/BRCA1/RAD51 expression variations associated with prognostic factors in Iranian sporadic breast cancer patients.

PubMed

Hallajian, Zeinab; Mahjoubi, Frouzandeh; Nafissi, Nahid

2017-07-01

DNA double-strand breaks (DSBs) as a serious lesion are repaired by non-homologous end-joining and homologous recombination pathways. ATM, BRCA1, RAD51 genes are involved in HR pathways. While some studies have revealed individual expression changes of these genes in different types of cancer, there are limited studies attempting to evaluate correlation of expression variations of these genes in breast cancer pathogenesis. This study aimed to determine RAD51, ATM and BRCA1 gene expression level and its association with clinicopathological factors in fresh breast cancer tissues. Moreover, this study evaluates potential correlations among expression levels of these genes. 50 breast cancer tissues were collected and examined for BRCA1, RAD51 and ATM gene expression by Real Time PCR. Expression changes were analyzed with REST software version 2009. mRNA expression was reduced in all these three genes when compared with β-Actin as a control gene (P value  < 0.001). Spearman's test demonstrated a significant positive correlation among ATM, BRCA1 and RAD51 gene down expression (P value  < 0.0001). There was a significant association between down expression of ATM with stage (P value  < 0.05), necrosis (P value  < 0.05), perineural invasion (P value  < 0.05), vascular invasion (P value  < 0.01), malignancy (P value  ≤ 0.001), PR (P value  < 0.05) and ER status (P value  < 0.01). In addition, there was a significant association between down expression of BRCA1 with Ki67 (P value  ≤ 0.001). Moreover, there was a significant association between down expression of RAD51 with lymph node involvement (P value  < 0.01), auxiliary lymph node metastasis (P value  = 0.01), age (P = 0.001), grade (P value  < 0.05) and PR status (P value  < 0.05). This study suggests association between expression changes in several DSB repair genes in a common functional pathway in breast cancer and the significant association between abnormal expression of these

Productive replication of human papillomavirus 31 requires DNA repair factor Nbs1.

PubMed

Anacker, Daniel C; Gautam, Dipendra; Gillespie, Kenric A; Chappell, William H; Moody, Cary A

2014-08-01

Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM

Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

PubMed Central

Anacker, Daniel C.; Gautam, Dipendra; Gillespie, Kenric A.; Chappell, William H.

2014-01-01

ABSTRACT Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. IMPORTANCE The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of

Rad GTPase Deficiency Leads to Cardiac Hypertrophy

PubMed Central

Tseng, Yu-Hua; Xie, Chang-Qing; Ilany, Jacob; Brüning, Jens C.; Sun, Zhongcui; Zhu, Xiaojun; Cui, Taixing; Youker, Keith A.; Yang, Qinglin; Day, Sharlene M.; Kahn, C. Ronald; Chen, Y. Eugene

2014-01-01

Background Rad (Ras associated with diabetes) GTPase is the prototypic member of a subfamily of Ras-related small G proteins. The aim of the present study was to define whether Rad plays an important role in mediating cardiac hypertrophy. Methods and Results We document for the first time that levels of Rad mRNA and protein were decreased significantly in human failing hearts (n=10) compared with normal hearts (n=3; P<0.01). Similarly, Rad expression was decreased significantly in cardiac hypertrophy induced by pressure overload and in cultured cardiomyocytes with hypertrophy induced by 10 μmol/L phenylephrine. Gain and loss of Rad function in cardiomyocytes significantly inhibited and increased phenylephrine-induced hypertrophy, respectively. In addition, activation of calcium-calmodulin–dependent kinase II (CaMKII), a strong inducer of cardiac hypertrophy, was significantly inhibited by Rad overexpression. Conversely, downregulation of CaMKIIδ by RNA interference technology attenuated the phenylephrine-induced hypertrophic response in cardiomyocytes in which Rad was also knocked down. To further elucidate the potential role of Rad in vivo, we generated Rad-deficient mice and demonstrated that they were more susceptible to cardiac hypertrophy associated with increased CaMKII phosphorylation than wild-type littermate controls. Conclusions The present data document for the first time that Rad is a novel mediator that inhibits cardiac hypertrophy through the CaMKII pathway. The present study will have significant implications for understanding the mechanisms of cardiac hypertrophy and setting the basis for the development of new strategies for treatment of cardiac hypertrophy. PMID:18056528

RadNet Databases and Reports

EPA Pesticide Factsheets

EPA’s RadNet data are available for viewing in a searchable database or as PDF reports. Historical and current RadNet monitoring data are used to estimate long-term trends in environmental radiation levels.

Laparoscopic Repair of Incisional Hernia Following Liver Transplantation-Early Experience of a Single Institution in Taiwan.

PubMed

Kuo, S-C; Lin, C-C; Elsarawy, A; Lin, Y-H; Wang, S-H; Wu, Y-J; Chen, C-L

2017-10-01

Ventral incisional hernia (VIH) is not uncommon following liver transplantation. Open repair was traditionally adopted for its management. Laparoscopic repair of VIH has been performed successfully in nontransplant patients with evidence of reduced recurrence rates and hospital stay. However, the application of VIH in post-transplantation patients has not been well established. Herein, we provide our initial experience with laparoscopic repair of post-transplantation VIH. From March 2015 to March 2016, 18 cases of post-transplantation VIH were subjected to laparoscopic repair (laparoscopy group). A historical control group of 17 patients who underwent conventional open repair (open group) from January 2013 to January 2015 were identified for comparison. The demographics and clinical outcomes were retrospectively compared. There were no significant differences among basic demographics between the 2 groups. No conversion was recorded in the laparoscopy group. Recurrence of VIH up to the end of the study period was not noted. In the laparoscopy group, the minor complications were lower (16.7% vs 52.9%; P = .035), the length of hospital stay was shorter (3 d vs 7 d, P = .007), but the median operative time was longer (137.5 min vs 106 min; P = .003). Laparoscopic repair of post-transplantation VIH is a safe and feasible procedure with shorter length of hospital stay. Copyright © 2017 Elsevier Inc. All rights reserved.

Whole pelvis megavoltage irradiation with single doses of 1000 rad to palliate advanced gynecologic cancers. [Incidence and severity of acute complications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boulware, R.J.; Caderao, J.B.; Delclos, L.

1979-03-01

This study reviews the experiences at M.D. Anderson Hospital of treating advanced gynecologic malignacies for palliation with single doses of 1000 rad per fraction. When feasible, this treatment was repeated twice (for a total of 3 treatments between intervals of 3 to 4 weeks. The patients who received 3 treatments had the best palliation; 2 treatments were more effective than 1. The palliative response was good in cervix, vagina, and vulva, poor in endometrial and ovarian carcinoma. The follow-up was short in some cases, but the acute complications appear minimal.

