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Sample records for gene repair mediated

  1. Mismatch-mediated error prone repair at the immunoglobulin genes.

    PubMed

    Chahwan, Richard; Edelmann, Winfried; Scharff, Matthew D; Roa, Sergio

    2011-12-01

    The generation of effective antibodies depends upon somatic hypermutation (SHM) and class-switch recombination (CSR) of antibody genes by activation induced cytidine deaminase (AID) and the subsequent recruitment of error prone base excision and mismatch repair. While AID initiates and is required for SHM, more than half of the base changes that accumulate in V regions are not due to the direct deamination of dC to dU by AID, but rather arise through the recruitment of the mismatch repair complex (MMR) to the U:G mismatch created by AID and the subsequent perversion of mismatch repair from a high fidelity process to one that is very error prone. In addition, the generation of double-strand breaks (DSBs) is essential during CSR, and the resolution of AID-generated mismatches by MMR to promote such DSBs is critical for the efficiency of the process. While a great deal has been learned about how AID and MMR cause hypermutations and DSBs, it is still unclear how the error prone aspect of these processes is largely restricted to antibody genes. The use of knockout models and mice expressing mismatch repair proteins with separation-of-function point mutations have been decisive in gaining a better understanding of the roles of each of the major MMR proteins and providing further insight into how mutation and repair are coordinated. Here, we review the cascade of MMR factors and repair signals that are diverted from their canonical error free role and hijacked by B cells to promote genetic diversification of the Ig locus. This error prone process involves AID as the inducer of enzymatically-mediated DNA mismatches, and a plethora of downstream MMR factors acting as sensors, adaptors and effectors of a complex and tightly regulated process from much of which is not yet well understood.

  2. Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides

    PubMed Central

    Bertoni, Carmen; Rustagi, Arjun; Rando, Thomas A.

    2009-01-01

    Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested. PMID:19854937

  3. NKX3.1 Suppresses TMPRSS2-ERG Gene Rearrangement and Mediates Repair of Androgen Receptor-Induced DNA Damage

    PubMed Central

    Bowen, Cai; Zheng, Tian; Gelmann, Edward P.

    2015-01-01

    TMPRSS2 gene rearrangements occur at DNA breaks formed during androgen receptor-mediated transcription and activate expression of ETS transcription factors at the early stages of more than half of prostate cancers. NKX3.1, a prostate tumor suppressor that accelerates the DNA repair response, binds to androgen receptor at the ERG gene breakpoint and inhibits both the juxtaposition of the TMPRSS2 and ERG gene loci and also their recombination. NKX3.1 acts by accelerating DNA repair after androgen-induced transcriptional activation. NKX3.1 influences the recruitment of proteins that promote homology-directed DNA repair. Loss of NKX3.1 favors recruitment to the ERG gene breakpoint of proteins that promote error-prone nonhomologous end-joining. Analysis of prostate cancer tissues showed that the presence of a TMPRSS2-ERG rearrangement was highly correlated with lower levels of NKX3.1 expression consistent with the role of NKX3.1 as a suppressor of the pathogenic gene rearrangement. PMID:25977336

  4. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  5. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    SciTech Connect

    Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie; Soliymani, Rabah; Tenhunen, Mikko; Ahtiainen, Laura; Hemminki, Akseli

    2012-05-01

    Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

  6. Gene Therapy for Cartilage Repair

    PubMed Central

    Madry, Henning; Orth, Patrick; Cucchiarini, Magali

    2011-01-01

    The concept of using gene transfer strategies for cartilage repair originates from the idea of transferring genes encoding therapeutic factors into the repair tissue, resulting in a temporarily and spatially defined delivery of therapeutic molecules to sites of cartilage damage. This review focuses on the potential benefits of using gene therapy approaches for the repair of articular cartilage and meniscal fibrocartilage, including articular cartilage defects resulting from acute trauma, osteochondritis dissecans, osteonecrosis, and osteoarthritis. Possible applications for meniscal repair comprise meniscal lesions, meniscal sutures, and meniscal transplantation. Recent studies in both small and large animal models have demonstrated the applicability of gene-based approaches for cartilage repair. Chondrogenic pathways were stimulated in the repair tissue and in osteoarthritic cartilage using genes for polypeptide growth factors and transcription factors. Although encouraging data have been generated, a successful translation of gene therapy for cartilage repair will require an ongoing combined effort of orthopedic surgeons and of basic scientists. PMID:26069580

  7. Retrovirus-mediated gene transfer corrects DNA repair defect of xeroderma pigmentosum cells of complementation groups A, B and C.

    PubMed

    Zeng, L; Quilliet, X; Chevallier-Lagente, O; Eveno, E; Sarasin, A; Mezzina, M

    1997-10-01

    With the aim to devise a long-term gene therapy protocol for skin cancers in individuals affected by the inherited autosomal recessive xeroderma pigmentosum we transferred the human DNA repair XPA, XPB/ERCC3 and XPC cDNAs, by using the recombinant retroviral vector LXSN, into primary and immortalized fibroblasts obtained from two XP-A, one XP-B (associated with Cockayne's syndrome) and two XP-C patients. After transduction, the complete correction of DNA repair deficiency and functional expression of the transgenes were monitored by UV survival, unscheduled DNA synthesis and recovery of RNA synthesis, and Western blots. The results show that the recombinant retroviruses are highly efficient vectors to transfer and stably express the human DNA repair genes in XP cells and correct the defect of DNA repair of group A, B and C. With our previous results with XPD/ERCC2, the present work extends further promising issues for the gene therapy strategy for most patients suffering from this cancer-prone syndrome. PMID:9415314

  8. Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts.

    PubMed

    Wei, Wang; Yushuang, Wang; Lanlan, Huang; Zijian, Jian; Xinhua, Wang; Shouren, Liu; Wenhui, Pi

    2016-09-01

    In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to effectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene provides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts. PMID:27644744

  9. Human mediator MED17 subunit plays essential roles in gene regulation by associating with the transcription and DNA repair machineries.

    PubMed

    Kikuchi, Yuko; Umemura, Hiroyasu; Nishitani, Saori; Iida, Satoshi; Fukasawa, Rikiya; Hayashi, Hiroto; Hirose, Yutaka; Tanaka, Aki; Sugasawa, Kaoru; Ohkuma, Yoshiaki

    2015-03-01

    In eukaryotes, holo-Mediator consists of four modules: head, middle, tail, and CDK/Cyclin. The head module performs an essential function involved in regulation of RNA polymerase II (Pol II). We studied the human head module subunit MED17 (hMED17). Recent structural studies showed that yeast MED17 may function as a hinge connecting the neck and movable jaw regions of the head module to the fixed jaw region. Luciferase assays in hMED17-knockdown cells showed that hMED17 supports transcriptional activation, and pulldown assays showed that hMED17 interacted with Pol II and the general transcription factors TFIIB, TBP, TFIIE, and TFIIH. In addition, hMED17 bound to a DNA helicase subunit of TFIIH, XPB, which is essential for both transcription and nucleotide excision repair (NER). Because hMED17 associates with p53 upon UV-C irradiation, we treated human MCF-7 cells with either UV-C or the MDM2 inhibitor Nutlin-3. Both treatments resulted in accumulation of p53 in the nucleus, but hMED17 remained concentrated in the nucleus in response to UV-C. hMED17 colocalized with the NER factors XPB and XPG following UV-C irradiation, and XPG and XPB bound to hMED17 in vitro. These findings suggest that hMED17 may play essential roles in switching between transcription and NER.

  10. Cardiac regeneration: epicardial mediated repair

    PubMed Central

    2015-01-01

    The hearts of lower vertebrates such as fish and salamanders display scarless regeneration following injury, although this feature is lost in adult mammals. The remarkable capacity of the neonatal mammalian heart to regenerate suggests that the underlying machinery required for the regenerative process is evolutionarily retained. Recent studies highlight the epicardial covering of the heart as an important source of the signalling factors required for the repair process. The developing epicardium is also a major source of cardiac fibroblasts, smooth muscle, endothelial cells and stem cells. Here, we examine animal models that are capable of scarless regeneration, the role of the epicardium as a source of cells, signalling mechanisms implicated in the regenerative process and how these mechanisms influence cardiomyocyte proliferation. We also discuss recent advances in cardiac stem cell research and potential therapeutic targets arising from these studies. PMID:26702046

  11. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  12. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases

    PubMed Central

    Yang, Diane; Scavuzzo, Marissa A; Chmielowiec, Jolanta; Sharp, Robert; Bajic, Aleksandar; Borowiak, Malgorzata

    2016-01-01

    Efficient gene editing is essential to fully utilize human pluripotent stem cells (hPSCs) in regenerative medicine. Custom endonuclease-based gene targeting involves two mechanisms of DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR is the preferred mechanism for common applications such knock-in, knock-out or precise mutagenesis, but remains inefficient in hPSCs. Here, we demonstrate that synchronizing synchronizing hPSCs in G2/M with ABT phase increases on-target gene editing, defined as correct targeting cassette integration, 3 to 6 fold. We observed improved efficiency using ZFNs, TALENs, two CRISPR/Cas9, and CRISPR/Cas9 nickase to target five genes in three hPSC lines: three human embryonic stem cell lines, neural progenitors and diabetic iPSCs. neural progenitors and diabetic iPSCs. Reversible synchronization has no effect on pluripotency or differentiation. The increase in on-target gene editing is locus-independent and specific to the cell cycle phase as G2/M phase enriched cells show a 6-fold increase in targeting efficiency compared to cells in G1 phase. Concurrently inhibiting NHEJ with SCR7 does not increase HDR or improve gene targeting efficiency further, indicating that HR is the major DNA repair mechanism after G2/M phase arrest. The approach outlined here makes gene editing in hPSCs a more viable tool for disease modeling, regenerative medicine and cell-based therapies. PMID:26887909

  13. DNA repair genes in the Megavirales pangenome.

    PubMed

    Blanc-Mathieu, Romain; Ogata, Hiroyuki

    2016-06-01

    The order 'Megavirales' represents a group of eukaryotic viruses with a large genome encoding a few hundred up to two thousand five hundred genes. Several members of Megavirales possess genes involved in major DNA repair pathways. Some of these genes were likely inherited from an ancient virus world and some others were derived from the genomes of their hosts. Here we examine molecular phylogenies of key DNA repair enzymes in light of recent hypotheses on the origin of Megavirales, and propose that the last common ancestors of the individual families of the order Megavirales already possessed DNA repair functions to achieve and maintain a moderately large genome and that this repair capacity gradually increased, in a family-dependent manner, during their recent evolution.

  14. Temporal activation of XRCC1-mediated DNA repair is essential for muscle differentiation

    PubMed Central

    Al-Khalaf, Mohammad H; Blake, Leanne E; Larsen, Brian D; Bell, Ryan A; Brunette, Steve; Parks, Robin J; Rudnicki, Michael A; McKinnon, Peter J; Jeffrey Dilworth, F; Megeney, Lynn A

    2016-01-01

    Transient DNA strand break formation has been identified as an effective means to enhance gene expression in living cells. In the muscle lineage, cell differentiation is contingent upon the induction of caspase-mediated DNA strand breaks, which act to establish the terminal gene expression program. This coordinated DNA nicking is rapidly resolved, suggesting that myoblasts may deploy DNA repair machinery to stabilize the genome and entrench the differentiated phenotype. Here, we identify the base excision repair pathway component XRCC1 as an indispensable mediator of muscle differentiation. Caspase-triggered XRCC1 repair foci form rapidly within differentiating myonuclei, and then dissipate as the maturation program proceeds. Skeletal myoblast deletion of Xrcc1 does not have an impact on cell growth, yet leads to perinatal lethality, with sustained DNA damage and impaired myofiber development. Together, these results demonstrate that XRCC1 manages a temporally responsive DNA repair process to advance the muscle differentiation program. PMID:27462438

  15. A GRHL3-regulated repair pathway suppresses immune-mediated epidermal hyperplasia

    PubMed Central

    Gordon, William M.; Zeller, Michael D.; Klein, Rachel H.; Swindell, William R.; Ho, Hsiang; Espetia, Francisco; Gudjonsson, Johann E.; Baldi, Pierre F.; Andersen, Bogi

    2014-01-01

    Dermal infiltration of T cells is an important step in the onset and progression of immune-mediated skin diseases such as psoriasis; however, it is not known whether epidermal factors play a primary role in the development of these diseases. Here, we determined that the prodifferentiation transcription factor grainyhead-like 3 (GRHL3), which is essential during epidermal development, is dispensable for adult skin homeostasis, but required for barrier repair after adult epidermal injury. Consistent with activation of a GRHL3-regulated repair pathway in psoriasis, we found that GRHL3 is upregulated in lesional skin and binds known epidermal differentiation gene targets. Using an imiquimod-induced model of immune-mediated epidermal hyperplasia, we found that mice lacking GRHL3 have an exacerbated epidermal damage response, greater sensitivity to disease induction, delayed resolution of epidermal lesions, and resistance to anti–IL-22 therapy compared with WT animals. ChIP-Seq and gene expression profiling of murine skin revealed that while GRHL3 regulates differentiation pathways both during development and during repair from immune-mediated damage, it targets distinct sets of genes in the 2 processes. In particular, GRHL3 suppressed a number of alarmin and other proinflammatory genes after immune injury. This study identifies a GRHL3-regulated epidermal barrier repair pathway that suppresses disease initiation and helps resolve existing lesions in immune-mediated epidermal hyperplasia. PMID:25347468

  16. Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.

    PubMed

    Ceccaldi, Raphael; Liu, Jessica C; Amunugama, Ravindra; Hajdu, Ildiko; Primack, Benjamin; Petalcorin, Mark I R; O'Connor, Kevin W; Konstantinopoulos, Panagiotis A; Elledge, Stephen J; Boulton, Simon J; Yusufzai, Timur; D'Andrea, Alan D

    2015-02-12

    Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ) in a pathway required for the repair of DNA double-strand breaks, referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway. Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy.

  17. Lambda Red Mediated Gap Repair Utilizes a Novel Replicative Intermediate in Escherichia coli

    PubMed Central

    Reddy, Thimma R.; Fevat, Léna M. S.; Munson, Sarah E.; Stewart, A. Francis; Cowley, Shaun M.

    2015-01-01

    The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction

  18. Caspase-1 mediates hyperlipidemia-weakened progenitor cell vessel repair

    PubMed Central

    Li, Ya-Feng; Huang, Xiao; Li, Xinyuan; Gong, Ren; Yin, Ying; Nelson, Jun; Gao, Erhe; Zhang, Hongyu; Hoffman, Nicholas E.; Houser, Steven R.; Madesh, Muniswamy; Tilley, Douglas G.; Choi, Eric T.; Jiang, Xiaohua; Huang, Cong-Xin; Wang, Hong; Yang, Xiao-Feng

    2015-01-01

    Caspase-1 activation senses metabolic danger-associated molecular patterns (DAMPs) and mediates the initiation of inflammation in endothelial cells. Here, we examined whether the caspase-1 pathway is responsible for sensing hyperlipidemia as a DAMP in bone marrow (BM)-derived Stem cell antigen-1 positive (Sca-1+) stem/progenitor cells and weakening their angiogenic ability. Using biochemical methods, gene knockout, cell therapy and myocardial infarction (MI) models, we had the following findings: 1) Hyperlipidemia induces caspase-1 activity in mouse Sca-1+ progenitor cells in vivo; 2) Caspase-1 contributes to hyperlipidemia-induced modulation of vascular cell death-related gene expression in vivo; 3) Injection of Sca-1+ progenitor cells from caspase-1−/− mice improves endothelial capillary density in heart and decreases cardiomyocyte death in a mouse model of MI; and 4) Caspase-1−/− Sca-1+ progenitor cell therapy improves mouse cardiac function after MI. Our results provide insight on how hyperlipidemia activates caspase-1 in Sca-1+ progenitor cells, which subsequently weakens Sca-1+ progenitor cell repair of vasculature injury. These results demonstrate the therapeutic potential of caspase-1 inhibition in improving progenitor cell therapy for MI. PMID:26709768

  19. RNA-mediated gene activation

    PubMed Central

    Jiao, Alan L; Slack, Frank J

    2014-01-01

    The regulation of gene expression by non-coding RNAs (ncRNAs) has become a new paradigm in biology. RNA-mediated gene silencing pathways have been studied extensively, revealing diverse epigenetic and posttranscriptional mechanisms. In contrast, the roles of ncRNAs in activating gene expression remains poorly understood. In this review, we summarize the current knowledge of gene activation by small RNAs, long non-coding RNAs, and enhancer-derived RNAs, with an emphasis on epigenetic mechanisms. PMID:24185374

  20. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  1. Macrophage-Mediated Injury and Repair After Ischemic Kidney Injury

    PubMed Central

    Huen, Sarah C.; Cantley, Lloyd G.

    2016-01-01

    Acute ischemic kidney injury is a common complication in hospitalized patients. Currently no treatment is available for augmenting kidney repair or preventing progressive kidney fibrosis. Animal models of acute kidney injury demonstrate that activation of the innate immune system plays a major role in the systemic response to ischemia/reperfusion injury. Macrophage depletion studies suggest that macrophages, key participants in the innate immune response, augment the initial injury after reperfusion, but also promote tubular repair and contribute to long-term kidney fibrosis after ischemic injury. The distinct functional outcomes seen following macrophage depletion at different time points after ischemia/reperfusion injury suggest heterogeneity in macrophage activation states. Identifying the pathways that regulate the transitions of macrophage activation is thus critical for understanding the mechanisms that govern both macrophage-mediated injury and repair in the post-ischemic kidney. This review examines our current understanding of the complex and intricately controlled pathways that determine monocyte recruitment, macrophage activation, and macrophage effector functions after renal ischemia/reperfusion injury. Careful delineation of repair and resolution pathways could provide therapeutic targets for the development of effective treatments to offer patients with acute kidney injury. PMID:24442822

  2. Nanoparticle-Mediated Gene Delivery

    NASA Astrophysics Data System (ADS)

    Jin, Sha; Leach, John C.; Ye, Kaiming

    Nonviral gene delivery has been gaining considerable attention recently. Although the efficacy of DNA transfection, which is a major concern, is low in nonviral vector-mediated gene transfer compared with viral ones, nonviral vectors are relatively easy to prepare, less immunogenic and oncogenic, and have no potential of virus recombination and no limitation on the size of a transferred gene. The ability to incorporate genetic materials such as plasmid DNA, RNA, and siRNA into functionalized nanoparticles with little toxicity demonstrates a new era in pharmacotherapy for delivering genes selectively to tissues and cells. In this chapter, we highlight the basic concepts and applications of nonviral gene delivery using super paramagnetic iron oxide nanoparticles and functionalized silica nanoparticles. The experimental protocols related to these topics are described in the chapter.

  3. Genomic landscape of DNA repair genes in cancer.

    PubMed

    Chae, Young Kwang; Anker, Jonathan F; Carneiro, Benedito A; Chandra, Sunandana; Kaplan, Jason; Kalyan, Aparna; Santa-Maria, Cesar A; Platanias, Leonidas C; Giles, Francis J

    2016-04-26

    DNA repair genes are frequently mutated in cancer, yet limited data exist regarding the overall genomic landscape and functional implications of these alterations in their entirety. We created comprehensive lists of DNA repair genes and indirect caretakers. Mutation, copy number variation (CNV), and expression frequencies of these genes were analyzed in COSMIC. Mutation co-occurrence, clinical outcomes, and mutation burden were analyzed in TCGA. We report the 20 genes most frequently with mutations (n > 19,689 tumor samples for each gene), CNVs (n > 1,556), or up- or down-regulated (n = 7,998). Mutual exclusivity was observed as no genes displayed both high CNV gain and loss or high up- and down-regulation, and CNV gain and loss positively correlated with up- and down-regulation, respectively. Co-occurrence of mutations differed between cancers, and mutations in many DNA repair genes were associated with higher total mutation burden. Mutation and CNV frequencies offer insights into which genes may play tumor suppressive or oncogenic roles, such as NEIL2 and RRM2B, respectively. Mutual exclusivities within CNV and expression frequencies, and correlations between CNV and expression, support the functionality of these genomic alterations. This study provides comprehensive lists of candidate genes as potential biomarkers for genomic instability, novel therapeutic targets, or predictors of immunotherapy efficacy.

  4. Genomic landscape of DNA repair genes in cancer

    PubMed Central

    Carneiro, Benedito A.; Chandra, Sunandana; Kaplan, Jason; Kalyan, Aparna; Santa-Maria, Cesar A.; Platanias, Leonidas C.; Giles, Francis J.

    2016-01-01

    DNA repair genes are frequently mutated in cancer, yet limited data exist regarding the overall genomic landscape and functional implications of these alterations in their entirety.  We created comprehensive lists of DNA repair genes and indirect caretakers.  Mutation, copy number variation (CNV), and expression frequencies of these genes were analyzed in COSMIC. Mutation co-occurrence, clinical outcomes, and mutation burden were analyzed in TCGA. We report the 20 genes most frequently with mutations (n > 19,689 tumor samples for each gene), CNVs (n > 1,556), or up- or down-regulated (n = 7,998).  Mutual exclusivity was observed as no genes displayed both high CNV gain and loss or high up- and down-regulation, and CNV gain and loss positively correlated with up- and down-regulation, respectively. Co-occurrence of mutations differed between cancers, and mutations in many DNA repair genes were associated with higher total mutation burden. Mutation and CNV frequencies offer insights into which genes may play tumor suppressive or oncogenic roles, such as NEIL2 and RRM2B, respectively.  Mutual exclusivities within CNV and expression frequencies, and correlations between CNV and expression, support the functionality of these genomic alterations. This study provides comprehensive lists of candidate genes as potential biomarkers for genomic instability, novel therapeutic targets, or predictors of immunotherapy efficacy. PMID:27004405

  5. Electroporation-mediated gene delivery.

    PubMed

    Young, Jennifer L; Dean, David A

    2015-01-01

    Electroporation has been used extensively to transfer DNA to bacteria, yeast, and mammalian cells in culture for the past 30 years. Over this time, numerous advances have been made, from using fields to facilitate cell fusion, delivery of chemotherapeutic drugs to cells and tissues, and most importantly, gene and drug delivery in living tissues from rodents to man. Electroporation uses electrical fields to transiently destabilize the membrane allowing the entry of normally impermeable macromolecules into the cytoplasm. Surprisingly, at the appropriate field strengths, the application of these fields to tissues results in little, if any, damage or trauma. Indeed, electroporation has even been used successfully in human trials for gene delivery for the treatment of tumors and for vaccine development. Electroporation can lead to between 100 and 1000-fold increases in gene delivery and expression and can also increase both the distribution of cells taking up and expressing the DNA as well as the absolute amount of gene product per cell (likely due to increased delivery of plasmids into each cell). Effective electroporation depends on electric field parameters, electrode design, the tissues and cells being targeted, and the plasmids that are being transferred themselves. Most importantly, there is no single combination of these variables that leads to greatest efficacy in every situation; optimization is required in every new setting. Electroporation-mediated in vivo gene delivery has proven highly effective in vaccine production, transgene expression, enzyme replacement, and control of a variety of cancers. Almost any tissue can be targeted with electroporation, including muscle, skin, heart, liver, lung, and vasculature. This chapter will provide an overview of the theory of electroporation for the delivery of DNA both in individual cells and in tissues and its application for in vivo gene delivery in a number of animal models. PMID:25620008

  6. Electroporation-mediated gene delivery.

    PubMed

    Young, Jennifer L; Dean, David A

    2015-01-01

    Electroporation has been used extensively to transfer DNA to bacteria, yeast, and mammalian cells in culture for the past 30 years. Over this time, numerous advances have been made, from using fields to facilitate cell fusion, delivery of chemotherapeutic drugs to cells and tissues, and most importantly, gene and drug delivery in living tissues from rodents to man. Electroporation uses electrical fields to transiently destabilize the membrane allowing the entry of normally impermeable macromolecules into the cytoplasm. Surprisingly, at the appropriate field strengths, the application of these fields to tissues results in little, if any, damage or trauma. Indeed, electroporation has even been used successfully in human trials for gene delivery for the treatment of tumors and for vaccine development. Electroporation can lead to between 100 and 1000-fold increases in gene delivery and expression and can also increase both the distribution of cells taking up and expressing the DNA as well as the absolute amount of gene product per cell (likely due to increased delivery of plasmids into each cell). Effective electroporation depends on electric field parameters, electrode design, the tissues and cells being targeted, and the plasmids that are being transferred themselves. Most importantly, there is no single combination of these variables that leads to greatest efficacy in every situation; optimization is required in every new setting. Electroporation-mediated in vivo gene delivery has proven highly effective in vaccine production, transgene expression, enzyme replacement, and control of a variety of cancers. Almost any tissue can be targeted with electroporation, including muscle, skin, heart, liver, lung, and vasculature. This chapter will provide an overview of the theory of electroporation for the delivery of DNA both in individual cells and in tissues and its application for in vivo gene delivery in a number of animal models.

  7. Targeting of AID-mediated sequence diversification to immunoglobulin genes.

    PubMed

    Kothapalli, Naga Rama; Fugmann, Sebastian D

    2011-04-01

    Activation-induced cytidine deaminase (AID) is a key enzyme for antibody-mediated immune responses. Antibodies are encoded by the immunoglobulin genes and AID acts as a transcription-dependent DNA mutator on these genes to improve antibody affinity and effector functions. An emerging theme in field is that many transcribed genes are potential targets of AID, presenting an obvious danger to genomic integrity. Thus there are mechanisms in place to ensure that mutagenic outcomes of AID activity are specifically restricted to the immunoglobulin loci. Cis-regulatory targeting elements mediate this effect and their mode of action is probably a combination of immunoglobulin gene specific activation of AID and a perversion of faithful DNA repair towards error-prone outcomes.

  8. Non-functional genes repaired at the RNA level.

    PubMed

    Burger, Gertraud

    2016-01-01

    Genomes and genes continuously evolve. Gene sequences undergo substitutions, deletions or nucleotide insertions; mobile genetic elements invade genomes and interleave in genes; chromosomes break, even within genes, and pieces reseal in reshuffled order. To maintain functional gene products and assure an organism's survival, two principal strategies are used - either repair of the gene itself or of its product. I will introduce common types of gene aberrations and how gene function is restored secondarily, and then focus on systematically fragmented genes found in a poorly studied protist group, the diplonemids. Expression of their broken genes involves restitching of pieces at the RNA-level, and substantial RNA editing, to compensate for point mutations. I will conclude with thoughts on how such a grotesquely unorthodox system may have evolved, and why this group of organisms persists and thrives since tens of millions of years.

  9. Hodgkin Lymphoma Risk: Role of Genetic Polymorphisms and Gene-Gene Interactions in DNA repair pathways

    PubMed Central

    Monroy, Claudia M.; Cortes, Andrea C.; Lopez, Mirtha; Rourke, Elizabeth; Etzel, Carol J.; Younes, Anas; Strom, Sara S.; El-Zein, Randa

    2011-01-01

    DNA repair variants may play a potentially important role in an individual’s susceptibility to developing cancer. Numerous studies have reported the association between genetic single nucleotide polymorphisms (SNPs) in DNA repair genes and different types of hematologic cancers. However, to date, the effects of such SNPs on modulating Hodgkin Lymphoma (HL) risk have not yet been investigated. We hypothesized that gene-gene interaction between candidate genes in Direct Reversal, Nucleotide excision repair (NER), Base excision repair (BER) and Double strand break (DSB) pathways may contribute to susceptibility to HL. To test this hypothesis, we conducted a study on 200 HL cases and 220 controls to assess associations between HL risk and 21 functional SNPs in DNA repair genes. We evaluated potential gene-gene interactions and the association of multiple polymorphisms in a chromosome region using a multi-analytic strategy combining logistic regression, multi-factor dimensionality reduction and classification and regression tree approaches. We observed that, in combination, allelic variants in the XPC Ala499Val, NBN Glu185Gln, XRCC3 Thr241Me, XRCC1 Arg194Trp and XRCC1 399Gln polymorphisms modify the risk for developing HL. Moreover, the cumulative genetic risk score revealed a significant trend where the risk for developing HL increases as the number of adverse alleles in BER and DSB genes increase. These findings suggest that DNA repair variants in BER and DSB pathways may play an important role in the development of HL. PMID:21374732

  10. Regulation of targeted gene repair by intrinsic cellular processes.

    PubMed

    Engstrom, Julia U; Suzuki, Takayuki; Kmiec, Eric B

    2009-02-01

    Targeted gene alteration (TGA) is a strategy for correcting single base mutations in the DNA of human cells that cause inherited disorders. TGA aims to reverse a phenotype by repairing the mutant base within the chromosome itself, avoiding the introduction of exogenous genes. The process of how to accurately repair a genetic mutation is elucidated through the use of single-stranded DNA oligonucleotides (ODNs) that can enter the cell and migrate to the nucleus. These specifically designed ODNs hybridize to the target sequence and act as a beacon for nucleotide exchange. The key to this reaction is the frequency with which the base is corrected; this will determine whether the approach becomes clinically relevant or not. Over the course of the last five years, workers have been uncovering the role played by the cells in regulating the gene repair process. In this essay, we discuss how the impact of the cell on TGA has evolved through the years and illustrate ways that inherent cellular pathways could be used to enhance TGA activity. We also describe the cost to cell metabolism and survival when certain processes are altered to achieve a higher frequency of repair.

  11. Bone tissue engineering and repair by gene therapy.

    PubMed

    Betz, Volker M; Betz, Oliver B; Harris, Mitchel B; Vrahas, Mark S; Evans, Christopher H

    2008-01-01

    Many clinical conditions require the stimulation of bone growth. The use of recombinant bone morphogenetic proteins does not provide a satisfying solution to these conditions due to delivery problems and high cost. Gene therapy has emerged as a very promising approach for bone repair that overcomes limitations of protein-based therapy. Several preclinical studies have shown that gene transfer technology has the ability to deliver osteogenic molecules to precise anatomical locations at therapeutic levels for sustained periods of time. Both in-vivo and ex-vivo transduction of cells can induce bone formation at ectopic and orthotopic sites. Genetic engineering of adult stem cells from various sources with osteogenic genes has led to enhanced fracture repair, spinal fusion and rapid healing of bone defects in animal models. This review describes current viral and non-viral gene therapy strategies for bone tissue engineering and repair including recent work from the author's laboratory. In addition, the article discusses the potential of gene-enhanced tissue engineering to enter widespread clinical use.

  12. uv excision-repair gene transfer in Chinese hamster ovary (CHO) cells

    SciTech Connect

    MacInnes, M.A.; Bingham, J.M.; Strniste, G.F.; Thompson, L.H.

    1983-01-01

    uvc-sensitive mutants of CHO cells provide a model system for molecular studies of DNA repair. We present our recent results which show that these mutants are competent recipients for plasmid marker gene transfer and incorporation of a putative CHO repair gene. The applicability and advantages of this system for interspecies human repair gene identification are discussed.

  13. The human CSB (ERCC6) gene corrects the transcription-coupled repair defect in the CHO cell mutant UV61.

    PubMed

    Orren, D K; Dianov, G L; Bohr, V A

    1996-09-01

    The human CSB gene, mutated in Cockayne's syndrome group B (partially defective in both repair and transcription) was previously cloned by virtue of its ability to correct the moderate UV sensitivity of the CHO mutant UV61. To determine whether the defect in UV61 is the hamster equivalent of Cockayne's syndrome, the RNA polymerase II transcription and DNA repair characteristics of a repair-proficient CHO cell line (AA8), UV61 and a CSB transfectant of UV61 were compared. In each cell line, formation and removal of UV-induced cyclobutane pyrimidine dimers (CPDs) were measured in the individual strands of the actively transcribed DHFR gene and in a transcriptionally inactive region downstream of DHFR. AA8 cells efficiently remove CPDs from the transcribed strand, but not from either the non-transcribed strand or the inactive region. There was no detectable repair of CPDs in any region of the genome in UV61. Transfection of the human CSB gene into UV61 restores the normal repair pattern (CPD removal in only the transcribed strand), demonstrating that the DNA repair defect in UV61 is homologous to that in Cockayne's syndrome (complementation group B) cells. However, we observe no significant deficiency in RNA polymerase II-mediated transcription in UV61, suggesting that the CSB protein has independent roles in DNA repair and RNA transcription pathways. PMID:8811084

  14. Phonological Mediation in Visual Masked Priming: Evidence from Phonotactic Repair

    ERIC Educational Resources Information Center

    Halle, Pierre A.; Dominguez, Alberto; Cuetos, Fernando; Segui, Juan

    2008-01-01

    In a series of 4 experiments, the authors show that phonological repair mechanisms, known to operate in the auditory modality, are directly translated in the visual modality. This holds with the provision that printed stimuli are presented for a very brief duration and that the effect of phonological repair is tested after a delay of some 100 ms…

  15. Expression of the excision repair gene, ERCC3 (excision repair cross-complementing), during mouse development.

    PubMed

    Hubank, M; Mayne, L

    1994-08-12

    Expression of the human ERCC3 (excision repair cross-complementing) gene in cells from patients with xeroderma pigmentosum (XP) group B (XP-B) corrects the defect in repair of UV light-induced DNA damage. XP-B is one of three groups of XP which exhibit the clinical symptoms of both XP and Cockayne's Syndrome (CS). CS and XP-B/CS patients develop severe neurological dysfunction during development. In order to explore the link between the defective gene and the neurological deficits in XP/CS, we have studied the expression of ERCC3 mRNA in developing mice by in situ hybridisation. ERCC3 was found to be ubiquitously expressed in cells from all regions and all developmental stages, from 9 day post-coitum embryo, to 15 day post-natal brain. In post-natal brain, regional differences in expression correlated with cell density and there was no evidence of cell specific or developmental alterations in levels of expression. These results indicate that the constitutively expressed gene does not perform a discrete developmental function. The neurological defects apparent in XP-B are likely to arise pleiotypically from the participation of ERCC3 in interactions with other elements involved in particular aspects of neurodevelopmental control. These results emphasise the developmental importance of genes whose primary functions are apparently unconnected with development. PMID:7805288

  16. Double-strand break damage and associated DNA repair genes predispose smokers to gene methylation

    PubMed Central

    Leng, Shuguang; Stidley, Christine A.; Willink, Randy; Bernauer, Amanda; Do, Kieu; Picchi, Maria A.; Sheng, Xin; Frasco, Melissa, A.; Berg, David Van Den; Gilliland, Frank D.; Zima, Christopher; Crowell, Richard E.; Belinsky, Steven A.

    2008-01-01

    Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could impact strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of double-strand break repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as ≥ 3 of 8 genes methylated in sputum, compared to smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-Pkc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with ≥ 7 risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ATM was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify double-strand break DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, that is, in turn, associated with elevated risk for lung cancer. PMID:18413776

  17. Biomaterial-mediated strategies targeting vascularization for bone repair.

    PubMed

    García, José R; García, Andrés J

    2016-04-01

    Repair of non-healing bone defects through tissue engineering strategies remains a challenging feat in the clinic due to the aversive microenvironment surrounding the injured tissue. The vascular damage that occurs following a bone injury causes extreme ischemia and a loss of circulating cells that contribute to regeneration. Tissue-engineered constructs aimed at regenerating the injured bone suffer from complications based on the slow progression of endogenous vascular repair and often fail at bridging the bone defect. To that end, various strategies have been explored to increase blood vessel regeneration within defects to facilitate both tissue-engineered and natural repair processes. Developments that induce robust vascularization will need to consolidate various parameters including optimization of embedded therapeutics, scaffold characteristics, and successful integration between the construct and the biological tissue. This review provides an overview of current strategies as well as new developments in engineering biomaterials to induce reparation of a functional vascular supply in the context of bone repair.

  18. 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.

  19. DNA mismatch repair gene mutations in human cancer.

    PubMed Central

    Peltomäki, P

    1997-01-01

    A new pathogenetic mechanism leading to cancer has been delineated in the past 3 years when human homologues of DNA mismatch repair (MMR) genes have been identified and shown to be involved in various types of cancer. Germline mutations of MMR genes cause susceptibility to a hereditary form of colon cancer, hereditary nonpolyposis colon cancer (HNPCC), which represents one of the most common syndromes associated with cancer predisposition in man. Tumors from HNPCC patients are hypermutable and show length variation at short tandem repeat sequences, a phenomenon referred to as microsatellite instability or replication errors. A similar abnormality is found in a proportion of sporadic tumors of the colorectum as well as a variety of other organs; acquired mutations in MMR genes or other endogenous or exogenous causes may underlie these cases. Genetic and biochemical characterization of the functions of normal and mutated MMR genes elucidates mechanisms of cancer development and provides tools for diagnostic applications. PMID:9255561

  20. The Schizosaccharomyces pombe rhp3+ gene required for DNA repair and cell viability is functionally interchangeable with the RAD3 gene of Saccharomyces cerevisiae.