High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells

PubMed Central

Abe, Takuya; Branzei, Dana

2014-01-01

Transient induction or suppression of target genes is useful to study the function of toxic or essential genes in cells. Here we apply a Tet-On 3G system to DT40 lymphoma B cell lines, validating it for three different genes. Using this tool, we then show that overexpression of the chicken BRC4 repeat of the tumor suppressor BRCA2 impairs cell proliferation and induces chromosomal breaks. Mechanistically, high levels of BRC4 suppress double strand break-induced homologous recombination, inhibit the formation of RAD51 recombination repair foci, reduce cellular resistance to DNA damaging agents and induce a G2 damage checkpoint-mediated cell-cycle arrest. The above phenotypes are mediated by BRC4 capability to bind and inhibit RAD51. The toxicity associated with BRC4 overexpression is exacerbated by chemotherapeutic agents and reversed by RAD51 overexpression, but it is neither aggravated nor suppressed by a deficit in the non-homologous end-joining pathway of double strand break repair. We further find that the endogenous BRCA2 mediates the cytotoxicity associated with BRC4 induction, thus underscoring the possibility that BRC4 or other domains of BRCA2 cooperate with ectopic BRC4 in regulating repair activities or mitotic cell division. In all, the results demonstrate the utility of the Tet-On 3G system in DT40 research and underpin a model in which BRC4 role on cell proliferation and chromosome repair arises primarily from its suppressive role on RAD51 functions. PMID:25218467

High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells.

PubMed

Abe, Takuya; Branzei, Dana

2014-10-01

Transient induction or suppression of target genes is useful to study the function of toxic or essential genes in cells. Here we apply a Tet-On 3G system to DT40 lymphoma B cell lines, validating it for three different genes. Using this tool, we then show that overexpression of the chicken BRC4 repeat of the tumor suppressor BRCA2 impairs cell proliferation and induces chromosomal breaks. Mechanistically, high levels of BRC4 suppress double strand break-induced homologous recombination, inhibit the formation of RAD51 recombination repair foci, reduce cellular resistance to DNA damaging agents and induce a G2 damage checkpoint-mediated cell-cycle arrest. The above phenotypes are mediated by BRC4 capability to bind and inhibit RAD51. The toxicity associated with BRC4 overexpression is exacerbated by chemotherapeutic agents and reversed by RAD51 overexpression, but it is neither aggravated nor suppressed by a deficit in the non-homologous end-joining pathway of double strand break repair. We further find that the endogenous BRCA2 mediates the cytotoxicity associated with BRC4 induction, thus underscoring the possibility that BRC4 or other domains of BRCA2 cooperate with ectopic BRC4 in regulating repair activities or mitotic cell division. In all, the results demonstrate the utility of the Tet-On 3G system in DT40 research and underpin a model in which BRC4 role on cell proliferation and chromosome repair arises primarily from its suppressive role on RAD51 functions. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination.

PubMed

Marin-Vicente, Consuelo; Domingo-Prim, Judit; Eberle, Andrea B; Visa, Neus

2015-03-15

The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs. © 2015. Published by The Company of Biologists Ltd.

Comprehensive Pathway-Based Association Study of DNA Repair Gene Variants and the Risk of Nasopharyngeal Carcinoma

PubMed Central

Qin, Hai-De; Shugart, Yin Yao; Bei, Jin-Xin; Pan, Qing-Hua; Chen, Lina; Feng, Qi-Sheng; Chen, Li-Zhen; Huang, Wei; Liu, Jian Jun; Jorgensen, Timothy J.; Zeng, Yi-Xin; Jia, Wei-Hua

2011-01-01

DNA repair plays a central role in protecting against environmental carcinogenesis, and genetic variants of DNA repair genes have been reported to be associated with several human malignancies. To assess whether DNA gene variants were associated with nasopharyngeal carcinoma (NPC) risk, a candidate gene association study was conducted among the Cantonese population within the Guangdong Province, China --the ethnic group with the highest risk for NPC. A two-stage study design was utilized. In the discovery stage, 676 tagging SNPs covering 88 DNA repair genes were genotyped in a matched case-control study (cases/controls = 755/755). Eleven SNPs with Ptrend <0.01 were identified. Seven of these SNPs were located within three genes, RAD51L1, BRCA2 and TP53BP1. In the validation stage, these 11 SNPs were genotyped in a separate Cantonese population (cases/controls = 1,568/1,297). Two of the SNPs (rs927220 and rs11158728) – both in RAD51L1 – remained strongly associated with NPC. The SNP rs927220 had a significant Pcombined of 5.55 × 10−5, with OR = 1.20 (95%CI = 1.10 to 1.30), Bonferroni corrected P = 0.0381. The other SNP (rs11158728), which is in strong LD with rs927220 (r2 = 0.7), had a significant Pcombined of 2.0 × 10−4, Bonferroni corrected P = 0.1372. Gene-environment interaction analysis suggested that the exposures of salted-fish consumption and cigarette smoking had potential interactions with DNA repair gene variations, but need to be further investigated. Our findings support the notion that DNA repair genes, in particular RAD51L1, play a role in NPC etiology and development. PMID:21368091

Phenothiazine Inhibitors of TLKs Affect Double-Strand Break Repair and DNA Damage Response Recovery and Potentiate Tumor Killing with Radiomimetic Therapy

PubMed Central

Ronald, Sharon; Awate, Sanket; Rath, Abhijit; Carroll, Jennifer; Galiano, Floyd; Dwyer, Donard; Kleiner-Hancock, Heather; Mathis, J. Michael; Vigod, Simone

2013-01-01

The Tousled-like kinases (TLKs) are involved in chromatin assembly, DNA repair, and transcription. Two TLK genes exist in humans, and their expression is often dysregulated in cancer. TLKs phosphorylate Asf1 and Rad9, regulating double-strand break (DSB) repair and the DNA damage response (DDR). TLKs maintain genomic stability and are important therapeutic intervention targets. We identified specific inhibitors of TLKs from several compound libraries, some of which belong to the family of phenothiazine antipsychotics. The inhibitors prevented the TLK-mediated phosphorylation of Rad9(S328) and impaired checkpoint recovery and DSB repair. The inhibitor thioridazine (THD) potentiated tumor killing with chemotherapy and also had activity alone. Staining for γ-H2AX revealed few positive cells in untreated tumors, but large numbers in mice treated with low doxorubicin or THD alone, possibly the result of the accumulation of DSBs that are not promptly repaired as they may occur in the harsh tumor growth environment. PMID:23946870

DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids.

PubMed

Ahmed, Emad A; Scherthan, Harry; de Rooij, Dirk G

2015-12-16

Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale

PubMed Central

Yu, Xiong; Egelman, Edward H.

2010-01-01

Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single stranded DNA with ∼ 9 subunits per turn in contrast to the filaments formed on double stranded DNA with ∼ 6.4 subunits per turn, and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy (STEM) that the Dmc1 filament formed on single stranded DNA has a mass per unit length expected from ∼ 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions, and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or STEM to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. PMID:20600108

BRIT1/MCPH1 is essential for mitotic and meiotic recombination DNA repair and maintaining genomic stability in mice.