    PubMed Central

    Reynolds, P R; Biggar, S; Prakash, L; Prakash, S

    1992-01-01

    The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA.RNA helicase activities. Mutational studies have indicated a requirement for the RAD3 helicase activities in excision repair. To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, we have cloned the RAD3 homolog, rhp3+, from the distantly related yeast Schizosaccharomyces pombe. RAD3 and rhp3+ encoded proteins are highly similar, sharing 67% identical amino acids. We show that like RAD3, rhp3+ is indispensable for excision repair and cell viability, and our studies indicate a requirement of the putative rhp3+ DNA helicase activity in DNA repair. We find that the RAD3 and rhp3+ genes can functionally substitute for one another. The level of complementation provided by the rhp3+ gene in S.cerevisiae rad3 mutants or by the RAD3 gene in S.pombe rhp3 mutants is remarkable in that both the excision repair and viability defects in both yeasts are restored to wild type levels. These observations suggest a parallel evolutionary conservation of other protein components with which RAD3 interacts in mediating its DNA repair and viability functions. Images PMID:1534406

  1. Substrate-mediated reprogramming of human fibroblasts into neural crest stem-like cells and their applications in neural repair.

    PubMed

    Tseng, Ting-Chen; Hsieh, Fu-Yu; Dai, Niann-Tzyy; Hsu, Shan-Hui

    2016-09-01

    Cell- and gene-based therapies have emerged as promising strategies for treating neurological diseases. The sources of neural stem cells are limited while the induced pluripotent stem (iPS) cells have risk of tumor formation. Here, we proposed the generation of self-renewable, multipotent, and neural lineage-related neural crest stem-like cells by chitosan substrate-mediated gene transfer of a single factor forkhead box D3 (FOXD3) for the use in neural repair. A simple, non-toxic, substrate-mediated method was applied to deliver the naked FOXD3 plasmid into human fibroblasts. The transfection of FOXD3 increased cell proliferation and up-regulated the neural crest marker genes (FOXD3, SOX2, and CD271), stemness marker genes (OCT4, NANOG, and SOX2), and neural lineage-related genes (Nestin, β-tubulin and GFAP). The expression levels of stemness marker genes and neural crest maker genes in the FOXD3-transfected fibroblasts were maintained until the fifth passage. The FOXD3 reprogrammed fibroblasts based on the new method significantly rescued the neural function of the impaired zebrafish. The chitosan substrate-mediated delivery of naked plasmid showed feasibility in reprogramming somatic cells. Particularly, the FOXD3 reprogrammed fibroblasts hold promise as an easily accessible cellular source with neural crest stem-like behavior for treating neural diseases in the future. PMID:27341268

  2. The theory of RNA-mediated gene evolution

    PubMed Central

    Morris, Kevin V

    2015-01-01

    Observations over the last decade suggest that some RNA transcripts, such as non-coding RNAs, function in regulating the transcriptional and epigenetic state of gene expression. DNA methylation appears to be operative in non-coding RNA regulation of gene expression. Interestingly, methylated cytosines undergo deamination to remove the methylation, which if not properly repaired results in the methylated cytosine being recognized by the cell as a thymine. This observation suggests that the process of non-coding RNA-directed epigenetic targeting also has the potential to alter the genomic landscape of the cell by changing cytosines to thymines and ultimately influence the evolution of the cell. This proposed theory of “RNA-mediated gene evolution” might be one possible mechanism of action whereby RNA participates in the natural selective process to drive cellular and possibly organismal evolution. PMID:25581873

  3. Dysferlin-mediated phosphatidylserine sorting engages macrophages in sarcolemma repair.

    PubMed

    Middel, Volker; Zhou, Lu; Takamiya, Masanari; Beil, Tanja; Shahid, Maryam; Roostalu, Urmas; Grabher, Clemens; Rastegar, Sepand; Reischl, Markus; Nienhaus, Gerd Ulrich; Strähle, Uwe

    2016-01-01

    Failure to repair the sarcolemma leads to muscle cell death, depletion of stem cells and myopathy. Hence, membrane lesions are instantly sealed by a repair patch consisting of lipids and proteins. It has remained elusive how this patch is removed to restore cell membrane integrity. Here we examine sarcolemmal repair in live zebrafish embryos by real-time imaging. Macrophages remove the patch. Phosphatidylserine (PS), an 'eat-me' signal for macrophages, is rapidly sorted from adjacent sarcolemma to the repair patch in a Dysferlin (Dysf) dependent process in zebrafish and human cells. A previously unrecognized arginine-rich motif in Dysf is crucial for PS accumulation. It carries mutations in patients presenting with limb-girdle muscular dystrophy 2B. This underscores the relevance of this sequence and uncovers a novel pathophysiological mechanism underlying this class of myopathies. Our data show that membrane repair is a multi-tiered process involving immediate, cell-intrinsic mechanisms as well as myofiber/macrophage interactions. PMID:27641898

  4. Dysferlin-mediated phosphatidylserine sorting engages macrophages in sarcolemma repair.

    PubMed

    Middel, Volker; Zhou, Lu; Takamiya, Masanari; Beil, Tanja; Shahid, Maryam; Roostalu, Urmas; Grabher, Clemens; Rastegar, Sepand; Reischl, Markus; Nienhaus, Gerd Ulrich; Strähle, Uwe

    2016-09-19

    Failure to repair the sarcolemma leads to muscle cell death, depletion of stem cells and myopathy. Hence, membrane lesions are instantly sealed by a repair patch consisting of lipids and proteins. It has remained elusive how this patch is removed to restore cell membrane integrity. Here we examine sarcolemmal repair in live zebrafish embryos by real-time imaging. Macrophages remove the patch. Phosphatidylserine (PS), an 'eat-me' signal for macrophages, is rapidly sorted from adjacent sarcolemma to the repair patch in a Dysferlin (Dysf) dependent process in zebrafish and human cells. A previously unrecognized arginine-rich motif in Dysf is crucial for PS accumulation. It carries mutations in patients presenting with limb-girdle muscular dystrophy 2B. This underscores the relevance of this sequence and uncovers a novel pathophysiological mechanism underlying this class of myopathies. Our data show that membrane repair is a multi-tiered process involving immediate, cell-intrinsic mechanisms as well as myofiber/macrophage interactions.

  5. Dysferlin-mediated phosphatidylserine sorting engages macrophages in sarcolemma repair

    PubMed Central

    Middel, Volker; Zhou, Lu; Takamiya, Masanari; Beil, Tanja; Shahid, Maryam; Roostalu, Urmas; Grabher, Clemens; Rastegar, Sepand; Reischl, Markus; Nienhaus, Gerd Ulrich; Strähle, Uwe

    2016-01-01

    Failure to repair the sarcolemma leads to muscle cell death, depletion of stem cells and myopathy. Hence, membrane lesions are instantly sealed by a repair patch consisting of lipids and proteins. It has remained elusive how this patch is removed to restore cell membrane integrity. Here we examine sarcolemmal repair in live zebrafish embryos by real-time imaging. Macrophages remove the patch. Phosphatidylserine (PS), an ‘eat-me' signal for macrophages, is rapidly sorted from adjacent sarcolemma to the repair patch in a Dysferlin (Dysf) dependent process in zebrafish and human cells. A previously unrecognized arginine-rich motif in Dysf is crucial for PS accumulation. It carries mutations in patients presenting with limb-girdle muscular dystrophy 2B. This underscores the relevance of this sequence and uncovers a novel pathophysiological mechanism underlying this class of myopathies. Our data show that membrane repair is a multi-tiered process involving immediate, cell-intrinsic mechanisms as well as myofiber/macrophage interactions. PMID:27641898

  6. Protein phosphatase 5 is necessary for ATR-mediated DNA repair

    SciTech Connect

    Kang, Yoonsung; Cheong, Hyang-Min; Lee, Jung-Hee; Song, Peter I.; Lee, Kwang-Ho; Kim, Sang-Yong; Jun, Jae Yeoul; You, Ho Jin

    2011-01-07

    Research highlights: {yields} Serine/threonine protein phosphatase 5 (PP5) has been shown to participate in ataxia telangiectasia-mutated (ATM)- and ATR (ATM- and Rad3-related)-mediated checkpoint pathways, which plays an important role in the DNA damage response and maintenance of genomic stability. {yields} However, it is not clear exactly how PP5 participates in this process. {yields} Our results indicate that PP5 is more closely related with ATR-mediated pathway than ATM-mediated pathway in DNA damage repair. -- Abstract: Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.

  7. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

    PubMed Central

    Richardson, C. D.; Ray, G. J.; Bray, N. L.; Corn, J. E.

    2016-01-01

    The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9–sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption. PMID:27530320

  8. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes.

    PubMed

    Richardson, C D; Ray, G J; Bray, N L; Corn, J E

    2016-01-01

    The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption. PMID:27530320

  9. Mating-type suppression of the DNA-repair defect of the yeast rad6 delta mutation requires the activity of genes in the RAD52 epistasis group.

    PubMed

    Yan, Y X; Schiestl, R H; Prakash, L

    1995-06-01

    The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MATa rad6 delta strain can be suppressed by the MAT alpha 2 gene carried on a multicopy plasmid. The a1-alpha 2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-alpha 2 suppression of the rad6 delta mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 delta mutant into the RAD52 recombinational repair pathway. The a1-alpha 2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.

  10. CRISPR-Cas9-Mediated Single-Gene and Gene Family Disruption in Trypanosoma cruzi

    PubMed Central

    Peng, Duo; Kurup, Samarchith P.; Yao, Phil Y.; Minning, Todd A.

    2014-01-01

    ABSTRACT Trypanosoma cruzi is a protozoan parasite of humans and animals, affecting 10 to 20 million people and innumerable animals, primarily in the Americas. Despite being the largest cause of infection-induced heart disease worldwide, even among the neglected tropical diseases (NTDs) T. cruzi is considered one of the least well understood and understudied. The genetic complexity of T. cruzi as well as the limited set of efficient techniques for genome engineering contribute significantly to the relative lack of progress in and understanding of this pathogen. Here, we adapted the CRISPR-Cas9 system for the genetic engineering of T. cruzi, demonstrating rapid and efficient knockout of multiple endogenous genes, including essential genes. We observed that in the absence of a template, repair of the Cas9-induced double-stranded breaks (DSBs) in T. cruzi occurs exclusively by microhomology-mediated end joining (MMEJ) with various-sized deletions. When a template for DNA repair is provided, DSB repair by homologous recombination is achieved at an efficiency several orders of magnitude higher than that in the absence of CRISPR-Cas9-induced DSBs. We also demonstrate the high multiplexing capacity of CRISPR-Cas9 in T. cruzi by knocking down expression of an enzyme gene family consisting of 65 members, resulting in a significant reduction of enzymatic product with no apparent off-target mutations. Lastly, we show that Cas9 can mediate disruption of its own coding sequence, rescuing a growth defect in stable Cas9-expressing parasites. These results establish a powerful new tool for the analysis of gene functions in T. cruzi, enabling the study of essential genes and their functions and analysis of the many large families of related genes that occupy a substantial portion of the T. cruzi genome. PMID:25550322

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

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

    1995-05-25

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

  13. Kidney injury molecule-1 (KIM-1) mediates renal epithelial cell repair via ERK MAPK signaling pathway

    PubMed Central

    Zhang, Zhiwei; Cai, Cindy X

    2016-01-01

    The expression of kidney injury molecule-1 (KIM-1), a very promising sensitive and specific urinary biomarker for acute renal injury, is markedly upregulated in injured and regenerating renal proximal tubular epithelial cells following ischemic or toxic insults, suggesting a possible role for this molecule in renal repair process. In the present study we report that expression of KIM-1 facilitates renal tubular epithelial cell repair by promoting cell migration and proliferation. KIM-1 expression also enhances ERK MAPK activation, and the modulatory effect of KIM-1 on cellular repair process is likely mediated via ERK MAPK signaling pathway. PMID:27084535

  14. Review: Clinical aspects of hereditary DNA Mismatch repair gene mutations.

    PubMed

    Sijmons, Rolf H; Hofstra, Robert M W

    2016-02-01

    Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive Constitutional Mismatch Repair Deficiency syndrome. Both conditions are important to recognize clinically as their identification has direct consequences for clinical management and allows targeted preventive actions in mutation carriers. Lynch syndrome is one of the more common adult-onset hereditary tumor syndromes, with thousands of patients reported to date. Its tumor spectrum is well established and includes colorectal cancer, endometrial cancer and a range of other cancer types. However, surveillance for cancers other than colorectal cancer is still of uncertain value. Prophylactic surgery, especially for the uterus and its adnexa is an option in female mutation carriers. Chemoprevention of colorectal cancer with aspirin is actively being investigated in this syndrome and shows promising results. In contrast, the Constitutional Mismatch Repair Deficiency syndrome is rare, features a wide spectrum of childhood onset cancers, many of which are brain tumors with high mortality rates. Future studies are very much needed to improve the care for patients with this severe disorder. PMID:26746812

  15. Simulated microgravity influenced the expression of DNA damage repair genes

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Sun, Yeqing; Jiawei, Liu; Wang, Ting

    2016-07-01

    Ionizing radiation and microgravity were considered to be the most important stress factors of space environmental the respective study of the biological effects of the radiation and microgravity carried out earlier, but the interaction of the effects of radiation with microgravity started later, and due to difference of the materials and methods the result of this experiment were not consistent. To further investigate the influence of microgravity on the expression of the radiation damage repair genes, the seed of Arabidopsis (Col) and its gravity-insensitive mutant (PIN2) were exposed to 0.1Gy of the dose of energetic carbon-ion beam radiation (LET = 30KeV / μm), and the germinated seed were than fixed in the 3D random positioning apparatus immediately for a 10-day simulated microgravity. By measuring the deflection angle of root tip and the changes of the expression of Ku70 and RAD51 protein, we investigated the impact of microgravity effect on radiation damage repair systems. The results shown that radiation, microgravity and microgravity with radiation could increase the angle of the root of the Col significantly, but no obvious effect on PIN2 type. The radiation could increase the expression of Ku70 significantly in both Col and PIN2, microgravity does not affect the expression, but the microgravity with radiation could decrease the expression of Ku70. This result shown that the microgravity could influence the radiation damage repair systems in molecular level. Moreover, our findings were important to understand the molecular mechanism of the impact of microgravity effect on radiation damage repair systems in vivo.

  16. DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks.

    PubMed

    van Overbeek, Megan; Capurso, Daniel; Carter, Matthew M; Thompson, Matthew S; Frias, Elizabeth; Russ, Carsten; Reece-Hoyes, John S; Nye, Christopher; Gradia, Scott; Vidal, Bastien; Zheng, Jiashun; Hoffman, Gregory R; Fuller, Christopher K; May, Andrew P

    2016-08-18

    The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits. PMID:27499295

  17. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    PubMed Central

    Wojcik, Katarzyna A.; Synowiec, Ewelina; Sobierajczyk, Katarzyna; Izdebska, Justyna; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.

    2014-01-01

    Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease. PMID:25356504

  18. [Role of HMGB1 in Inflammatory-mediated Injury Caused by Digestive System Diseases and Its Repair].

    PubMed

    Wang, Fucai; Xie, Yong

    2015-08-01

    High mobility group box 1 protein (HMGB1), a damage-associated molecular pattern, exists ubiquitously in the cells of mammals. It contributes to maintaining the structure of nucleosome and modulating transcription of gene in nuclei. Extracellular HMGB1 plays two-way roles in promoting inflammatory and tissue repair. Released actively as well as passively following cytokine stimulation during cell death, HMGB1 may act as a late inflammatory factor and an endogenous damage-associated molecular pattern recognized by its receptors. And it may mediate the occurrence, development and outcome of the inflammatory injury of digestive system diseases, such as gastric mucosal injury, inflammatory bowel-disease, liver injury, pancreatitis, and so on. This review mainly concerns the research progresses of HMGB1 in the inflammatory injury of digestive system diseases. At the same time, HMGB1 itself, or as a therapeutic target, can promote tissue repair.

  19. Transplantation of human telomerase reverse transcriptase gene-transfected Schwann cells for repairing spinal cord injury

    PubMed Central

    Zhang, Shu-quan; Wu, Min-fei; Liu, Jia-bei; Li, Ye; Zhu, Qing-san; Gu, Rui

    2015-01-01

    Transfection of the human telomerase reverse transcriptase (hTERT) gene has been shown to increase cell proliferation and enhance tissue repair. In the present study, hTERT was transfected into rat Schwann cells. A rat model of acute spinal cord injury was established by the modified free-falling method. Retrovirus PLXSN was injected at the site of spinal cord injury as a vector to mediate hTERT gene-transfected Schwann cells (1 × 1010/L; 10 μL) or Schwann cells (1 × 1010/L; 10 μL) without hTERT gene transfection. Between 1 and 4 weeks after model establishment, motor function of the lower limb improved in the hTERT-transfected group compared with the group with non-transfected Schwann cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and reverse transcription-polymerase chain reaction results revealed that the number of apoptotic cells, and gene expression of aquaporin 4/9 and matrix metalloproteinase 9/2 decreased at the site of injury in both groups; however, the effect improved in the hTERT-transfected group compared with the Schwann cells without hTERT transfection group. Hematoxylin and eosin staining, PKH26 fluorescent labeling, and electrophysiological testing demonstrated that compared with the non-transfected group, spinal cord cavity and motor and sensory evoked potential latencies were reduced, while the number of PKH26-positive cells and the motor and sensory evoked potential amplitude increased at the site of injury in the hTERT-transfected group. These findings suggest that transplantation of hTERT gene-transfected Schwann cells repairs the structure and function of the injured spinal cord. PMID:26889196

  20. The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes

    PubMed Central

    Pierandrei, Silvia; Luchetti, Andrea; Sanchez, Massimo; Novelli, Giuseppe; Sangiuolo, Federica; Lucarelli, Marco

    2016-01-01

    Cellular responses and molecular mechanisms activated by exogenous DNA that invades cells are only partially understood. This limits the practical use of gene targeting strategies. Small fragment homologous replacement (SFHR) uses a small exogenous wild-type DNA fragment to restore the endogenous wild-type sequence; unfortunately, this mechanism has a low frequency of correction. In this study, we used a mouse embryonic fibroblast cell line with a stably integrated mutated gene for enhanced green fluorescence protein. The restoration of a wild-type sequence can be detected by flow cytometry analysis. We quantitatively analyzed the expression of 84 DNA repair genes and 84 cell cycle control genes. Peculiar temporal gene expression patterns were observed for both pathways. Different DNA repair pathways, not only homologous recombination, as well as the three main cell cycle checkpoints appeared to mediate the cellular response. Eighteen genes were selected as highly significant target/effectors of SFHR. We identified a wide interconnection between SFHR, DNA repair, and cell cycle control. Our results increase the knowledge of the molecular mechanisms involved in cell invasion by exogenous DNA and SFHR. Specific molecular targets of both the cell cycle and DNA repair machineries were selected for manipulation to enhance the practical application of SFHR. PMID:27045208

  1. Gene transfer mediated by alpha2-macroglobulin.

    PubMed Central

    Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

    1996-01-01

    alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

  2. Role of paracrine factors in stem and progenitor cell mediated cardiac repair and tissue fibrosis

    PubMed Central

    Burchfield, Jana S; Dimmeler, Stefanie

    2008-01-01

    A new era has begun in the treatment of ischemic disease and heart failure. With the discovery that stem cells from diverse organs and tissues, including bone marrow, adipose tissue, umbilical cord blood, and vessel wall, have the potential to improve cardiac function beyond that of conventional pharmacological therapy comes a new field of research aiming at understanding the precise mechanisms of stem cell-mediated cardiac repair. Not only will it be important to determine the most efficacious cell population for cardiac repair, but also whether overlapping, common mechanisms exist. Increasing evidence suggests that one mechanism of action by which cells provide tissue protection and repair may involve paracrine factors, including cytokines and growth factors, released from transplanted stem cells into the surrounding tissue. These paracrine factors have the potential to directly modify the healing process in the heart, including neovascularization, cardiac myocyte apoptosis, inflammation, fibrosis, contractility, bioenergetics, and endogenous repair. PMID:19014650

  3. SUMO-mediated regulation of DNA damage repair and responses

    PubMed Central

    Sarangi, Prabha; Zhao, Xiaolan

    2015-01-01

    Sumoylation plays important roles during DNA damage repair and responses. Recent broad-scope and substrate-based studies have shed light on the regulation and significance of sumoylation during these processes. An emerging paradigm is that sumoylation of many DNA metabolism proteins is controlled by DNA engagement. Such “on-site modification” can explain low substrate modification levels and has important implications in sumoylation mechanisms and effects. New studies also suggest that sumoylation can regulate a process through an ensemble effect or via major substrates. Additionally, we describe new trends in the functional effects of sumoylation, such as bi-directional changes in biomolecule binding and multi-level coordination with other modifications. These emerging themes and models will stimulate our thinking and research in sumoylation and genome maintenance. PMID:25778614

  4. Solvent-mediated repair and patterning of surfaces by AFM

    SciTech Connect

    Elhadj, S; Chernov, A; De Yoreo, J

    2007-10-30

    A tip-based approach to shaping surfaces of soluble materials with nanometer-scale control is reported. The proposed method can be used, for example, to eliminate defects and inhomogeneities in surface shape, repair mechanical or laser-induced damage to surfaces, or perform 3D lithography on the length scale of an AFM tip. The phenomenon that enables smoothing and repair of surfaces is based on the transport of material from regions of high- to low-curvature within the solution meniscus formed in a solvent-containing atmosphere between the surface in question and an AFM tip scanned over the surface. Using in situ AFM measurements of the kinetics of surface remodeling on KDP (KH{sub 2}PO{sub 4}) crystals in humid air, we show that redistribution of solute material during relaxation of grooves and mounds is driven by a reduction in surface free energy as described by the Gibbs-Thomson law. We find that the perturbation from a flat interface evolves according to the diffusion equation where the effective diffusivity is determined by the product of the surface stiffness and the step kinetic coefficient. We also show that, surprisingly, if the tip is instead scanned over or kept stationary above an atomically flat area of the surface, a convex structure is formed with a diameter that is controlled by the dimensions of the meniscus, indicating that the presence of the tip and meniscus reduces the substrate chemical potential beneath that of the free surface. This allows one to create nanometer-scale 3D structures of arbitrary shape without the removal of substrate material or the use of extrinsic masks or chemical compounds. Potential applications of these tip-based phenomena are discussed.

  5. Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA.

    PubMed

    Tsukioka, Haruna; Takahashi, Masateru; Mon, Hiroaki; Okano, Kazuhiro; Mita, Kazuei; Shimada, Toru; Lee, Jae Man; Kawaguchi, Yutaka; Koga, Katsumi; Kusakabe, Takahiro

    2006-01-01

    The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair.

  6. Immortalized neural progenitor cells for CNS gene transfer and repair.

    PubMed

    Martínez-Serrano, A; Björklund, A

    1997-11-01

    Immortalized multipotent neural stem and progenitor cells have emerged as a highly convenient source of tissue for genetic manipulation and ex vivo gene transfer to the CNS. Recent studies show that these cells, which can be maintained and genetically transduced as cell lines in culture, can survive, integrate and differentiate into both neurons and glia after transplantation to the intact or damaged brain. Progenitors engineered to secrete trophic factors, or to produce neurotransmitter-related or metabolic enzymes can be made to repopulate diseased or injured brain areas, thus providing a new potential therapeutic tool for the blockade of neurodegenerative processes and reversal of behavioural deficits in animal models of neurodegenerative diseases. With further technical improvements, the use of immortalized neural progenitors may bring us closer to the challenging goal of targeted and effective CNS repair.

  7. Gene Delivery Strategies to Promote Spinal Cord Repair

    PubMed Central

    Walthers, Christopher M; Seidlits, Stephanie K

    2015-01-01

    Gene therapies hold great promise for the treatment of many neurodegenerative disorders and traumatic injuries in the central nervous system. However, development of effective methods to deliver such therapies in a controlled manner to the spinal cord is a necessity for their translation to the clinic. Although essential progress has been made to improve efficiency of transgene delivery and reduce the immunogenicity of genetic vectors, there is still much work to be done to achieve clinical strategies capable of reversing neurodegeneration and mediating tissue regeneration. In particular, strategies to achieve localized, robust expression of therapeutic transgenes by target cell types, at controlled levels over defined time periods, will be necessary to fully regenerate functional spinal cord tissues. This review summarizes the progress over the last decade toward the development of effective gene therapies in the spinal cord, including identification of appropriate target genes, improvements to design of genetic vectors, advances in delivery methods, and strategies for delivery of multiple transgenes with synergistic actions. The potential of biomaterials to mediate gene delivery while simultaneously providing inductive scaffolding to facilitate tissue regeneration is also discussed. PMID:25922572

  8. Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes

    PubMed Central

    Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

    2015-01-01

    DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

  9. Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes.

    PubMed

    Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

    2015-01-01

    DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

  10. MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

    PubMed Central

    Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

    2014-01-01

    Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

  11. ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    PubMed Central

    Gracheva, Ekaterina; Chitale, Shalaka; Wilhelm, Thomas; Rapp, Alexander; Byrne, Jonathan; Stadler, Jens; Medina, Rebeca; Cardoso, M. Cristina

    2016-01-01

    Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites. PMID:27091446

  12. Isolation of a human DNA repair gene by selection in Chinese hamster ovary cells

    SciTech Connect

    Ding, R.C.; Eastman, A.; Bresnick, E.

    1987-05-01

    Alkylation of DNA at the O/sup 6/-position of guanine represents a potent mutagenic and carcinogenic lesion. O/sup 6/-Methylguanine DNA methyltransferase is the repair system responsible for catalyzing the transfer of the methyl group to a cysteine of the protein in a suicide reaction. The gene controlling its expression in mammalian systems is designated mex. Resistance to chloroethylnitrosourea (CNU) is also mediated by this protein; this was used to select cells into which the max gene has been introduced. DNA purified from human liver has been transfected into mex/sup -/ CHO cells by the CaPO/sub 4/ method. pSV2gpt, containing a marker gene, gpt, was cotransfected. The transformed cells were initially selected for the expression of gpt (mycophenolic acid resistance) and reselected in CNU for mex/sup +/. Several clones were resistant to both demonstrating the linkage of these genes. A cosmid library was made from a mex/sup +/gpt/sup +/ clone and grown in a gpt/sup -/ strain of E. coli. gpt/sup +/ colonies were selected and the cosmid DNA rescued. One of the tested cosmid DNA's produced CNU resistance upon introduction into CHO cells. This cosmid was subcloned, restriction endonuclease-treated and a 5.3 kb fragment showed mex activity. This fragment is being further characterized and the DNA sequenced.

  13. A new nucleotide-excision-repair gene associated with the disorder trichothiodystrophy

    SciTech Connect

    Stefanini, M.; Giliani, S. ); Vermuelen, W.; Weeda, G.; Hoeijmakers, H.J.; Mezzina, M.; Sarasin, A.; Harper, J.I.; Arlett, C.F.; Lehmann, A.R.

    1993-10-01

    The sun-sensitive, cancer-prone genetic disorder xeroderma pigmentosum (XP) is associated in most cases with a defect in the ability to carry out excision repair of UV damage. Seven genetically distinct complementation groups (i.e., A-G) have been identified. A large proportion of patients with the unrelated disorder trichothiodystrophy (TTD), which is characterized by hair-shaft abnormalities, as well as by physical and mental retardation, are also deficient in excision repair of UV damage. In most of these cases the repair deficiency is in the same complementation group as is XP group D. The authors report here on cells from a patient, TTD1BR, in which the repair defect complements all known XP groups (including XP-D). Furthermore, microinjection of various cloned human repair genes fails to correct the repair defect in this cell strain. The defect in TTD1BR cells is therefore in a new gene involved in excision repair in human cells. The finding of a second DNA repair gene that is associated with the clinical features of TTD argues strongly for an involvement of repair proteins in hair-shaft development. 20 refs., 2 figs., 1 tab.

  14. Triple Negative Breast Cancers Have a Reduced Expression of DNA Repair Genes

    PubMed Central

    Andreis, Daniele; Bertoni, Ramona; Giardini, Roberto; Fox, Stephen B.; Broggini, Massimo; Bottini, Alberto; Zanoni, Vanessa; Bazzola, Letizia; Foroni, Chiara; Generali, Daniele; Damia, Giovanna

    2013-01-01

    DNA repair is a key determinant in the cellular response to therapy and tumor repair status could play an important role in tailoring patient therapy. Our goal was to evaluate the mRNA of 13 genes involved in different DNA repair pathways (base excision, nucleotide excision, homologous recombination, and Fanconi anemia) in paraffin embedded samples of triple negative breast cancer (TNBC) compared to luminal A breast cancer (LABC). Most of the genes involved in nucleotide excision repair and Fanconi Anemia pathways, and CHK1 gene were significantly less expressed in TNBC than in LABC. PARP1 levels were higher in TNBC than in LABC. In univariate analysis high level of FANCA correlated with an increased overall survival and event free survival in TNBC; however multivariate analyses using Cox regression did not confirm FANCA as independent prognostic factor. These data support the evidence that TNBCs compared to LABCs harbour DNA repair defects. PMID:23825533

  15. Regulation of Ceramide Synthase-Mediated Crypt Epithelium Apoptosis by DNA Damage Repair Enzymes

    PubMed Central

    Rotolo, Jimmy A.; Mesicek, Judith; Maj, Jerzy; Truman, Jean-Philip; Haimovitz-Friedman, Adriana; Kolesnick, Richard; Fuks, Zvi

    2015-01-01

    Acute endothelial cell apoptosis and microvascular compromise couple GI tract irradiation to reproductive death of intestinal crypt stem cell clonogens (SCCs) following high-dose radiation. Genetic or pharmacologic inhibition of endothelial apoptosis prevents intestinal damage, but as the radiation dose is escalated, SCCs become directly susceptible to an alternate cell death mechanism, mediated via ceramide synthase (CS)-stimulated de novo synthesis of the pro-apoptotic sphingolipid ceramide, and p53-independent apoptosis of crypt SCCs. We previously reported that ATM deficiency resets the primary radiation lethal pathway, allowing CS-mediated apoptosis at the low-dose range of radiation. The mechanism for this event, termed target reordering, remains unknown. Here we show that inactivation of DNA damage repair pathways signal CS-mediated apoptosis in crypt SCCs, presumably via persistent unrepaired DNA double strand breaks (DSBs). Genetic loss-of-function of sensors and transducers of DNA DSB repair confers the CS-mediated lethal pathway in intestines of sv129/B6Mre11ATLD1/ATLD1 and C57BL/6Prkdc/SCID (SCID) mice exposed to low-dose radiation. In contrast, CS-mediated SCC lethality was mitigated in irradiated gain-of-function Rad50S/S mice, and epistasis studies order Rad50 upstream of Mre11. These studies suggest unrepaired DNA DSBs as causative in target re-ordering in intestinal SCCs. As such, we provide an in vivo model of DNA damage repair that is standardized, can be exploited to understand allele-specific regulation in intact tissue, and is pharmacologically tractable. PMID:20086180

  16. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair.

    PubMed

    Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A

    2015-01-01

    Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3' side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4(Cdt2). Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4(Cdt2) for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.

  17. Ku-Mediated Coupling of DNA Cleavage and Repair during Programmed Genome Rearrangements in the Ciliate Paramecium tetraurelia

    PubMed Central

    Marmignon, Antoine; Bischerour, Julien; Silve, Aude; Fojcik, Clémentine; Dubois, Emeline; Arnaiz, Olivier; Kapusta, Aurélie; Malinsky, Sophie; Bétermier, Mireille

    2014-01-01

    During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage

  18. Expression profiles of DNA repair-related genes in rat target organs under subchronic cadmium exposure.

    PubMed

    Lei, Y X; Lu, Q; Shao, C; He, C C; Lei, Z N; Lian, Y Y

    2015-01-01

    We aimed to evaluate the toxicity of long-term exposure to different cadmium (Cd) doses in rats and expression profiles of DNA repair-related genes. The model rats were exposed to different concentrations of CdCl2 for 3 months, and 5 DNA repair-related genes - hMSH2, MLH1, XRCC1, hOGG1, ERCC1 - were cloned in different tissues, including the liver, kidney, heart, and lung. Accumulated amounts of Cd were detected in the tissues. Gene and protein detections were conducted via fluorescence quantitative real-time polymerase chain reaction and Western blotting, respectively. Methylated sequences of the 5 DNA repair-related gene promoters were used to investigate whether the low expression levels of the genes were related to methylation of the promoter. In the Cd-exposed group, 3 DNA repair genes (i.e., XRCC1, hOGG1, and ERCC1) significantly decreased in the rat liver, kidney, heart, and lung according to the β-actin internal standard (P < 0.01). Western blotting indicated the same trend for the different tissues. Each of the DNA repair genes had special characteristics; for example, hOGG1 gene expression decreased by 75% in the kidney, and XRCC1 gene expression decreased by 5% in the liver and heart when compared to the control group (P < 0.01). A negative correlation between the DNA repair gene expression levels and the cumulative levels of Cd was also suggested by malignancy pathology. The expression levels of 3 DNA repair genes (i.e., ERCC1, XRCC1, and hOGG1) played an important role in the rat response to Cd exposure but not DNA methylated protection. PMID:25729986

  19. The role of DNA damage and repair in decitabine-mediated apoptosis in multiple myeloma.

    PubMed

    Maes, Ken; De Smedt, Eva; Lemaire, Miguel; De Raeve, Hendrik; Menu, Eline; Van Valckenborgh, Els; McClue, Steve; Vanderkerken, Karin; De Bruyne, Elke

    2014-05-30

    DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) are under investigation for the treatment of cancer, including the plasma cell malignancy multiple myeloma (MM). Evidence exists that DNA damage and repair contribute to the cytotoxicity mediated by the DNMTi decitabine. Here, we investigated the DNA damage response (DDR) induced by decitabine in MM using 4 human MM cell lines and the murine 5T33MM model. In addition, we explored how the HDACi JNJ-26481585 affects this DDR. Decitabine induced DNA damage (gamma-H2AX foci formation), followed by a G0/G1- or G2/M-phase arrest and caspase-mediated apoptosis. JNJ-26481585 enhanced the anti-MM effect of decitabine both in vitro and in vivo. As JNJ-26481585 did not enhance decitabine-mediated gamma-H2AX foci formation, we investigated the DNA repair response towards decitabine and/or JNJ-26481585. Decitabine augmented RAD51 foci formation (marker for homologous recombination (HR)) and/or 53BP1 foci formation (marker for non-homologous end joining (NHEJ)). Interestingly, JNJ-26481585 negatively affected basal or decitabine-induced RAD51 foci formation. Finally, B02 (RAD51 inhibitor) enhanced decitabine-mediated apoptosis. Together, we report that decitabine-induced DNA damage stimulates HR and/or NHEJ. JNJ-26481585 negatively affects RAD51 foci formation, thereby providing an additional explanation for the combinatory effect between decitabine and JNJ-26481585.

  20. Angiogenic activity mediates bone repair from human pluripotent stem cell-derived osteogenic cells

    PubMed Central

    Zou, Li; Chen, Qingshan; Quanbeck, Zachary; Bechtold, Joan E.; Kaufman, Dan S.

    2016-01-01

    Human pluripotent stem cells provide a standardized resource for bone repair. However, criteria to determine which exogenous cells best heal orthopedic injuries remain poorly defined. We evaluated osteogenic progenitor cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Phenotypic and genotypic analyses demonstrated that these hESCs/hiPSCs are similar in their osteogenic differentiation efficiency and they generate osteogenic cells comparable to osteogenic cells derived from mesenchymal stromal cells (BM-MSCs). However, expression of angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor in these osteogenic progenitor cells are markedly different, suggesting distinct pro-angiogenic potential of these stem cell derivatives. Studies to repair a femur non-union fracture demonstrate only osteogenic progenitor cells with higher pro-angiogenic potential significantly enhance bone repair in vivo. Together, these studies highlight a key role of pro-angiogenic potential of transplanted osteogenic cells for effective cell-mediated bone repair. PMID:26980556

  1. Genes and Junk in Plant Mitochondria—Repair Mechanisms and Selection

    PubMed Central

    Christensen, Alan C.