PubMed

Liang, Yulong; Gao, Hong; Lin, Shiaw-Yih; Peng, Guang; Huang, Xingxu; Zhang, Pumin; Goss, John A; Brunicardi, Francis C; Multani, Asha S; Chang, Sandy; Li, Kaiyi

2010-01-22

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1(-/-) mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1(-/-) mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to gamma-irradiation. BRIT1(-/-) MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1(-/-) mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2's function and as a result leads to infertility and genomic instability in mice.

Breast Imaging-Reporting and Data System (BI-RADS) classification in 51 excised palpable pediatric breast masses.

PubMed

Koning, Jeffrey L; Davenport, Katherine P; Poole, Patricia S; Kruk, Peter G; Grabowski, Julia E

2015-10-01

The American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) classification was developed to risk stratify breast lesions and guide surgical management based on imaging. Previous studies validating BI-RADS for US do not include pediatric patients. Most pediatric breast masses present as palpable lesions and frequently undergo ultrasound, which is often accompanied with a BI-RADS classification. Our study aimed to correlate BI-RADS with pathology findings to assess applicability of the classification system to pediatric patients. We performed a retrospective review of all patients who underwent excision of a breast mass at a single center from July 2010 to November 2013. We identified all patients who underwent preoperative ultrasound with BI-RADS classification. Demographic data, imaging results, and surgical pathology were analyzed and correlated. A total of 119 palpable masses were excised from 105 pediatric patients during the study period. Of 119 masses, 81 had preoperative ultrasound, and BI-RADS categories were given to 51 masses. Of these 51, all patients were female and the average age was 15.9 years. BI-RADS 4 was given to 25 of 51 masses (49%), and 100% of these lesions had benign pathology, the most common being fibroadenoma. Treatment algorithm based on BI-RADS classification may not be valid in pediatric patients. In this study, all patients with a BI-RADS 4 lesion had benign pathology. BI-RADS classification may overstate the risk of malignancy or need for biopsy in this population. Further validation of BI-RADS classification with large scale studies is needed in pediatric and adolescent patients. Copyright © 2015 Elsevier Inc. All rights reserved.

Rad-deletion Phenocopies Tonic Sympathetic Stimulation of the Heart.

PubMed

Levitan, Bryana M; Manning, Janet R; Withers, Catherine N; Smith, Jeffrey D; Shaw, Robin M; Andres, Douglas A; Sorrell, Vincent L; Satin, Jonathan

2016-12-01

Sympathetic stimulation modulates L-type calcium channel (LTCC) gating to contribute to increased systolic heart function. Rad is a monomeric G-protein that interacts with LTCC. Genetic deletion of Rad (Rad -/- ) renders LTCC in a sympathomimetic state. The study goal was to use a clinically inspired pharmacological stress echocardiography test, including analysis of global strain, to determine whether Rad -/- confers tonic positive inotropic heart function. Sarcomere dynamics and strain showed partial parallel isoproterenol (ISO) responsiveness for wild-type (WT) and for Rad -/- . Rad -/- basal inotropy was elevated compared to WT but was less responsiveness to ISO. Rad protein levels were lower in human patients with end-stage non-ischemic heart failure. These results show that Rad reduction provides a stable inotropic response rooted in sarcomere level function. Thus, reduced Rad levels in heart failure patients may be a compensatory response to need for increased output in the setting of HF. Rad deletion suggests a future therapeutic direction for inotropic support.

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

PubMed Central

Prakash, Louise; Prakash, Satya

1977-01-01

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad6, rad52, rad55 and rad57 were found among these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev ) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups. PMID:195865

Three New Structures of Left-Handed RadA Helical Filaments: Structural Flexibility of N-Terminal Domain Is Critical for Recombinase Activity

PubMed Central

Chang, Yu-Wei; Ko, Tzu-Ping; Lee, Chien-Der; Chang, Yuan-Chih; Lin, Kuei-Ann; Chang, Chia-Seng; Wang, Andrew H.-J.; Wang, Ting-Fang

2009-01-01

RecA family proteins, including bacterial RecA, archaeal RadA, and eukaryotic Dmc1 and Rad51, mediate homologous recombination, a reaction essential for maintaining genome integrity. In the presence of ATP, these proteins bind a single-strand DNA to form a right-handed nucleoprotein filament, which catalyzes pairing and strand exchange with a homologous double-stranded DNA (dsDNA), by as-yet unknown mechanisms. We recently reported a structure of RadA left-handed helical filament, and here present three new structures of RadA left-handed helical filaments. Comparative structural analysis between different RadA/Rad51 helical filaments reveals that the N-terminal domain (NTD) of RadA/Rad51, implicated in dsDNA binding, is highly flexible. We identify a hinge region between NTD and polymerization motif as responsible for rigid body movement of NTD. Mutant analysis further confirms that structural flexibility of NTD is essential for RadA's recombinase activity. These results support our previous hypothesis that ATP-dependent axial rotation of RadA nucleoprotein helical filament promotes homologous recombination. PMID:19295907

Biomechanics of Polyhydroxyalkanoate Mesh-Augmented Single-Row Rotator Cuff Repairs.

PubMed

Tashjian, Robert Z; Kolz, Christopher W; Suter, Thomas; Henninger, Heath B

Polyhydroxyalkanoate (PHA) mesh is a bioresorbable scaffold used to reinforce the suture-tendon interface in rotator cuff repairs (RCRs). We conducted a study of cyclic and ultimate failure properties of PHA mesh-augmented single-row RCRs and nonaugmented RCRs. Eight pairs of fresh-frozen cadaver humeri (6 male, 2 female) were tested. Mean (SD) age was 61 (9) years. The supraspinatus tendon was resected and reattached in a single-row configuration using 2 triple-loaded suture anchors and 6 simple stitches. The opposite humerus underwent RCR augmented with 2 strips of 13-mm × 23-mm PHA mesh. Humeri were mounted in an Instron load frame, cycled 1000 times to 1.0 MPa of effective stress, and loaded to failure. Construct gapping and ultimate failure loads/displacements were recorded. Paired t tests compared augmented and nonaugmented RCRs (P ≤ .05 was significant). There was no difference in gapping over 1000 cycles (P = .879). Mean (SD) failure load was higher for PHA mesh-augmented RCRs, 571 (173) N, than for nonaugmented (control) RCRs, 472 (120) N (P = .042), and failures were consistent within pairs because of tissue failure at the knots or anchor pullout. This technique for arthroscopic augmentation can be used to improve initial biomechanical repair strength in tears at risk for failure.

Knotless single-row rotator cuff repair: a comparative biomechanical study of 2 knotless suture anchors.