    2014-01-01

    Plant mitochondrial genomes have very low mutation rates. In contrast, they also rearrange and expand frequently. This is easily understood if DNA repair in genes is accomplished by accurate mechanisms, whereas less accurate mechanisms including nonhomologous end joining or break-induced replication are used in nongenes. An important question is how different mechanisms of repair predominate in coding and noncoding DNA, although one possible mechanism is transcription-coupled repair (TCR). This work tests the predictions of TCR and finds no support for it. Examination of the mutation spectra and rates in genes and junk reveals what DNA repair mechanisms are available to plant mitochondria, and what selective forces act on the repair products. A model is proposed that mismatches and other DNA damages are repaired by converting them into double-strand breaks (DSBs). These can then be repaired by any of the DSB repair mechanisms, both accurate and inaccurate. Natural selection will eliminate coding regions repaired by inaccurate mechanisms, accounting for the low mutation rates in genes, whereas mutations, rearrangements, and expansions generated by inaccurate repair in noncoding regions will persist. Support for this model includes the structure of the mitochondrial mutS homolog in plants, which is fused to a double-strand endonuclease. The model proposes that plant mitochondria do not distinguish a damaged or mismatched DNA strand from the undamaged strand, they simply cut both strands and perform homology-based DSB repair. This plant-specific strategy for protecting future generations from mitochondrial DNA damage has the side effect of genome expansions and rearrangements. PMID:24904012

  2. Decreased DNA repair gene expression among individuals exposed to arsenic in United States drinking water.

    PubMed

    Andrew, Angeline S; Karagas, Margaret R; Hamilton, Joshua W

    2003-04-10

    Arsenic is well established as a human carcinogen, but its precise mechanism of action remains unknown. Arsenic does not directly damage DNA, but may act as a carcinogen through inhibition of DNA repair mechanisms, leading indirectly to increased mutations from other DNA damaging agents. The molecular mechanism underlying arsenic inhibition of nucleotide excision repair after UV irradiation (Hartwig et al., Carcinogenesis 1997;18:399-405) is unknown, but could be due to decreased expression of critical genes involved in nucleotide excision repair of damaged DNA. This hypothesis was tested by analyzing expression of repair genes and arsenic exposure in a subset of 16 individuals enrolled in a population based case-control study investigating arsenic exposure and cancer risk in New Hampshire. Toenail arsenic levels were inversely correlated with expression of critical members of the nucleotide excision repair complex, ERCC1 (r(2) = 0.82, p < 0.0001), XPF (r(2) = 0.56, p < 0.002), and XPB (r(2) = 0.75, p < 0.0001). The internal dose marker, toenail arsenic level, was more strongly associated with changes in expression of these genes than drinking water arsenic concentration. Our findings, based on human exposure to arsenic in a US population, show an association between biomarkers of arsenic exposure and expression of DNA repair genes. Although our findings need verification in a larger study group, they are consistent with the hypothesis that inhibition of DNA repair capacity is a potential mechanism for the co-carcinogenic activity of arsenic.

  3. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    PubMed

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

  4. DNA Double-Strand Break Repair Genes and Oxidative Damage in Brain Metastasis of Breast Cancer

    PubMed Central

    Evans, Lynda; Duchnowska, Renata; Reed, L. Tiffany; Palmieri, Diane; Qian, Yongzhen; Badve, Sunil; Sledge, George; Gril, Brunilde; Aladjem, Mirit I.; Fu, Haiqing; Flores, Natasha M.; Gökmen-Polar, Yesim; Biernat, Wojciech; Szutowicz-Zielińska, Ewa; Mandat, Tomasz; Trojanowski, Tomasz; Och, Waldemar; Czartoryska-Arlukowicz, Bogumiła; Jassem, Jacek; Mitchell, James B.

    2014-01-01

    Background Breast cancer frequently metastasizes to the brain, colonizing a neuro-inflammatory microenvironment. The molecular pathways facilitating this colonization remain poorly understood. Methods Expression profiling of 23 matched sets of human resected brain metastases and primary breast tumors by two-sided paired t test was performed to identify brain metastasis–specific genes. The implicated DNA repair genes BARD1 and RAD51 were modulated in human (MDA-MB-231-BR) and murine (4T1-BR) brain-tropic breast cancer cell lines by lentiviral transduction of cDNA or short hairpin RNA (shRNA) coding sequences. Their functional contribution to brain metastasis development was evaluated in mouse xenograft models (n = 10 mice per group). Results Human brain metastases overexpressed BARD1 and RAD51 compared with either matched primary tumors (1.74-fold, P < .001; 1.46-fold, P < .001, respectively) or unlinked systemic metastases (1.49-fold, P = .01; 1.44-fold, P = .008, respectively). Overexpression of either gene in MDA-MB-231-BR cells increased brain metastases by threefold to fourfold after intracardiac injections, but not lung metastases upon tail-vein injections. In 4T1-BR cells, shRNA-mediated RAD51 knockdown reduced brain metastases by 2.5-fold without affecting lung metastasis development. In vitro, BARD1- and RAD51-overexpressing cells showed reduced genomic instability but only exhibited growth and colonization phenotypes upon DNA damage induction. Reactive oxygen species were present in tumor cells and elevated in the metastatic neuro-inflammatory microenvironment and could provide an endogenous source of genotoxic stress. Tempol, a brain-permeable oxygen radical scavenger suppressed brain metastasis promotion induced by BARD1 and RAD51 overexpression. Conclusions BARD1 and RAD51 are frequently overexpressed in brain metastases from breast cancer and may constitute a mechanism to overcome reactive oxygen species–mediated genotoxic stress in the metastatic

  5. N-cadherin coordinates AMP kinase-mediated lung vascular repair.

    PubMed

    Jian, Ming-Yuan; Liu, Yanping; Li, Qian; Wolkowicz, Paul; Alexeyev, Mikhail; Zmijewski, Jaroslaw; Creighton, Judy

    2016-01-01

    Injury to the pulmonary circulation compromises endothelial barrier function and increases lung edema. Resolution of lung damage involves restoring barrier integrity, a process requiring reestablishment of endothelial cell-cell adhesions. However, mechanisms underlying repair in lung endothelium are poorly understood. In pulmonary microvascular endothelium, AMP kinase α1 (AMPKα1) stimulation enhances recovery of the endothelial barrier after LPS-induced vascular damage. AMPKα1 colocalizes to a discrete membrane compartment with the adhesion protein neuronal cadherin (N-cadherin). This study sought to determine N-cadherin's role in the repair process. Short-hairpin RNA against full-length N-cadherin or a C-terminally truncated N-cadherin, designed to disrupt the cadherin's interactions with intracellular proteins, were expressed in lung endothelium. Disruption of N-cadherin's intracellular domain caused translocation of AMPK away from the membrane and attenuated AMPK-mediated restoration of barrier function in LPS-treated endothelium. AMPK activity measurements indicated that lower basal AMPK activity in cells expressing the truncated N-cadherin compared with controls. Moreover, the AMPK stimulator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to increase AMPK activity in cells expressing the modified N-cadherin, indicating uncoupling of a functional association between AMPK and the cadherin. Isolated lung studies confirmed a physiologic role for this pathway in vivo. AMPK activation reversed LPS-induced increase in permeability, whereas N-cadherin inhibition hindered AMPK-mediated repair. Thus N-cadherin coordinates the vascular protective actions of AMPK through a functional link with the kinase. This study provides insight into intrinsic repair mechanisms in the lung and supports AMPK stimulation as a modality for treating vascular disease.

  6. DNA Damage Response Factors from Diverse Pathways, Including DNA Crosslink Repair, Mediate Alternative End Joining

    PubMed Central

    Howard, Sean M.; Yanez, Diana A.; Stark, Jeremy M.

    2015-01-01

    Alternative end joining (Alt-EJ) chromosomal break repair involves bypassing classical non-homologous end joining (c-NHEJ), and such repair causes mutations often with microhomology at the repair junction. Since the mediators of Alt-EJ are not well understood, we have sought to identify DNA damage response (DDR) factors important for this repair event. Using chromosomal break reporter assays, we surveyed an RNAi library targeting known DDR factors for siRNAs that cause a specific decrease in Alt-EJ, relative to an EJ event that is a composite of Alt-EJ and c-NHEJ (Distal-EJ between two tandem breaks). From this analysis, we identified several DDR factors that are specifically important for Alt-EJ relative to Distal-EJ. While these factors are from diverse pathways, we also found that most of them also promote homologous recombination (HR), including factors important for DNA crosslink repair, such as the Fanconi Anemia factor, FANCA. Since bypass of c-NHEJ is likely important for both Alt-EJ and HR, we disrupted the c-NHEJ factor Ku70 in Fanca-deficient mouse cells and found that Ku70 loss significantly diminishes the influence of Fanca on Alt-EJ. In contrast, an inhibitor of poly ADP-ribose polymerase (PARP) causes a decrease in Alt-EJ that is enhanced by Ku70 loss. Additionally, the helicase/nuclease DNA2 appears to have distinct effects from FANCA and PARP on both Alt-EJ, as well as end resection. Finally, we found that the proteasome inhibitor Bortezomib, a cancer therapeutic that has been shown to disrupt FANC signaling, causes a significant reduction in both Alt-EJ and HR, relative to Distal-EJ, as well as a substantial loss of end resection. We suggest that several distinct DDR functions are important for Alt-EJ, which include promoting bypass of c-NHEJ and end resection. PMID:25629353

  7. The effect of acute dose charge particle radiation on expression of DNA repair genes in mice.

    PubMed

    Tariq, Muhammad Akram; Soedipe, Ayodotun; Ramesh, Govindarajan; Wu, Honglu; Zhang, Ye; Shishodia, Shishir; Gridley, Daila S; Pourmand, Nader; Jejelowo, Olufisayo

    2011-03-01

    The space radiation environment consists of trapped particle radiation, solar particle radiation, and galactic cosmic radiation (GCR), in which protons are the most abundant particle type. During missions to the moon or to Mars, the constant exposure to GCR and occasional exposure to particles emitted from solar particle events (SPE) are major health concerns for astronauts. Therefore, in order to determine health risks during space missions, an understanding of cellular responses to proton exposure is of primary importance. The expression of DNA repair genes in response to ionizing radiation (X-rays and gamma rays) has been studied, but data on DNA repair in response to protons is lacking. Using qPCR analysis, we investigated changes in gene expression induced by positively charged particles (protons) in four categories (0, 0.1, 1.0, and 2.0 Gy) in nine different DNA repair genes isolated from the testes of irradiated mice. DNA repair genes were selected on the basis of their known functions. These genes include ERCC1 (5' incision subunit, DNA strand break repair), ERCC2/NER (opening DNA around the damage, Nucleotide Excision Repair), XRCC1 (5' incision subunit, DNA strand break repair), XRCC3 (DNA break and cross-link repair), XPA (binds damaged DNA in preincision complex), XPC (damage recognition), ATA or ATM (activates checkpoint signaling upon double strand breaks), MLH1 (post-replicative DNA mismatch repair), and PARP1 (base excision repair). Our results demonstrate that ERCC1, PARP1, and XPA genes showed no change at 0.1 Gy radiation, up-regulation at 1.0 Gy radiation (1.09 fold, 7.32 fold, 0.75 fold, respectively), and a remarkable increase in gene expression at 2.0 Gy radiation (4.83 fold, 57.58 fold and 87.58 fold, respectively). Expression of other genes, including ATM and XRCC3, was unchanged at 0.1 and 1.0 Gy radiation but showed up-regulation at 2.0 Gy radiation (2.64 fold and 2.86 fold, respectively). We were unable to detect gene expression for the

  8. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene

    PubMed Central

    Hollywood, Jennifer A.; Lee, Ciaran M.; Scallan, Martina F.; Harrison, Patrick T.

    2016-01-01

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of ~80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair. PMID:27557525

  9. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene.

    PubMed

    Hollywood, Jennifer A; Lee, Ciaran M; Scallan, Martina F; Harrison, Patrick T

    2016-01-01

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of ~80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair. PMID:27557525

  10. Preferential Repair of DNA Double-strand Break at the Active Gene in Vivo*

    PubMed Central

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K.; Bhaumik, Sukesh R.

    2012-01-01

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3′ end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells. PMID:22910905

  11. Modulation of Wound Healing and Scar Formation by MG53 Protein-mediated Cell Membrane Repair*

    PubMed Central

    Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M.; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

    2015-01-01

    Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53−/− mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. PMID:26306047

  12. Modulation of wound healing and scar formation by MG53 protein-mediated cell membrane repair.

    PubMed

    Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

    2015-10-01

    Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53(-/-) mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing.

  13. Plant transformation via pollen tube-mediated gene transfer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT ...

  14. Interaction of DNA repair gene polymorphisms and aflatoxin B1 in the risk of hepatocellular carcinoma

    PubMed Central

    Yao, Jin-Guang; Huang, Xiao-Ying; Long, Xi-Dai

    2014-01-01

    Aflatoxin B1 (AFB1) is an important environmental carcinogen and can induce DNA damage and involve in the carcinogenesis of hepatocellular carcinoma (HCC). The deficiency of DNA repair capacity related to the polymorphisms of DNA repair genes might play a central role in the process of HCC tumorigenesis. However, the interaction of DNA repair gene polymorphisms and AFB1 in the risk of hepatocellular carcinoma has not been elucidated. In this study, we investigated whether six polymorphisms (including rs25487, rs861539, rs7003908, rs28383151, rs13181, and rs2228001) in DNA repair genes (XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and XRCC3) interacted with AFB1, and the gene-environmental interactive role in the risk of HCC using hospital-based case-control study (including 1486 HCC cases and 1996 controls). Genotypes of DNA repair genes were tested using TaqMan-PCR technique. Higher AFB1 exposure was observed among HCC patients versus the control group [odds ratio (OR) = 2.08 for medium AFB1 exposure level and OR = 6.52 for high AFB1 exposure level]. Increasing risk of HCC was also observed in these with the mutants of DNA repair genes (risk values were from 1.57 to 5.86). Furthermore, these risk roles would be more noticeable under the conditions of two variables, and positive interactive effects were proved in the followed multiplicative interaction analysis. These results suggested that DNA repair risk genotypes might interact with AFB1 in the risk of HCC. PMID:25337275

  15. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

    PubMed Central

    McAllister, Katherine A.; Yasseen, Akeel A.; McKerr, George; Downes, C. S.; McKelvey-Martin, Valerie J.

    2014-01-01

    Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents. PMID:25152750

  16. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair.

    PubMed

    McAllister, Katherine A; Yasseen, Akeel A; McKerr, George; Downes, C S; McKelvey-Martin, Valerie J

    2014-01-01

    Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1(+) and TK1(-) clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1(+) compared to TK1(-) cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK(+) cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1(+) cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents. PMID:25152750

  17. Epidermal growth factor receptor distribution in burn wounds. Implications for growth factor-mediated repair.

    PubMed Central

    Wenczak, B A; Lynch, J B; Nanney, L B

    1992-01-01

    Epidermal growth factor (EGF) along with several related peptide growth factors has been shown both in vivo and in vitro to accelerate events associated with epidermal wound repair. EGF and transforming growth factor alpha act by binding to a common EGF receptor tyrosine kinase thereby initiating a series of events which ultimately regulate cell proliferation. This study examined the immunohistochemical localization of EGF receptor (EGF-R) in burn wound margins, adjacent proliferating epithelium, and closely associated sweat ducts, sebaceous glands, and hair follicles. Tissue specimens removed during surgical debridement were obtained from full and partial thickness burn wounds in 32 patients with total body surface area burns ranging from 2 to 88%. In the early postburn period (days 2-4), prominent staining for EGF-R was found in undifferentiated, marginal keratinocytes, adjacent proliferating, hypertrophic epithelium, and both marginal and nonmarginal hair follicles, sweat ducts, and sebaceous glands. During the late postburn period (days 5-16), EGF-R was depleted along leading epithelial margins; however, immunoreactive EGF-R remained intensely positive in the hypertrophic epithelium and all skin appendages. Increased detection of immunoreactive EGF-R and the presence of [125I]EGF binding in the hypertrophic epithelium correlated positively with proliferating cell nuclear antigen distributions. Thus, the presence of EGF-R in the appropriate keratinocyte populations suggests a functional role for this receptor during wound repair. Dynamic modulation in EGF receptor distribution during the temporal sequence of repair provides further evidence that an EGF/transforming growth factor alpha/EGF-R-mediated pathway is activated during human wound repair. Images PMID:1361495

  18. Insulin-glycerolipid mediators and gene expression

    SciTech Connect

    Standaert, M.L.; Pollet, R.J. )

    1988-06-01

    Insulin is an anabolic polypeptide hormone with pleiotrophic effects. During the decades since the initial description by Banting and Best, the acute effects of insulin have been widely studied with particular focus on the mechanism or mechanisms of insulin activation of hexose transport and regulation of metabolic enzyme activity. However, recently there has been a major expansion of investigation to include insulin regulation of gene expression with multiple insulin-sensitive specific mRNAs now reported. In this review, we explore the involvement of insulin-induced changes in plasma membrane glycerolipid metabolism in the transmembrane signaling process required for insulin regulation of mRNA levels. Insulin increase diacylglycerol levels in insulin-responsive cells, and synthetic diacylglycerols or their phorbol ester diacylglycerol analogs, such as 4{beta}, 9{alpha}, 12{beta}, 13{alpha}, 20-pentahydroxytiglia-1,6-dien-3-one 12{beta}-myristate 13-acetate (TPA), mimic insulin regulation of ornithine decarboxylase mRNA, c-fos mRNA, and phosphoenolpyruvate carboxykinase mRNA levels. This suggests that insulin regulation of specific mRNA levels may be mediated by insulin-induced changes in phospholipid metabolism and that diacylglycerol may play a pivotal role in insulin regulation of gene expression.

  19. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme.

    PubMed Central

    Xanthoudakis, S; Miao, G; Wang, F; Pan, Y C; Curran, T

    1992-01-01

    The DNA binding activity of Fos and Jun is regulated in vitro by a post-translational mechanism involving reduction-oxidation. Redox regulation occurs through a conserved cysteine residue located in the DNA binding domain of Fos and Jun. Reduction of this residue by chemical reducing agents or by a ubiquitous nuclear redox factor (Ref-1) recently purified from Hela cells, stimulates AP-1 DNA binding activity in vitro, whereas oxidation or chemical modification of the cysteine has an inhibitory effect on DNA binding activity. Here we demonstrate that the protein product of the ref-1 gene stimulates the DNA binding activity of Fos-Jun heterodimers, Jun-Jun homodimers and Hela cell AP-1 proteins as well as that of several other transcription factors including NF-kappa B, Myb and members of the ATF/CREB family. Furthermore, immunodepletion analysis indicates that Ref-1 is the major AP-1 redox activity in Hela nuclear extracts. Interestingly, Ref-1 is a bifunctional protein; it also possesses an apurinic/apyrimidinic (AP) endonuclease DNA repair activity. However, the redox and DNA repair activities of Ref-1 can, in part, be distinguished biochemically. This study suggests a novel link between transcription factor regulation, oxidative signalling and DNA repair processes in higher eukaryotes. Images PMID:1380454

  20. A new approach to tissue repair: gene therapy.

    PubMed

    Wei, Kuanhai; Pei, Guoxian; Hu, Basheng

    2000-11-15

    The process of tissue repair involves a complex tissue response to injury in which growth factors, playing a major role in this process, trigger, control and terminate soakage of inflammatory cells, cells proliferation, secretion of matrix and scars formation by autocrine, paracrine or both. Thus, growth factors can be used to alter the microenvironment of the wounded tissues and to promote their repair. But, there are notable disadvantages in using purified recombination growth factors, 1) the source is so limited that their prices are expensive; 2) the ir half-lives are short and easy to be destroyed by wound proteases; 3) there is no perfect carrier; 4) high initial doses are required but easy to bring toxicity; 5) it is difficult to apply growth factors in deep wounded tissues again and again, their function cannot be played enough accordingly; 6) most of growth factors are the products of recombination. All above-mentioned disadvantages result in a low activity.

  1. Polymorphisms in DNA repair genes, recreational physical activity and breast cancer risk.

    PubMed

    McCullough, Lauren E; Santella, Regina M; Cleveland, Rebecca J; Millikan, Robert C; Olshan, Andrew F; North, Kari E; Bradshaw, Patrick T; Eng, Sybil M; Terry, Mary Beth; Shen, Jing; Crew, Katherine D; Rossner, Pavel; Teitelbaum, Susan L; Neugut, Alfred I; Gammon, Marilie D

    2014-02-01

    The mechanisms driving the inverse association between recreational physical activity (RPA) and breast cancer risk are complex. While exercise is associated with increased reactive oxygen species production it may also improve damage repair systems, particularly those that operate on single-strand breaks including base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). Of these repair pathways, the role of MMR in breast carcinogenesis is least investigated. Polymorphisms in MMR or other DNA repair gene variants may modify the association between RPA and breast cancer incidence. We investigated the individual and joint effects of variants in three MMR pathway genes (MSH3, MLH1 and MSH2) on breast cancer occurrence using resources from the Long Island Breast Cancer Study Project. We additionally characterized interactions between RPA and genetic polymorphisms in MMR, BER and NER pathways. We found statistically significant multiplicative interactions (p < 0.05) between MSH2 and MLH1, as well as between postmenopausal RPA and four variants in DNA repair (XPC-Ala499Val, XPF-Arg415Gln, XPG-Asp1104His and MLH1-lle219Val). Significant risk reductions were observed among highly active women with the common genotype for XPC (OR = 0.54; 95% CI, 0.36-0.81) and XPF (OR = 0.62; 95% CI, 0.44-0.87), as well as among active women who carried at least one variant allele in XPG (OR = 0.46; 95% CI, 0.29-0.77) and MLH1 (OR = 0.46; 95% CI, 0.30-0.71). Our data show that women with minor alleles in both MSH2 and MLH1 could be at increased breast cancer risk. RPA may be modified by genes in the DNA repair pathway, and merit further investigation.

  2. Polymorphisms in DNA Repair Genes, Recreational Physical Activity and Breast Cancer Risk

    PubMed Central

    McCullough, Lauren E.; Santella, Regina M.; Cleveland, Rebecca J.; Millikan, Robert C.; Olshan, Andrew F.; North, Kari E.; Bradshaw, Patrick T.; Eng, Sybil M.; Terry, Mary Beth; Shen, Jing; Crew, Katherine D.; Rossner, Pavel; Teitelbaum, Susan L.; Neugut, Alfred I.; Gammon, Marilie D.

    2013-01-01

    The mechanisms driving the inverse association between recreational physical activity (RPA) and breast cancer risk are complex. While exercise is associated with increased reactive oxygen species production it may also improve damage repair systems, particularly those that operate on single-strand breaks including base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). Of these repair pathways, the role of MMR in breast carcinogenesis is least investigated. Polymorphisms in MMR or other DNA repair gene variants may modify the association between RPA and breast cancer incidence. We investigated the individual and joint effects of variants in three MMR pathway genes (MSH3, MLH1 and MSH2) on breast cancer occurrence using resources from the Long Island Breast Cancer Study Project. We additionally characterized interactions between RPA and genetic polymorphisms in MMR, BER and NER pathways. We found statistically significant multiplicative interactions (p<0.05) between MSH2 and MLH1, as well as between postmenopausal RPA and four variants in DNA repair (XPC-Ala499Val, XPF-Arg415Gln, XPG-Asp1104His and MLH1-lle219Val). Significant risk reductions were observed among highly active women with the common genotype for XPC (OR=0.54; 95% CI, 0.36–0.81) and XPF (OR=0.62; 95% CI, 0.44–0.87), as well as among active women who carried at least one variant allele in XPG (OR=0.46; 95% CI, 0.29–0.77) and MLH1 (OR=0.46; 95% CI, 0.30–0.71). Our data show that women with minor alleles in both MSH2 and MLH1 could be at increased breast cancer risk. RPA may be modified by genes in the DNA repair pathway, and merit further investigation. PMID:23852586

  3. Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques

    PubMed Central

    Bethea, Cynthia L.; Reddy, Arubala P.

    2015-01-01

    Depression often accompanies the peri-menopausal transition and it often precedes overt symptomology in common neurodegenerative diseases (NDD; Alzheimer's, Parkinson's, Huntington, ALS). Serotonin dysfunction is frequently found in the different etiologies of depression. We have shown that ovariectomized (Ovx) monkeys treated with estradiol (E) for 28 days supplement with placebo or progesterone (P) on days 14-28 had reduced DNA fragmentation in serotonin neurons of the dorsal raphe nucleus; and long-term Ovx monkeys had fewer serotonin neurons than intact controls. We questioned the effect of E alone or E+P on gene expression related to DNA repair, protein folding (chaperones), the ubiquitin proteosome, axon transport, and NDD specific genes in serotonin neurons. Ovx macaques were treated with placebo, E or E+P (n=3/group) for 1 month. Serotonin neurons were laser captured and subjected to microarray analysis and qRT-PCR. Increases were confirmed with qRT-PCR in 5 genes that code for proteins involved in repair of strand breaks and nucleotide excision. NBN1, PCNA, GADD45A, RAD23A and GTF2H5 significantly increased with E or E+P treatment (all ANOVA p< 0.01). Chaperone genes HSP70, HSP60 and HSP27 significantly increased with E or E+P treatment (all ANOVA p<0.05). HSP90 showed a similar trend. Ubiquinase coding genes UBEA5, UBE2D3 and UBE3A (Parkin) increased with E or E+P (all ANOVA p<0.003). Transport related genes coding kinesin, dynein, and dynactin increased with E or E+P (all ANOVA p<0.03). SCNA (α synuclein) and ADAM10 (α secretase) increased (both ANOVA p<0.02), but PSEN1 (presenilin1) decreased (ANOVA p<0.02) with treatment. APP decreased 10-fold with E or E+P administration. Newman-Keuls posthoc comparisons indicated variation in the response to E alone versus E+P across the different genes. In summary, E or E+P increased gene expression for DNA repair mechanisms in serotonin neurons, thereby rendering them less vulnerable to stress-induced DNA

  4. Gene Expression in Experimental Aortic Coarctation and Repair: Candidate Genes for Therapeutic Intervention?

    PubMed Central

    LaDisa, John F.; Bozdag, Serdar; Olson, Jessica; Ramchandran, Ramani; Kersten, Judy R.; Eddinger, Thomas J.

    2015-01-01

    Coarctation of the aorta (CoA) is a constriction of the proximal descending thoracic aorta and is one of the most common congenital cardiovascular defects. Treatments for CoA improve life expectancy, but morbidity persists, particularly due to the development of chronic hypertension (HTN). Identifying the mechanisms of morbidity is difficult in humans due to confounding variables such as age at repair, follow-up duration, coarctation severity and concurrent anomalies. We previously developed an experimental model that replicates aortic pathology in humans with CoA without these confounding variables, and mimics correction at various times using dissolvable suture. Here we present the most comprehensive description of differentially expressed genes (DEGs) to date from the pathology of CoA, which were obtained using this model. Aortic samples (n=4/group) from the ascending aorta that experiences elevated blood pressure (BP) from induction of CoA, and restoration of normal BP after its correction, were analyzed by gene expression microarray, and enriched genes were converted to human orthologues. 51 DEGs with >6 fold-change (FC) were used to determine enriched Gene Ontology terms, altered pathways, and association with National Library of Medicine Medical Subject Headers (MeSH) IDs for HTN, cardiovascular disease (CVD) and CoA. The results generated 18 pathways, 4 of which (cell cycle, immune system, hemostasis and metabolism) were shared with MeSH ID’s for HTN and CVD, and individual genes were associated with the CoA MeSH ID. A thorough literature search further uncovered association with contractile, cytoskeletal and regulatory proteins related to excitation-contraction coupling and metabolism that may explain the structural and functional changes observed in our experimental model, and ultimately help to unravel the mechanisms responsible for persistent morbidity after treatment for CoA. PMID:26207811

  5. The Convergence of Fracture Repair and Stem Cells: Interplay of Genes, Aging, Environmental Factors and Disease

    PubMed Central

    Hadjiargyrou, Michael; O’Keefe, Regis J

    2015-01-01

    The complexity of fracture repair makes it an ideal process for studying the interplay between the molecular, cellular, tissue, and organ level events involved in tissue regeneration. Additionally, as fracture repair recapitulates many of the processes that occur during embryonic development, investigations of fracture repair provide insights regarding skeletal embryogenesis. Specifically, inflammation, signaling, gene expression, cellular proliferation and differentiation, osteogenesis, chondrogenesis, angiogenesis, and remodeling represent the complex array of interdependent biological events that occur during fracture repair. Here we review studies of bone regeneration in genetically modified mouse models, during aging, following environmental exposure, and in the setting of disease that provide insights regarding the role of multipotent cells and their regulation during fracture repair. Complementary animal models and ongoing scientific discoveries define an increasing number of molecular and cellular targets to reduce the morbidity and complications associated with fracture repair. Last, some new and exciting areas of stem cell research such as the contribution of mitochondria function, limb regeneration signaling, and microRNA (miRNA) posttranscriptional regulation are all likely to further contribute to our understanding of fracture repair as an active branch of regenerative medicine. PMID:25264148

  6. Repair of UV damaged DNA, genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1991-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, as well as studying the human homologs of yeast excision repair and postreplication repair proteins. In addition to its single-stranded DNA-dependent A TPase and DNA helicase activities, we have found that RAD3 protein also possesses DNA-RNA helicase activity, and that like RAD3, the Schizosaccharomyces pombe RAD3 homolog, rhp3{sup +}, is an essential gene. We have overexpressed the human RAD3 homolog, ERCC2, in yeast to facilitate its purification. The RAD10 protein was purified to homogeneity and shown to bind DNA. ERCC3y, the yeast homolog of the human ERCC-3/XP-B gene, has been sequenced and shown to be essential for viability. The Drosophila and human homologs of RAD6, required for postreplication repair and UV induced mutagenesis, were shown to complement the rad6 {Delta} mutation of yeast. Since defective DNA repair and enhanced neoplasia characterize several human genetic diseases, and repair proteins are highly conserved between yeast and man, a thorough understanding of the molecular mechanisms of DNA repir in yeast should provide a better understanding of the causes of carcinogenesis.

  7. Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa.

    PubMed

    Berger, Adeline; Lorain, Stéphanie; Joséphine, Charlène; Desrosiers, Melissa; Peccate, Cécile; Voit, Thomas; Garcia, Luis; Sahel, José-Alain; Bemelmans, Alexis-Pierre

    2015-05-01

    The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission.

  8. Optimising plasticity: environmental and training associated factors in transplant-mediated brain repair.

    PubMed

    Döbrössy, Màtè Daniel; Dunnett, Stephen B

    2005-01-01

    With progressively ageing populations, degeneration of nerve cells of the brain, due to accident or disease, represents one of the major problems for health and welfare in the developed world. The molecular environment in the adult brain promotes stability limiting its ability to regenerate or to repair itself following injury. Cell transplantation aims to repair the nervous system by introducing new cells that can replace the function of the compromised or lost cells. Alternatives to primary embryonic tissue are actively being sought but this is at present the only source that has been shown reliably to survive grafting into the adult brain and spinal cord, connect with the host nervous system, and influence behaviour. Based on animal studies, several clinical trials have now shown that embryonic tissue grafts can partially alleviate symptoms in Parkinson's disease, and related strategies are under evaluation for Huntington's disease, spinal cord injury, stroke and other CNS disorders. The adult brain is at its most plastic in the period following injury, offering a window of opportunity for therapeutic intervention. Enriched environment, behavioural experience and grafting can each separately influence neuronal plasticity and recovery of function after brain damage, but the extent to which these factors interact is at present unknown. To improve the outcome following brain damage, transplantation must make use of the endogenous potential for plasticity of both the host and the graft and optimise the external circumstances associated with graft-mediated recovery. Our understanding of mechanisms of brain plasticity subsequent to brain damage needs to be associated with what we know about enhancing intrinsic recovery processes in order to improve neurobiological and surgical strategies for repair at the clinical level. With the proof of principle beginning to emerge from clinical trials, a rich area for innovative research with profound therapeutic application, even

  9. Coordination of cell cycle, DNA repair and muscle gene expression in myoblasts exposed to genotoxic stress

    PubMed Central

    Minetti, Giulia Claudia

    2011-01-01

    Upon exposure to genotoxic stress, skeletal muscle progenitors coordinate DNA repair and the activation of the differentiation program through the DNA damage-activated differentiation checkpoint, which holds the transcription of differentiation genes while the DNA is repaired. A conceptual hurdle intrinsic to this process relates to the coordination of DNA repair and muscle-specific gene transcription within specific cell cycle boundaries (cell cycle checkpoints) activated by different types of genotoxins. Here, we show that, in proliferating myoblasts, the inhibition of muscle gene transcription occurs by either a G1- or G2-specific differentiation checkpoint. In response to genotoxins that induce G1 arrest, MyoD binds target genes but is functionally inactivated by a c-Abl-dependent phosphorylation. In contrast, DNA damage-activated G2 checkpoint relies on the inability of MyoD to bind the chromatin at the G2 phase of the cell cycle. These results indicate an intimate relationship between DNA damage-activated cell cycle checkpoints and the control of tissue-specific gene expression to allow DNA repair in myoblasts prior to the activation of the differentiation program. PMID:21685725

  10. Repair of rDNA in Saccharomyces cerevisiae: RAD4-independent strand-specific nucleotide excision repair of RNA polymerase I transcribed genes.

    PubMed Central

    Verhage, R A; Van de Putte, P; Brouwer, J

    1996-01-01

    Removal of UV-induced pyrimidine dimers from the individual strands of the rDNA locus in Saccharomyces cerevisiae was studied. Yeast rDNA, that is transcribed by RNA polymerase I(RNA pol I), is repaired efficiently, slightly strand-specific and independently of RAD26, which has been implicated in transcription-coupled repair of the RNA pol II transcribed RPB2 gene. No repair of rDNA is observed in rad1,2,3 and 14 mutants, demonstrating that dimer removal from this highly repetitive DNA is accomplished by nucleotide excision repair (NER). In rad7 and rad16 mutants, which are specifically deficient in repair of non-transcribed DNA, there is a clear preferential repair of the transcribed strand of rDNA, indicating that strand-specific and therefore probably transcription-coupled repair of RNA pol I transcribed genes does exist in yeast. Unexpectedly, the transcribed but not the non-transcribed strand of rDNA can be repaired in rad4 mutants, which seem otherwise completely NER-deficient. PMID:8604332

  11. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations

    PubMed Central

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M.; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A.F.V.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  12. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.

    PubMed

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A F V

    2016-02-18

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  13. Xilei San Ameliorates Experimental Colitis in Rats by Selectively Degrading Proinflammatory Mediators and Promoting Mucosal Repair

    PubMed Central

    Hori, Kazutoshi; Wang, Shenglan; Kogure, Yoko; Fukunaga, Ken; Kashiwamura, Shinichiro; Yamamoto, Satoshi; Nakamura, Shiro; Li, Junxiang; Miwa, Hiroto; Noguchi, Koichi

    2014-01-01

    Xilei san (XLS), a herbal preparation widely used in China for erosive and ulcerative diseases, has been shown to be effective in ulcerative colitis (UC). The present experiments were conducted to assess its efficacy and determine its mechanism of action in a rat model that resembles human UC. The model was induced by adding 4% dextran sulfate sodium (DSS) to the rats' drinking water for 7 days. XLS was administered daily by retention enema from day 2 to day 7; the rats were sacrificed on day 8. The colon tissues were obtained for further experiments. A histological damage score and the activity of tissue myeloperoxidase were used to evaluate the severity of the colitis. The colonic cytokine levels were detected in a suspension array, and epithelial proliferation was assessed using Ki-67 immunohistochemistry. Intrarectal administration of XLS attenuated the DSS-induced colitis, as evidenced by a reduction in both the histological damage score and myeloperoxidase activity. It also decreased the levels of proinflammatory cytokines, but increased the mucosal repair-related cytokines. In addition, the epithelial Ki-67 expression was upregulated by XLS. These results suggest that XLS attenuates DSS-induced colitis by degrading proinflammatory mediators and promoting mucosal repair. XLS could be a potential topical treatment for human UC. PMID:25120575

  14. Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion.

    PubMed

    Wang, Harris H; Xu, George; Vonner, Ashley J; Church, George

    2011-09-01

    Genome engineering using single-stranded oligonucleotides is an efficient method for generating small chromosomal and episomal modifications in a variety of host organisms. The efficiency of this allelic replacement strategy is highly dependent on avoidance of the endogenous mismatch repair (MMR) machinery. However, global MMR inactivation generally results in significant accumulation of undesired background mutations. Here, we present a novel strategy using oligos containing chemically modified bases (2'-Fluoro-Uridine, 5-Methyl-deoxyCytidine, 2,6-Diaminopurine or Iso-deoxyGuanosine) in place of the standard T, C, A or G to avoid mismatch detection and repair, which we tested in Escherichia coli. This strategy increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level. We further show that the mismatched bases between the full length oligo and the chromosome are often not incorporated at the target site, probably due to nuclease activity at the 5' and 3' termini of the oligo. These results further elucidate the mechanism of oligo-mediated allelic replacement (OMAR) and enable improved methodologies for efficient, large-scale engineering of genomes.

  15. Enhanced infarct myocardium repair mediated by thermosensitive copolymer hydrogel-based stem cell transplantation

    PubMed Central

    Xia, Yu; Zhu, Kai; Lai, Hao; Lang, Meidong; Xiao, Yan; Lian, Sheng

    2015-01-01

    Mesenchymal stem cell (MSC) transplantation by intramyocardial injection has been proposed as a promising therapy strategy for cardiac repair after myocardium infarction. However, low retention and survival of grafted MSCs hinder its further application. In this study, copolymer with N-isopropylacrylamide/acrylic acid/2-hydroxylethyl methacrylate-poly(ɛ-caprolactone) ratio of 88:9.6:2.4 was bioconjugated with type I collagen to construct a novel injectable thermosensitive hydrogel. The injectable and biocompatible hydrogel-mediated MSC transplantation could enhance the grafted cell survival in the myocardium, which contributed to the increased neovascularization, decreased interstitial fibrosis, and ultimately improved heart function to a significantly greater degree than regular MSC transplantation. We suggest that this novel hydrogel has the potential for future stem cell transplantation. PMID:25432986

  16. Gene expression profiling of archival tongue squamous cell carcinomas provides sub-classification based on DNA repair genes.

    PubMed

    Rentoft, Matilda; Laurell, Göran; Coates, Philip John; Sjöström, Björn; Nylander, Karin

    2009-12-01

    A subgroup of patients with squamous cell carcinoma of the head and neck (SCCHN) comprise young persons under the age of 40, who have not been heavily exposed to the classical risk factors, smoking and alcohol. The number of SCCHN in young adults, particularly tongue tumours, is increasing in several parts of the world. Here we employed a novel gene expression array methodology specifically developed for analysis of degraded RNA and investigated the expression of 502 cancer-related genes in archival paraffin-embedded SCCHN of the tongue from young (< or =40) and elderly patients (> or =50). Genes detected as de-regulated in tumours compared to non-malignant controls were in concordance with results from earlier studies of fresh frozen material. No genes were detected as significantly differentially expressed between young and old patients suggesting that the overall pathobiology of SCCHN is similar in young and old. Unsupervised clustering divided tumours into three groups, irrespective of age, where several differentially expressed DNA repair genes were a prominent separation factor. High levels of DNA repair genes associated with impaired therapeutic response to radiation, suggesting that DNA repair genes play a role in clinical outcome after radiotherapy.

  17. Higher expression of somatic repair genes in long-lived ant queens than workers

    PubMed Central

    Lucas, Eric R.; Privman, Eyal; Keller, Laurent

    2016-01-01

    Understanding why organisms senesce is a fundamental question in biology. One common explanation is that senescence results from an increase in macromolecular damage with age. The tremendous variation in lifespan between genetically identical queen and worker ants, ranging over an order of magnitude, provides a unique system to study how investment into processes of somatic maintenance and macromolecular repair influence lifespan. Here we use RNAseq to compare patterns of expression of genes involved in DNA and protein repair of age-matched queens and workers. There was no difference between queens and workers in 1-day-old individuals, but the level of expression of these genes increased with age and this up-regulation was greater in queens than in workers, resulting in significantly queen-biased expression in 2-month-old individuals in both legs and brains. Overall, these differences are consistent with the hypothesis that higher longevity is associated with increased investment into somatic repair. PMID:27617474

  18. Expression of DNA repair and metabolic genes in response to a flavonoid-rich diet.

    PubMed

    Guarrera, Simonetta; Sacerdote, Carlotta; Fiorini, Laura; Marsala, Rosa; Polidoro, Silvia; Gamberini, Sara; Saletta, Federica; Malaveille, Christian; Talaska, Glenn; Vineis, Paolo; Matullo, Giuseppe

    2007-09-01

    A diet rich in fruit and vegetables can be effective in the reduction of oxidative stress, through the antioxidant effects of phytochemicals and other mechanisms. Protection against the carcinogenic effects of chemicals may also be exerted by an enhancement of detoxification and DNA damage repair mechanisms. To investigate a putative effect of flavonoids, a class of polyphenols, on the regulation of the gene expression of DNA repair and metabolic genes, a 1-month flavonoid-rich diet was administered to thirty healthy male smokers, nine of whom underwent gene expression analysis. We postulated that tobacco smoke is a powerful source of reactive oxygen species. The expression level of twelve genes (APEX, ERCC1, ERCC2, ERCC4, MGMT, OGG1, XPA, XPC, XRCC1, XRCC3, AHR, CYP1A1) was investigated. We found a significant increase (P < 0.001) in flavonoid intake. Urinary phenolic content and anti-mutagenicity did not significantly change after diet, nor was a correlation found between flavonoid intake and urinary phenolic levels or anti-mutagenicity. Phenolic levels showed a significant positive correlation with urinary anti-mutagenicity. AHR levels were significantly reduced after the diet (P = 0.038), whereas the other genes showed a generalized up regulation, significant for XRCC3 gene (P = 0.038). Also in the context of a generalized up regulation of DNA repair genes, we found a non-significant negative correlation between flavonoid intake and the expression of all the DNA repair genes. Larger studies are needed to clarify the possible effects of flavonoids in vivo; our preliminary results could help to better plan new studies on gene expression and diet.

  19. DNA repair mutant pso2 of Saccharomyces cerevisiae is sensitive to intracellular acetaldehyde accumulated by disulfiram-mediated inhibition of acetaldehyde dehydrogenase.

    PubMed

    Brendel, M; Marisco, G; Ganda, I; Wolter, R; Pungartnik, C

    2010-01-12

    Blocking aldehyde dehydrogenase with the drug disulfiram leads to an accumulation of intracellular acetaldehyde, which negatively affects the viability of the yeast Saccharomyces cerevisiae. Mutants of the yeast gene PSO2, which encodes a protein specific for repair of DNA interstrand cross-links, showed higher sensitivity to disulfiram compared to the wild type. This leads us to suggest that accumulated acetaldehyde induces DNA lesions, including highly deleterious interstrand cross-links. Acetaldehyde induced the expression of a PSO2-lacZ reporter construct that is specifically inducible by bi- or poly-functional mutagens, e.g., nitrogen mustard and photo-activated psoralens. Chronic exposure of yeast cells to disulfiram and acute exposure to acetaldehyde induced forward mutagenesis in the yeast CAN1 gene. Disulfiram-induced mutability of a pso2Delta mutant was significantly increased over that of the isogenic wild type; however, this was not found for acetaldehyde-induced mutagenesis. Spontaneous mutability at the CAN1 locus was elevated in pso2Delta, suggesting that growth of glucose-repressed yeast produces DNA lesions that, in the absence of Pso2p-mediated crosslink repair, are partially removed by an error-prone DNA repair mechanism. The use of disulfiram in the control of human alcohol abuse increases cellular acetaldehyde pools, which, based on our observations, enhances the risk of mutagenesis and of other genetic damage.

  20. MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response

    PubMed Central

    Pothof, Joris; Verkaik, Nicole S; van IJcken, Wilfred; Wiemer, Erik A C; Ta, Van T B; van der Horst, Gijsbertus T J; Jaspers, Nicolaas G J; van Gent, Dik C; Hoeijmakers, Jan H J; Persengiev, Stephan P

    2009-01-01

    DNA damage provokes DNA repair, cell-cycle regulation and apoptosis. This DNA-damage response encompasses gene-expression regulation at the transcriptional and post-translational levels. We show that cellular responses to UV-induced DNA damage are also regulated at the post-transcriptional level by microRNAs. Survival and checkpoint response after UV damage was severely reduced on microRNA-mediated gene-silencing inhibition by knocking down essential components of the microRNA-processing pathway (Dicer and Ago2). UV damage triggered a cell-cycle-dependent relocalization of Ago2 into stress granules and various microRNA-expression changes. Ago2 relocalization required CDK activity, but was independent of ATM/ATR checkpoint signalling, whereas UV-responsive microRNA expression was only partially ATM/ATR independent. Both microRNA-expression changes and stress-granule formation were most pronounced within the first hours after genotoxic stress, suggesting that microRNA-mediated gene regulation operates earlier than most transcriptional responses. The functionality of the microRNA response is illustrated by the UV-inducible miR-16 that downregulates checkpoint-gene CDC25a and regulates cell proliferation. We conclude that microRNA-mediated gene regulation adds a new dimension to the DNA-damage response. PMID:19536137

  1. Semiconservative replication, genetic repair, and many-gened genomes: Extending the quasispecies paradigm to living systems

    NASA Astrophysics Data System (ADS)

    Tannenbaum, Emmanuel; Shakhnovich, Eugene I.

    2005-12-01

    Quasispecies theory has emerged as an important tool for modeling the evolutionary dynamics of biological systems. We review recent advances in the field, with an emphasis on the quasispecies dynamics of semiconservatively replicating genomes. Applications to cancer and adult stem cell growth are discussed. Additional topics, such as genetic repair and many-gene genomes, are covered as well.

  2. Recent Trends of Polymer Mediated Liposomal Gene Delivery System

    PubMed Central

    Lee, Sang-Soo; George Priya Doss, C.; Yagihara, Shin; Kim, Do-Young

    2014-01-01

    Advancement in the gene delivery system have resulted in clinical successes in gene therapy for patients with several genetic diseases, such as immunodeficiency diseases, X-linked adrenoleukodystrophy (X-ALD) blindness, thalassemia, and many more. Among various delivery systems, liposomal mediated gene delivery route is offering great promises for gene therapy. This review is an attempt to depict a portrait about the polymer based liposomal gene delivery systems and their future applications. Herein, we have discussed in detail the characteristics of liposome, importance of polymer for liposome formulation, gene delivery, and future direction of liposome based gene delivery as a whole. PMID:25250340

  3. Short and long-term changes in gene expression mediated by the activation of TLR9

    PubMed Central

    Klaschik, Sven; Tross, Debra; Shirota, Hidekazu; Klinman, Dennis M.

    2009-01-01

    CpG DNA binds to Toll-like receptor 9 to stimulate a strong innate immune response. The magnitude, duration and scope of CpG-induced changes in gene expression is incompletely understood despite extensive studies of TLR9 mediated signal transduction pathways. In particular, the prolonged effects of CpG DNA on gene activation have not been investigated despite evidence that a single dose of CpG DNA alters immune reactivity for several weeks. This study used gene expression analysis to monitor changes in mRNA levels for 14 days, and identified the genes, pathways and functional groups triggered in vivo following CpG DNA administration. Two discrete peaks of gene activation (at 3 hr and 5 days) were observed after CpG injection. Both the behavior and function of genes activated during the second peak differed from those triggered shortly after CpG administration. Initial gene up-regulation corresponded to a period when TLR9 ligation stimulated genes functionally associated with the generation of innate and adaptive immune responses (e.g. the NF-kB and B-cell receptor pathways). The second peak reflected processes associated with cell division (e.g., cell cycle and DNA replication & repair). The complex bimodal pattern of gene expression elicited by CpG DNA administration provides novel insights into the long term effects of TLR9 engagement on genes associated with immunity and cell proliferation. PMID:20005572

  4. Lin− Cells Mediate Tissue Repair by Regulating MCP-1/CCL-2

    PubMed Central

    Schatteman, Gina C.; Awad, Ola; Nau, Eric; Wang, Chunlin; Jiao, Chunhua; Tomanek, Robert J.; Dunnwald, Martine

    2010-01-01

    Exogenous bone marrow-derived cells (BMDCs) are promising therapeutic agents for the treatment of tissue ischemia and traumatic injury. However, until we identify the molecular mechanisms that underlie their actions, there can be no rational basis for the design of therapeutic strategies using BMDCs. The pro-healing effects of BMDCs are apparent very shortly after treatment, which suggests that they may exert their effects by the modulation of acute inflammation. We investigated this hypothesis by taking advantage of the fact that BMDCs from healthy, young, but not obese, diabetic mice stimulate vascular growth. By comparing both in vitro secretion and in vivo local induction of acute phase inflammatory cytokines by these cells, we identified monocyte chemoattractant factor 1 and tumor necrosis factor α as potential mediators of BMDC-induced tissue repair. In vivo analysis of BMDC-treated ischemic limbs and cutaneous wounds revealed that the production of monocyte chemoattractant factor 1 by exogenous and endogenous BMDCs is essential for BMDC-mediated vascular growth and tissue healing, while the inability of BMDCs to produce tumor necrosis factor α appears to play a lesser but still meaningful role. Thus, measurements of the secretion of cytokines by BMDCs may allow us to identify a priori individuals who would or would not be good candidates for BMDC-based therapies. PMID:20813969

  5. Polymorphisms in DNA Repair Genes and MDR1 and the Risk for Non-Hodgkin Lymphoma

    PubMed Central

    Kim, Hee Nam; Kim, Nan Young; Yu, Li; Kim, Yeo-Kyeoung; Lee, Il-Kwon; Yang, Deok-Hwan; Lee, Je-Jung; Shin, Min-Ho; Park, Kyeong-Soo; Choi, Jin-Su; Kim, Hyeoung-Joon

    2014-01-01

    The damage caused by oxidative stress and exposure to cigarette smoke and alcohol necessitate DNA damage repair and transport by multidrug resistance-1 (MDR1). To explore the association between polymorphisms in these genes and non-Hodgkin lymphoma risk, we analyzed 15 polymorphisms of 12 genes in a population-based study in Korea (694 cases and 1700 controls). Four genotypes of DNA repair pathway genes (XRCC1 399 GA, OGG1 326 GG, BRCA1 871 TT, and WRN 787 TT) were associated with a decreased risk for NHL [odds ratio (OR)XRCC1 GA = 0.80, p = 0.02; OROGG1 GG = 0.70, p = 0.008; ORBRCA1 TT = 0.71, p = 0.048; ORWRN TT = 0.68, p = 0.01]. Conversely, the MGMT 115 CT genotype was associated with an increased risk for NHL (OR = 1.25, p = 0.04). In the MDR1 gene, the 1236 CC genotype was associated with a decreased risk for NHL (OR = 0.74, p = 0.04), and the 3435 CT and TT genotypes were associated with an increased risk (OR3435CT = 1.50, p < 0.0001; OR3435TT = 1.43, p = 0.02). These results suggest that polymorphisms in the DNA repair genes XRCC1, OGG1, BRCA1, WRN1, and MGMT and in the MDR1 gene may affect the risk for NHL in Korean patients. PMID:24756092

  6. Polymorphisms in DNA repair genes and MDR1 and the risk for non-Hodgkin lymphoma.

    PubMed

    Kim, Hee Nam; Kim, Nan Young; Yu, Li; Kim, Yeo-Kyeoung; Lee, Il-Kwon; Yang, Deok-Hwan; Lee, Je-Jung; Shin, Min-Ho; Park, Kyeong-Soo; Choi, Jin-Su; Kim, Hyeoung-Joon

    2014-04-21

    The damage caused by oxidative stress and exposure to cigarette smoke and alcohol necessitate DNA damage repair and transport by multidrug resistance-1 (MDR1). To explore the association between polymorphisms in these genes and non-Hodgkin lymphoma risk, we analyzed 15 polymorphisms of 12 genes in a population-based study in Korea (694 cases and 1700 controls). Four genotypes of DNA repair pathway genes (XRCC1 399 GA, OGG1 326 GG, BRCA1 871 TT, and WRN 787 TT) were associated with a decreased risk for NHL [odds ratio (OR)XRCC1 GA=0.80, p=0.02; OROGG1 GG=0.70, p=0.008; ORBRCA1 TT=0.71, p=0.048; ORWRN TT=0.68, p=0.01]. Conversely, the MGMT 115 CT genotype was associated with an increased risk for NHL (OR=1.25, p=0.04). In the MDR1 gene, the 1236 CC genotype was associated with a decreased risk for NHL (OR=0.74, p=0.04), and the 3435 CT and TT genotypes were associated with an increased risk (OR3435CT=1.50, p<0.0001; OR3435TT=1.43, p=0.02). These results suggest that polymorphisms in the DNA repair genes XRCC1, OGG1, BRCA1, WRN1, and MGMT and in the MDR1 gene may affect the risk for NHL in Korean patients.

  7. 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

  8. Biochemical studies of DNA strand break repair and molecular characterization of mei-41, a gene involved in DNA break repair

    SciTech Connect

    Oliveri, D.R.

    1989-01-01

    The ability to repair X-irradiation induced single-strand DNA breaks was examined in mutagen-sensitive mutants of Drosophila melanogaster. This analysis demonstrated that examined stocks possess a normal capacity to repair X-ray induced single-strand breaks. One of the mutants in this study, mei-41, has been shown to be involved in a number of DNA metabolizing functions. A molecular characterization of this mutant is presented. A cDNA hybridizing to genomic DNA both proximal and distal to a P element inducing a mei-41 mutation was isolated from both embryonic and adult female recombinant lambda phage libraries. A 2.2 kilobase embryonic cDNA clone was sequenced; the sequence of an open reading frame was identified which would predict a protein of 384 amino acids with a molecular weight of 43,132 daltons. An examination of homologies to sequences in protein and nucleic acid data bases revealed no sequences with significant homology to mei-41, however, two potential Zinc-finger domains were identified. Analysis of RNA hybridizing to the embryonic cDNA demonstrated the existence of a major 2.2 kilobase transcript expressed primarily in embryos and adult flies. An examination of the transcription of this gene in mei-41 mutants revealed significant variation from wild-type, an indication that the embryonic cDNA does represent a mei-41 transcript. Expression in tissues from adult animals demonstrated that the 2.2 kilobase RNA is expressed primarily in reproductive tissues. A 3.8kb transcript is the major species of RNA in the adult head and thorax. Evidence is presented which implies that expression of the mei-41 gene is strongly induced by exposure of certain cells to mutagens.

  9. Transcription-coupled and global genome repair in the Saccharomyces cerevisiae RPB2 gene at nucleotide resolution.

    PubMed Central

    Tijsterman, M; Tasseron-de Jong, J G; van de Putte, P; Brouwer, J

    1996-01-01

    Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was examined at single nucleotide resolution in the yeast Saccharomyces cerevisiae, using an improved protocol for genomic end-labelling. To obtain the sensitivity required for adduct detection in yeast, an oligonucleotide-directed enrichment step was introduced into the current methodology developed for adduct detection in Escherichia coli. With this method, heterogeneous repair of CPDs within the RPB2 locus is observed. Individual CPDs positioned in the transcribed strand are removed very efficiently with identical kinetics. This fast repair starts within 23 bases downstream of the transcription initiation site. The non-transcribed strand of the active gene exhibits slow repair without detectable repair variations between individual lesions. In contrast, CPDs positioned in the promoter region show profound repair heterogeneity. Here, CPDs at specific sites are removed very quickly, with comparable rates to CPDs positioned in the transcribed strand, while at other positions lesions are not repaired at all during the period studied. Interestingly, the fast repair in the promoter region is dependent on the RAD7 and RAD16 genes, as are the slowly repaired CPDs in this region and in the non-transcribed strand. This indicates that the global genome repair pathway is not intrinsically slow and at specific positions can be as efficient as the transcription-coupled repair pathway. PMID:8836174

  10. Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

    PubMed

    da Silva Neto Trajano, Larissa Alexsandra; Stumbo, Ana Carolina; da Silva, Camila Luna; Mencalha, Andre Luiz; Fonseca, Adenilson S

    2016-08-01

    Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

  11. Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

    PubMed

    da Silva Neto Trajano, Larissa Alexsandra; Stumbo, Ana Carolina; da Silva, Camila Luna; Mencalha, Andre Luiz; Fonseca, Adenilson S

    2016-08-01

    Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions. PMID:27220530

  12. Irreversible UV inactivation of Cryptosporidium spp. despite the presence of UV repair genes.

    PubMed

    Rochelle, Paul A; Fallar, Daffodil; Marshall, Marilyn M; Montelone, Beth A; Upton, Steve J; Woods, Keith

    2004-01-01

    Ultraviolet light is being considered as a disinfectant by the water industry because it appears to be very effective for inactivating pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms for repairing ultraviolet light-induced DNA damage, which may limit the utility of this disinfection technology. Inactivation of C. parvum was assessed by measuring infectivity in cells of the human ileocecal adenocarcinoma HCT-8 cell line, with an assay targeting a heat shock protein gene and using a reverse transcriptase polymerase chain reaction to detect infections. Oocysts of five different isolates displayed similar sensitivity to ultraviolet light. An average dosage of 7.6 mJ/cm2 resulted in 99.9% inactivation, providing the first evidence that multiple isolates of C. parvum are equally sensitive to ultraviolet disinfection. Irradiated oocysts were unable to regain pre-irradiation levels of infectivity, following exposure to a broad array of potential repair conditions, such as prolonged incubation, pre-infection excystation triggers, and post-ultraviolet holding periods. A combination of data-mining and sequencing was used to identify genes for all of the major components of a nucleotide excision repair complex in C. parvum and Cryptosporidium hominis. The average similarity between the two organisms for the various genes was 96.4% (range, 92-98%). Thus, while Cryptosporidum spp. may have the potential to repair ultraviolet light-induced damage, oocyst reactivation will not occur under the standard conditions used for storage and distribution of treated drinking water.

  13. Antioxidative Dietary Compounds Modulate Gene Expression Associated with Apoptosis, DNA Repair, Inhibition of Cell Proliferation and Migration

    PubMed Central

    Wang, Likui; Gao, Shijuan; Jiang, Wei; Luo, Cheng; Xu, Maonian; Bohlin, Lars; Rosendahl, Markus; Huang, Wenlin

    2014-01-01

    Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2) called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE), which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix) polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen) and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair. PMID:25226533

  14. Melatonin enhances DNA repair capacity possibly by affecting genes involved in DNA damage responsive pathways

    PubMed Central

    2013-01-01

    Background Melatonin, a hormone-like substance involved in the regulation of the circadian rhythm, has been demonstrated to protect cells against oxidative DNA damage and to inhibit tumorigenesis. Results In the current study, we investigated the effect of melatonin on DNA strand breaks using the alkaline DNA comet assay in breast cancer (MCF-7) and colon cancer (HCT-15) cell lines. Our results demonstrated that cells pretreated with melatonin had significantly shorter Olive tail moments compared to non-melatonin treated cells upon mutagen (methyl methanesulfonate, MMS) exposure, indicating an increased DNA repair capacity after melatonin treatment. We further examined the genome-wide gene expression in melatonin pretreated MCF-7 cells upon carcinogen exposure and detected altered expression of many genes involved in multiple DNA damage responsive pathways. Genes exhibiting altered expression were further analyzed for functional interrelatedness using network- and pathway-based bioinformatics analysis. The top functional network was defined as having relevance for “DNA Replication, Recombination, and Repair, Gene Expression, [and] Cancer”. Conclusions These findings suggest that melatonin may enhance DNA repair capacity by affecting several key genes involved in DNA damage responsive pathways. PMID:23294620

  15. Transcriptional and Post-Transcriptional Regulation of Nucleotide Excision Repair Genes in Human Cells

    PubMed Central

    Lefkofsky, Hailey B.; Veloso, Artur; Ljungman, Mats

    2014-01-01

    Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death. PMID:26255935

  16. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    PubMed

    Dupuy, Aurélie; Sarasin, Alain

    2015-06-01

    Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients. PMID:26255934

  17. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    PubMed

    Dupuy, Aurélie; Sarasin, Alain

    2015-06-01

    Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  18. The barley EST DNA Replication and Repair Database (bEST-DRRD) as a tool for the identification of the genes involved in DNA replication and repair

    PubMed Central

    2012-01-01

    Background The high level of conservation of genes that regulate DNA replication and repair indicates that they may serve as a source of information on the origin and evolution of the species and makes them a reliable system for the identification of cross-species homologs. Studies that had been conducted to date shed light on the processes of DNA replication and repair in bacteria, yeast and mammals. However, there is still much to be learned about the process of DNA damage repair in plants. Description These studies, which were conducted mainly using bioinformatics tools, enabled the list of genes that participate in various pathways of DNA repair in Arabidopsis thaliana (L.) Heynh to be outlined; however, information regarding these mechanisms in crop plants is still very limited. A similar, functional approach is particularly difficult for a species whose complete genomic sequences are still unavailable. One of the solutions is to apply ESTs (Expressed Sequence Tags) as the basis for gene identification. For the construction of the barley EST DNA Replication and Repair Database (bEST-DRRD), presented here, the Arabidopsis nucleotide and protein sequences involved in DNA replication and repair were used to browse for and retrieve the deposited sequences, derived from four barley (Hordeum vulgare L.) sequence databases, including the “Barley Genome version 0.05” database (encompassing ca. 90% of barley coding sequences) and from two databases covering the complete genomes of two monocot models: Oryza sativa L. and Brachypodium distachyon L. in order to identify homologous genes. Sequences of the categorised Arabidopsis queries are used for browsing the repositories, which are located on the ViroBLAST platform. The bEST-DRRD is currently used in our project during the identification and validation of the barley genes involved in DNA repair. Conclusions The presented database provides information about the Arabidopsis genes involved in DNA replication and

  19. Energy and Technology Review: Unlocking the mysteries of DNA repair

    SciTech Connect

    Quirk, W.A.

    1993-04-01

    DNA, the genetic blueprint, has the remarkable property of encoding its own repair following diverse types of structural damage induced by external agents or normal metabolism. We are studying the interplay of DNA damaging agents, repair genes, and their protein products to decipher the complex biochemical pathways that mediate such repair. Our research focuses on repair processes that correct DNA damage produced by chemical mutagens and radiation, both ionizing and ultraviolet. The most important type of DNA repair in human cells is called excision repair. This multistep process removes damaged or inappropriate pieces of DNA -- often as a string of 29 nucleotides containing the damage -- and replaces them with intact ones. We have isolated, cloned, and mapped several human repair genes associated with the nucleotide excision repair pathway and involved in the repair of DNA damage after exposure to ultraviolet light or mutagens in cooked food. We have shown that a defect in one of these repair genes, ERCC2, is responsible for the repair deficiency in one of the groups of patients with the recessive genetic disorder xeroderma pigmentosum (XP group D). We are exploring ways to purify sufficient quantities (milligrams) of the protein products of these and other repair genes so that we can understand their functions. Our long-term goals are to link defective repair proteins to human DNA repair disorders that predispose to cancer, and to produce DNA-repair-deficient mice that can serve as models for the human disorders.

  20. Low intensity infrared laser affects expression of oxidative DNA repair genes in mitochondria and nucleus

    NASA Astrophysics Data System (ADS)

    Fonseca, A. S.; Magalhães, L. A. G.; Mencalha, A. L.; Geller, M.; Paoli, F.

    2014-11-01

    Practical properties and physical characteristics of low intensity lasers have made possible their application to treat soft tissue diseases. Excitation of intracellular chromophores by red and infrared radiation at low energy fluences with increase of mitochondrial metabolism is the basis of the biostimulation effect but free radicals can be produced. DNA lesions induced by free radicals are repaired by the base excision repair pathway. In this work, we evaluate the expression of POLγ and APEX2 genes related to repair of mitochondrial and nuclear DNA, respectively. Skin and muscle tissue of Wistar rats were exposed to low intensity infrared laser at different fluences. One hour and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of POLγ and APEX2 mRNA expression by real time quantitative polymerase chain reaction. Skin and muscle tissue of Wistar rats exposed to laser radiation show different expression of POLγ and APEX2 mRNA depending of the fluence and time after exposure. Our study suggests that a low intensity infrared laser affects expression of genes involved in repair of oxidative lesions in mitochondrial and nuclear DNA.

  1. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

    PubMed Central

    Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  2. Particle-mediated gene transfer into murine livers using a newly developed gene gun.

    PubMed

    Kuriyama, S; Mitoro, A; Tsujinoue, H; Nakatani, T; Yoshiji, H; Tsujimoto, T; Yamazaki, M; Fukui, H

    2000-07-01

    Although particle-mediated gene transfer using gene gun technology has been applied for gene transfer into epidermis, applications of this technology to visceral tissues have not been well investigated. Although all helium gas-driven gene gun instruments have used macrocarriers to discharge DNA-coated microprojectiles so far, we used a newly developed gene gun instrument, in which a hammering bullet is used to discharge microprojectiles. With the gene gun, gold particles coated with lacZ expression plasmid were discharged to murine livers. LacZ expression was induced much more profoundly in the liver by particle-mediated gene transfer than by simple plasmid injection and electroporation-mediated gene transfer. LacZ expression was broadly and randomly distributed throughout the bombarded livers, indicating that particle-mediated gene transfer can induce transgene expression even at relatively distant areas from the surface of the bombarded tissue. Furthermore, although transgene expression was at its peak on day 2 after the bombardment, it was still detectable even on day 28. These results indicate that particle-mediated gene transfer with a newly developed gene gun may provide a new approach to gene therapy for human diseases.

  3. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    SciTech Connect

    Perez, C.F.

    1984-08-01

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

  4. Differences in Expression of Key DNA Damage Repair Genes after Epigenetic-Induced BRCAness Dictate Synthetic Lethality with PARP1 Inhibition.

    PubMed

    Wiegmans, Adrian P; Yap, Pei-Yi; Ward, Ambber; Lim, Yi Chieh; Khanna, Kum Kum

    2015-10-01

    The triple-negative breast cancer (TNBC) subtype represents a cancer that is highly aggressive with poor patient outcome. Current preclinical success has been gained through synthetic lethality, targeting genome instability with PARP inhibition in breast cancer cells that harbor silencing of the homologous recombination (HR) pathway. Histone deacetylase inhibitors (HDACi) are a class of drugs that mediate epigenetic changes in expression of HR pathway genes. Here, we compare the activity of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), the class I/IIa HDAC inhibitor valproic acid (VPA), and the HDAC1/2-specific inhibitor romidepsin (ROMI) for their capability to regulate DNA damage repair gene expression and in sensitizing TNBC to PARPi. We found that two of the HDACis tested, SAHA and ROMI, but not VPA, indeed inhibit HR repair and that RAD51, BARD1, and FANCD2 represent key proteins whose inhibition is required for HDACi-mediated therapy with PARP inhibition in TNBC. We also observed that restoration of BRCA1 function stabilizes the genome compared with mutant BRCA1 that results in enhanced polyploid population after combination treatment with HDACi and PARPi. Furthermore, we found that overexpression of the key HR protein RAD51 represents a mechanism for this resistance, promoting aberrant repair and the enhanced polyploidy observed. These findings highlight the key components of HR in guiding synthetic lethality with PARP inhibition and support the rationale for utilizing the novel combination of HDACi and PARPi against TNBC in the clinical setting. PMID:26294743

  5. Repair in ribosomal RNA genes is deficient in xeroderma pigmentosum group C and in Cockayne's syndrome cells.

    PubMed

    Christians, F C; Hanawalt, P C

    1994-04-01

    Previous studies have demonstrated transcription-coupled DNA repair in mammalian genes transcribed by RNA polymerase II but not in ribosomal RNA genes (rDNA), which are transcribed by RNA polymerase I. The removal of UV-induced cyclobutane pyrimidine dimers (CPD) from rDNA in repair-proficient human cells has been shown to be slow but detectable and apparently not coupled to transcription. We studied the induction and removal of CPD from rDNA in cultured cells from two repair-deficient human disorders. Primary xeroderma pigmentosum complementation group C (XP-C) cells, whether proliferating or nondividing, removed no CPD from either rDNA strand in 24 h post-UV, a result which supports earlier conclusions that XP-C cells lack the general, transcription-independent pathway of nucleotide excision repair. We also observed lower than normal repair of rDNA in Cockayne's syndrome (CS) cells from complementation groups A and B. In agreement with previous findings, the repair of both strands of the RNA polymerase II-transcribed dihydrofolate reductase gene was also deficient relative to that of normal cells. This strongly suggests that the defect in CS cells is not limited to a deficiency in a transcription-repair coupling factor. Rather, the defect may interfere with the ability of repair proteins to gain access to all expressed genes. PMID:7512688

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

    PubMed

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

    2005-02-01

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

  7. Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.

    PubMed

    Petermann, Eva; Orta, Manuel Luís; Issaeva, Natalia; Schultz, Niklas; Helleday, Thomas

    2010-02-26

    Faithful DNA replication is essential to all life. Hydroxyurea (HU) depletes the cells of dNTPs, which initially results in stalled replication forks that, after prolonged treatment, collapse into DSBs. Here, we report that stalled replication forks are efficiently restarted in a RAD51-dependent process that does not trigger homologous recombination (HR). The XRCC3 protein, which is required for RAD51 foci formation, is also required for replication restart of HU-stalled forks, suggesting that RAD51-mediated strand invasion supports fork restart. In contrast, replication forks collapsed by prolonged replication blocks do not restart, and global replication is rescued by new origin firing. We find that RAD51-dependent HR is triggered for repair of collapsed replication forks, without apparent restart. In conclusion, our data suggest that restart of stalled replication forks and HR repair of collapsed replication forks require two distinct RAD51-mediated pathways.

  8. Targeted gene knockout in chickens mediated by TALENs.

    PubMed

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-09-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  9. Gene therapy for cardiovascular disease mediated by ultrasound and microbubbles

    PubMed Central

    2013-01-01

    Gene therapy provides an efficient approach for treatment of cardiovascular disease. To realize the therapeutic effect, both efficient delivery to the target cells and sustained expression of transgenes are required. Ultrasound targeted microbubble destruction (UTMD) technique has become a potential strategy for target-specific gene and drug delivery. When gene-loaded microbubble is injected, the ultrasound-mediated microbubble destruction may spew the transported gene to the targeted cells or organ. Meanwhile, high amplitude oscillations of microbubbles increase the permeability of capillary and cell membrane, facilitating uptake of the released gene into tissue and cell. Therefore, efficiency of gene therapy can be significantly improved. To date, UTMD has been successfully investigated in many diseases, and it has achieved outstanding progress in the last two decades. Herein, we discuss the current status of gene therapy of cardiovascular diseases, and reviewed the progress of the delivery of genes to cardiovascular system by UTMD. PMID:23594865

  10. DNA Repair Gene Expression and Risk of Locoregional Relapse in Breast Cancer Patients

    SciTech Connect

    Le Scodan, Romuald; Cizeron-Clairac, Geraldine

    2010-10-01

    Purpose: Radiation therapy appears to kill cells mainly by inducing DNA double-strand breaks. We investigated whether the DNA repair gene expression status might influence the risk of locoregional recurrence (LRR) in breast cancer patients. Methods and Materials: We used a quantitative reverse transcriptase PCR-based approach to measure messenger RNA levels of 20 selected DNA repair genes in tumor samples from 97 breast cancer patients enrolled in a phase III trial (Centre Rene Huguenin cohort). Normalized mRNA levels were tested for an association with LRR-free survival (LRR-FS) and overall survival (OS). The findings were validated in comparison with those of an independent cohort (Netherlands Cancer Institute (NKI) cohort). Multivariate analysis encompassing known prognostic factors was used to assess the association between DNA repair gene expression and patient outcome. Results: RAD51 was the only gene associated with LRR in both cohorts. With a median follow-up of 126 months in the CRH cohort, the 5-year LRR-FS and OS rates were 100% and 95% in the 61 patients with low RAD51 expression, compared with 70% and 69% in the 36 patients with high RAD51 expression, respectively (p < 0.001). RAD51 overexpression was associated with a higher risk of LRR (hazard ratio [HR], 12.83; 95% confidence interval [CI], 3.6-45.6) and death (HR, 4.10; 95% CI, 1.7-9.7). RAD51 overexpression was also significantly associated with shorter LRR-FS and OS in the NKI cohort. Conclusions: Overexpression of RAD51, a key component of the homologous DNA repair pathway, is associated with poor breast cancer outcome. This finding warrants prospective studies of RAD51 as a prognosticator and therapeutic target.

  11. Rearrangement of Rag-1 recombinase gene in DNA-repair deficient/immunodeficient ``wasted`` mice

    SciTech Connect

    Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M.; Libertin, C.R.

    1992-11-01

    Mice recessive for the autosomal gene ``wasted`` (wst) display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (Rag-l/Rag-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed that in thymus tissue, a small Rag-I transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/{sm_bullet} mice, a two-fold increase in Rag-1 mRNA was evident in thymus tissue. Rag-2 mRNA could only be detected in thymus tissue from wst/{sm_bullet} and not from wst/wst or parental control BCF, mice. Southern blots revealed a rearrangement or deletion within the Rag-1 gene of affected wasted mice that was not evident in known strain-specific parental or littermate controls. These results support the idea that the Rag-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  12. Rearrangement of Rag-1 recombinase gene in DNA-repair deficient/immunodeficient wasted'' mice

    SciTech Connect

    Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M. ); Libertin, C.R. )

    1992-01-01

    Mice recessive for the autosomal gene wasted'' (wst) display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (Rag-l/Rag-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed that in thymus tissue, a small Rag-I transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/[sm bullet] mice, a two-fold increase in Rag-1 mRNA was evident in thymus tissue. Rag-2 mRNA could only be detected in thymus tissue from wst/[sm bullet] and not from wst/wst or parental control BCF, mice. Southern blots revealed a rearrangement or deletion within the Rag-1 gene of affected wasted mice that was not evident in known strain-specific parental or littermate controls. These results support the idea that the Rag-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  13. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

    PubMed

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H; Yi, Henry; Collarini, Ellen J; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds. PMID:27099923

  14. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

    PubMed Central

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H.; Yi, Henry; Collarini, Ellen J.; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A.