PubMed

Efird, Chad; Traub, Shaun; Baldini, Todd; Rioux-Forker, Dana; Spalazzi, Jeffrey P; Davisson, Twana; Hawkins, Monica; McCarty, Eric

2013-08-01

The purpose of this study was to compare the gap formation during cyclic loading, maximum repair strength, and failure mode of single-row full-thickness supraspinatus repairs performed using 2 knotless suture anchors with differing internal suture-retention mechanisms in a human cadaver model. Nine matched pairs of cadaver shoulders were used. Full-thickness tears were induced by detaching the supraspinatus tendon from the greater tuberosity. Single-row repairs were performed with either type I (Opus Magnum PI; ArthroCare, Austin, Texas) or type II (ReelX STT; Stryker, Mahwah, New Jersey) knotless suture anchors. The repaired tendon was cycled from 10 to 90 N for 500 cycles, followed by load to failure. Gap formation was measured at 5, 100, 200, 300, 400, and 500 cycles with a video digitizing system. Anchor type or location (anterior or posterior) had no effect on gap formation during cyclic loading regardless of position (anterior, P=.385; posterior, P=.389). Maximum load to failure was significantly greater (P=.018) for repairs performed with type II anchors (288±62 N) compared with type I anchors (179±39 N). Primary failure modes were anchor pullout and tendon tearing for type II anchors and suture slippage through the anchor for type I anchors. The internal ratcheting suture-retention mechanism of type II anchors may have helped this anchor outperform the suture-cinching mechanism of type I anchors by supporting significantly higher loads before failure and minimizing suture slippage, potentially leading to stronger repairs clinically. Copyright 2013, SLACK Incorporated.

ERCC1-XPF Endonuclease Facilitates DNA Double-Strand Break Repair▿ †

PubMed Central

Ahmad, Anwaar; Robinson, Andria Rasile; Duensing, Anette; van Drunen, Ellen; Beverloo, H. Berna; Weisberg, David B.; Hasty, Paul; Hoeijmakers, Jan H. J.; Niedernhofer, Laura J.

2008-01-01

ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and γH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1−/− Ku86−/− fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3′ overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent. PMID:18541667

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

PubMed Central

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

2016-01-01

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

Comparison of low-dose spinal anesthesia and single-shot femoral block combination with conventional dose spinal anesthesia in outpatient arthroscopic meniscus repair.

PubMed

Turhan, K S Cakar; Akmese, R; Ozkan, F; Okten, F F

2015-04-01

In the current prospective, randomized study, we aimed to compare the effects of low dose selective spinal anesthesia with 5 mg of hyperbaric bupivacaine and single-shot femoral nerve block combination with conventional dose selective spinal anesthesia in terms of intraoperative anesthesia characteristics, block recovery characteristics, and postoperative analgesic consumption. After obtaining institutional Ethics Committee approval, 52 ASA I-II patients aged 25-65, undergoing arthroscopic meniscus repair were randomly assigned to Group S (conventional dose selective spinal anesthesia with 10 mg bupivacaine) and Group FS (low-dose selective spinal anesthesia with 5mg bupivacaine +single-shot femoral block with 0.25% bupivacaine). Primary endpoints were time to reach T12 sensory block level, L2 regression, and complete motor block regression. Secondary endpoints were maximum sensory block level (MSBL); time to reach MSBL, time to first urination, time to first analgesic consumption and pain severity at the time of first mobilization. Demographic characteristics were similar in both groups (p > 0.05). MSBL and time to reach T12 sensory level were similar in both groups (p > 0.05). Time to reach L2 regression, complete motor block regression, and time to first micturition were significantly shorter; time to first analgesic consumption was significantly longer; and total analgesic consumption and severity of pain at time of first mobilization were significantly lower in Group FS (p < 0.05). The findings of the current study suggest that addition of single-shot femoral block to low dose spinal anesthesia could be an alternative to conventional dose spinal anesthesia in outpatient arthroscopic meniscus repair. NCT02322372.

Amplification of unscheduled DNA synthesis signal enables fluorescence-based single cell quantification of transcription-coupled nucleotide excision repair

PubMed Central

Wienholz, Franziska; Vermeulen, Wim

2017-01-01

Abstract Nucleotide excision repair (NER) comprises two damage recognition pathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER), which remove a wide variety of helix-distorting lesions including UV-induced damage. During NER, a short stretch of single-stranded DNA containing damage is excised and the resulting gap is filled by DNA synthesis in a process called unscheduled DNA synthesis (UDS). UDS is measured by quantifying the incorporation of nucleotide analogues into repair patches to provide a measure of NER activity. However, this assay is unable to quantitatively determine TC-NER activity due to the low contribution of TC-NER to the overall NER activity. Therefore, we developed a user-friendly, fluorescence-based single-cell assay to measure TC-NER activity. We combined the UDS assay with tyramide-based signal amplification to greatly increase the UDS signal, thereby allowing UDS to be quantified at low UV doses, as well as DNA-repair synthesis of other excision-based repair mechanisms such as base excision repair and mismatch repair. Importantly, we demonstrated that the amplified UDS is sufficiently sensitive to quantify TC-NER-derived repair synthesis in GG-NER-deficient cells. This assay is important as a diagnostic tool for NER-related disorders and as a research tool for obtaining new insights into the mechanism and regulation of excision repair. PMID:28088761

Ultrasound evaluation of arthroscopic full-thickness supraspinatus rotator cuff repair: single-row versus double-row suture bridge (transosseous equivalent) fixation. Results of a prospective, randomized study.

PubMed

Gartsman, Gary M; Drake, Gregory; Edwards, T Bradley; Elkousy, Hussein A; Hammerman, Steven M; O'Connor, Daniel P; Press, Cyrus M

2013-11-01

The purpose of this study was to compare the structural outcomes of a single-row rotator cuff repair and double-row suture bridge fixation after arthroscopic repair of a full-thickness supraspinatus rotator cuff tear. We evaluated with diagnostic ultrasound a consecutive series of ninety shoulders in ninety patients with full-thickness supraspinatus tears at an average of 10 months (range, 6-12) after operation. A single surgeon at a single hospital performed the repairs. Inclusion criteria were full-thickness supraspinatus tears less than 25 mm in their anterior to posterior dimension. Exclusion criteria were prior operations on the shoulder, partial thickness tears, subscapularis tears, infraspinatus tears, combined supraspinatus and infraspinatus repairs and irreparable supraspinatus tears. Forty-three shoulders were repaired with single-row technique and 47 shoulders with double-row suture bridge technique. Postoperative rehabilitation was identical for both groups. Ultrasound criteria for healed repair included visualization of a tendon with normal thickness and length, and a negative compression test. Eighty-three patients were available for ultrasound examination (40 single-row and 43 suture-bridge). Thirty of 40 patients (75%) with single-row repair demonstrated a healed rotator cuff repair compared to 40/43 (93%) patients with suture-bridge repair (P = .024). Arthroscopic double-row suture bridge repair (transosseous equivalent) of an isolated supraspinatus rotator cuff tear resulted in a significantly higher tendon healing rate (as determined by ultrasound examination) when compared to arthroscopic single-row repair. Copyright © 2013 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.

PI-RADS v2: Current standing and future outlook.

PubMed

Smith, Clayton P; Türkbey, Barış

2018-05-01

The Prostate Imaging-Reporting and Data System (PI-RADS) was created in 2012 to establish standardization in prostate multiparametric magnetic resonance imaging (mpMRI) acquisition, interpretation, and reporting. In hopes of improving upon some of the PI-RADS v1 shortcomings, the PI-RADS Steering Committee released PI-RADS v2 in 2015. This paper reviews the accuracy, interobserver agreement, and clinical outcomes of PI-RADS v2 and comments on the limitations of the current literature. Overall, PI-RADS v2 shows improved sensitivity and similar specificity compared to PI-RADS v1. However, concerns exist regarding interobserver agreement and the heterogeneity of the study methodology.