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds. PMID:27099923

  15. Vector-mediated antibody gene transfer for infectious diseases.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2015-01-01

    This chapter discusses the emerging field of vector-mediated antibody gene transfer as an alternative vaccine for infectious disease, with a specific focus on HIV. However, this methodology need not be confined to HIV-1; the general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets like hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. This approach is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. With vector-mediated gene transfer, the antibody gene is delivered to the host, via a recombinant adeno-associated virus (rAAV) vector; this in turn results in long-term endogenous antibody expression from the injected muscle that confers protective immunity. Vector-mediated antibody gene transfer can rapidly move existing, potent broadly cross-neutralizing HIV-1-specific antibodies into the clinic. The gene transfer products demonstrate a potency and breadth identical to the original product. This strategy eliminates the need for immunogen design and interaction with the adaptive immune system to generate protection, a strategy that so far has shown limited promise.

  16. Programmed genetic instability: a tumor-permissive mechanism for maintaining the evolvability of higher species through methylation-dependent mutation of DNA repair genes in the male germ line.

    PubMed

    Zhao, Yongzhong; Epstein, Richard J

    2008-08-01

    Tumor suppressor genes are classified by their somatic behavior either as caretakers (CTs) that maintain DNA integrity or as gatekeepers (GKs) that regulate cell survival, but the germ line role of these disease-related gene subgroups may differ. To test this hypothesis, we have used genomic data mining to compare the features of human CTs (n = 38), GKs (n = 36), DNA repair genes (n = 165), apoptosis genes (n = 622), and their orthologs. This analysis reveals that repair genes are numerically less common than apoptosis genes in the genomes of multicellular organisms (P < 0.01), whereas CT orthologs are commoner than GK orthologs in unicellular organisms (P < 0.05). Gene targeting data show that CTs are less essential than GKs for survival of multicellular organisms (P < 0.0005) and that CT knockouts often permit offspring viability at the cost of male sterility. Patterns of human familial oncogenic mutations confirm that isolated CT loss is commoner than is isolated GK loss (P < 0.00001). In sexually reproducing species, CTs appear subject to less efficient purifying selection (i.e., higher Ka/Ks) than GKs (P = 0.000003); the faster evolution of CTs seems likely to be mediated by gene methylation and reduced transcription-coupled repair, based on differences in dinucleotide patterns (P = 0.001). These data suggest that germ line CT/repair gene function is relatively dispensable for survival, and imply that milder (e.g., epimutational) male prezygotic repair defects could enhance sperm variation-and hence environmental adaptation and speciation-while sparing fertility. We submit that CTs and repair genes are general targets for epigenetically initiated adaptive evolution, and propose a model in which human cancers arise in part as an evolutionarily programmed side effect of age- and damage-inducible genetic instability affecting both somatic and germ line lineages. PMID:18535014

  17. Adenoviral vector-mediated gene transfer for human gene therapy.

    PubMed

    Breyer, B; Jiang, W; Cheng, H; Zhou, L; Paul, R; Feng, T; He, T C

    2001-07-01

    Human gene therapy promises to change the practice of medicine by treating the causes of disease rather than the symptoms. Since the first clinical trial made its debut ten years ago, there are over 400 approved protocols in the United States alone, most of which have failed to show convincing data of clinical efficacy. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanisms of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes or targets that are available for gene therapy. Therefore, the urgency and need for efficacious gene therapies are greater than ever. Clearly, the current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Of late, there has been a remarkable increase in adenoviral vector-based clinical trials. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of virus tropism, accommodation of larger genes, increase in stability and control of transgene expression, and down-modulation of host immune responses. These modifications and continued improvements in adenoviral vectors will provide a great opportunity for human gene therapy to live up to its enormous potential in the second decade.

  18. Incomplete complementation of the DNA repair defect in cockayne syndrome cells by the denV gene from bacteriophage T4 suggests a deficiency in base excision repair.

    PubMed

    Francis, M A; Bagga, P S; Athwal, R S; Rainbow, A J

    1997-10-01

    Endonuclease V (denV) from bacteriophage T4 has been examined for its ability to complement the repair defect in Cockayne syndrome (CS) cells of complementation groups A and B. CS is an autosomal recessive disorder characterized by hypersensitivity to UV light and a defect in the preferential repair of UV-induced lesions in transcriptionally active DNA by the nucleotide excision repair (NER) pathway. The denV gene was introduced into non-transformed normal and CS fibroblasts transiently via a recombinant adenovirus (Ad) vector and into SV40-transformed normal and CS cells via a retroviral vector. Expression of denV in CS-A cells resulted in partial correction of the UV-sensitive phenotype in assays of gene-specific repair and cell viability, while correction of CS-B cells by expression of denV in the same assays was minimal or non-existent. In contrast, denV expression led to enhanced host cell reactivation (HCR) of viral DNA synthesis in both CS complementation groups to near normal levels. DenV is a glycosylase which is specific for cyclobutane-pyrimidine dimers (CPDs) but does not recognize other UV-induced lesions. Previous work has indicated that CS cells can efficiently repair all non-CPD UV-induced transcription blocking lesions (S.F. Barrett et al.. Mutation Res. 255 (1991) 281-291 [1]) and that denV incised lesions are believed to be processed via the base excision repair (BER) pathway. The inability of denV to complement the NER defect in CS cells to normal levels implies an impaired ability to process denV incised lesions by the BER pathway, and suggests a role for the CS genes, particularly the CS-B gene, in BER. PMID:9372849

  19. Novel hyperbranched polyamidoamine nanoparticles for transfecting skeletal myoblasts with vascular endothelial growth factor gene for cardiac repair.

    PubMed

    Zhu, Kai; Guo, Changfa; Lai, Hao; Yang, Wuli; Xia, Yu; Zhao, Dong; Wang, Chunsheng

    2011-11-01

    We investigated the feasibility and efficacy of hyperbranched polyamidoamine (hPAMAM) mediated human vascular endothelial growth factor-165 (hVEGF(165)) gene transfer into skeletal myoblasts for cardiac repair. The hPAMAM was synthesized using a modified one-pot method. Encapsulated DNA was protected by hPAMAM from degradation for over 120 min. The transfection efficiency of hPAMAM in myoblasts was 82.6 ± 7.0% with cell viability of 94.6 ± 1.4% under optimal conditions. The hPAMAM showed much higher transfection efficiency (P < 0.05) than polyetherimide and Lipofectamine 2000 with low cytotoxicity. The transfected skeletal myoblasts gave stable hVEGF(165) expression for 18 days. After transplantation of hPAMAM-hVEGF(165) transfected cells, apoptotic myocardial cells decreased at day 1 and heart function improved at day 28, with increased neovascularization (P < 0.05). These results indicate that hPAMAM-based gene delivery into myoblasts is feasible and effective and may serve as a novel and promising non-viral DNA vehicle for gene therapy in myocardial infarction.

  20. The RASSF1A Tumor Suppressor Regulates XPA-Mediated DNA Repair

    PubMed Central

    Donninger, Howard; Clark, Jennifer; Rinaldo, Francesca; Nelson, Nicholas; Barnoud, Thibaut; Schmidt, M. Lee; Hobbing, Katharine R.; Vos, Michele D.; Sils, Brian

    2014-01-01

    RASSF1A may be the most frequently inactivated tumor suppressor identified in human cancer so far. It is a proapoptotic Ras effector and plays an important role in the apoptotic DNA damage response (DDR). We now show that in addition to DDR regulation, RASSF1A also plays a key role in the DNA repair process itself. We show that RASSF1A forms a DNA damage-regulated complex with the key DNA repair protein xeroderma pigmentosum A (XPA). XPA requires RASSF1A to exert full repair activity, and RASSF1A-deficient cells exhibit an impaired ability to repair DNA. Moreover, a cancer-associated RASSF1A single-nucleotide polymorphism (SNP) variant exhibits differential XPA binding and inhibits DNA repair. The interaction of XPA with other components of the repair complex, such as replication protein A (RPA), is controlled in part by a dynamic acetylation/deacetylation cycle. We found that RASSF1A and its SNP variant differentially regulate XPA protein acetylation, and the SNP variant hyperstabilizes the XPA-RPA70 complex. Thus, we identify two novel functions for RASSF1A in the control of DNA repair and protein acetylation. As RASSF1A modulates both apoptotic DDR and DNA repair, it may play an important and unanticipated role in coordinating the balance between repair and death after DNA damage. PMID:25368379

  1. Microsatellites in the Eukaryotic DNA Mismatch Repair Genes as Modulators of Evolutionary Mutation Rate

    NASA Technical Reports Server (NTRS)

    Chang, Dong Kyung; Metzgar, David; Wills, Christopher; Boland, C. Richard

    2003-01-01

    All "minor" components of the human DNA mismatch repair (MMR) system-MSH3, MSH6, PMS2, and the recently discovered MLH3-contain mononucleotide microsatellites in their coding sequences. This intriguing finding contrasts with the situation found in the major components of the DNA MMR system-MSH2 and MLH1-and, in fact, most human genes. Although eukaryotic genomes are rich in microsatellites, non-triplet microsatellites are rare in coding regions. The recurring presence of exonal mononucleotide repeat sequences within a single family of human genes would therefore be considered exceptional.

  2. A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

    PubMed Central

    Leclerc, Xavier; Danos, Olivier; Scherman, Daniel; Kichler, Antoine

    2009-01-01

    Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments. PMID:19379497

  3. The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair

    SciTech Connect

    Lehmann, A.R.; Walicka, M.; Griffiths, D.J.F.; Carr, A.M.

    1995-12-01

    This report describes the cloning and sequencing of the rad18 gene of Schizosaccharomyces pombe and its essential role in cell proliferation. It also describes the isolation and sequencing of its homolog from Saccharomyces cerevisiae, designated RHC18. Genetic radiation effects were explored and results indicate the gene product`s importance in a DNA repair pathway that is distinct from classical nucleotide excision repair. 57 refs., 20 figs., 1 tab.

  4. Alternative-splicing-mediated gene expression

    NASA Astrophysics Data System (ADS)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

    Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

  5. Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes.

    PubMed Central

    Sia, E A; Kokoska, R J; Dominska, M; Greenwell, P; Petes, T D

    1997-01-01

    We examined the stability of microsatellites of different repeat unit lengths in Saccharomyces cerevisiae strains deficient in DNA mismatch repair. The msh2 and msh3 mutations destabilized microsatellites with repeat units of 1, 2, 4, 5, and 8 bp; a poly(G) tract of 18 bp was destabilized several thousand-fold by the msh2 mutation and about 100-fold by msh3. The msh6 mutations destabilized microsatellites with repeat units of 1 and 2 bp but had no effect on microsatellites with larger repeats. These results argue that coding sequences containing repetitive DNA tracts will be preferred target sites for mutations in human tumors with mismatch repair defects. We find that the DNA mismatch repair genes destabilize microsatellites with repeat units from 1 to 13 bp but have no effect on the stability of minisatellites with repeat units of 16 or 20 bp. Our data also suggest that displaced loops on the nascent strand, resulting from DNA polymerase slippage, are repaired differently than loops on the template strand. PMID:9111357

  6. Nucleotide excision repair of the 5 S ribosomal RNA gene assembled into a nucleosome.

    PubMed

    Liu, X; Smerdon, M J

    2000-08-01

    A-175-base pair fragment containing the Xenopus borealis somatic 5 S ribosomal RNA gene was used as a model system to determine the effect of nucleosome assembly on nucleotide excision repair (NER) of the major UV photoproduct (cyclobutane pyrimidine dimer (CPD)) in DNA. Xenopus oocyte nuclear extracts were used to carry out repair in vitro on reconstituted, positioned 5 S rDNA nucleosomes. Nucleosome structure strongly inhibits NER at many CPD sites in the 5 S rDNA fragment while having little effect at a few sites. The time course of CPD removal at 35 different sites indicates that >85% of the CPDs in the naked DNA fragment have t(12) values <2 h, whereas <26% of the t(12) values in nucleosomes are <2 h, and 15% are >8 h. Moreover, removal of histone tails from these mononucleosomes has little effect on the repair rates. Finally, nucleosome inhibition of repair shows no correlation with the rotational setting of a 14-nucleotide-long pyrimidine tract located 30 base pairs from the nucleosome dyad. These results suggest that inhibition of NER by mononucleosomes is not significantly influenced by the rotational orientation of CPDs on the histone surface, and histone tails play little (or no) role in this inhibition. PMID:10821833

  7. Assessment by Southern blot analysis of UV-induced damage and repair in human immunoglobulin genes.

    PubMed

    Bianchi, M S; Bianchi, N O; de la Chapelle, A

    1990-09-01

    Irradiation of DNA with UV light induces pyrimidine dimers and (6-4) photoproducts. The presence of one of these photolesions in the restriction site of a given endonuclease inhibits DNA cleavage and induces the formation of fragments by incomplete DNA digestion which appear as additional, facultative bands in Southern hybridization autoradiograms. The number and size of these fragments show a positive correlation with the UV dose. The response to UV light of immunoglobulin light-chain constant kappa and heavy-chain constant mu genes was analyzed with 2 specific probes. Constant kappa and mu genes when irradiated as part of the chromatin of living lymphocytes showed a UV sensitivity similar to that of naked DNA. The same genes from granulocytes had 50-60 times lower UV sensitivity. When cells were allowed to repair photolesions for 24 h the facultative bands from granulocytes disappeared indicating that these cells were able to remove photolesions from constant kappa and mu genes. Facultative bands from lymphocytes showed a smaller decrease of density after 24 h repair. This suggests that lymphocytes are less efficient than granulocytes in removing UV damage from constant kappa and mu genes.

  8. Existence and expression of photoreactivation repair genes in various yeast species.

    PubMed

    Yasui, A; Eker, A P; Koken, M

    1989-01-01

    Photoreactivation repair (Phr) activities in cell extracts of 13 different yeast species were measured by the Haemophilus influenzae transformation assay. Five species including Schizosaccharomyces pombe showed no or low enzymatic activity. In contrast to the other species, chromosomal DNAs of these 5 species did not show detectable hybridization using a DNA fragment of the photolyase PHR1 gene of Saccharomyces cervisiae as a probe even at a low stringency condition. When the PHR1 gene was attached to the 5'-flanking sequence of the iso-1-cytochrome c (CYC-1) gene of S. cerevisiae and introduced into S. pombe cells, the transformants acquired a high Phr activity, indicating that the PHR1 gene alone can provide a Phr-negative species with this repair activity and the light-absorbing cofactor(s) must be present in S. pombe. Our results also demonstrated that the 5'-flanking sequence of the S. cerevisiae CYC-1 gene works in S. pombe as a regulatory element. PMID:2911265

  9. The Polycomb Group Protein EZH2 Impairs DNA Damage Repair Gene Expression in Human Uterine Fibroids.

    PubMed

    Yang, Qiwei; Nair, Sangeeta; Laknaur, Archana; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

    2016-03-01

    Uterine fibroids are benign, smooth muscle tumors that occur in approximately 70%-80% of women by age 50 yr. The cellular and molecular mechanism(s) by which uterine fibroids (UFs) develop are not fully understood. Accumulating evidence demonstrates that several genetic abnormalities, including deletions, rearrangements, translocations, as well as mutations, have been found in UFs. These genetic anomalies suggest that low DNA damage repair capacity may be involved in UF formation. The objective of this study was to determine whether expression levels of DNA damage repair-related genes were altered, and how they were regulated in the pathogenesis of UFs. Expression levels of DNA repair-related genes RAD51 and BRCA1 were deregulated in fibroid tissues as compared to adjacent myometrial tissues. Expression levels of chromatin protein enhancer of zeste homolog 2 (EZH2) were higher in a subset of fibroids as compared to adjacent myometrial tissues by both immunohistochemistry and Western blot analysis. Treatment with an inhibitor of EZH2 markedly increased expression levels of RAD51 and BRCA1 in fibroid cells and inhibited cell proliferation paired with cell cycle arrest. Restoring the expression of RAD51 and BRCA1 by treatment with EZH2 inhibitor was dependent on reducing the enrichment of trimethylation of histone 3 lysine 27 epigenetic mark in their promoter regions. This study reveals the important role of EZH2-regulated DNA damage-repair genes via histone methylation in fibroid biology, and may provide novel therapeutic targets for the medical treatment of women with symptomatic UFs. PMID:26888970

  10. Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins.

    PubMed

    Hegde, Muralidhar L; Hegde, Pavana M; Bellot, Larry J; Mandal, Santi M; Hazra, Tapas K; Li, Guo-Min; Boldogh, Istvan; Tomkinson, Alan E; Mitra, Sankar

    2013-08-13

    Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a "cowcatcher" and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.

  11. p53-mediated control of gene expression via mRNA translation during Endoplasmic Reticulum stress.

    PubMed

    López, Ignacio; Tournillon, Anne-Sophie; Nylander, Karin; Fåhraeus, Robin

    2015-01-01

    p53 is activated by different stress and damage pathways and regulates cell biological responses including cell cycle arrest, repair pathways, apoptosis and senescence. Following DNA damage, the levels of p53 increase and via binding to target gene promoters, p53 induces expression of multiple genes including p21(CDKN1A) and mdm2. The effects of p53 on gene expression during the DNA damage response are well mimicked by overexpressing p53 under normal conditions. However, stress to the Endoplasmic Reticulum (ER) and the consequent Unfolded Protein Response (UPR) leads to the induction of the p53/47 isoform that lacks the first 40 aa of p53 and to an active suppression of p21(CDKN1A) transcription and mRNA translation. We now show that during ER stress p53 also suppresses MDM2 protein levels via a similar mechanism. These observations not only raise questions about the physiological role of MDM2 during ER stress but it also reveals a new facet of p53 as a repressor toward 2 of its major target genes during the UPR. As suppression of p21(CDKN1A) and MDM2 protein synthesis is mediated via their coding sequences, it raises the possibility that p53 controls mRNA translation via a common mechanism that might play an important role in how p53 regulates gene expression during the UPR, as compared to the transcription-dependent gene regulation taking place during the DNA damage response.

  12. Nanoparticle mediated silencing of DNA repair sensitizes pediatric brain tumor cells to γ-irradiation

    PubMed Central

    Kievit, Forrest M.; Stephen, Zachary R.; Wang, Kui; Dayringer, Christopher J.; Sham, Jonathan G.; Ellenbogen, Richard G.; Silber, John R.; Zhang, Miqin

    2015-01-01

    Medulloblastoma (MB) and ependymoma (EP) are the most common pediatric brain tumors, afflicting 3,000 children annually. Radiotherapy (RT) is an integral component in the treatment of these tumors; however, the improvement in survival is often accompanied by radiation-induced adverse developmental and psychosocial sequelae. Therefore, there is an urgent need to develop strategies that can increase the sensitivity of brain tumors cells to RT while sparing adjacent healthy brain tissue. Apurinic endonuclease 1 (Ape1), an enzyme in the base excision repair pathway, has been implicated in radiation resistance in cancer. Pharmacological and specificity limitations inherent to small molecule inhibitors of Ape1 have hindered their clinical development. Here we report on a nanoparticle (NP) based siRNA delivery vehicle for knocking down Ape1 expression and sensitizing pediatric brain tumor cells to RT. The NP comprises a superparamagnetic iron oxide core coated with a biocompatible, biodegradable coating of chitosan, polyethylene glycol (PEG), and polyethyleneimine (PEI) that is able to bind and protect siRNA from degradation and to deliver siRNA to the perinuclear region of target cells. NPs loaded with siRNA against Ape1 (NP:siApe1) knocked down Ape1 expression over 75% in MB and EP cells, and reduced Ape1 activity by 80%. This reduction in Ape1 activity correlated with increased DNA damage post-irradiation, which resulted in decreased cell survival in clonogenic assays. The sensitization was specific to therapies generating abasic lesions as evidenced by NP:siRNA not increasing sensitivity to paclitaxel, a microtubule disrupting agent. Our results indicate NP-mediated delivery of siApe1 is a promising strategy for circumventing pediatric brain tumor resistance to RT. PMID:25681012

  13. RNA-Mediated Gene Silencing in Hematopoietic Cells

    PubMed Central

    Venturini, Letizia; Eder, Matthias; Scherr, Michaela

    2006-01-01

    In the past few years, the discovery of RNA-mediated gene silencing mechanisms, like RNA interference (RNAi), has revolutionized our understanding of eukaryotic gene expression. These mechanisms are activated by double-stranded RNA (dsRNA) and mediate gene silencing either by inducing the sequence-specific degradation of complementary mRNA or by inhibiting mRNA translation. RNAi now provides a powerful experimental tool to elucidate gene function in vitro and in vivo, thereby opening new exciting perspectives in the fields of molecular analysis and eventually therapy of several diseases such as infections and cancer. In hematology, numerous studies have described the successful application of RNAi to better define the role of oncogenic fusion proteins in leukemogenesis and to explore therapeutic approaches in hematological malignancies. In this review, we highlight recent advances and caveats relating to the application of this powerful new methodology to hematopoiesis. PMID:17057372

  14. Genetic Polymorphisms in DNA Repair Genes as Modulators of Hodgkin Disease Risk

    PubMed Central

    El-Zein, Randa; Monroy, Claudia M.; Etzel, Carol J.; Cortes, Andrea C.; Xing, Yun; Collier, Amanda L.; Strom, Sara S.

    2009-01-01

    BACKGROUND Although the pathogenesis of Hodgkin disease (HD) remains unknown, the results of epidemiologic studies suggest that heritable factors are important in terms of susceptibility. Polymorphisms in DNA repair genes may contribute to individual susceptibility for development of different cancers. However, to the authors’ knowledge, few studies to date have investigated the role of such polymorphisms as risk factors for development of HD. METHODS The authors evaluated the relation between polymorphisms in 3 nucleotide excision repair pathway genes (XPD [Lys751Gln], XPC [Lys939Gln], and XPG [Asp1104His]), the base excision repair XRCC1 (Arg399Gln), and double-strand break repair XRCC3 (Thr241Met) in a population of 200 HD cases and 220 matched controls. Variants were investigated independently and in combination; odd ratios (OR) were calculated. RESULTS A positive association was found for XRCC1 gene polymorphism Arg399Gln (OR, 1.77; 95% confidence interval [95% CI], 1.16−2.71) and risk of HD. The combined analysis demonstrated that XRCC1/XRCC3 and XRCC1/XPC polymorphisms were associated with a significant increase in HD risk. XRCC1 Arg/Arg and XRCC3 Thr/Met genotypes combined were associated with an OR of 2.38 (95% CI, 1.24−4.55). The XRCC1 Arg/Gln and XRCC3 Thr/Thr, Thr/Met, and Met/Met genotypes had ORs of 1.88 (95% CI, 1.02−4.10), 1.97 (95% CI, 1.05−3.73), and 4.13 (95% CI, 1.50−11.33), respectively. XRCC1 Gln/Gln and XRCC3 Thr/Thr variant led to a significant increase in risk, with ORs of 3.00 (95% CI, 1.15−7.80). Similarly, XRCC1 Arg/Gln together with XPC Lys/Lys was found to significantly increase the risk of HD (OR, 2.14; 95% CI, 1.09−4.23). CONCLUSIONS These data suggest that genetic polymorphisms in DNA repair genes may modify the risk of HD, especially when interactions between the pathways are considered. PMID:19280628

  15. TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1

    PubMed Central

    Zeng, Zhihong; Sharma, Abhishek; Ju, Limei; Murai, Junko; Umans, Lieve; Vermeire, Liesbeth; Pommier, Yves; Takeda, Shunichi; Huylebroeck, Danny; Caldecott, Keith W.; El-Khamisy, Sherif F.

    2012-01-01

    The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3′- or 5′-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase ‘poisons’. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3′-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3′-tyrosyl DNA phosphodiesterase (3′-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1−/− cells, suggesting that Tdp2 contributes to cellular 3′-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1−/−/Tdp2−/−/− DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1−/− DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a

  16. Polymorphisms in DNA repair genes, traffic-related polycyclic aromatic hydrocarbon exposure and breast cancer incidence.

    PubMed

    Mordukhovich, Irina; Beyea, Jan; Herring, Amy H; Hatch, Maureen; Stellman, Steven D; Teitelbaum, Susan L; Richardson, David B; Millikan, Robert C; Engel, Lawrence S; Shantakumar, Sumitra; Steck, Susan E; Neugut, Alfred I; Rossner, Pavel; Santella, Regina M; Gammon, Marilie D

    2016-07-15

    Vehicular traffic polycyclic aromatic hydrocarbons (PAHs) have been associated with breast cancer incidence in epidemiologic studies, including our own. Because PAHs damage DNA by forming adducts and oxidative lesions, genetic polymorphisms that alter DNA repair capacity may modify associations between PAH-related exposures and breast cancer risk. Our goal was to examine the association between vehicular traffic exposure and breast cancer incidence within strata of a panel of nine biologically plausible nucleotide excision repair (NER) and base excision repair (BER) genotypes. Residential histories of 1,508 cases and 1,556 controls were assessed in the Long Island Breast Cancer Study Project between 1996 and 1997 and used to reconstruct residential traffic exposures to benzo[a]pyrene, as a proxy for traffic-related PAHs. Likelihood ratio tests from adjusted unconditional logistic regression models were used to assess multiplicative interactions. A gene-traffic interaction was evident (p = 0.04) for ERCC2 (Lys751); when comparing the upper and lower tertiles of 1995 traffic exposure estimates, the odds ratio (95% confidence interval) was 2.09 (1.13, 3.90) among women with homozygous variant alleles. Corresponding odds ratios for 1960-1990 traffic were also elevated nearly 2-3-fold for XRCC1(Arg194Trp), XRCC1(Arg399Gln) and OGG1(Ser326Cys), but formal multiplicative interaction was not evident. When DNA repair variants for ERCC2, XRCC1 and OGG1 were combined, among women with 4-6 variants, the odds ratios were 2.32 (1.22, 4.49) for 1995 traffic and 2.96 (1.06, 8.21) for 1960-1990 traffic. Our study is first to report positive associations between traffic-related PAH exposure and breast cancer incidence among women with select biologically plausible DNA repair genotypes.

  17. Cloning and molecular characterization of the Chinese hamster ERCC2 nucleotide excision repair gene

    SciTech Connect

    Kirchner, J.M.; Salazar, E.P.; Lamerdin, J.E.

    1994-10-01

    The Chinese hamster ERCC2 nucleotide excision repair gene, encoding a presumed ATP-dependent DNA helicase, was cloned from the V79 cell line, and its nucleotide sequence was determined. The {approximately}15-kb gene comprises 23 exons with a 2283-base open reading frame. The predicted 760-amino-acid protein is 98% identical to the human ERCC2/EXP (760 amino acids), 51% identical to the Saccharomyces cerevisiae RAD3 (778 amino acids), and 54% identical to the Schizosaccharomyces pombe rad15 (772 amino acids) proteins. The promoter region of the hamster ERCC2 gene contains a pyrimidine-rich stretch (42 nucleotides, 88% C+T) similar to sequences found in the promoter regions of two other nucleotide excision repair genes, a GC box, a putative {alpha}-Pal transcription factor binding site, and two CAAT boxes. There is no apparent TAATA box. No consensus polyadenylation sequence (AATAAA or its variants) was found with 663 bases 3{prime} of the translation termination codon. 54 refs., 2 figs., 2 tabs.

  18. Tissue repair genes: the TiRe database and its implication for skin wound healing.

    PubMed

    Yanai, Hagai; Budovsky, Arie; Tacutu, Robi; Barzilay, Thomer; Abramovich, Amir; Ziesche, Rolf; Fraifeld, Vadim E

    2016-04-19

    Wound healing is an inherent feature of any multicellular organism and recent years have brought about a huge amount of data regarding regular and abnormal tissue repair. Despite the accumulated knowledge, modulation of wound healing is still a major biomedical challenge, especially in advanced ages. In order to collect and systematically organize what we know about the key players in wound healing, we created the TiRe (Tissue Repair) database, an online collection of genes and proteins that were shown to directly affect skin wound healing. To date, TiRe contains 397 entries for four organisms: Mus musculus, Rattus norvegicus, Sus domesticus, and Homo sapiens. Analysis of the TiRe dataset of skin wound healing-associated genes showed that skin wound healing genes are (i) over-conserved among vertebrates, but are under-conserved in invertebrates; (ii) enriched in extracellular and immuno-inflammatory genes; and display (iii) high interconnectivity and connectivity to other proteins. The latter may provide potential therapeutic targets. In addition, a slower or faster skin wound healing is indicative of an aging or longevity phenotype only when assessed in advanced ages, but not in the young. In the long run, we aim for TiRe to be a one-station resource that provides researchers and clinicians with the essential data needed for a better understanding of the mechanisms of wound healing, designing new experiments, and the development of new therapeutic strategies. TiRe is freely available online at http://www.tiredb.org.

  19. Double-strand gap repair in a mammalian gene targeting reaction.

    PubMed Central

    Valancius, V; Smithies, O

    1991-01-01

    To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments. Images PMID:1875928

  20. A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy.

    PubMed

    Weeda, G; Eveno, E; Donker, I; Vermeulen, W; Chevallier-Lagente, O; Taïeb, A; Stary, A; Hoeijmakers, J H; Mezzina, M; Sarasin, A

    1997-02-01

    Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and nails, mental retardation, impaired sexual development, and ichthyosis. Photosensitivity has been reported in approximately 50% of the cases, but no skin cancer is associated with TTD. Virtually all photosensitive TTD patients have a deficiency in the nucleotide excision repair (NER) of UV-induced DNA damage that is indistinguishable from that of xeroderma pigmentosum (XP) complementation group D (XP-D) patients. DNA repair defects in XP-D are associated with two additional, quite different diseases; XP, a sun-sensitive and cancer-prone repair disorder, and Cockayne syndrome (CS), a photosensitive condition characterized by physical and mental retardation and wizened facial appearance. One photosensitive TTD case constitutes a new repair-deficient complementation group, TTD-A. Remarkably, both TTD-A and XP-D defects are associated with subunits of TFIIH, a basal transcription factor with a second function in DNA repair. Thus, mutations in TFIIH components may, on top of a repair defect, also cause transcriptional insufficiency, which may explain part of the non-XP clinical features of TTD. Besides XPD and TTDA, the XPB gene product is also part of TFIIH. To date, three patients with the remarkable conjunction of XP and CS but not TTD have been assigned to XP complementation group B (XP-B). Here we present the characterization of the NER defect in two mild TTD patients (TTD6VI and TTD4VI) and confirm the assignment to X-PB. The causative mutation was found to be a single base substitution resulting in a missense mutation (T119P) in a region of the XPB protein completely conserved in yeast, Drosophila, mouse, and man. These findings define a third TTD complementation group, extend the clinical heterogeneity associated with XP-B, stress the exclusive relationship between TTD and mutations in subunits of repair/transcription factor TFIIH, and strongly

  1. Molecular genetic and biochemical analyses of a DNA repair gene from Serratia marcescens

    SciTech Connect

    Murphy, K.E.

    1989-01-01

    In Escherichia coli, the SOS response and two 3-methyladenine DNA glycosylases (TagI and TagII) are required for repair of DNA damaged by alkylating agents such as methyl methanesulfonate (MMS). Mutations of the recA gene eliminate the SOS response. TagI and TagII are encoded by the tag and alkA genes, respectively. A gene (rpr) encoding 3-methyladenine DNA glycosylase activity was isolated from the Gram-negative bacterium Serratia marcescens. The gene, localized to a 1.5-kilobase pair SmaI-HindIII restriction fragment, was cloned into plasmid pUC18. The clone complemented E. coli tag alkA and recA mutations for MMS resistance. The rpr gene did not, however, complement recA mutations for resistance to ultraviolet light or the ability to perform homologous recombination reactions, nor did it complement E. coli ada or alkB mutations. Two proteins of molecular weights 42,000 and 16,000 were produced from the rpr locus. Analysis of deletion and insertion mutants of rpr suggested that the 42kD molecule is the active protein. The 16kD protein may either be a breakdown product of the 42kD species or may be encoded by another gene overlapping the reading frame of the rpr gene. Biochemical assays showed that the rpr gene product (Rpr) possesses 3-methyladenine DNA glycosylase activity.

  2. Reduced Activity of Double-Strand Break Repair Genes in Prostate Cancer Patients With Late Normal Tissue Radiation Toxicity

    SciTech Connect

    Oorschot, Bregje van; Hovingh, Suzanne E.; Moerland, Perry D.; Medema, Jan Paul; Stalpers, Lukas J.A.; Vrieling, Harry; Franken, Nicolaas A.P.

    2014-03-01

    Purpose: To investigate clinical parameters and DNA damage response as possible risk factors for radiation toxicity in the setting of prostate cancer. Methods and Materials: Clinical parameters of 61 prostate cancer patients, 34 with (overresponding, OR) and 27 without (non-responding, NR) severe late radiation toxicity were assembled. In addition, for a matched subset the DNA damage repair kinetics (γ-H2AX assay) and expression profiles of DNA repair genes were determined in ex vivo irradiated lymphocytes. Results: Examination of clinical data indicated none of the considered clinical parameters to be correlated with the susceptibility of patients to develop late radiation toxicity. Although frequencies of γ-H2AX foci induced immediately after irradiation were similar (P=.32), significantly higher numbers of γ-H2AX foci were found 24 hours after irradiation in OR compared with NR patients (P=.03). Patient-specific γ-H2AX foci decay ratios were significantly higher in NR patients than in OR patients (P<.0001). Consequently, NR patients seem to repair DNA double-strand breaks (DSBs) more efficiently than OR patients. Moreover, gene expression analysis indicated several genes of the homologous recombination pathway to be stronger induced in NR compared with OR patients (P<.05). A similar trend was observed in genes of the nonhomologous end-joining repair pathway (P=.09). This is congruent with more proficient repair of DNA DSBs in patients without late radiation toxicity. Conclusions: Both gene expression profiling and DNA DSB repair kinetics data imply that less-efficient repair of radiation-induced DSBs may contribute to the development of late normal tissue damage. Induction levels of DSB repair genes (eg, RAD51) may potentially be used to assess the risk for late radiation toxicity.

  3. DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae.

    PubMed

    Meyer, Damon; Fu, Becky Xu Hua; Heyer, Wolf-Dietrich

    2015-12-15

    Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ. PMID:26607450

  4. Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Gridley, Daila S.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

  5. Development of an intein-mediated split–Cas9 system for gene therapy

    PubMed Central

    Truong, Dong-Jiunn Jeffery; Kühner, Karin; Kühn, Ralf; Werfel, Stanislas; Engelhardt, Stefan; Wurst, Wolfgang; Ortiz, Oskar

    2015-01-01

    Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split–Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV. PMID:26082496

  6. Divergent genes in potential inoculant Sinorhizobium strains are related to DNA replication, recombination, and repair.

    PubMed

    Penttinen, Petri; Greco, Dario; Muntyan, Victoria; Terefework, Zewdu; De Lajudie, Philippe; Roumiantseva, Marina; Becker, Anke; Auvinen, Petri; Lindström, Kristina

    2016-06-01

    To serve as inoculants of legumes, nitrogen-fixing rhizobium strains should be competitive and tolerant of diverse environments. We hybridized the genomes of symbiotically efficient and salt tolerant Sinorhizobium inoculant strains onto the Sinorhizobium meliloti Rm1021 microarray. The number of variable genes, that is, divergent or putatively multiplied genes, ranged from 503 to 1556 for S. meliloti AK23, S. meliloti STM 1064 and S. arboris HAMBI 1552. The numbers of divergent genes affiliated with the symbiosis plasmid pSymA and related to DNA replication, recombination and repair were significantly higher than expected. The variation was mainly in the accessory genome, implying that it was important in shaping the adaptability of the strains.

  7. The swi4+ gene of Schizosaccharomyces pombe encodes a homologue of mismatch repair enzymes.

    PubMed Central

    Fleck, O; Michael, H; Heim, L

    1992-01-01

    The swi4+ gene of Schizosaccharomyces pombe is involved in termination of copy-synthesis during mating-type switching. The gene was cloned by functional complementation of a swi4 mutant transformed with a genomic library. Determination of the nucleotide sequence revealed an open reading frame of 2979 nucleotides which is interrupted by a 68 bp long intron. The putative Swi4 protein shows homology to Duc-1 (human), Rep-3 (mouse), HexA (Streptococcus pneumoniae) and MutS (Salmonella typhimurium). The prokaryotic proteins are known as essential components involved in mismatch repair. A strain with a disrupted swi4+ gene was constructed and analysed with respect to the switching process. As in swi4 mutants duplications occur in the mating-type region of the swi4 (null) strain, reducing the efficiency of switching. Images PMID:1317550

  8. Sox2-mediated regulation of adult neural crest precursors and skin repair.

    PubMed

    Johnston, Adam P W; Naska, Sibel; Jones, Karen; Jinno, Hiroyuki; Kaplan, David R; Miller, Freda D

    2013-01-01

    Nerve-derived neural crest cells are essential for regeneration in certain animals, such as newts. Here, we asked whether they play a similar role during mammalian tissue repair, focusing on Sox2-positive neural crest precursors in skin. In adult skin, Sox2 was expressed in nerve-terminal-associated neural crest precursor cells (NCPCs) around the hair follicle bulge, and following injury was induced in nerve-derived cells, likely dedifferentiated Schwann cell precursors. At later times postinjury, Sox2-positive cells were scattered throughout the regenerating dermis, and lineage tracing showed that these were all neural-crest-derived NCPCs. These Sox2-positive NCPCs were functionally important, since acute deletion of Sox2 prior to injury caused a decrease of NCPCs in the wound and aberrant skin repair. These data demonstrate that Sox2 regulates skin repair, likely by controlling NCPCs, and raise the possibility that nerve-derived NCPCs may play a general role in mammalian tissue repair.