PI-RADS v2: Current standing and future outlook

PubMed Central

Smith, Clayton P.

2018-01-01

The Prostate Imaging-Reporting and Data System (PI-RADS) was created in 2012 to establish standardization in prostate multiparametric magnetic resonance imaging (mpMRI) acquisition, interpretation, and reporting. In hopes of improving upon some of the PI-RADS v1 shortcomings, the PI-RADS Steering Committee released PI-RADS v2 in 2015. This paper reviews the accuracy, interobserver agreement, and clinical outcomes of PI-RADS v2 and comments on the limitations of the current literature. Overall, PI-RADS v2 shows improved sensitivity and similar specificity compared to PI-RADS v1. However, concerns exist regarding interobserver agreement and the heterogeneity of the study methodology. PMID:29733790

Construction of two whole genome radiation hybrid panels for dromedary (Camelus dromedarius): 5000RAD and 15000RAD.

PubMed

Perelman, Polina L; Pichler, Rudolf; Gaggl, Anna; Larkin, Denis M; Raudsepp, Terje; Alshanbari, Fahad; Holl, Heather M; Brooks, Samantha A; Burger, Pamela A; Periasamy, Kathiravan

2018-01-31

The availability of genomic resources including linkage information for camelids has been very limited. Here, we describe the construction of a set of two radiation hybrid (RH) panels (5000 RAD and 15000 RAD ) for the dromedary (Camelus dromedarius) as a permanent genetic resource for camel genome researchers worldwide. For the 5000 RAD panel, a total of 245 female camel-hamster radiation hybrid clones were collected, of which 186 were screened with 44 custom designed marker loci distributed throughout camel genome. The overall mean retention frequency (RF) of the final set of 93 hybrids was 47.7%. For the 15000 RAD panel, 238 male dromedary-hamster radiation hybrid clones were collected, of which 93 were tested using 44 PCR markers. The final set of 90 clones had a mean RF of 39.9%. This 15000 RAD panel is an important high-resolution complement to the main 5000 RAD panel and an indispensable tool for resolving complex genomic regions. This valuable genetic resource of dromedary RH panels is expected to be instrumental for constructing a high resolution camel genome map. Construction of the set of RH panels is essential step toward chromosome level reference quality genome assembly that is critical for advancing camelid genomics and the development of custom genomic tools.

52. Virginia Route 666. This single span structure, built in ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

52. Virginia Route 666. This single span structure, built in 1962, is an example of a spandrel arch grade separation structure with a roman arch over a state secondary road. The bridge is constructed of reinforced concrete, backfilled with earth and has a thick stone veneer. Looking east-northeast at elevation. - Blue Ridge Parkway, Between Shenandoah National Park & Great Smoky Mountains, Asheville, Buncombe County, NC

Budding yeast mms4 is epistatic with rad52 and the function of Mms4 can be replaced by a bacterial Holliday junction resolvase.

PubMed

Odagiri, Nao; Seki, Masayuki; Onoda, Fumitoshi; Yoshimura, Akari; Watanabe, Sei; Enomoto, Takemi

2003-03-01

MMS4 of Saccharomyces cerevisiae was originally identified as the gene responsible for one of the collection of methyl methanesulfonate (MMS)-sensitive mutants, mms4. Recently it was identified as a synthetic lethal gene with an SGS1 mutation. Epistatic analyses revealed that MMS4 is involved in a pathway leading to homologous recombination requiring Rad52 or in the recombination itself, in which SGS1 is also involved. MMS sensitivity of mms4 but not sgs1, was suppressed by introducing a bacterial Holliday junction (HJ) resolvase, RusA. The frequencies of spontaneously occurring unequal sister chromatid recombination (SCR) and loss of marker in the rDNA in haploid mms4 cells and interchromosomal recombination between heteroalleles in diploid mms4 cells were essentially the same as those of wild-type cells. Although UV- and MMS-induced interchromosomal recombination was defective in sgs1 diploid cells, hyper-induction of interchromosomal recombination was observed in diploid mms4 cells, indicating that the function of Mms4 is dispensable for this type of recombination.

Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates.

PubMed

Ashley, T; Plug, A W; Xu, J; Solari, A J; Reddy, G; Golub, E I; Ward, D C

1995-10-01

Antibodies against human Rad51 protein were used to examine the distribution of Rad51 on meiotic chromatin in mouse spermatocytes and oocytes as well as chicken oocytes during sequential stages of meiosis. We observed the following dynamic changes in distribution of Rad51 during meiosis: (1) in early leptotene nuclei there are multiple, apparently randomly distributed, foci that by late leptonema become organized into tracks of foci. (2) These foci persist into zygonema, but most foci are now localized on Rad51-positive axes that correspond to lateral elements of the synaptonemal complex. As homologs synapse foci from homologous axes fuse. The distribution and involvement of Rad51 foci as contact points between homologs suggest that they may be components to early recombination nodules. (3) As pachynema progresses the number of foci drops dramatically; the temporal occurrence (mice) and physical and numerical distribution of foci on axes (chickens) suggest that they may be a component of late recombination nodules. (4) In early pachynema there are numerous Rad51 foci on the single axis of the X (mouse spermatocytes) or the Z (chicken oocytes) chromosomes that neither pair, nor recombine. (5) In late pachynema in mouse spermatocytes, but not oocytes, the Rad51 signal is preferentially enhanced at both ends of all the bivalents. As bivalents in spermatocytes, but not oocytes, begin to desynapse at diplonema they are often held together at these Rad51-positive termini. These observations parallel observations that recombination rates are exceptionally high near chromosome ends in male but not female eutherian mammals. (6) From diakinesis through metaphase I, Rad51 protein is detected as low-intensity fluorescent doublets that localize with CREST-specific antigens (kinetochores), suggesting that Rad51 participates, at least as a structural component of the materials involved, in sister kinetochore cohesiveness. Finally, the changes in Rad51 distribution during meiosis

RadVel: The Radial Velocity Modeling Toolkit

NASA Astrophysics Data System (ADS)

Fulton, Benjamin J.; Petigura, Erik A.; Blunt, Sarah; Sinukoff, Evan

2018-04-01

RadVel is an open-source Python package for modeling Keplerian orbits in radial velocity (RV) timeseries. RadVel provides a convenient framework to fit RVs using maximum a posteriori optimization and to compute robust confidence intervals by sampling the posterior probability density via Markov Chain Monte Carlo (MCMC). RadVel allows users to float or fix parameters, impose priors, and perform Bayesian model comparison. We have implemented real-time MCMC convergence tests to ensure adequate sampling of the posterior. RadVel can output a number of publication-quality plots and tables. Users may interface with RadVel through a convenient command-line interface or directly from Python. The code is object-oriented and thus naturally extensible. We encourage contributions from the community. Documentation is available at http://radvel.readthedocs.io.