  9. Rearrangement of RAG-1 recombinase gene in DNA-repair deficient ``wasted`` mice

    SciTech Connect

    Woloschak, G.E.; Libertin, C.R.; Weaver, P.; Churchill, M.; Chang-Liu, C.M.

    1993-11-01

    Mice recessive for the autosomal gene ``wasted`` wst display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (RAG-l/RAG-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed expression of RAG-1 mRNA in spinal cord (but not brain) of control mice; no expression of RAG-1 mRNA was detected in spinal cord or brain from wst/wst mice or their normal littermates (wst/{center_dot}mice). In thymus tissue, a small RAG-1 transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/{center_dot}mice, a two-fold increase in RAG-1 mRNA was evident in thymus tissue. RAG-2 mRNA could only be detected in thymus tissue from wst/{center_dot} and not from wst/wst or parental control BCF{sub 1} mice. Southern blots revealed a rearrangement/deletion within the RAG-1 gene of affected wasted mice, not evident in known strain-specific parental or littermate controls. These results support the idea that the RAG-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  10. Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

    PubMed Central

    Allgayer, Julia; Kitsera, Nataliya; Bartelt, Solveig; Epe, Bernd; Khobta, Andriy

    2016-01-01

    DNA damage can significantly modulate expression of the affected genes either by direct structural interference with transcription components or as a collateral outcome of cellular repair attempts. Thus, DNA glycosylases of the base excision repair (BER) pathway have been implicated in negative transcriptional response to several spontaneously generated DNA base modifications, including a common oxidative DNA base modification 8-oxoguanine (8-oxoG). Here, we report that single 8-oxoG situated in the non-transcribed DNA strand of a reporter gene has a pronounced negative effect on transcription, driven by promoters of various strength and with different structural properties, including viral, human, and artificial promoters. We further show that the magnitude of the negative effect on the gene expression correlates with excision of the modified base by OGG1 in all promoter constructs tested. Moreover, by using expression vectors with nuclease resistant backbone modifications, we demonstrate that OGG1 does not catalyse DNA strand cleavage in vivo. Rather, cleavage of the phosphate bond 5′ to 8-oxodG (catalysed by APE1) is essential and universally required for the onset of transcriptional silencing, regardless of the promoter structure. Hence, induction of transcriptional silencing emerges as a ubiquitous mode of biological response to 8-oxoG in DNA. PMID:27220469

  11. Are SNP-Smoking Association Studies Needed in Controls? DNA Repair Gene Polymorphisms and Smoking Intensity

    PubMed Central

    Verde, Zoraida; Reinoso, Luis; Chicharro, Luis Miguel; Resano, Pilar; Sánchez-Hernández, Ignacio; Rodríguez González-Moro, Jose Miguel; Bandrés, Fernando

    2015-01-01

    Variations in tobacco-related cancers, incidence and prevalence reflect differences in tobacco consumption in addition to genetic factors. Besides, genes related to lung cancer risk could be related to smoking behavior. Polymorphisms altering DNA repair capacity may lead to synergistic effects with tobacco carcinogen-induced lung cancer risk. Common problems in genetic association studies, such as presence of gene-by-environment (G x E) correlation in the population, may reduce the validity of these designs. The main purpose of this study was to evaluate the independence assumption for selected SNPs and smoking behaviour in a cohort of 320 healthy Spanish smokers. We found an association between the wild type alleles of XRCC3 Thr241Met or KLC3 Lys751Gln and greater smoking intensity (OR = 12.98, 95% CI = 2.86–58.82 and OR=16.90, 95% CI=2.09-142.8; respectively). Although preliminary, the results of our study provide evidence that genetic variations in DNA-repair genes may influence both smoking habits and the development of lung cancer. Population-specific G x E studies should be carried out when genetic and environmental factors interact to cause the disease. PMID:26017978

  12. Are SNP-Smoking Association Studies Needed in Controls? DNA Repair Gene Polymorphisms and Smoking Intensity.

    PubMed

    Verde, Zoraida; Reinoso, Luis; Chicharro, Luis Miguel; Resano, Pilar; Sánchez-Hernández, Ignacio; Rodríguez González-Moro, Jose Miguel; Bandrés, Fernando; Gómez-Gallego, Félix; Santiago, Catalina

    2015-01-01

    Variations in tobacco-related cancers, incidence and prevalence reflect differences in tobacco consumption in addition to genetic factors. Besides, genes related to lung cancer risk could be related to smoking behavior. Polymorphisms altering DNA repair capacity may lead to synergistic effects with tobacco carcinogen-induced lung cancer risk. Common problems in genetic association studies, such as presence of gene-by-environment (G x E) correlation in the population, may reduce the validity of these designs. The main purpose of this study was to evaluate the independence assumption for selected SNPs and smoking behaviour in a cohort of 320 healthy Spanish smokers. We found an association between the wild type alleles of XRCC3 Thr241Met or KLC3 Lys751Gln and greater smoking intensity (OR = 12.98, 95% CI = 2.86-58.82 and OR=16.90, 95% CI=2.09-142.8; respectively). Although preliminary, the results of our study provide evidence that genetic variations in DNA-repair genes may influence both smoking habits and the development of lung cancer. Population-specific G x E studies should be carried out when genetic and environmental factors interact to cause the disease.

  13. Expression of DNA repair genes in burned skin exposed to low-level red laser.

    PubMed

    Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

    2014-11-01

    Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin.

  14. Increased expression of p53 enhances transcription-coupled repair and global genomic repair of a UVC-damaged reporter gene in human cells.

    PubMed

    Dregoesc, Diana; Rybak, Adrian P; Rainbow, Andrew J

    2007-05-01

    Ultraviolet (UV) light-induced DNA damage is repaired by nucleotide excision repair, which is divided into two sub-pathways: global genome repair (GGR) and transcription-coupled repair (TCR). While it is well established that the GGR pathway is dependent on the p53 tumour suppressor protein in human cells, both p53-dependent and p53-independent pathways have been reported for TCR. In the present work, we investigated the role of p53 in both GGR and TCR of a UVC-damaged reporter gene in human fibroblasts. We employed a non-replicating recombinant human adenovirus, AdCA17lacZ, that can efficiently infect human fibroblasts and express the beta-galactosidase (beta-gal) reporter gene under the control of the human cytomegalovirus promoter. We examined host cell reactivation (HCR) of beta-gal expression for the UVC-treated reporter construct in normal fibroblasts and in xeroderma pigmentosum (XP) and Cockayne syndrome (CS) fibroblasts deficient in GGR, TCR, or both. HCR was examined in fibroblasts that had been pre-infected with Ad5p53wt, which expresses wild-type p53, or a control adenovirus, AdCA18luc, which expresses the luciferase gene. We show that increased expression of p53 results in enhanced HCR of the UVC-damaged reporter gene in both untreated and UVC-treated cells for normal, CS-B (TCR-deficient), and XP-C (GGR-deficient), but not XP-A (TCR- and GGR-deficient) fibroblasts. These results indicate an involvement of p53 in both TCR and GGR of the UV-damaged reporter gene in human cells. PMID:17196445

  15. A systematic gene-gene and gene-environment interaction analysis of DNA repair genes XRCC1, XRCC2, XRCC3, XRCC4, and oral cancer risk.

    PubMed

    Yang, Cheng-Hong; Lin, Yu-Da; Yen, Ching-Yui; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2015-04-01

    Oral cancer is the sixth most common cancer worldwide with a high mortality rate. Biomarkers that anticipate susceptibility, prognosis, or response to treatments are much needed. Oral cancer is a polygenic disease involving complex interactions among genetic and environmental factors, which require multifaceted analyses. Here, we examined in a dataset of 103 oral cancer cases and 98 controls from Taiwan the association between oral cancer risk and the DNA repair genes X-ray repair cross-complementing group (XRCCs) 1-4, and the environmental factors of smoking, alcohol drinking, and betel quid (BQ) chewing. We employed logistic regression, multifactor dimensionality reduction (MDR), and hierarchical interaction graphs for analyzing gene-gene (G×G) and gene-environment (G×E) interactions. We identified a significantly elevated risk of the XRCC2 rs2040639 heterozygous variant among smokers [adjusted odds ratio (OR) 3.7, 95% confidence interval (CI)=1.1-12.1] and alcohol drinkers [adjusted OR=5.7, 95% CI=1.4-23.2]. The best two-factor based G×G interaction of oral cancer included the XRCC1 rs1799782 and XRCC2 rs2040639 [OR=3.13, 95% CI=1.66-6.13]. For the G×E interaction, the estimated OR of oral cancer for two (drinking-BQ chewing), three (XRCC1-XRCC2-BQ chewing), four (XRCC1-XRCC2-age-BQ chewing), and five factors (XRCC1-XRCC2-age-drinking-BQ chewing) were 32.9 [95% CI=14.1-76.9], 31.0 [95% CI=14.0-64.7], 49.8 [95% CI=21.0-117.7] and 82.9 [95% CI=31.0-221.5], respectively. Taken together, the genotypes of XRCC1 rs1799782 and XRCC2 rs2040639 DNA repair genes appear to be significantly associated with oral cancer. These were enhanced by exposure to certain environmental factors. The observations presented here warrant further research in larger study samples to examine their relevance for routine clinical care in oncology.

  16. HTLV-I Tax-Mediated Inactivation of Cell Cycle Checkpoints and DNA Repair Pathways Contribute to Cellular Transformation: “A Random Mutagenesis Model”

    PubMed Central

    Nicot, Christophe

    2015-01-01

    To achieve cellular transformation, most oncogenic retroviruses use transduction by proto-oncogene capture or insertional mutagenesis, whereby provirus integration disrupts expression of tumor suppressors or proto-oncogenes. In contrast, the Human T-cell leukemia virus type 1 (HTLV-I) has been classified in a separate class referred to as “transactivating retroviruses”. Current views suggest that the viral encoded Tax protein transactivates expression of cellular genes leading to deregulated growth and transformation. However, if Tax-mediated transactivation was indeed sufficient for cellular transformation, a fairly high frequency of infected cells would eventually become transformed. In contrast, the frequency of transformation by HTLV-I is very low, likely less than 5%. This review will discuss the current understanding and recent discoveries highlighting critical functions of Tax in cellular transformation. HTLV-I Tax carries out essential functions in order to override cell cycle checkpoints and deregulate cellular division. In addition, Tax expression is associated with increased DNA damage and genome instability. Since Tax can inhibit multiple DNA repair pathways and stimulate unfaithful DNA repair or bypass checkpoints, these processes allow accumulation of genetic mutations in the host genome. Given this, a “Random Mutagenesis” transformation model seems more suitable to characterize the oncogenic activities of HTLV-I. PMID:26835512

  17. Chromosome territory relocation during DNA repair requires nuclear myosin 1 recruitment to chromatin mediated by ϒ-H2AX signaling

    PubMed Central

    Kulashreshtha, Mugdha; Mehta, Ishita S.; Kumar, Pradeep; Rao, Basuthkar J.

    2016-01-01

    During DNA damage response (DDR), certain gene rich chromosome territories (CTs) relocate to newer positions within interphase nuclei and revert to their native locations following repair. Such dynamic relocation of CTs has been observed under various cellular conditions, however, the underlying mechanistic basis of the same has remained largely elusive. In this study, we aim to understand the temporal and molecular details of such crosstalk between DDR signaling and CT relocation dynamics. We demonstrate that signaling at DNA double strand breaks (DSBs) by the phosphorylated histone variant (ϒ-H2AX) is a pre-requisite for damage induced CT relocation, as cells deficient in ϒ-H2AX signaling fail to exhibit such a response. Inhibition of Rad51 or DNA Ligase IV mediated late steps of double strand break repair does not seem to abrogate CT relocation completely. Upon DNA damage, an increase in the levels of chromatin bound motor protein nuclear myosin 1 (NM1) ensues, which appears to be functionally linked to ϒ-H2AX signaling. Importantly, the motor function of NM1 is essential for its recruitment to chromatin and CT relocation following damage. Taking these observations together, we propose that early DDR sensing and signaling result in NM1 recruitment to chromosomes which in turn guides DNA damage induced CT relocation. PMID:27365048

  18. Correction of the Crb1rd8 Allele and Retinal Phenotype in C57BL/6N Mice Via TALEN-Mediated Homology-Directed Repair

    PubMed Central

    Low, Benjamin E.; Krebs, Mark P.; Joung, J. Keith; Tsai, Shengdar Q.; Nishina, Patsy M.; Wiles, Michael V.

    2014-01-01

    Purpose. We directly corrected the mouse Crb1rd8 gene mutation, which is present in many inbred laboratory strains derived from C57BL/6N and complicates genetic studies of retinal disease in mice. Methods. Fertilized C57BL/6NJ oocytes were coinjected with mRNAs encoding a transcription activator-like effector nuclease (TALEN) targeting the Crb1rd8 allele plus single-stranded oligonucleotides to correct the allele. The oligonucleotides included additional nucleotide changes to distinguish the corrected allele (Crb1em1Mvw) from wild-type Crb1 and to minimize TALEN recutting. Oligonucleotide length, concentration of injected oligonucleotides and TALEN mRNAs were varied to optimize homology-directed repair of the locus. Following microinjection, embryos were carried to term in pseudopregnant females. Correction efficiency was assessed by PCR analysis of the Crb1em1Mvw allele. Phenotypic correction was demonstrated by fundus imaging and optical coherence tomography of live mice, and by confocal fluorescence microscopy of retinal flat mounts. Results. Under optimal conditions, homology-directed repair was observed in 27% (8/30) of live-born animals and showed minimal illegitimate recombination of donor DNA. However, extensive founder mosaicism was evident, emphasizing the need to analyze offspring of founder animals. Unlike C57BL/6NJ mice, which exhibited external limiting membrane fragmentation and regional retinal dysplasia, heterozygous Crb1em1Mvw/Crb1rd8 mice showed a normal retinal phenotype. Conclusions. The C57BL/6NJ-Crb1rd8 mutation and its associated retinal phenotypes were corrected efficiently by TALEN-mediated homology-directed repair. The C57BL/6NJ-Crb1em1Mvw mice generated by this strategy will enhance ocular phenotyping efforts based on the C57BL/6N background, such as those implemented by the International Mouse Phenotyping Consortium (IMPC) project. PMID:24346171

  19. Adenoviral-vector-mediated gene transfer to dendritic cells.

    PubMed

    Song, W; Crystal, R G

    2001-01-01

    Dendritic cells (DC) are the most potent antigen presenting cells capable of initiating T-cell-dependent immune responses (1-5). This biologic potential can be harnessed to elicit effective antigen-specific immune responses by transferring the relevant antigens to the DC. Once the DC have been mobilized and purified, the relevant antigens can be transferred to the DC as intact proteins, or as peptides representing specific epitopes, or with gene transfer using sequences of DNA or RNA coding for the pertinent antigen(s) (6-15). Theoretically, genetically modifying DC with genes coding for specific antigens has potential advantages over pulsing the DC with peptides repeating the antigen or antigen fragment. First, the genetically modified DC may present previously unknown epitopes in association with different MHC molecules. Second, gene transfer to DC ensures that the gene product is endogenously processed, leading to the generation of MHC class I-restricted cytotoxic T lymphocytes (CTL), the effector arm of cell-mediated immune responses. Finally, in addition to genes coding for the antigen(s), genetic modification of the DC can induce genes coding for mediators relevant to generation of the immune response to the antigen(s), further boosting host responses to the antigens presented by the modified DC. Different gene transfer approaches have been explored to genetically modify DC, including retroviral vectors (16-18), recombinant vaccinia virus vectors (19), and recombinant adenovirus (Ad) vectors (19-23). The focus of this chapter is on using recombinant Ad vectors to transfer genes to murine DC. We have used a similar strategy to transfer genes to human DC (24). As an example of the power of this technology, we will describe the use of Ad-vector-modified DC to suppress the growth of tumor cells modified to express a specific antigen.

  20. Toll-Like Receptor 4 Mediates the Regenerative Effects of Bone Grafts for Calvarial Bone Repair

    PubMed Central

    Wang, Dan; Gilbert, James R.; Shaw, Melissa A.; Shakir, Sameer; Losee, Joseph E.; Billiar, Timothy R.

    2015-01-01

    Craniofacial trauma is difficult to repair and presents a significant burden to the healthcare system. The inflammatory response following bone trauma is critical to initiate healing, serving to recruit inflammatory and progenitor cells and to promote angiogenesis. A role for inflammation in graft-induced bone regeneration has been suggested, but is still not well understood. The current study assessed the impact of Toll-like receptor (TLR4) signaling on calvarial repair in the presence of morselized bone components. Calvarial defects in wild-type and global TLR4−/− knockout mouse strains were treated with fractionated bone components in the presence or absence of a TLR4 neutralizing peptide. Defect healing was subsequently evaluated over 28 days by microcomputed tomography and histology. The matrix-enriched fraction of morselized bone stimulated calvarial bone repair comparably with intact bone graft, although the capacity for grafts to induce calvarial bone repair was significantly diminished by inhibition or genetic ablation of TLR4. Overall, our findings suggest that the matrix component of bone graft stimulates calvarial bone repair in a TLR4-dependent manner. These results support the need to better understand the role of inflammation in the design and implementation of strategies to improve bone healing. PMID:25603990

  1. Toll-like receptor 4 mediates the regenerative effects of bone grafts for calvarial bone repair.

    PubMed

    Wang, Dan; Gilbert, James R; Shaw, Melissa A; Shakir, Sameer; Losee, Joseph E; Billiar, Timothy R; Cooper, Gregory M

    2015-04-01

    Craniofacial trauma is difficult to repair and presents a significant burden to the healthcare system. The inflammatory response following bone trauma is critical to initiate healing, serving to recruit inflammatory and progenitor cells and to promote angiogenesis. A role for inflammation in graft-induced bone regeneration has been suggested, but is still not well understood. The current study assessed the impact of Toll-like receptor (TLR4) signaling on calvarial repair in the presence of morselized bone components. Calvarial defects in wild-type and global TLR4(-/-) knockout mouse strains were treated with fractionated bone components in the presence or absence of a TLR4 neutralizing peptide. Defect healing was subsequently evaluated over 28 days by microcomputed tomography and histology. The matrix-enriched fraction of morselized bone stimulated calvarial bone repair comparably with intact bone graft, although the capacity for grafts to induce calvarial bone repair was significantly diminished by inhibition or genetic ablation of TLR4. Overall, our findings suggest that the matrix component of bone graft stimulates calvarial bone repair in a TLR4-dependent manner. These results support the need to better understand the role of inflammation in the design and implementation of strategies to improve bone healing.

  2. Global genome repair of 8-oxoG in hamster cells requires a functional CSB gene product.

    PubMed

    Sunesen, Morten; Stevnsner, Tinna; Brosh, Robert M; Dianov, Grigory L; Bohr, Vilhelm A

    2002-05-16

    Cockayne syndrome (CS) is an autosomal recessive human disease characterized by UV-sensitivity as well as neurological and developmental abnormalities. Two complementation groups have been established, designated CS-A and CS-B. Traditionally, CSA and CSB have been ascribed a function in the transcription-coupled repair (TCR) pathway of nucleotide excision repair (NER) that efficiently removes bulky lesions from the transcribed strand of RNA polymerase II transcribed genes. To assess the role of the CSB protein in the repair of the highly mutagenic base lesion 7,8-dihydro-8-oxoguanine (8-oxoG), we have investigated the removal of this lesion using an in vitro incision approach with cell extracts as well as an in vivo approach with a modified protocol of the gene-specific repair assay, which allows the measurement of base lesion repair in intragenomic sequences. Our results demonstrate that the integrity of the CSB protein is pivotal for processes leading to incision at the site of 8-oxoG and that the global genome repair (GGR) of this lesion requires a functional CSB gene product in vivo. PMID:12032859

  3. Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows.

    PubMed

    Liu, Xu; Wang, Yongsheng; Guo, Wenjiang; Chang, Bohao; Liu, Jun; Guo, Zekun; Quan, Fusheng; Zhang, Yong

    2013-01-01

    Zinc-finger nickases (ZFNickases) are a type of programmable nuclease that can be engineered from zinc-finger nucleases to induce site-specific single-strand breaks or nicks in genomic DNA, which result in homology-directed repair. Although zinc-finger nuclease-mediated gene disruption has been demonstrated in pigs and cattle, they have not been used to target gene addition into an endogenous gene locus in any large domestic species. Here we show in bovine fetal fibroblasts that targeting ZFNickases to the endogenous β-casein (CSN2) locus stimulates lysostaphin gene addition by homology-directed repair. We find that ZFNickase-treated cells can be successfully used in somatic cell nuclear transfer, resulting in live-born gene-targeted cows. Furthermore, the gene-targeted cows secrete lysostaphin in their milk and in vitro assays demonstrate the milk's ability to kill Staphylococcus aureus. Our success with this strategy will facilitate new transgenic technologies beneficial to both agriculture and biomedicine.

  4. Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows

    PubMed Central

    Liu, Xu; Wang, Yongsheng; Guo, Wenjiang; Chang, Bohao; Liu, Jun; Guo, Zekun; Quan, Fusheng; Zhang, Yong

    2013-01-01

    Zinc-finger nickases (ZFNickases) are a type of programmable nuclease that can be engineered from zinc-finger nucleases to induce site-specific single-strand breaks or nicks in genomic DNA, which result in homology-directed repair. Although zinc-finger nuclease-mediated gene disruption has been demonstrated in pigs and cattle, they have not been used to target gene addition into an endogenous gene locus in any large domestic species. Here we show in bovine fetal fibroblasts that targeting ZFNickases to the endogenous β-casein (CSN2) locus stimulates lysostaphin gene addition by homology-directed repair. We find that ZFNickase-treated cells can be successfully used in somatic cell nuclear transfer, resulting in live-born gene-targeted cows. Furthermore, the gene-targeted cows secrete lysostaphin in their milk and in vitro assays demonstrate the milk’s ability to kill Staphylococcus aureus. Our success with this strategy will facilitate new transgenic technologies beneficial to both agriculture and biomedicine. PMID:24121612

  5. MRN, CtIP, and BRCA1 mediate repair of topoisomerase II–DNA adducts

    PubMed Central

    Aparicio, Tomas; Baer, Richard; Gottesman, Max

    2016-01-01

    Repair of DNA double-strand breaks (DSBs) with complex ends poses a special challenge, as additional processing is required before DNA ligation. For example, protein–DNA adducts must be removed to allow repair by either nonhomologous end joining or homology-directed repair. Here, we investigated the processing of topoisomerase II (Top2)–DNA adducts induced by treatment with the chemotherapeutic agent etoposide. Through biochemical analysis in Xenopus laevis egg extracts, we establish that the MRN (Mre11, Rad50, and Nbs1) complex, CtIP, and BRCA1 are required for both the removal of Top2–DNA adducts and the subsequent resection of Top2-adducted DSB ends. Moreover, the interaction between CtIP and BRCA1, although dispensable for resection of endonuclease-generated DSB ends, is required for resection of Top2-adducted DSBs, as well as for cellular resistance to etoposide during genomic DNA replication. PMID:26880199

  6. Repairing DNA damage in xeroderma pigmentosum: T4N5 lotion and gene therapy.

    PubMed

    Zahid, Sarwar; Brownell, Isaac

    2008-04-01

    Patients with xeroderma pigmentosum (XP) have defective DNA repair and are at a high risk for cutaneous malignancies. Standard treatments for XP are limited in scope and effectiveness. Understanding the molecular etiology of XP has led to the development of novel therapeutic approaches, including enzyme and gene therapies. One new topical treatment utilizing bacteriophage T4 endonuclease 5 (T4N5) in a liposomal lotion is currently in clinical trials and has received a Fast Track designation from the FDA. Gene therapy for XP, while making leaps in preclinical studies, has been slower to develop due to tactical hurdles, but seems to have much potential for future treatment. If these treatments prove effective in lowering the risk of cancer in patients with XP, they may also be found useful in reducing skin cancers in other at-risk patient populations.

  7. p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

    PubMed Central

    Gurdziel, Katherine; Bell, George W.; Jacks, Tyler; Flores, Elsa R.

    2009-01-01

    The p53 family activates many of the same genes in response to DNA damage. Because p63 and p73 have structural differences from p53 and play distinct biological functions in development and metastasis, it is likely that they activate a unique transcriptional network. Therefore, we performed a genome-wide analysis using cells lacking the p53 family members after treatment with DNA damage. We identified over 100 genes involved in multiple pathways that were uniquely regulated by p63 or p73, and not p53. Further validation indicated that BRCA2, Rad51, and mre11 are direct transcriptional targets of p63 and p73. Additionally, cells deficient for p63 and p73 are impaired in DNA repair and p63+/−;p73+/− mice develop mammary tumors suggesting a novel mechanism whereby p63 and p73 suppress tumorigenesis. PMID:19816568

  8. RAD6/sup +/ gene of Saccharomyces cerevisiae codes for two mutationally separable deoxyribonucleic acid repair functions

    SciTech Connect

    Tuite, M.F.; Cox, B.S.

    1981-02-01

    The response of two mutant alleles of the RAD6/sup +/ gene of Saccharomyces cerevisiae to the ochre translational suppressor SUQ5 was determined. Both the ultraviolet sensitivity phenotype and the deficiency in ultraviolet-induced mutagenesis phenotype of the rad6-1 allelle were suppressed in a (psi/sup +/) background. For the rad6-3 allelle, only the ultraviolet-sensitivity phenotype was suppressible in a (psi/sup +/) background. An SUQ5 rad6-3 (psi/sup +/) strain that was examined showed the normal rad6-3 deficiency in ultraviolet-induced mutagenesis. The authors propose that the RAD6/sup +/ gene is divided into two cistrons, RAD6A and RAD6B. RAD6A codes for an activity responsible for the error-prone repair of ultraviolet-induced lesions in deoxyribonucleic acid but is not involved in a cell's resistance to the lethal effects of ultraviolet light. RAD6B codes for an activity essential for error-free repair of potentially lethal mutagenic damage.

  9. Adenovirus serotype 5 hexon mediates liver gene transfer.

    PubMed

    Waddington, Simon N; McVey, John H; Bhella, David; Parker, Alan L; Barker, Kristeen; Atoda, Hideko; Pink, Rebecca; Buckley, Suzanne M K; Greig, Jenny A; Denby, Laura; Custers, Jerome; Morita, Takashi; Francischetti, Ivo M B; Monteiro, Robson Q; Barouch, Dan H; van Rooijen, Nico; Napoli, Claudio; Havenga, Menzo J E; Nicklin, Stuart A; Baker, Andrew H

    2008-02-01

    Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo. PMID:18267072

  10. Tissue repair genes: the TiRe database and its implication for skin wound healing

    PubMed Central

    Yanai, Hagai; Budovsky, Arie; Tacutu, Robi; Barzilay, Thomer; Abramovich, Amir; Ziesche, Rolf; Fraifeld, Vadim E.

    2016-01-01

    Wound healing is an inherent feature of any multicellular organism and recent years have brought about a huge amount of data regarding regular and abnormal tissue repair. Despite the accumulated knowledge, modulation of wound healing is still a major biomedical challenge, especially in advanced ages. In order to collect and systematically organize what we know about the key players in wound healing, we created the TiRe (Tissue Repair) database, an online collection of genes and proteins that were shown to directly affect skin wound healing. To date, TiRe contains 397 entries for four organisms: Mus musculus, Rattus norvegicus, Sus domesticus, and Homo sapiens. Analysis of the TiRe dataset of skin wound healing-associated genes showed that skin wound healing genes are (i) over-conserved among vertebrates, but are under-conserved in invertebrates; (ii) enriched in extracellular and immuno-inflammatory genes; and display (iii) high interconnectivity and connectivity to other proteins. The latter may provide potential therapeutic targets. In addition, a slower or faster skin wound healing is indicative of an aging or longevity phenotype only when assessed in advanced ages, but not in the young. In the long run, we aim for TiRe to be a one-station resource that provides researchers and clinicians with the essential data needed for a better understanding of the mechanisms of wound healing, designing new experiments, and the development of new therapeutic strategies. TiRe is freely available online at http://www.tiredb.org. PMID:27049721

  11. Tissue-engineering strategies to repair joint tissue in osteoarthritis: nonviral gene-transfer approaches.

    PubMed

    Madry, Henning; Cucchiarini, Magali

    2014-10-01

    Loss of articular cartilage is a common clinical consequence of osteoarthritis (OA). In the past decade, substantial progress in tissue engineering, nonviral gene transfer, and cell transplantation have provided the scientific foundation for generating cartilaginous constructs from genetically modified cells. Combining tissue engineering with overexpression of therapeutic genes enables immediate filling of a cartilage defect with an engineered construct that actively supports chondrogenesis. Several pioneering studies have proved that spatially defined nonviral overexpression of growth-factor genes in constructs of solid biomaterials or hydrogels is advantageous compared with gene transfer or scaffold alone, both in vitro and in vivo. Notably, these investigations were performed in models of focal cartilage defects, because advanced cartilage-repair strategies based on the principles of tissue engineering have not advanced sufficiently to enable resurfacing of extensively degraded cartilage as therapy for OA. These studies serve as prototypes for future technological developments, because they raise the possibility that cartilage constructs engineered from genetically modified chondrocytes providing autocrine and paracrine stimuli could similarly compensate for the loss of articular cartilage in OA. Because cartilage-tissue-engineering strategies are already used in the clinic, combining tissue engineering and nonviral gene transfer could prove a powerful approach to treat OA.

  12. Common variants in mismatch repair genes associated with increased risk of sperm DNA damage and male infertility

    PubMed Central

    2012-01-01

    Background The mismatch repair (MMR) pathway plays an important role in the maintenance of the genome integrity, meiotic recombination and gametogenesis. This study investigated whether genetic variations in MMR genes are associated with an increased risk of sperm DNA damage and male infertility. Methods We selected and genotyped 21 tagging single nucleotide polymorphisms (SNPs) in five MMR genes (MLH1, MLH3, PMS2, MSH4 and MSH5) using the SNPstream 12-plex platform in a case-control study of 1,292 idiopathic infertility patients and 480 fertile controls in a Chinese population. Sperm DNA damage levels were detected with the Tdt-mediated dUTP nick end labelling (TUNEL) assay in 450 cases. Fluorescence resonance energy transfer (FRET) and co-immunoprecipitation techniques were employed to determine the effects of functional variants. Results One intronic SNP in MLH1 (rs4647269) and two non-synonymous SNPs in PMS2 (rs1059060, Ser775Asn) and MSH5 (rs2075789, Pro29Ser) seem to be risk factors for the development of azoospermia or oligozoospermia. Meanwhile, we also identified a possible contribution of PMS2 rs1059060 to the risk of male infertility with normal sperm count. Among patients with normal sperm count, MLH1 rs4647269 and PMS2 rs1059060 were associated with increased sperm DNA damage. Functional analysis revealed that the PMS2 rs1059060 can affect the interactions between MLH1 and PMS2. Conclusions Our results provide evidence supporting the involvement of genetic polymorphisms in MMR genes in the aetiology of male infertility. PMID:22594646

  13. Reduced XPC DNA repair gene mRNA levels in clinically normal parents of xeroderma pigmentosum patients.

    PubMed

    Khan, Sikandar G; Oh, Kyu-Seon; Shahlavi, Tala; Ueda, Takahiro; Busch, David B; Inui, Hiroki; Emmert, Steffen; Imoto, Kyoko; Muniz-Medina, Vanessa; Baker, Carl C; DiGiovanna, John J; Schmidt, Deborah; Khadavi, Arash; Metin, Ahmet; Gozukara, Engin; Slor, Hanoch; Sarasin, Alain; Kraemer, Kenneth H

    2006-01-01

    Xeroderma pigmentosum group C (XP-C) is a rare autosomal recessive disorder. Patients with two mutant alleles of the XPC DNA repair gene have sun sensitivity and a 1000-fold increase in skin cancers. Clinically normal parents of XP-C patients have one mutant allele and one normal allele. As a step toward evaluating cancer risk in these XPC heterozygotes we characterized cells from 16 XP families. We identified 15 causative mutations (5 frameshift, 6 nonsense and 4 splicing) in the XPC gene in cells from 16 XP probands. All had premature termination codons (PTC) and absence of normal XPC protein on western blotting. The cell lines from 26 parents were heterozygous for the same mutations. We employed a real-time quantitative reverse transcriptase-PCR assay as a rapid and sensitive method to measure XPC mRNA levels. The mean XPC mRNA levels in the cell lines from the XP-C probands were 24% (P<10(-7)) of that in 10 normal controls. This reduced XPC mRNA level in cells from XP-C patients was caused by the PTC that induces nonsense-mediated mRNA decay. The mean XPC mRNA levels in cell lines from the heterozygous XP-C carriers were intermediate (59%, P=10(-4)) between the values for the XP patients and the normal controls. This study demonstrates reduced XPC mRNA levels in XP-C patients and heterozygotes. Thus, XPC mRNA levels may be evaluated as a marker of cancer susceptibility in carriers of mutations in the XPC gene. PMID:16081512

  14. Topoisomerase II-Mediated DNA Damage Is Differently Repaired during the Cell Cycle by Non-Homologous End Joining and Homologous Recombination

    PubMed Central

    de Campos-Nebel, Marcelo; Larripa, Irene; González-Cid, Marcela

    2010-01-01

    Topoisomerase II (Top2) is a nuclear enzyme involved in several metabolic processes of DNA. Chemotherapy agents that poison Top2 are known to induce persistent protein-mediated DNA double strand breaks (DSB). In this report, by using knock down experiments, we demonstrated that Top2α was largely responsible for the induction of γH2AX and cytotoxicity by the Top2 poisons idarubicin and etoposide in normal human cells. As DSB resulting from Top2 poisons-mediated damage may be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), we aimed to analyze both DNA repair pathways. We found that DNA-PKcs was rapidly activated in human cells, as evidenced by autophosphorylation at serine 2056, following Top2-mediated DNA damage. The chemical inhibition of DNA-PKcs by wortmannin and vanillin resulted in an increased accumulation of DNA DSB, as evaluated by the comet assay. This was supported by a hypersensitive phenotype to Top2 poisons of Ku80- and DNA-PKcs- defective Chinese hamster cell lines. We also showed that Rad51 protein levels, Rad51 foci formation and sister chromatid exchanges were increased in human cells following Top2-mediated DNA damage. In support, BRCA2- and Rad51C- defective Chinese hamster cells displayed hypersensitivity to Top2 poisons. The analysis by immunofluorescence of the DNA DSB repair response in synchronized human cell cultures revealed activation of DNA-PKcs throughout the cell cycle and Rad51 foci formation in S and late S/G2 cells. Additionally, we found an increase of DNA-PKcs-mediated residual repair events, but not Rad51 residual foci, into micronucleated and apoptotic cells. Therefore, we conclude that in human cells both NHEJ and HR are required, with cell cycle stage specificity, for the repair of Top2-mediated reversible DNA damage. Moreover, NHEJ-mediated residual repair events are more frequently associated to irreversibly damaged cells. PMID:20824055

  15. The study of the relation of DNA repair pathway genes SNPs and the sensitivity to radiotherapy and chemotherapy of NSCLC

    PubMed Central

    Wang, Chunbo; Nie, Huan; Li, Yiqun; Liu, Guiyou; Wang, Xu; Xing, Shijie; Zhang, Liping; Chen, Xin; Chen, Yue; Li, Yu

    2016-01-01

    To analyze the relation between SNPs in DNA repair pathway-related genes and sensitivity of tumor radio-chemotherapy, 26 SNPs in 20 DNA repair genes were genotyped on 176 patients of NSCLC undertaking radio-chemotherapy treatment. In squamous cell carcinoma (SCC), as the rs2228000, rs2228001 (XPC), rs2273953 (TP73), rs2279744 (MDM2), rs2299939 (PTEN) and rs8178085, rs12334811 (DNA-PKcs) affected the sensitivity to chemotherapy, so did the rs8178085, rs12334811 to radiotherapy. Moreover rs344781, rs2273953 and rs12334811 were related with the survival time of SCC. In general, the “good” genotype GG (rs12334811) showed greater efficacy of radio-chemotherapy and MSF (24 months) on SCC. In adenocarcinoma, as the rs2699887 (PIK3), rs12334811 (DNA-PKcs) influenced the sensitivity to chemotherapy, so did the rs2299939, rs2735343 (PTEN) to radiotherapy. And rs402710, rs80270, rs2279744 and rs2909430 impacted the survival time of the adenocarcinoma patients. Both GG (rs2279744) and AG (rs2909430) showed a shorter survival time (MFS = 6). Additionally, some SNPs such as rs2228000, rs2228001 and rs344781 were found to regulate the expression of DNA repair pathway genes through eQTLs dataset analysis. These results indicate that SNPs in DNA repair pathway genes might regulate the expression and affect the DNA damage repair, and thereby impact the efficacy of radio-chemotherapy and the survival time of NSCLC. PMID:27246533

  16. Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification.