RadNet Air Quality (Fixed Station) Data

EPA Pesticide Factsheets

RadNet is a national network of monitoring stations that regularly collect air for analysis of radioactivity. The RadNet network, which has stations in each State, has been used to track environmental releases of radioactivity from nuclear weapons tests and nuclear accidents. RadNet also documents the status and trends of environmental radioactivity

Chromosome Synapsis Alleviates Mek1-Dependent Suppression of Meiotic DNA Repair

PubMed Central

Subramanian, Vijayalakshmi V.; MacQueen, Amy J.; Vader, Gerben; Shinohara, Miki; Sanchez, Aurore; Borde, Valérie; Shinohara, Akira; Hochwagen, Andreas

2016-01-01

Faithful meiotic chromosome segregation and fertility require meiotic recombination between homologous chromosomes rather than the equally available sister chromatid, a bias that in Saccharomyces cerevisiae depends on the meiotic kinase, Mek1. Mek1 is thought to mediate repair template bias by specifically suppressing sister-directed repair. Instead, we found that when Mek1 persists on closely paired (synapsed) homologues, DNA repair is severely delayed, suggesting that Mek1 suppresses any proximal repair template. Accordingly, Mek1 is excluded from synapsed homologues in wild-type cells. Exclusion requires the AAA+-ATPase Pch2 and is directly coupled to synaptonemal complex assembly. Stage-specific depletion experiments further demonstrate that DNA repair in the context of synapsed homologues requires Rad54, a repair factor inhibited by Mek1. These data indicate that the sister template is distinguished from the homologue primarily by its closer proximity to inhibitory Mek1 activity. We propose that once pairing or synapsis juxtaposes homologues, exclusion of Mek1 is necessary to avoid suppression of all templates and accelerate repair progression. PMID:26870961

RadMap

EPA Pesticide Factsheets

RadMap is an interactive desktop tool featuring a nationwide geographic information systems (GIS) map of long-term radiation monitoring locations across the United States with access to key information about the monitor and the area surrounding it.

Ring-shaped architecture of RecR: implications for its role in homologous recombinational DNA repair

PubMed Central

Lee, Byung Il; Kim, Kyoung Hoon; Park, Soo Jeong; Eom, Soo Hyun; Song, Hyun Kyu; Suh, Se Won

2004-01-01

RecR, together with RecF and RecO, facilitates RecA loading in the RecF pathway of homologous recombinational DNA repair in procaryotes . The human Rad52 protein is a functional counterpart of RecFOR. We present here the crystal structure of RecR from Deinococcus radiodurans (DR RecR). A monomer of DR RecR has a two-domain structure: the N-terminal domain with a helix–hairpin–helix (HhH) motif and the C-terminal domain with a Cys4 zinc-finger motif, a Toprim domain and a Walker B motif. Four such monomers form a ring-shaped tetramer of 222 symmetry with a central hole of 30−35 Å diameter. In the crystal, two tetramers are concatenated, implying that the RecR tetramer is capable of opening and closing. We also show that DR RecR binds to both dsDNA and ssDNA, and that its HhH motif is essential for DNA binding. PMID:15116069

The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

PubMed

Blaikley, Elizabeth J; Tinline-Purvis, Helen; Kasparek, Torben R; Marguerat, Samuel; Sarkar, Sovan; Hulme, Lydia; Hussey, Sharon; Wee, Boon-Yu; Deegan, Rachel S; Walker, Carol A; Pai, Chen-Chun; Bähler, Jürg; Nakagawa, Takuro; Humphrey, Timothy C

2014-05-01

DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability. © The Author(s) 2014. Published by Oxford University Press.

Biomechanical and magnetic resonance imaging evaluation of a single- and double-row rotator cuff repair in an in vivo sheep model.

PubMed

Baums, Mike H; Spahn, Gunter; Buchhorn, Gottfried H; Schultz, Wolfgang; Hofmann, Lars; Klinger, Hans-Michael

2012-06-01

To investigate the biomechanical and magnetic resonance imaging (MRI)-derived morphologic changes between single- and double-row rotator cuff repair at different time points after fixation. Eighteen mature female sheep were randomly assigned to either a single-row treatment group using arthroscopic Mason-Allen stitches or a double-row treatment group using a combination of arthroscopic Mason-Allen and mattress stitches. Each group was analyzed at 1 of 3 survival points (6 weeks, 12 weeks, and 26 weeks). We evaluated the integrity of the cuff repair using MRI and biomechanical properties using a mechanical testing machine. The mean load to failure was significantly higher in the double-row group compared with the single-row group at 6 and 12 weeks (P = .018 and P = .002, respectively). At 26 weeks, the differences were not statistically significant (P = .080). However, the double-row group achieved a mean load to failure similar to that of a healthy infraspinatus tendon, whereas the single-row group reached only 70% of the load of a healthy infraspinatus tendon. No significant morphologic differences were observed based on the MRI results. This study confirms that in an acute repair model, double-row repair may enhance the speed of mechanical recovery of the tendon-bone complex when compared with single-row repair in the early postoperative period. Double-row rotator cuff repair enables higher mechanical strength that is especially sustained during the early recovery period and may therefore improve clinical outcome. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

RadNet Sampling and Analyses Schedules

EPA Pesticide Factsheets

RadNet air monitors operate continuously and samples of air, precipitation and drinking water and analyzed on a routine schedule. RadNet can send deployable monitors to any U.S. location in the case of a radiological emergency.

Modified classification and single-stage microsurgical repair of posttraumatic infected massive bone defects in lower extremities.

PubMed

Yang, Yun-fa; Xu, Zhong-he; Zhang, Guang-ming; Wang, Jian-wei; Hu, Si-wang; Hou, Zhi-qi; Xu, Da-chuan

2013-11-01

Posttraumatic infected massive bone defects in lower extremities are difficult to repair because they frequently exhibit massive bone and/or soft tissue defects, serious bone infection, and excessive scar proliferation. This study aimed to determine whether these defects could be classified and repaired at a single stage. A total of 51 cases of posttraumatic infected massive bone defect in lower extremity were included in this study. They were classified into four types on the basis of the conditions of the bone defects, soft tissue defects, and injured limb length, including Type A (without soft tissue defects), Type B (with soft tissue defects of 10 × 20 cm or less), Type C (with soft tissue defects of 10 × 20 cm or more), and Type D (with the limb shortening of 3 cm or more). Four types of single-stage microsurgical repair protocols were planned accordingly and implemented respectively. These protocols included the following: Protocol A, where vascularized fibular graft was implemented for Type A; Protocol B, where vascularized fibular osteoseptocutaneous graft was implemented for Type B; Protocol C, where vascularized fibular graft and anterior lateral thigh flap were used for Type C; and Protocol D, where limb lengthening and Protocols A, B, or C were used for Type D. There were 12, 33, 4, and 2 cases of Types A, B, C, and D, respectively, according to this classification. During the surgery, three cases of planned Protocol B had to be shifted into Protocol C; however, all microsurgical repairs were completed. With reference to Johner-Wruhs evaluation method, the total percentage of excellent and good results was 82.35% after 6 to 41 months of follow-up. It was concluded that posttraumatic massive bone defects could be accurately classified into four types on the basis of the conditions of bone defects, soft tissue coverage, and injured limb length, and successfully repaired with the single-stage repair protocols after thorough debridement. Thieme Medical

Structured reporting platform improves CAD-RADS assessment.