    PubMed

    Henriksson, Sara; Shaposhnikov, Sergey; Nilsson, Mats; Collins, Andrew

    2011-04-25

    We used padlock probes to study the rate of gene specific repair of three genes, OGG1 (8-oxoguanine-DNA glycosylase-1), XPD (xeroderma pigmentosum group D), and HPRT (hypoxanthine-guanine phosphoribosyltransferase) in human lymphocytes, in relation to the repair rate of Alu repeats and total genomic DNA. Padlock probes offer highly specific detection of short target sequences by combining detection by ligation and signal amplification. In this approach only genes in sequences containing strand breaks, which become single-stranded in the tail, are available for hybridisation. Thus the total number of signals from the padlock probes per comet gives a direct measure of the amount of damage (strand-breaks) present and allows the repair process to be monitored. This method could provide insights on the organisation of genomic DNA in the comet tail. Alu repeat containing DNA was repaired rapidly in comparison with total genomic DNA, and the studied genes were generally repaired more rapidly than the Alu repeats.

  17. PCNA is involved in the EndoQ-mediated DNA repair process in Thermococcales

    PubMed Central

    Shiraishi, Miyako; Ishino, Sonoko; Yoshida, Kotaro; Yamagami, Takeshi; Cann, Isaac; Ishino, Yoshizumi

    2016-01-01

    To maintain genome integrity for transfer to their offspring, and to maintain order in cellular processes, all living organisms have DNA repair systems. Besides the well-conserved DNA repair machineries, organisms thriving in extreme environments are expected to have developed efficient repair systems. We recently discovered a novel endonuclease, which cleaves the 5′ side of deoxyinosine, from the hyperthermophilic archaeon, Pyrococcus furiosus. The novel endonuclease, designated as Endonulcease Q (EndoQ), recognizes uracil, abasic site and xanthine, as well as hypoxanthine, and cuts the phosphodiester bond at their 5′ sides. To understand the functional process involving EndoQ, we searched for interacting partners of EndoQ and identified Proliferating Cell Nuclear Angigen (PCNA). The EndoQ activity was clearly enhanced by addition of PCNA in vitro. The physical interaction between the two proteins through a PIP-motif of EndoQ and the toroidal structure of PCNA are critical for the stimulation of the endonuclease activity. These findings provide us a clue to elucidate a unique DNA repair system in Archaea. PMID:27150116

  18. Cockayne syndrome exhibits dysregulation of p21 and other gene products that may be independent of transcription-coupled repair.

    PubMed

    Cleaver, J E; Hefner, E; Laposa, R R; Karentz, D; Marti, T

    2007-04-14

    Cockayne syndrome (CS) is a progressive childhood neurodegenerative disorder associated with a DNA repair defect caused by mutations in either of two genes, CSA and CSB. These genes are involved in nucleotide excision repair (NER) of DNA damage from ultraviolet (UV) light, other bulky chemical adducts and reactive oxygen in transcriptionally active genes (transcription-coupled repair, TCR). For a long period it has been assumed that the symptoms of CS patients are all due to reduced TCR of endogenous DNA damage in the brain, together with unexplained unique sensitivity of specific neural cells in the cerebellum. Not all the symptoms of CS patients are however easily related to repair deficiencies, so we hypothesize that there are additional pathways relevant to the disease, particularly those that are downstream consequences of a common defect in the E3 ubiquitin ligase associated with the CSA and CSB gene products. We have found that the CSB defect results in altered expression of anti-angiogenic and cell cycle genes and proteins at the level of both gene expression and protein lifetime. We find an over-abundance of p21 due to reduced protein turnover, possibly due to the loss of activity of the CSA/CSB E3 ubiquitylation pathway. Increased levels of p21 can result in growth inhibition, reduced repair from the p21-PCNA interaction, and increased generation of reactive oxygen. Consistent with increased reactive oxygen levels we find that CS-A and -B cells grown under ambient oxygen show increased DNA breakage, as compared with xeroderma pigmentosum cells. Thus the complex symptoms of CS may be due to multiple, independent downstream targets of the E3 ubiquitylation system that results in increased DNA damage, reduced transcription coupled repair, and inhibition of cell cycle progression and growth. PMID:17055654

  19. Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes

    PubMed Central

    Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana; Horikoshi, Nobuo; Pandita, Raj K.; Sarker, Altaf H.; Sarkar, Partha S.; Pandita, Tej K.; Hazra, Tapas K.

    2016-01-01

    DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome. PMID:27703167

  20. Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription

    PubMed Central

    Liu, Zhihui; Lam, Norris; Thiele, Carol J.

    2015-01-01

    The zinc finger transcription factor CASZ1 has been found to control neural fate-determination in flies, regulate murine and frog cardiac development, control murine retinal cell progenitor expansion and function as a tumor suppressor gene in humans. However, the molecular mechanism by which CASZ1 regulates gene transcription to exert these diverse biological functions has not been described. Here we identify co-factors that are recruited by CASZ1b to regulate gene transcription using co-immunoprecipitation (co-IP) and mass spectrometry assays. We find that CASZ1b binds to the nucleosome remodeling and histone deacetylase (NuRD) complex, histones and DNA repair proteins. Mutagenesis of the CASZ1b protein assay demonstrates that the N-terminus of CASZ1b is required for NuRD binding, and a poly(ADP-ribose) binding motif in the CASZ1b protein is required for histone H3 and DNA repair proteins binding. The N-terminus of CASZ1b fused to an artificial DNA-binding domain (GAL4DBD) causes a significant repression of transcription (5xUAS-luciferase assay), which could be blocked by treatment with an HDAC inhibitor. Realtime PCR results show that the transcriptional activity of CASZ1b mutants that abrogate NuRD or histone H3/DNA binding is significantly decreased. This indicates a model in which CASZ1b binds to chromatin and recruits NuRD complexes to orchestrate epigenetic-mediated transcriptional programs. PMID:26296975

  1. DNA repair gene XPD and susceptibility to arsenic-induced hyperkeratosis.

    PubMed

    Ahsan, Habibul; Chen, Yu; Wang, Qiao; Slavkovich, Vesna; Graziano, Joseph H; Santella, Regina M

    2003-07-20

    Chronic exposure to inorganic arsenic is known to cause non-melanocytic skin and internal cancers in humans. An estimated 50-70 million people in Bangladesh have been chronically exposed to arsenic from drinking water and are at risk of skin and other cancers. We undertook the first study to examine whether genetic susceptibility, as determined by the codon 751 SNP (A-->C) of the DNA repair gene XPD, influences the risk of arsenic-induced hyperkeratotic skin lesions, precursors of skin cancer, in a case-control study of 29 hyperkeratosis cases and 105 healthy controls from the same community in an area of Bangladesh. As expected, there was a monotonic increase in risk of hyperkeratosis in relation to urinary arsenic measures but the XPD genotype was not independently associated with the risk. However, the increase in hyperkeratosis risk in relation to urinary arsenic measures genotype was borderline significant for urinary total arsenic (P for trend=0.06) and statistically significant for urinary creatinine adjusted arsenic (P for trend=0.01) among subjects with the XPD A allele (AA) but not among subjects with the other XPD genotypes. Among AA carriers, the risk for the highest arsenic exposed group compared with the lowest was more than 7-fold for urinary total arsenic and about 11-fold for urinary creatinine adjusted arsenic. In conclusion, our findings suggest that the DNA repair gene XPD may influence the risk of arsenic-induced premalignant hyperkeratotic skin lesions. Future larger studies are needed to confirm this novel finding and investigate how combinations of different candidate genes and/or other host and environmental factors may influence the risk of arsenic induced skin and other cancers.

  2. Dimethylarsinic acid in drinking water changed the morphology of urinary bladder but not the expression of DNA repair genes of bladder transitional epithelium in F344 rats.

    PubMed

    Wang, Amy; Wolf, Douglas C; Sen, Banalata; Knapp, Geremy W; Holladay, Steven D; Huckle, William R; Caceci, Thomas; Robertson, John L

    2009-06-01

    Inorganic arsenic increases urinary bladder transitional cell carcinoma in humans. In F344 rats, dimethylarsinic acid (DMA[V]) increases transitional cell carcinoma. Arsenic-induced inhibition of DNA repair has been reported in cultured cell lines and in lymphocytes of arsenic-exposed humans, but it has not been studied in urinary bladder. Should inhibition of DNA damage repair in transitional epithelium occur, it may contribute to carcinogenesis or cocarcinogenesis. We investigated morphology and expression of DNA repair genes in F344 rat transitional cells following up to 100 ppm DMA(V) in drinking water for four weeks. Mitochondria were very sensitive to DMA(V), and swollen mitochondria appeared to be the main source of vacuoles in the transitional epithelium. Real-time reverse transcriptase polymerase chain reaction (Real-Time RT PCR) showed the mRNA levels of tested DNA repair genes, ataxia telangectasia mutant (ATM), X-ray repair cross-complementing group 1 (XRCC1), excision repair cross-complementing group 3/xeroderma pigmentosum B (ERCC3/XPB), and DNA polymerase beta (Polbeta), were not altered by DMA(V). These data suggested that either DMA(V) does not affect DNA repair in the bladder or DMA(V) affects DNA repair without affecting baseline mRNA levels of repair genes. The possibility remains that DMA(V) may lower damage-induced increases in repair gene expression or cause post-translational modification of repair enzymes.

  3. Modulation of adenovirus-mediated gene transfer by nitric oxide.

    PubMed

    Haddad, I Y; Sorscher, E J; Garver, R I; Hong, J; Tzeng, E; Matalon, S

    1997-05-01

    We assessed the role of .NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 microM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N(G)-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of .NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 microM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of .NO, did not alter the efficacy of gene transfer. .NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting .NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled .NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 microl of saline) and exposed to .NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n > or = 8). We concluded that

  4. Evidence for presence of mismatch repair gene expression positive Lynch syndrome cases in India.

    PubMed

    Bashyam, Murali D; Kotapalli, Viswakalyan; Raman, Ratheesh; Chaudhary, Ajay K; Yadav, Brijesh K; Gowrishankar, Swarnalata; Uppin, Shantveer G; Kongara, Ravikanth; Sastry, Regulagadda A; Vamsy, Mohana; Patnaik, Sujit; Rao, Satish; Dsouza, Shoba; Desai, Devendra; Tester, Ashavaid

    2015-12-01

    Lynch syndrome (LS), the most common form of familial CRC predisposition that causes tumor onset at a young age, is characterized by the presence of microsatellite instability (MSI) in tumors due to germline inactivation of mismatch repair (MMR) system. Two MMR genes namely MLH1 and MSH2 account for majority of LS cases while MSH6 and PMS2 may account for a minor proportion. In order to identify MMR genes causing LS in India, we analyzed MSI and determined expression status of the four MMR genes in forty eight suspected LS patient colorectal tumor samples. Though a majority exhibited MSI, only 58% exhibited loss of MMR expression, a significantly low proportion compared to reports from other populations. PCR-DNA sequencing and MLPA-based mutation and exonic deletion/duplication screening respectively, revealed genetic lesions in samples with and without MMR gene expression. Interestingly, tumor samples with and without MMR expression exhibited significant differences with respect to histological (mucin content) and molecular (instability exhibited by mononucleotide microsatellites) features. The study has revealed for the first time a significant proportion of LS tumors not exhibiting loss of MMR expression.

  5. Resveratrol improves bone repair by modulation of bone morphogenetic proteins and osteopontin gene expression in rats.

    PubMed

    Casarin, R C; Casati, M Z; Pimentel, S P; Cirano, F R; Algayer, M; Pires, P R; Ghiraldini, B; Duarte, P M; Ribeiro, F V

    2014-07-01

    This study investigated the effect of resveratrol on bone healing and its influence on the gene expression of osteogenic markers. Two calvarial defects were created and one screw-shaped titanium implant was inserted in the tibia of rats that were assigned to daily administration of placebo (control group, n=15) or 10mg/kg of resveratrol (RESV group, n=15) for 30 days. The animals were then sacrificed. One of the calvarial defects was processed for histomorphometric analysis and the tissue relative to the other was collected for mRNA quantification of bone morphogenetic protein (BMP)-2, BMP-7, osteopontin (OPN), bone sialoprotein (BSP), osteoprotegrin (OPG), and receptor activator of NF-κB ligand (RANKL). Implants were removed by applying a counter-torque force. Histomorphometric analysis revealed higher remaining defect in the calvarial defects of the control group than the RESV group (P=0.026). Resveratrol increased the counter-torque values of implant removal when compared to control therapy (P=0.031). Gene expression analysis showed a higher expression of BMP-2 (P=0.011), BMP-7 (P=0.049), and OPN (P=0.002) genes in the RESV group than in the control group. In conclusion, resveratrol improved the repair of critical-sized bone defects and the biomechanical retention of implants. Indeed, this natural agent may up-regulate the gene expression of important osteogenic markers. PMID:24530035

  6. Hypoxia-mediated regulation of gene expression in mammalian cells

    PubMed Central

    Shih, Shu-Ching; Claffey, Kevin P.

    1998-01-01

    The molecular mechanism underlying oxygen sensing in mammalian cells has been extensively investigated in the areas of glucose transport, glycolysis, erythropoiesis, angiogenesis and catecholamine metabolism. Expression of functionally operative representative proteins in these specific areas, such as the glucose transporter 1, glycolytic enzymes, erythropoietin, vascular endothelial growth factor and tyrosine hydroxylase are all induced by hypoxia. Recent studies demonstrated that both transcriptional activation and post-transcriptional mechanisms are important to the hypoxia-mediated regulation of gene expression. In this article, the cis-acting elements and trans-acting factors involved in the transcriptional activation of gene expression will be reviewed. In addition, the mechanisms of post-transcriptional mRNA stabilization will also be addressed. We will discuss whether these two processes of regulation of hypoxia-responsive genes are mechanistically linked and co-operative in nature. PMID:10319016

  7. Fibrin patch-based insulin-like growth factor-1 gene-modified stem cell transplantation repairs ischemic myocardium

    PubMed Central

    Li, Jun; Zhu, Kai; Yang, Shan; Wang, Yulin; Guo, Changfa; Yin, Kanhua; Wang, Chunsheng

    2015-01-01

    Bone marrow mesenchymal stem cells (BMSCs), tissue-engineered cardiac patch, and therapeutic gene have all been proposed as promising therapy strategies for cardiac repair after myocardial infarction. In our study, BMSCs were modified with insulin-like growth factor-1 (IGF-1) gene, loaded into a fibrin patch, and then transplanted into a porcine model of ischemia/reperfusion (I/R) myocardium injury. The results demonstrated that IGF-1 gene overexpression could promote proliferation of endothelial cells and cardiomyocyte-like differentiation of BMSCs in vitro. Four weeks after transplantation of fibrin patch loaded with gene-modified BMSCs, IGF-1 overexpression could successfully promote angiogenesis, inhibit remodeling, increase grafted cell survival and reduce apoptosis. In conclusion, the integrated strategy, which combined fibrin patch with IGF-1 gene modified BMSCs, could promote the histological cardiac repair for a clinically relevant porcine model of I/R myocardium injury. PMID:25767192

  8. Changes in the expression of DNA double strand break repair genes in primordial follicles from immature and aged rats.

    PubMed

    Govindaraj, Vijayakumar; Keralapura Basavaraju, Rajani; Rao, Addicam Jagannadha

    2015-03-01

    Oocytes present at birth undergo a progressive process of apoptosis in humans and other mammals as they age. Accepted opinion is that no fresh oocytes are produced other than those present at the time of birth. Studies have shown that DNA repair genes in oocytes of mice and women decline with age, and lack of these genes show higher DNA breaks and increased oocyte death rates. In contrast to the ethical problems associated with monitoring the changes in DNA double-strand breaks in oocytes from young and old humans, it is relatively easy to carry out such a study using a rodent model. In this study, the mRNA levels of DNA repair genes are compared with protein products of some of the genes in the primordial follicles isolated from immature (18-20 days) and aged (400-450 days) female rats. Results revealed a significant decline in mRNA levels of BRAC1 (P < 0.01), RAD51 (P < 0.05), ERCC2 (P < 0.05), and H2AX (P < 0.01) of DNA repair genes and phospho-protein levels of BRAC1 (P < 0.01) and H2AX (P < 0.05) in primordial follicles of aged rats. Impaired DNA repair is confirmed as a mechanism of oocyte ageing.

  9. Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.

    PubMed

    Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena

    2002-02-15

    Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

  10. Benzo(a)pyrene induces similar gene expression changes in testis of DNA repair proficient and deficient mice

    PubMed Central

    2010-01-01

    Background Benzo [a]pyrene (B[a]P) exposure induces DNA adducts at all stages of spermatogenesis and in testis, and removal of these lesions is less efficient in nucleotide excision repair deficient Xpc-/- mice than in wild type mice. In this study, we investigated by using microarray technology whether compromised DNA repair in Xpc-/- mice may lead to a transcriptional reaction of the testis to cope with increased levels of B[a]P induced DNA damage. Results Two-Way ANOVA revealed only 4 genes differentially expressed between wild type and Xpc-/- mice, and 984 genes between testes of B[a]P treated and untreated mice irrespective of the mouse genotype. However, the level in which these B[a]P regulated genes are expressed differs between Wt and Xpc-/- mice (p = 0.000000141), and were predominantly involved in the regulation of cell cycle, translation, chromatin structure and spermatogenesis, indicating a general stress response. In addition, analysis of cell cycle phase dependent gene expression revealed that expression of genes involved in G1-S and G2-M phase arrest was increased after B[a]P exposure in both genotypes. A slightly higher induction of average gene expression was observed at the G2-M checkpoint in Xpc-/- mice, but this did not reach statistical significance (P = 0.086). Other processes that were expected to have changed by exposure, like apoptosis and DNA repair, were not found to be modulated at the level of gene expression. Conclusion Gene expression in testis of untreated Xpc-/- and wild type mice were very similar, with only 4 genes differentially expressed. Exposure to benzo(a)pyrene affected the expression of genes that are involved in cell cycle regulation in both genotypes, indicating that the presence of unrepaired DNA damage in testis blocks cell proliferation to protect DNA integrity in both DNA repair proficient and deficient animals. PMID:20504355

  11. Revisiting DNA damage repair, p53-mediated apoptosis and cisplatin sensitivity in germ cell tumors.

    PubMed

    Cavallo, Francesca; Feldman, Darren R; Barchi, Marco

    2013-01-01

    Testicular germ cell tumors (TGCTs), ie, seminomas and nonseminomas, account for 1% to 3% of all neoplasms in men. They are the most common cancer in young white males and are unique in their responsiveness to cisplatin-based chemotherapy. For this reason, TGCTs are considered a model for curative disease. However, up to now, the molecular mechanisms behind this exceptional responsiveness to DNA-damaging agents have remained unclear. A hypersensitive apoptotic response, as well as a reduction in the proficiency to repair cisplatin-induced DNA damage might account for this behavior. In this review, building on recent findings of p53-induced apoptosis and DNA-repair mechanisms in TGCTs, we will discuss the molecular bases that drive tumor sensitivity to cisplatin, emphasizing the new therapeutic approaches proposed to eventually constrain tumor recurrence, and target TGCTs which are unresponsive to standard therapies. PMID:23784838

  12. NADPH Oxidase 1 and Its Derived Reactive Oxygen Species Mediated Tissue Injury and Repair

    PubMed Central

    Fu, Xiu-Jun; Peng, Ying-Bo; Hu, Yi-Ping; Shi, You-Zhen; Yao, Min; Zhang, Xiong

    2014-01-01

    Reactive oxygen species are mostly viewed to cause oxidative damage to various cells and induce organ dysfunction after ischemia-reperfusion injury. However, they are also considered as crucial molecules for cellular signal transduction in biology. NADPH oxidase, whose only function is reactive oxygen species production, has been extensively investigated in many cell types especially phagocytes. The deficiency of NADPH oxidase extends the process of inflammation and delays tissue repair, which causes chronic granulomatous disease in patients. NADPH oxidase 1, one member of the NADPH oxidase family, is not only constitutively expressed in a variety of tissues, but also induced to increase expression in both mRNA and protein levels under many circumstances. NADPH oxidase 1 and its derived reactive oxygen species are suggested to be able to regulate inflammation reaction, cell proliferation and migration, and extracellular matrix synthesis, which contribute to the processes of tissue injury and repair. PMID:24669283

  13. Mismatch repair genes expression defects & association with clinicopathological characteristics in colorectal carcinoma

    PubMed Central

    Kaur, Gurjeet; Masoud, Abdelhafid; Raihan, N.; Radzi, M.; Khamizar, W.; Kam, Lee Suk

    2011-01-01

    Background & objectives: DNA mismatch repair gene (MMR) abnormalities are seen in 95 per cent of hereditary nonpolyposis colorectal cancer (HNPCC) and 10-15 per cent of sporadic colorectal cancers. There are no data on MMR abnormalities in Malaysian colorectal cancer patients. This study was aimed to determine the frequency of abnormal MMR gene protein expression in colorectal carcinoma in Northern Peninsular Malaysia using immunohistochemistry. Methods: Clinicopathological information was obtained from 148 patients’ records who underwent bowel resection for colorectal cancer (CRC) at the three hospitals in Malaysia. Immunohistochemistry for MLH1, MSH2, MSH6 and PMS2 proteins were performed on paraffin embedded tissue containing carcinoma. Results: A total of 148 subjects and 150 colorectal carcinomas of sporadic and hereditary types were assessed. Three patients had synchronous tumours. Twenty eight cancers (18.6%) from 26 subjects (17.6%) had absent immunohistochemical expression of any one of the MMR gene proteins. This comprised absent MLH1 only – 3 cancers, absent MSH2 only – 3, absent MSH6 only – 2, absent PMS2 only – 3, absent MLH1 and PMS2 – 14, absent MSH2 and MSH6 – 2 and absent MLH1, MSH6 and PMS2 – 1. There was significant association between abnormal MMR gene protein expression and proximal colon cancers, mucinous, signet ring and poorly differentiated morphology. Interpretation & conclusions: Cancers with abnormal MMR gene expression were associated with microsatellite instability-high (MSI-H) phenotype. About 15 per cent demonstrated absent MSH2, MSH6 and PMS2 protein expression in isolation or in combination with other MMR genes, which often predicts a germline mutation, synonymous with a diagnosis of HNPCC. This appears to be high frequency compared to reported data. PMID:21911971

  14. The mechanism of nucleotide excision repair-mediated UV-induced mutagenesis in nonproliferating cells.

    PubMed

    Kozmin, Stanislav G; Jinks-Robertson, Sue

    2013-03-01

    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.

  15. Repair of dense connective tissues via biomaterial-mediated matrix reprogramming of the wound interface.

    PubMed

    Qu, Feini; Pintauro, Michael P; Haughan, Joanne E; Henning, Elizabeth A; Esterhai, John L; Schaer, Thomas P; Mauck, Robert L; Fisher, Matthew B

    2015-01-01

    Repair of dense connective tissues in adults is limited by their intrinsic hypocellularity and is exacerbated by a dense extracellular matrix (ECM) that impedes cellular migration to and local proliferation at the wound site. Conversely, healing in fetal tissues occurs due in part to an environment conducive to cell mobility and division. Here, we investigated whether the application of a degradative enzyme, collagenase, could reprogram the adult wound margin to a more fetal-like state, and thus abrogate the biophysical impediments that hinder migration and proliferation. We tested this concept using the knee meniscus, a commonly injured structure for which few regenerative approaches exist. To focus delivery and degradation to the wound interface, we developed a system in which collagenase was stored inside poly(ethylene oxide) (PEO) electrospun nanofibers and released upon hydration. Through a series of in vitro and in vivo studies, our findings show that partial digestion of the wound interface improves repair by creating a more compliant and porous microenvironment that expedites cell migration to and/or proliferation at the wound margin. This innovative approach of targeted manipulation of the wound interface, focused on removing the naturally occurring barriers to adult tissue repair, may find widespread application in the treatment of injuries to a variety of dense connective tissues.

  16. Repair of Dense Connective Tissues via Biomaterial-Mediated Matrix Reprogramming of the Wound Interface

    PubMed Central

    Qu, Feini; Pintauro, Michael P.; Haughan, Joanne; Henning, Elizabeth A.; Esterhai, John L.; Schaer, Thomas P.; Mauck, Robert L.; Fisher, Matthew B.

    2014-01-01

    Repair of dense connective tissues in adults is limited by their intrinsic hypocellularity and is exacerbated by a dense extracellular matrix (ECM) that impedes cellular migration to and local proliferation at the wound site. Conversely, healing in fetal tissues occurs due in part to an environment conducive to cell mobility and division. Here, we investigated whether the application of a degradative enzyme, collagenase, could reprogram the adult wound margin to a more fetal-like state, and thus abrogate the biophysical impediments that hinder migration and proliferation. We tested this concept using the knee meniscus, a commonly injured structure for which few regenerative approaches exist. To focus delivery and degradation to the wound interface, we developed a system in which collagenase was stored inside poly(ethylene oxide) (PEO) electrospun nanofibers and released upon hydration. Through a series of in vitro and in vivo studies, our findings show that partial digestion of the wound interface improves repair by creating a more compliant and porous microenvironment that expedites cell migration to and/or proliferation at the wound margin. This innovative approach of targeted manipulation of the wound interface, focused on removing the naturally occurring barriers to adult tissue repair, may find widespread application in the treatment of injuries to a variety of dense connective tissues. PMID:25477175

  17. Myeloid-derived growth factor (C19orf10) mediates cardiac repair following myocardial infarction.

    PubMed

    Korf-Klingebiel, Mortimer; Reboll, Marc R; Klede, Stefanie; Brod, Torben; Pich, Andreas; Polten, Felix; Napp, L Christian; Bauersachs, Johann; Ganser, Arnold; Brinkmann, Eva; Reimann, Ines; Kempf, Tibor; Niessen, Hans W; Mizrahi, Jacques; Schönfeld, Hans-Joachim; Iglesias, Antonio; Bobadilla, Maria; Wang, Yong; Wollert, Kai C

    2015-02-01

    Paracrine-acting proteins are emerging as a central mechanism by which bone marrow cell-based therapies improve tissue repair and heart function after myocardial infarction (MI). We carried out a bioinformatic secretome analysis in bone marrow cells from patients with acute MI to identify novel secreted proteins with therapeutic potential. Functional screens revealed a secreted protein encoded by an open reading frame on chromosome 19 (C19orf10) that promotes cardiac myocyte survival and angiogenesis. We show that bone marrow-derived monocytes and macrophages produce this protein endogenously to protect and repair the heart after MI, and we named it myeloid-derived growth factor (MYDGF). Whereas Mydgf-deficient mice develop larger infarct scars and more severe contractile dysfunction compared to wild-type mice, treatment with recombinant Mydgf reduces scar size and contractile dysfunction after MI. This study is the first to assign a biological function to MYDGF, and it may serve as a prototypical example for the development of protein-based therapies for ischemic tissue repair. PMID:25581518

  18. Repair of dense connective tissues via biomaterial-mediated matrix reprogramming of the wound interface.

    PubMed

    Qu, Feini; Pintauro, Michael P; Haughan, Joanne E; Henning, Elizabeth A; Esterhai, John L; Schaer, Thomas P; Mauck, Robert L; Fisher, Matthew B

    2015-01-01

    Repair of dense connective tissues in adults is limited by their intrinsic hypocellularity and is exacerbated by a dense extracellular matrix (ECM) that impedes cellular migration to and local proliferation at the wound site. Conversely, healing in fetal tissues occurs due in part to an environment conducive to cell mobility and division. Here, we investigated whether the application of a degradative enzyme, collagenase, could reprogram the adult wound margin to a more fetal-like state, and thus abrogate the biophysical impediments that hinder migration and proliferation. We tested this concept using the knee meniscus, a commonly injured structure for which few regenerative approaches exist. To focus delivery and degradation to the wound interface, we developed a system in which collagenase was stored inside poly(ethylene oxide) (PEO) electrospun nanofibers and released upon hydration. Through a series of in vitro and in vivo studies, our findings show that partial digestion of the wound interface improves repair by creating a more compliant and porous microenvironment that expedites cell migration to and/or proliferation at the wound margin. This innovative approach of targeted manipulation of the wound interface, focused on removing the naturally occurring barriers to adult tissue repair, may find widespread application in the treatment of injuries to a variety of dense connective tissues. PMID:25477175

  19. Concise Review: The Bystander Effect: Mesenchymal Stem Cell-Mediated Lung Repair.

    PubMed

    Savukinas, Ulrika Blank; Enes, Sara Rolandsson; Sjöland, Annika Andersson; Westergren-Thorsson, Gunilla

    2016-06-01

    Mesenchymal stem or stromal cells (MSCs), a heterogeneous subset of adult stem/progenitor cells, have surfaced as potential therapeutic units with significant clinical benefit for a wide spectrum of disease conditions, including those affecting the lung. Although MSCs carry both self-renewal and multilineage differentiation abilities, current dogma holds that MSCs mainly contribute to tissue regeneration and repair by modulating the host tissue via secreted cues. Thus, the therapeutic benefit of MSCs is thought to derive from so called bystander effects. The regenerative mechanisms employed by MSCs in the lung include modulation of the immune system as well as promotion of epithelial and endothelial repair. Apart from secreted factors, a number of recent findings suggest that MSCs engage in mitochondrial transfer and shedding of membrane vesicles as a means to enhance tissue repair following injury. Furthermore, it is becoming increasingly clear that MSCs are an integral component of epithelial lung stem cell niches. As such, MSCs play an important role in coupling information from the environment to stem and progenitor populations, such that homeostasis can be ensured even in the face of injury. It is the aim of this review to outline the major mechanisms by which MSCs contribute to lung regeneration, synthesizing recent preclinical findings with data from clinical trials and potential for future therapy. Stem Cells 2016;34:1437-1444. PMID:26991735

  20. DqsIR quorum sensing-mediated gene regulation of the extremophilic bacterium Deinococcus radiodurans in response to oxidative stress.

    PubMed

    Lin, Lin; Dai, Shang; Tian, Bing; Li, Tao; Yu, Jiangliu; Liu, Chengzhi; Wang, Liangyan; Xu, Hong; Zhao, Ye; Hua, Yuejin

    2016-05-01

    Here, we show that AHLs can be employed by Deinococcus radiodurans, which belongs to the unique phylum Deinococcus-Thermus and is known for its cellular resistance to environmental stresses. An AHL-mediated quorum-sensing system (DqsI/DqsR) was identified in D. radiodurans. We found that under non-stress conditions, the AHL level was "shielded" by quorum quenching enzymes, whereas AHLs accumulated when D. radiodurans was exposed to oxidative stress. Upon exposure to H2 O2 , AHL synthetic enzymes (DqsI) were immediately induced, while the expression of quorum-quenching enzymes began to increase approximately 30 min after exposure to H2 O2 , as shown by time-course analyses of gene expression. Both dqsI mutant (DMDqsI) and dqsR mutant (MDqsR) were more sensitive to oxidative stress compared with the wild-type strain. Exogenous AHLs (5 μM) could completely restore the survival fraction of DMDqsI under oxidative stress. RNA-seq analysis showed that a number of genes involved in stress-response, cellular cleansing, and DNA repair had altered transcriptional levels in MDqsR. The DqsR, acting as a regulator of quorum sensing, controls gene expression along with AHLs. Hence, the DqsIR-mediated quorum sensing that mediates gene regulation is an adaptive strategy for D. radiodurans in response to oxidative stresses and is conserved in the extremophilic Deinococcus bacteria.

  1. Insulin-Like Growth Factor-1 Contributes to Mucosal Repair by β-Arrestin2-Mediated Extracellular Signal-Related Kinase Signaling in Experimental Colitis.

    PubMed

    Chen, Tingting; Zheng, Fengping; Tao, Jin; Tan, Siwei; Zeng, Lixian; Peng, Xiaojie; Wu, Bin

    2015-09-01

    Insulin-like growth factor-1 (IGF-1) possesses the ability to attenuate intestinal damage and promote mucosal repair of colitis. β-Arrestins, as the scaffolding proteins of G protein-coupled receptors or non-G protein-coupled receptors signaling, can be involved in IGF-1-mediated signaling pathways. However, the interaction of IGF-1 and β-arrestin2 in the mucosal repair of experimental colitis remains unexplored. Ulcerative colitis was induced in β-arrestin2 wild-type mice and β-arrestin2 knockout littermates by using 3% dextran sulfate sodium for 5 days, followed by regular water consumption for 1, 2, 3, and 4 weeks to analyze the mucosal repair from experimental colitis. Disease activity index and histologic score analyses were performed. Apoptosis and proliferation were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Ki-67 staining, respectively. The expressions of β-arrestin2, phospho (p)-IGF-1R, and p-extracellular signal-regulated kinase (ERK)1/2 were examined. Furthermore, β-arrestin2 was overexpressed or altered in HCT116 cells by transfection before IGF-1 treatment in vitro. IGF-1 and β-arrestin2 expression was up-regulated in the repairing phase of experimental colitis. Targeted deletion of β-arrestin2 delayed the repair of colitis by inhibiting cell proliferation without affecting the levels of IGF-1 and p-IGF-1R. The β-arrestin2/ERK signaling pathway was involved in IGF-1-mediated mucosal repair through promoting epithelial cell and goblet cell regeneration from experimental colitis. These results indicate that IGF-1 contributes to the mucosal repair by β-arrestin2-mediated ERK signaling in experimental colitis.

  2. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

    PubMed

    He, Xiangjun; Tan, Chunlai; Wang, Feng; Wang, Yaofeng; Zhou, Rui; Cui, Dexuan; You, Wenxing; Zhao, Hui; Ren, Jianwei; Feng, Bo

    2016-05-19

    CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. PMID:26850641

  3. 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

  4. Detoxification and repair process of ozone injury: from O3 uptake to gene expression adjustment.

    PubMed

    Castagna, A; Ranieri, A

    2009-05-01

    Plants react to O(3) threat by setting up a variety of defensive strategies involving the co-ordinated modulation of stress perception, signalling and metabolic responses. Although stomata largely controls O(3) uptake, differences in O(3) tolerance cannot always be ascribed to changes in stomatal conductance but cell protective and repair processes should be taken into account. O(3)-driven ROS production in the apoplast induces a secondary, active, self-propagating generation of ROS, whose levels must be finely tuned, by many enzymatic and non-enzymatic antioxidant systems, to induce gene activation without determining uncontrolled cell death. Additional signalling molecules, as ethylene, jasmonic and salicylic acid are also crucial to determine the spreading and the containment of leaf lesions. The main recent results obtained on O(3) sensing, signal transduction, ROS formation and detoxification mechanisms are here discussed.

  5. Evaluating biological containment strategies for pollen-mediated gene flow.

    PubMed

    Hüsken, Alexandra; Prescher, Sabine; Schiemann, Joachim

    2010-01-01

    Several biological containment methods have been developed to reduce pollen dispersal; many of them only have a proof of concept in a model plant species. This review focuses on biological containment measures which were tested for their long-term efficiency at the greenhouse or field scale level, i.e. plastid transformation, transgene excission, cleistogamy and cytoplasmic male sterility (CMS). Pollen-mediated gene transfer in transplastomic tobacco could occur at very low frequencies if the predominant mode of inheritance is maternal. Transgene excision from tobacco pollen can be made highly efficient by coexpression of two recombinases. For cleistogamous oilseed rape it was shown that some flowers were partially open depending on genotypes, environment and recording dates. Reports on the stability of CMS in maize and sunflower indicated that there is a high variability for different genotypes under different environmental conditions and over successive years. But for both crop types some stable lines could be selected. These data demonstrate that the biological containment methods discussed are very promising for reducing gene flow but that no single containment strategy provides 100% reduction. However, the necessary efficiency of biological containment methods depends on the level of containment required. The containment level may need to be higher for safety purposes (e.g. production of special plant-made pharmaceuticals), while much lower containment levels may already be sufficient to reach coexistence goals. It is concluded that where pollen-mediated gene flow must be prevented altogether, combinations of complementary containment systems will be required.