PubMed

Szilveszter, Bálint; Kolossváry, Márton; Karády, Júlia; Jermendy, Ádám L; Károlyi, Mihály; Panajotu, Alexisz; Bagyura, Zsolt; Vecsey-Nagy, Milán; Cury, Ricardo C; Leipsic, Jonathon A; Merkely, Béla; Maurovich-Horvat, Pál

2017-11-01

Structured reporting in cardiac imaging is strongly encouraged to improve quality through consistency. The Coronary Artery Disease - Reporting and Data System (CAD-RADS) was recently introduced to facilitate interdisciplinary communication of coronary CT angiography (CTA) results. We aimed to assess the agreement between manual and automated CAD-RADS classification using a structured reporting platform. Five readers prospectively interpreted 500 coronary CT angiographies using a structured reporting platform that automatically calculates the CAD-RADS score based on stenosis and plaque parameters manually entered by the reader. In addition, all readers manually assessed CAD-RADS blinded to the automatically derived results, which was used as the reference standard. We evaluated factors influencing reader performance including CAD-RADS training, clinical load, time of the day and level of expertise. Total agreement between manual and automated classification was 80.2%. Agreement in stenosis categories was 86.7%, whereas the agreement in modifiers was 95.8% for "N", 96.8% for "S", 95.6% for "V" and 99.4% for "G". Agreement for V improved after CAD-RADS training (p = 0.047). Time of the day and clinical load did not influence reader performance (p > 0.05 both). Less experienced readers had a higher total agreement as compared to more experienced readers (87.0% vs 78.0%, respectively; p = 0.011). Even though automated CAD-RADS classification uses data filled in by the readers, it outperforms manual classification by preventing human errors. Structured reporting platforms with automated calculation of the CAD-RADS score might improve data quality and support standardization of clinical decision making. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair.

PubMed

Wong, J M; Ingles, C J

2001-02-01

Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions. Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome.

Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

2009-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale.

PubMed

Yu, Xiong; Egelman, Edward H

2010-08-20

Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single-stranded DNA with approximately 9 subunits per turn in contrast to the filaments formed on double-stranded DNA with approximately 6.4 subunits per turn and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy that the Dmc1 filament formed on single-stranded DNA has a mass per unit length expected from approximately 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or scanning transmission electron microscopy, to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

A cost analysis of single-row versus double-row and suture bridge rotator cuff repair methods.

PubMed

Bisson, Leslie; Zivaljevic, Nikola; Sanders, Samuel; Pula, David

2015-02-01

To calculate the costs to the US healthcare system of transition from single-row (SR) to double-row (DR) rotator cuff repair (RCR) and to calculate the decrease in re-operations for re-tear that DR RCR would need to accomplish in order to render the transition cost-neutral. Standard accounting methods were used to determine the cost of a single RCR, the annual cost to the US healthcare system of rotator cuff surgery, the cost of a single-revision RCR, and the decrease in revision for re-tear rate necessary to make DR or suture bridge (SB) methods cost-neutral in comparison with SR methods. We varied tear size, operating room cost, time required for implant placement, annual tear size distribution, and repair method. The cost of RCR ranged from $7,572 (SR, <1 cm tear) to $12,979 (DR, >5 cm tear). Complete conversion from SR RCR to a DR technique without an associated decrease in revision surgeries would increase the annual US healthcare cost between $80 million and $262 million per year. To obtain cost neutrality, use of DR or SB methods would need to result in one fewer revision in every 17 primary repairs (for tears <1 cm) to one fewer in every four primary repairs (for tears >5 cm). Conversion from SR to DR or SB RCR techniques would result in considerable increases in healthcare expenditures. Since the large decreases in revision surgery rates necessary to justify DR or SB repairs purely on a cost basis may not be realistic or even possible, the use of these methods should be supported by evidence of improved structural healing rates and quality-adjusted life years in comparison with SR methods. IV.

The germline variants in DNA repair genes in pediatric medulloblastoma: a challenge for current therapeutic strategies.

PubMed

Trubicka, Joanna; Żemojtel, Tomasz; Hecht, Jochen; Falana, Katarzyna; Piekutowska-Abramczuk, Dorota; Płoski, Rafał; Perek-Polnik, Marta; Drogosiewicz, Monika; Grajkowska, Wiesława; Ciara, Elżbieta; Moszczyńska, Elżbieta; Dembowska-Bagińska, Bożenna; Perek, Danuta; Chrzanowska, Krystyna H; Krajewska-Walasek, Małgorzata; Łastowska, Maria

2017-04-04

The defects in DNA repair genes are potentially linked to development and response to therapy in medulloblastoma. Therefore the purpose of this study was to establish the spectrum and frequency of germline variants in selected DNA repair genes and their impact on response to chemotherapy in medulloblastoma patients. The following genes were investigated in 102 paediatric patients: MSH2 and RAD50 using targeted gene panel sequencing and NBN variants (p.I171V and p.K219fs*19) by Sanger sequencing. In three patients with presence of rare life-threatening adverse events (AE) and no detected variants in the analyzed genes, whole exome sequencing was performed. Based on combination of molecular and immunohistochemical evaluations tumors were divided into molecular subgroups. Presence of variants was tested for potential association with the occurrence of rare life-threatening AE and other clinical features. We have identified altogether six new potentially pathogenic variants in MSH2 (p.A733T and p.V606I), RAD50 (p.R1093*), FANCM (p.L694*), ERCC2 (p.R695C) and EXO1 (p.V738L), in addition to two known NBN variants. Five out of twelve patients with defects in either of MSH2, RAD50 and NBN genes suffered from rare life-threatening AE, more frequently than in control group (p = 0.0005). When all detected variants were taken into account, the majority of patients (8 out of 15) suffered from life-threatening toxicity during chemotherapy. Our results, based on the largest systematic study performed in a clinical setting, provide preliminary evidence for a link between defects in DNA repair genes and treatment related toxicity in children with medulloblastoma. The data suggest that patients with DNA repair gene variants could need special vigilance during and after courses of chemotherapy.

Genetic Interactions Between the Meiosis-Specific Cohesin Components, STAG3, REC8, and RAD21L.

PubMed

Ward, Ayobami; Hopkins, Jessica; Mckay, Matthew; Murray, Steve; Jordan, Philip W

2016-06-01

Cohesin is an essential structural component of chromosomes that ensures accurate chromosome segregation during mitosis and meiosis. Previous studies have shown that there are cohesin complexes specific to meiosis, required to mediate homologous chromosome pairing, synapsis, recombination, and segregation. Meiosis-specific cohesin complexes consist of two structural maintenance of chromosomes proteins (SMC1α/SMC1β and SMC3), an α-kleisin protein (RAD21, RAD21L, or REC8), and a stromal antigen protein (STAG1, 2, or 3). STAG3 is exclusively expressed during meiosis, and is the predominant STAG protein component of cohesin complexes in primary spermatocytes from mouse, interacting directly with each α-kleisin subunit. REC8 and RAD21L are also meiosis-specific cohesin components. Stag3 mutant spermatocytes arrest in early prophase ("zygotene-like" stage), displaying failed homolog synapsis and persistent DNA damage, as a result of unstable loading of cohesin onto the chromosome axes. Interestingly, Rec8, Rad21L double mutants resulted in an earlier "leptotene-like" arrest, accompanied by complete absence of STAG3 loading. To assess genetic interactions between STAG3 and α-kleisin subunits RAD21L and REC8, our lab generated Stag3, Rad21L, and Stag3, Rec8 double knockout mice, and compared them to the Rec8, Rad21L double mutant. These double mutants are phenotypically distinct from one another, and more severe than each single knockout mutant with regards to chromosome axis formation, cohesin loading, and sister chromatid cohesion. The Stag3, Rad21L, and Stag3, Rec8 double mutants both progress further into prophase I than the Rec8, Rad21L double mutant. Our genetic analysis demonstrates that cohesins containing STAG3 and REC8 are the main complex required for centromeric cohesion, and RAD21L cohesins are required for normal clustering of pericentromeric heterochromatin. Furthermore, the STAG3/REC8 and STAG3/RAD21L cohesins are the primary cohesins required for

Endovascular repair or open repair for ruptured abdominal aortic aneurysm: a Cochrane systematic review

PubMed Central

Badger, S A; Harkin, D W; Blair, P H; Ellis, P K; Kee, F; Forster, R

2016-01-01

Objectives Emergency endovascular aneurysm repair (eEVAR) may improve outcomes for patients with ruptured abdominal aortic aneurysm (RAAA). The study aim was to compare the outcomes for eEVAR with conventional open surgical repair for the treatment of RAAA. Setting A systematic review of relevant publications was performed. Randomised controlled trials (RCTs) comparing eEVAR with open surgical repair for RAAA were included. Participants 3 RCTs were included, with a total of 761 patients with RAAA. Interventions Meta-analysis was performed with fixed-effects models with ORs and 95% CIs for dichotomous data and mean differences with 95% CIs for continuous data. Primary and secondary outcome measures Primary outcome was short-term mortality. Secondary outcome measures included aneurysm-specific and general complication rates, quality of life and economic analysis. Results Overall risk of bias was low. There was no difference between the 2 interventions on 30-day (or in-hospital) mortality, OR 0.91 (95% CI 0.67 to 1.22; p=0.52). 30-day complications included myocardial infarction, stroke, composite cardiac complications, renal complications, severe bowel ischaemia, spinal cord ischaemia, reoperation, amputation and respiratory failure. Reporting was incomplete, and no robust conclusion was drawn. For complication outcomes that did include at least 2 studies in the meta-analysis, there was no clear evidence to support a difference between eEVAR and open repair. Longer term outcomes and cost per patient were evaluated in only a single study, thus precluding definite conclusions. Conclusions Outcomes between eEVAR and open repair, specifically 30-day mortality, are similar. However, further high-quality trials are required, as the paucity of data currently limits the conclusions. PMID:26873043

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ko, Jen-Chung; Department of Nursing, Yuanpei University, HsinChu, Taiwan; Graduate Institute of Technology Law, National Chiao Tung University, Taiwan

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)more » 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

Photodetachment and UV-Vis spectral properties of Cl2rad -·nHO clusters: Extrapolation to bulk

NASA Astrophysics Data System (ADS)

Pathak, A. K.; Mukherjee, T.; Maity, D. K.

2008-03-01

Vertical detachment energy (VDE) and UV-Vis spectra of Cl2rad -·nHO clusters ( n = 1-11) are reported based on first principle electronic structure calculations. VDE of the hydrated clusters are calculated following second order Moller-Plesset perturbation (MP2) as well as coupled cluster theory with 6-311++G(d,p) set of basis function. The excess electron in these hydrated clusters is mainly localized over the solute Cl atoms. A linear relationship is obtained for VDE vs. ( n + 2.6) -1/3 and bulk VDE of Cl2rad - aqueous solution is calculated as 10.61 eV at CCSD(T) level of theory. UV-Vis spectra of these hydrated clusters are calculated applying CI with single electron (CIS) excitation procedure. Simulated UV-Vis spectra of Cl2rad -·10HO cluster is noted to be in excellent agreement with the reported spectra of Cl2rad - (aq) system, λmax for Cl2rad -·11HO system is calculated to be red shifted though.

The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K.

Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair ismore » suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.« less

Use of Single-Cysteine Variants for Trapping Transient States in DNA Mismatch Repair.

PubMed

Friedhoff, Peter; Manelyte, Laura; Giron-Monzon, Luis; Winkler, Ines; Groothuizen, Flora S; Sixma, Titia K

2017-01-01

DNA mismatch repair (MMR) is necessary to prevent incorporation of polymerase errors into the newly synthesized DNA strand, as they would be mutagenic. In humans, errors in MMR cause a predisposition to cancer, called Lynch syndrome. The MMR process is performed by a set of ATPases that transmit, validate, and couple information to identify which DNA strand requires repair. To understand the individual steps in the repair process, it is useful to be able to study these large molecular machines structurally and functionally. However, the steps and states are highly transient; therefore, the methods to capture and enrich them are essential. Here, we describe how single-cysteine variants can be used for specific cross-linking and labeling approaches that allow trapping of relevant transient states. Analysis of these defined states in functional and structural studies is instrumental to elucidate the molecular mechanism of this important DNA MMR process. © 2017 Elsevier Inc. All rights reserved.

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae.

PubMed

Galli, Alvaro; Cervelli, Tiziana; Schiestl, Robert H

2003-05-01

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanly, J.G.; Hassan, J.; Moriarty, M.

1986-01-01

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

Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity

PubMed Central

Wang, Shu-Huei; Lin, Pei-Ya; Chiu, Ya-Chen; Huang, Ju-Sui; Kuo, Yi-Tsen; Wu, Jen-Chine; Chen, Chin-Chuan

2015-01-01

Chemo- and radiotherapy cause multiple forms of DNA damage and lead to the death of cancer cells. Inhibitors of the DNA damage response are candidate drugs for use in combination therapies to increase the efficacy of such treatments. In this study, we show that curcumin, a plant polyphenol, sensitizes budding yeast to DNA damage by counteracting the DNA damage response. Following DNA damage, the Mec1-dependent DNA damage checkpoint is inactivated and Rad52 recombinase is degraded by curcumin, which results in deficiencies in double-stand break repair. Additive effects on damage-induced apoptosis and the inhibition of damage-induced autophagy by curcumin were observed. Moreover, rpd3 mutants were found to mimic the curcumin-induced suppression of the DNA damage response. In contrast, hat1 mutants were resistant to DNA damage, and Rad52 degradation was impaired following curcumin treatment. These results indicate that the histone deacetylase inhibitor activity of curcumin is critical to DSB repair and DNA damage sensitivity. PMID:26218133