  6. ABCB5 is a limbal stem cell gene required for corneal development and repair

    PubMed Central

    Ksander, Bruce R.; Kolovou, Paraskevi E.; Wilson, Brian J.; Saab, Karim R.; Guo, Qin; Ma, Jie; McGuire, Sean P.; Gregory, Meredith S.; Vincent, William J. B.; Perez, Victor L.; Cruz-Guilloty, Fernando; Kao, Winston W. Y.; Call, Mindy K.; Tucker, Budd A.; Zhan, Qian; Murphy, George F.; Lathrop, Kira L.; Alt, Clemens; Mortensen, Luke J.; Lin, Charles P.; Zieske, James D.; Frank, Markus H.; Frank, Natasha Y.

    2014-01-01

    Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs)1–3, and LSC deficiency is a major cause of blindness worldwide4. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts5, a gene allowing for prospective LSC enrichment has not been identified so far5. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5)6,7 marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs2 in mice and p63α-positive LSCs8 in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency. PMID:25030174

  7. Characterization of Chondrocyte Scaffold Carriers for Cell-based Gene Therapy in Articular Cartilage Repair

    PubMed Central

    Shui, Wei; Yin, Liangjun; Luo, Jeffrey; Li, Ruidong; Zhang, Wenwen; Zhang, Jiye; Huang, Wei; Hu, Ning; Liang, Xi; Deng, Zhong-Liang; Hu, Zhenming; Shi, Lewis; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan; Ho, Sherwin

    2014-01-01

    Articular cartilage lesions in the knee are common injuries. Chondrocyte transplant represents a promising therapeutic modality for articular cartilage injuries. Here, we characterize the viability and transgene expression of articular chondrocytes cultured in 3-D scaffolds provided by four types of carriers. Articular chondrocytes are isolated from rabbit knees and cultured in four types of scaffolds: type I collagen sponge, fibrin glue, hyaluronan, and Open-cell PolyLactic Acid (OPLA). The cultured cells are transduced with adenovirus expressing green fluorescence protein (AdGFP) and luciferase (AdGL3-Luc). The viability and gene expression in the chondrocytes are determined with fluorescence microscopy and luciferase assay. Cartilage matrix production is assessed by Alcian blue staining. Rabbit articular chondrocytes are effectively infected by AdGFP and exhibited sustained GFP expression. All tested scaffolds support the survival and gene expression of the infected chondrocytes. However, the highest transgene expression is observed in the OPLA carrier. At four weeks, Alcian blue-positive matrix materials are readily detected in OPLA cultures. Thus, our results indicate that, while all tested carriers can support the survival of chondrocytes, OPLA supports the highest transgene expression and is the most conductive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repairs. PMID:23629940

  8. The ATPase domain but not the acidic region of Cockayne syndrome group B gene product is essential for DNA repair.

    PubMed

    Brosh, R M; Balajee, A S; Selzer, R R; Sunesen, M; Proietti De Santis, L; Bohr, V A

    1999-11-01

    Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways. PMID:10564257

  9. Stem and progenitor cell-mediated tumor selective gene therapy.

    PubMed

    Aboody, K S; Najbauer, J; Danks, M K

    2008-05-01

    The poor prognosis for patients with aggressive or metastatic tumors and the toxic side effects of currently available treatments necessitate the development of more effective tumor-selective therapies. Stem/progenitor cells display inherent tumor-tropic properties that can be exploited for targeted delivery of anticancer genes to invasive and metastatic tumors. Therapeutic genes that have been inserted into stem cells and delivered to tumors with high selectivity include prodrug-activating enzymes (cytosine deaminase, carboxylesterase, thymidine kinase), interleukins (IL-2, IL-4, IL-12, IL-23), interferon-beta, apoptosis-promoting genes (tumor necrosis factor-related apoptosis-inducing ligand) and metalloproteinases (PEX). We and others have demonstrated that neural and mesenchymal stem cells can deliver therapeutic genes to elicit a significant antitumor response in animal models of intracranial glioma, medulloblastoma, melanoma brain metastasis, disseminated neuroblastoma and breast cancer lung metastasis. Most studies reported reduction in tumor volume (up to 90%) and increased survival of tumor-bearing animals. Complete cures have also been achieved (90% disease-free survival for >1 year of mice bearing disseminated neuroblastoma tumors). As we learn more about the biology of stem cells and the molecular mechanisms that mediate their tumor-tropism and we identify efficacious gene products for specific tumor types, the clinical utility of cell-based delivery strategies becomes increasingly evident.

  10. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes.

    PubMed

    Troelstra, C; van Gool, A; de Wit, J; Vermeulen, W; Bootsma, D; Hoeijmakers, J H

    1992-12-11

    Cells from patients with the UV-sensitive nucleotide excision repair disorder Cockayne's syndrome (CS) have a specific defect in preferential repair of lesions from the transcribed strand of active genes. This system permits quick resumption of transcription after UV exposure. Here we report the characterization of ERCC6, a gene involved in preferential repair in eukaryotes. ERCC6 corrects the repair defect of CS complementation group B (CS-B). It encodes a protein of 1493 amino acids, containing seven consecutive domains conserved between DNA and RNA helicases. The entire helicase region bears striking homology to segments in recently discovered proteins involved in transcription regulation, chromosome stability, and DNA repair. Mutation analysis of a CS-B patient indicates that the gene is not essential for cell viability and is specific for preferential repair of transcribed sequences. PMID:1339317

  11. SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint

    PubMed Central

    Carvalho, Sílvia; Vítor, Alexandra C; Sridhara, Sreerama C; Martins, Filipa B; Raposo, Ana C; Desterro, Joana MP; Ferreira, João; de Almeida, Sérgio F

    2014-01-01

    Histone modifications establish the chromatin states that coordinate the DNA damage response. In this study, we show that SETD2, the enzyme that trimethylates histone H3 lysine 36 (H3K36me3), is required for ATM activation upon DNA double-strand breaks (DSBs). Moreover, we find that SETD2 is necessary for homologous recombination repair of DSBs by promoting the formation of RAD51 presynaptic filaments. In agreement, SETD2-mutant clear cell renal cell carcinoma (ccRCC) cells displayed impaired DNA damage signaling. However, despite the persistence of DNA lesions, SETD2-deficient cells failed to activate p53, a master guardian of the genome rarely mutated in ccRCC and showed decreased cell survival after DNA damage. We propose that this novel SETD2-dependent role provides a chromatin bookmarking instrument that facilitates signaling and repair of DSBs. In ccRCC, loss of SETD2 may afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for additional genetic mutations in TP53. DOI: http://dx.doi.org/10.7554/eLife.02482.001 PMID:24843002

  12. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting. PMID:14970588

  13. Repair of uv damaged DNA: Genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  14. Photochemical internalization-mediated nonviral gene transfection: polyamine core-shell nanoparticles as gene carrier

    NASA Astrophysics Data System (ADS)

    Zamora, Genesis; Wang, Frederick; Sun, Chung-Ho; Trinidad, Anthony; Kwon, Young Jik; Cho, Soo Kyung; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2014-10-01

    The overall objective of the research was to investigate the utility of photochemical internalization (PCI) for the enhanced nonviral transfection of genes into glioma cells. The PCI-mediated introduction of the tumor suppressor gene phosphatase and tensin homolog (PTEN) or the cytosine deaminase (CD) pro-drug activating gene into U87 or U251 glioma cell monolayers and multicell tumor spheroids were evaluated. In the study reported here, polyamine-DNA gene polyplexes were encapsulated in a nanoparticle (NP) with an acid degradable polyketal outer shell. These NP synthetically mimic the roles of viral capsid and envelope, which transport and release the gene, respectively. The effects of PCI-mediated suppressor and suicide genes transfection efficiency employing either "naked" polyplex cores alone or as NP-shelled cores were compared. PCI was performed with the photosensitizer AlPcS2a and λ=670-nm laser irradiance. The results clearly demonstrated that the PCI can enhance the delivery of both the PTEN or CD genes in human glioma cell monolayers and multicell tumor spheroids. The transfection efficiency, as measured by cell survival and inhibition of spheroid growth, was found to be significantly greater at suboptimal light and DNA levels for shelled NPs compared with polyamine-DNA polyplexes alone.

  15. Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks

    PubMed Central

    Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

    2016-01-01

    Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress. PMID:26765540

  16. Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks.

    PubMed

    Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

    2016-01-01

    Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.

  17. Treatment of acute lung injury by targeting MG53-mediated cell membrane repair

    PubMed Central

    Lieber, Gissela; Nishi, Miyuki; Yan, Rosalie; Wang, Zhen; Yao, Yonggang; Li, Yu; Whitson, Bryan A.; Duann, Pu; Li, Haichang; Zhou, Xinyu; Zhu, Hua; Takeshima, Hiroshi; Hunter, John C.; McLeod, Robbie L.; Weisleder, Noah; Zeng, Chunyu; Ma, Jianjie

    2014-01-01

    Injury to lung epithelial cells has a role in multiple lung diseases. We previously identified mitsugumin 53 (MG53) as a component of the cell membrane repair machinery in striated muscle cells. Here we show that MG53 also has a physiological role in the lung and may be used as a treatment in animal models of acute lung injury. Mice lacking MG53 show increased susceptibility to ischemia-reperfusion and over-ventilation induced injury to the lung when compared with wild type mice. Extracellular application of recombinant human MG53 (rhMG53) protein protects cultured lung epithelial cells against anoxia/reoxygenation-induced injuries. Intravenous delivery or inhalation of rhMG53 reduces symptoms in rodent models of acute lung injury and emphysema. Repetitive administration of rhMG53 improves pulmonary structure associated with chronic lung injury in mice. Our data indicate a physiological function for MG53 in the lung and suggest that targeting membrane repair may be an effective means for treatment or prevention of lung diseases. PMID:25034454

  18. Minireview: mechanisms of growth hormone-mediated gene regulation.

    PubMed

    Chia, Dennis J

    2014-07-01

    GH exerts a diverse array of physiological actions that include prominent roles in growth and metabolism, with a major contribution via stimulating IGF-1 synthesis. GH achieves its effects by influencing gene expression profiles, and Igf1 is a key transcriptional target of GH signaling in liver and other tissues. This review examines the mechanisms of GH-mediated gene regulation that begin with signal transduction pathways activated downstream of the GH receptor and continue with chromatin events at target genes and additionally encompasses the topics of negative regulation and cross talk with other cellular inputs. The transcription factor, signal transducer and activator of transcription 5b, is regarded as the major signaling pathway by which GH achieves its physiological effects, including in stimulating Igf1 gene transcription in liver. Recent studies exploring the mechanisms of how activated signal transducer and activator of transcription 5b accomplishes this are highlighted, which begin to characterize epigenetic features at regulatory domains of the Igf1 locus. Further research in this field offers promise to better understand the GH-IGF-1 axis in normal physiology and disease and to identify strategies to manipulate the axis to improve human health.

  19. Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

    SciTech Connect

    Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2009-12-18

    The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

  20. Mutational analysis of the Drosophila DNA repair and recombination gene mei-9.

    PubMed Central

    Yildiz, Ozlem; Kearney, Hutton; Kramer, Benjamin C; Sekelsky, Jeff J

    2004-01-01

    Drosophila mei-9 is essential for several DNA repair and recombination pathways, including nucleotide excision repair (NER), interstrand crosslink repair, and meiotic recombination. To better understand the role of MEI-9 in these processes, we characterized 10 unique mutant alleles of mei-9. These include a P-element insertion that disrupts repair functions but not the meiotic function; three nonsense mutations, one of which has nearly wild-type levels of protein; three missense mutations, one of which disrupts the meiotic function but not repair functions; two small in-frame deletions; and one frameshift. PMID:15166153

  1. Cell and gene therapy for arrhythmias: Repair of cardiac conduction damage

    PubMed Central

    Xiao, Yong-Fu

    2011-01-01

    Action potentials generated in the sinoatrial node (SAN) dominate the rhythm and rate of a healthy human heart. Subsequently, these action potentials propagate to the whole heart via its conduction system. Abnormalities of impulse generation and/or propagation in a heart can cause arrhythmias. For example, SAN dysfunction or conduction block of the atrioventricular node can lead to serious bradycardia which is currently treated with an implanted electronic pacemaker. On the other hand, conduction damage may cause reentrant tachyarrhythmias which are primarily treated pharmacologically or by medical device-based therapies, including defibrillation and tissue ablation. However, drug therapies sometimes may not be effective or are associated with serious side effects. Device-based therapies for cardiac arrhythmias, even with well developed technology, still face inadequacies, limitations, hardware complications, and other challenges. Therefore, scientists are actively seeking other alternatives for antiarrhythmic therapy. In particular, cells and genes used for repairing cardiac conduction damage/defect have been investigated in various studies both in vitro and in vivo. Despite the complexities of the excitation and conduction systems of the heart, cell and gene-based strategies provide novel alternatives for treatment or cure of cardiac arrhythmias. This review summarizes some highlights of recent research progress in this field. PMID:22783301

  2. Influence of functional polymorphisms in DNA repair genes of myelodysplastic syndrome.

    PubMed

    Ribeiro, Howard Lopes; Soares Maia, Allan Rodrigo; Costa, Marília Braga; Farias, Izabelle Rocha; de Paula Borges, Daniela; de Oliveira, Roberta Taiane Germano; de Sousa, Juliana Cordeiro; Magalhães, Silvia Maria Meira; Pinheiro, Ronald Feitosa

    2016-09-01

    Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell (HSC) malignances characterized by peripheral cytopenias and predisposition to acute myeloid leukemia transformation. Several studies show that the MDS pathogenesis is a complex and heterogeneous process that involves multiple steps through a sequence of genetic lesions in the DNA which lead to functional changes in the cell and the emergence and subsequent evolution of pre-malignant clone. Double strand breaks (DSB) lesions are the most severe type of DNA damage in HSCs, which, if not properly repaired, might contribute to the development of chromosomal abnormalities, which in turn may lead to leukemia development. We assessed the mRNA expression levels of ATM, BRCA1, BRCA2, RAD51, XRCC5, XRCC6 and LIG4 genes in bone marrow samples of 47 MDS patients in order to evaluate the association with functional polymorphisms rs228593, rs4793191, rs9567623, rs1801320, rs3835, rs2267437 and rs1805388, respectively, and try to detect clinical associations. We found that the rs228593, rs2267437 and rs1805388 functional polymorphisms probably alter the level of expression of the ATM, XRCC6 and LIG4 genes, respectively, being important in the maintenance of genomic instability in MDS. PMID:27497341

  3. Variable continental distribution of polymorphisms in the coding regions of DNA-repair genes.

    PubMed

    Mathonnet, Géraldine; Labuda, Damian; Meloche, Caroline; Wambach, Tina; Krajinovic, Maja; Sinnett, Daniel

    2003-01-01

    DNA-repair pathways are critical for maintaining the integrity of the genetic material by protecting against mutations due to exposure-induced damages or replication errors. Polymorphisms in the corresponding genes may be relevant in genetic epidemiology by modifying individual cancer susceptibility or therapeutic response. We report data on the population distribution of potentially functional variants in XRCC1, APEX1, ERCC2, ERCC4, hMLH1, and hMSH3 genes among groups representing individuals of European, Middle Eastern, African, Southeast Asian and North American descent. The data indicate little interpopulation differentiation in some of these polymorphisms and typical FST values ranging from 10 to 17% at others. Low FST was observed in APEX1 and hMSH3 exon 23 in spite of their relatively high minor allele frequencies, which could suggest the effect of balancing selection. In XRCC1, hMSH3 exon 21 and hMLH1 Africa clusters either with Middle East and Europe or with Southeast Asia, which could be related to the demographic history of human populations, whereby human migrations and genetic drift rather than selection would account for the observed differences.

  4. DNA repair gene XRCC4 codon 247 polymorphism modified diffusely infiltrating astrocytoma risk and prognosis.

    PubMed

    Lin, Zhong-Hui; Chen, Jin-Chun; Wang, Yun-Sun; Huang, Teng-Jiao; Wang, Jin; Long, Xi-Dai

    2013-12-27

    The DNA repair gene X-ray cross-complementary group 4 (XRCC4), an important caretaker of the overall genome stability, is thought to play a major role in human tumorigenesis. We investigated the association between an important polymorphic variant of this gene at codon 247 (rs373409) and diffusely infiltrating astrocytoma (DIA) risk and prognosis. This hospital-based case-control study investigated this association in the Guangxi population. In total, 242 cases with DIA and 358 age-, sex-, and race-matched healthy controls were genotyped using TaqMan-PCR technique. We found a significant difference in the frequency of XRCC4 genotypes between cases and controls. Compared with the homozygote of XRCC4 codon 247 Ala alleles (XRCC4-AA), the genotypes of XRCC4 codon 247 Ser alleles (namely XRCC4-AS or -SS) increased DIA risk (odds ratios [OR], 1.82 and 2.89, respectively). Furthermore, XRCC4 polymorphism was correlated with tumor dedifferentiation of DIA (r = 0.261, p < 0.01). Additionally, this polymorphism modified the overall survival of DIA patients (the median survival times were 26, 14, and 8 months for patients with XRCC4-AA, -AS, and -SS, respectively). Like tumor grade, XRCC4 codon 247 polymorphism was an independent prognostic factor influencing the survival of DIA. These results suggest that XRCC4 codon 247 polymorphism may be associated with DIA risk and prognosis among the Guangxi population.

  5. DNA Repair, Redox Regulation and Modulation of Estrogen Receptor Alpha Mediated Transcription

    ERIC Educational Resources Information Center

    Curtis-Ducey, Carol Dianne

    2009-01-01

    Interaction of estrogen receptor [alpha] (ER[alpha]) with 17[beta]-estradiol (E[subscript 2]) facilitates binding of the receptor to estrogen response elements (EREs) in target genes, which in turn leads to recruitment of coregulatory proteins. To better understand how estrogen-responsive genes are regulated, our laboratory identified a number of…

  6. DNA repair gene ERCC1 polymorphisms may contribute to the risk of glioma.

    PubMed

    Yuan, Guanqian; Gao, Dandan; Ding, Shaofeng; Tan, Jun

    2014-05-01

    Polymorphisms in excision repair cross-complementing rodent repair deficiency complementation group 1 (ERCC1) gene have been shown to affect individual susceptibility to glioma, though studies have yielded conflicting results. This meta-analysis aims to derive a more precise estimation of the association between ERCC1 C8092A and C118T polymorphisms and glioma risk. A literature search of PubMed, Embase, Web of Science, Cochrane Library, and CBM databases was conducted to identify all eligible studies published before August 5, 2013. Crude odds ratios (ORs) with their corresponding confidence intervals (95% CIs) were used to assess the strength of this association. A meta-analysis was performed by reviewing seven studies on the C8092A polymorphism (2,978 cases and 4,051 controls) and four studies on the C118T polymorphism (1,390 Asian cases and 1,546 Asian controls). Pooled analysis yielded a significant association between the C8092A variant genotype and increased risk of glioma. As for ethnicity, the A allele was associated with increased risk of glioma in Asians, while no similar finding was observed in Caucasians. Stratified analyses by histological subtype indicated that the C8092A polymorphism showed a significant association with the risk of non-glioblastoma multiforme. For the C118T polymorphism, increased glioma susceptibility was also observed among Asians. Taken together, results from our meta-analysis support the view that common variants in ERCC1 may contribute to susceptibility to glioma, especially in Asians. However, further studies investigating the significance of these two polymorphisms as markers of susceptibility to and disease progression of glioma are still needed. PMID:24453030

  7. Cell survival after UV radiation stress in the unicellular chlorophyte Dunaliella tertiolecta is mediated by DNA repair and MAPK phosphorylation

    PubMed Central

    García-Gómez, Candela; Segovia, María

    2012-01-01

    Ultraviolet radiation (UVR) induces damage in a variety of organisms, and cells may adapt by developing repair or tolerance mechanisms to counteract such damage; otherwise, the cellular fate is cell death. Here, the effect of UVR-induced cell damage and the associated signalling and repair mechanisms by which cells are able to survive was studied in Dunaliella tertiolecta. UVR did not cause cell death, as shown by the absence of SYTOX Green-positive labelling cells. Ultrastructure analysis by transmission electron microscopy demonstrated that the cells were alive but were subjected to morphological changes such as starch accumulation, chromatin disaggregation, and chloroplast degradation. This behaviour paralleled a decrease in F v/F m and the formation of cyclobutane–pyrimidine dimers, showing a 10-fold increase at the end of the time course. There was a high accumulation of the repressor of transcriptional gene silencing (ROS1), as well as the cell proliferation nuclear antigen (PCNA) in UVR-treated cells, revealing activation of DNA repair mechanisms. The degree of phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinases was higher in UVR-exposed cells; however, the opposite occurred with the phosphorylated extracellular signal-regulated kinase (ERK). This confirmed that both JNK and p38 need to be phosphorylated to trigger the stress response, as well as the fact that cell division is arrested when an ERK is dephosphorylated. In parallel, both DEVDase and WEHDase caspase-like enzymatic activities were active even though the cells were not dead, suggesting that these proteases must be considered within a wider frame of stress proteins, rather than specifically being involved in cell death in these organisms. PMID:22859678

  8. Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA.

    PubMed

    de Vries, A; van Oostrom, C T; Hofhuis, F M; Dortant, P M; Berg, R J; de Gruijl, F R; Wester, P W; van Kreijl, C F; Capel, P J; van Steeg, H; Verbeek, S J

    1995-09-14

    Xeroderma pigmentosum patients with a defect in the nucleotide-excision repair gene XPA are characterized by, for example, a > 1,000-fold higher risk of developing sunlight-induced skin cancer. Nucleotide-excision repair (NER) is involved in the removal of a wide spectrum of DNA lesions. The XPA protein functions in a pre-incision step, the recognition of DNA damage. To permit the functional analysis of the XPA gene in vivo, we have generated XPA-deficient mice by gene targeting in embryonic stem cells. The XPA-/-mice appear normal, at least until the age of 13 months. XPA-/-mice are highly susceptible to ultraviolet (UV)-B-induced skin and eye tumours and to 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumours. We conclude that the XPA-deficient mice strongly mimic the phenotype of humans with xeroderma pigmentosum. PMID:7675086

  9. DNA repair and recovery of RNA synthesis following exposure to ultraviolet light are delayed in long genes.

    PubMed

    Andrade-Lima, Leonardo C; Veloso, Artur; Paulsen, Michelle T; Menck, Carlos F M; Ljungman, Mats

    2015-03-11

    The kinetics of DNA repair and RNA synthesis recovery in human cells following UV-irradiation were assessed using nascent RNA Bru-seq and quantitative long PCR. It was found that UV light inhibited transcription elongation and that recovery of RNA synthesis occurred as a wave in the 5'-3' direction with slow recovery and TC-NER at the 3' end of long genes. RNA synthesis resumed fully at the 3'-end of genes after a 24 h recovery in wild-type fibroblasts, but not in cells deficient in transcription-coupled nucleotide excision repair (TC-NER) or global genomic NER (GG-NER). Different transcription recovery profiles were found for individual genes but these differences did not fully correlate to differences in DNA repair of these genes. Our study gives the first genome-wide view of how UV-induced lesions affect transcription and how the recovery of RNA synthesis of large genes are particularly delayed by the apparent lack of resumption of transcription by arrested polymerases.

  10. Association of genomic instability, and the methylation status of imprinted genes and mismatch-repair genes, with neural tube defects.

    PubMed

    Liu, Zhuo; Wang, Zhigang; Li, Yuanyuan; Ouyang, Shengrong; Chang, Huibo; Zhang, Ting; Zheng, Xiaoying; Wu, Jianxin

    2012-05-01

    We studied the genomic instability and methylation status of the mismatch-repair (MMR) genes hMLH1 and hMSH2, and the imprinted genes H19/IGF2, in fetuses with neural tube defects (NTDs) to explore the pathogenesis of the disease. Microsatellite instability (MSI) was observed in 23 of 50 NTD patients. Five NTD patients showed high-degree MSI (MSI-H) and 18 showed low-degree MSI (MSI-L). The frequencies of mutated microsatellite loci were 3/50 (6%) for BatT-25, 10/50 (20%) for Bat-26, 3/50 (6%) for Bat34C4, 6/50 (12%) for D2S123, 4/50 (8%) for D2S119, and 3/50 (6%) for D3S1611. The promoter regions of the hMLH1 and hMSH2 genes were unmethylated in NTD patients, as determined by methylation-specific PCR. The hMLH1 and hMSH2 promoter methylation patterns, the methylation levels of H19 DMR1, and IGF2 DMR0 were detected by bisulfite sequencing PCR, sub-cloning, and sequencing. The hMSH2 promoter sequence was unmethylated, and the hMLH1 promoter showed a specific methylation pattern at two CpG sites. The methylation levels of H19 DMR1 in the NTD and control groups are 73.3% ± 15.9 and 58.3% ± 11.2, respectively. The methylation level of the NTD group was higher than that of the control group (Student's t-test, P<0.05). There is no significant difference in IGF2 DMR0 methylation level between the two groups. All of the results presented here suggest that genomic instability, the MMR system, and hyper-methylation of the H19 DMR1 may be correlated with the occurrence of NTDs.

  11. Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome.

    PubMed Central

    Leadon, S. A.; Cooper, P. K.

    1993-01-01

    Cells from patients with Cockayne syndrome (CS), which are sensitive to killing by UV although overall damage removal appears normal, are specifically defective in repair of UV damage in actively transcribed genes. Because several CS strains display cross-sensitivity to killing by ionizing radiation, we examined whether ionizing radiation-induced damage in active genes is preferentially repaired by normal cells and whether the radiosensitivity of CS cells can be explained by a defect in this process. We found that ionizing radiation-induced damage was repaired more rapidly in the transcriptionally active metallothionein IIA (MTIIA) gene than in the inactive MTIIB gene or in the genome overall in normal cells as a result of faster repair on the transcribed strand of MTIIA. Cells of the radiosensitive CS strain CS1AN are completely defective in this strand-selective repair of ionizing radiation-induced damage, although their overall repair rate appears normal. CS3BE cells, which are intermediate in radiosensitivity, do exhibit more rapid repair of the transcribed strand but at a reduced rate compared to normal cells. Xeroderma pigmentosum complementation group A cells, which are hypersensitive to UV light because of a defect in the nucleotide excision repair pathway but do not show increased sensitivity to ionizing radiation, preferentially repair ionizing radiation-induced damage on the transcribed strand of MTIIA. Thus, the ability to rapidly repair ionizing radiation-induced damage in actively transcribing genes correlates with cell survival. Our results extend the generality of preferential repair in active genes to include damage other than bulky lesions. Images Fig. 2 Fig. 3 PMID:8248136

  12. Single nucleotide polymorphisms in nucleotide excision repair genes, cancer treatment, and head and neck cancer survival

    PubMed Central

    Wyss, Annah B.; Weissler, Mark C.; Avery, Christy L.; Herring, Amy H.; Bensen, Jeannette T.; Barnholtz-Sloan, Jill S.; Funkhouser, William K.

    2014-01-01

    Purpose Head and neck cancers (HNC) are commonly treated with radiation and platinum-based chemotherapy, which produce bulky DNA adducts to eradicate cancerous cells. Because nucleotide excision repair (NER) enzymes remove adducts, variants in NER genes may be associated with survival among HNC cases both independently and jointly with treatment. Methods Cox proportional hazards models were used to estimate race-stratified (White, African American) hazard ratios (HRs) and 95 % confidence intervals for overall (OS) and disease-specific (DS) survival based on treatment (combinations of surgery, radiation, and chemotherapy) and 84 single nucleotide polymorphisms (SNPs) in 15 NER genes among 1,227 HNC cases from the Carolina Head and Neck Cancer Epidemiology Study. Results None of the NER variants evaluated were associated with survival at a Bonferroni-corrected alpha of 0.0006. However, rs3136038 [OS HR = 0.79 (0.65, 0.97), DS HR = 0.69 (0.51, 0.93)] and rs3136130 [OS HR = 0.78 (0.64, 0.96), DS HR = 0.68 (0.50, 0.92)] of ERCC4 and rs50871 [OS HR = 0.80 (0.64, 1.00), DS HR = 0.67 (0.48, 0.92)] of ERCC2 among Whites, and rs2607755 [OS HR = 0.62 (0.45, 0.86), DS HR = 0.51 (0.30, 0.86)] of XPC among African Americans were suggestively associated with survival at an uncorrected alpha of 0.05. Three SNP-treatment joint effects showed possible departures from additivity among Whites. Conclusions Our study, a large and extensive evaluation of SNPs in NER genes and HNC survival, identified mostly null associations, though a few variants were suggestively associated with survival and potentially interacted additively with treatment. PMID:24487794

  13. Nucleotide fluctuation of RecA repair gene in Siberian permafrost Psychrobacter cryohalolentis K5

    NASA Astrophysics Data System (ADS)

    Tremberger, George, Jr.; Holden, T.; Cheung, E.; Subramaniam, R.; Sullivan, R.; Schneider, P.; Flamholz, A.; Marchese, P.; Lieberman, D.; Cheung, T.

    2008-08-01

    A nucleotide sequence can be expressed as a numerical sequence when each nucleotide is assigned its proton number. A resulting gene numerical sequence can be investigated for its fractal dimension in terms of evolution and chemical properties for comparative studies. We have investigated such nucleotide fluctuation in the RecA repair gene of Psychrobacter cryohalolentis K5, Psychrobacter arcticus 273-4, and Psychrobacter sp. PRwf-1. The fractal dimension was found to correlate with the gene's operating temperature with the highest fractal dimension associated with P. cryohalolentis K5 living at the low temperatures found in Siberian permafrost. The CpG dinucleotide content was found to be about 5% for the three species of Psychrobacters, which is substantially lower than that of Deinococcus radiodurans at about 12%. The average nucleotide pair-wise free energy was found to be lowest for Psychrobacter sp. PRwf-1, the species with the lowest fractal dimension of the three, consistent with the recent finding that Psychrobacter sp. PRw-f1 has a temperature growth maximum of 15-20°C higher than P. arcticus 273-4 and P. cryohaloentis K5. The results suggest that microbial vitality in extreme environments is associated with fractal dimension as well as high CpG dinucleotide content, while the average nucleotide pair-wise free energy is related to the operating environment. Evidence that extreme temperature operation would impose constraints measurable by Shannon entropy is also discussed. A quantitative estimate of an entropy-based measure having the characteristics of a mechanical pressure shows that the Psychrobacter RecA sequence experiences lower pressure than that of the human HAR1 sequence.

  14. Bcl-2 and accelerated DNA repair mediates resistance of hair follicle bulge stem cells to DNA-damage-induced cell death.

    PubMed

    Sotiropoulou, Panagiota A; Candi, Aurélie; Mascré, Guilhem; De Clercq, Sarah; Youssef, Khalil Kass; Lapouge, Gaelle; Dahl, Ellen; Semeraro, Claudio; Denecker, Geertrui; Marine, Jean-Christophe; Blanpain, Cédric

    2010-06-01

    Adult stem cells (SCs) are at high risk of accumulating deleterious mutations because they reside and self-renew in adult tissues for extended periods. Little is known about how adult SCs sense and respond to DNA damage within their natural niche. Here, using mouse epidermis as a model, we define the functional consequences and the molecular mechanisms by which adult SCs respond to DNA damage. We show that multipotent hair-follicle-bulge SCs have two important mechanisms for increasing their resistance to DNA-damage-induced cell death: higher expression of the anti-apoptotic gene Bcl-2 and transient stabilization of p53 after DNA damage in bulge SCs. The attenuated p53 activation is the consequence of a faster DNA repair activity, mediated by a higher non-homologous end joining (NHEJ) activity, induced by the key protein DNA-PK. Because NHEJ is an error-prone mechanism, this novel characteristic of adult SCs may have important implications in cancer development and ageing.

  15. Ipr1 gene mediates innate immunity to tuberculosis

    PubMed Central

    Pan, Hui; Yan, Bo-Shiun; Rojas, Mauricio; Shebzukhov, Yuriy V.; Zhou, Hongwei; Kobzik, Lester; Higgins, Darren; Daly, Mark; Bloom, Barry R.; Kramnik, Igor

    2005-01-01

    An estimated 8 million people are infected each year with the pathogen, Mycobacterium tuberculosis, and over 2 million die annually1. Yet only about 10% of those infected develop tuberculosis. Genetic variation within host populations is known to play a significant role in humans and animals 2,3, but the nature of genetic control of host resistance to tuberculosis remains poorly understood. Previously we mapped a new genetic locus on mouse chromosome 1, designated sst1 (for supersusceptibility to tuberculosis1)4. Here we demonstrate in sst1 congenic mouse strains that this locus mediates innate immunity, and identify a candidate gene, Intracellular Pathogen Resistance 1 (Ipr1), within the sst1 locus. The Ipr1 gene is upregulated in the sst1 resistant macrophages upon activation and infection, but is not expressed in the sst1 susceptible macrophages. Expression of the Ipr1 transgene in the sst1 susceptible macrophages limits multiplication not only of MTB but also Listeria monocytogenes and switches a cell death pathway of the infected macrophages from necrosis to apoptosis. Our data suggest that the Ipr1 gene product may play a novel role in integrating signals generated by intracellular pathogens with mechanisms controlling innate immunity, cell death and pathogenesis. PMID:15815631

  16. Retroviral-mediated IL-2 gene transfer into murine neuroblastoma.

    PubMed

    Cho, H S; Song, J Y; Park, C Y; Lyu, C J; Kim, B S; Kim, K Y

    2000-02-01

    We used retroviral-mediated gene transfer of the human interleukin (IL)-2 gene into murine neuroblastoma cells to investigate whether locally-secreted IL-2 is able to influence the generation of anti-tumor immune responses. Supernatant obtained from cultures of approximately 1 x 10(6) IL-2 gene-transduced, G-418 selected neuro-2a cells was assayed for human IL-2 production by ELISA kit. First, to estimate whether the local secretion of IL-2 from the genetically-modified tumor cells would affect their tumorigenicity in vivo, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice and tumor growth was measured weekly. And to estimate whether IL-2 transfected neuroblastoma cells protect mice from tumor development after wild-type tumor cell challenge, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice. Seven days after IL-2 gene-transfected neuroblastoma cell injection, unmodified neuro-2a cells were s.c. injected into the contralateral site of A/J mice and tumor growth was measured weekly. Finally, to estimate IL-2 effect on pre-established large tumor burdens, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice with established tumor and its growth was measured weekly. The IL-2 gene-transduced neuro-2a clones secreted 120.25-177.3 IU of IL-2 per ml per 10(6) cells during 24 hr. None of the mice injected with IL-2-secreting neuro-2a cells developed tumors within 6 weeks, while all of the mice injected with wild-type neuro-2a cells developed tumors. Immunization of mice with IL-2 gene-transfected, irradiated neuro-2a cells protected these animals against a subsequent challenge with wild-type tumor cells. Finally, the size of large neuroblastomas decreased after IL-2-secreting neuro-2a cell injection into mice. Local secretion of IL-2 gene-transduced tumor cells abrogates their tumorigenicity and induces protective immunity and may inhibit the growth of neuroblastoma.

  17. TAT-mediated delivery of a DNA repair enzyme to skin cells rapidly initiates repair of UV-induced DNA damage

    PubMed Central

    Johnson, Jodi L.; Lowell, Brian C.; Ryabinina, Olga P.; Lloyd, R. Stephen; McCullough, Amanda K.

    2011-01-01

    Ultraviolet (UV) light causes DNA damage in skin cells, leading to more than one million cases of non-melanoma skin cancer diagnosed annually in the United States. Although human cells possess a mechanism (Nucleotide Excision Repair, NER) to repair UV-induced DNA damage, mutagenesis still occurs when DNA is replicated prior to repair of these photoproducts. While human cells have all the enzymes necessary to complete an alternate repair pathway, Base Excision Repair (BER), they lack a DNA glycosylase that can initiate BER of dipyrimidine photoproducts. Certain prokaryotes and viruses produce pyrimidine dimer-specific DNA glycosylases (pdgs) that initiate BER of cyclobutane pyrimidine dimers (CPDs), the predominant UV-induced lesions. Such a pdg was identified in the Chlorella virus PBCV-1 and termed Cv-pdg. The Cv-pdg protein was engineered to contain a nuclear localization sequence (NLS) and a membrane permeabilization peptide (TAT). Here, we demonstrate that the Cv-pdg-NLS-TAT protein was delivered to repair-proficient keratinocytes and fibroblasts, and to a human skin model, where it rapidly initiated removal of CPDs. These data suggest a potential strategy for prevention of human skin cancer. PMID:20927123

  18. Gypenosides causes DNA damage and inhibits expression of DNA repair genes of human oral cancer SAS cells.

    PubMed

    Lu, Kung-Wen; Chen, Jung-Chou; Lai, Tung-Yuan; Yang, Jai-Sing; Weng, Shu-Wen; Ma, Yi-Shih; Tang, Nou-Ying; Lu, Pei-Jung; Weng, Jing-Ru; Chung, Jing-Gung

    2010-01-01

    Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino, a Chinese medical plant. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage and DNA repair-associated gene expression in human oral cancer cells. Therefore, we investigated whether Gyp induced DNA damage and DNA repair gene expression in human oral cancer SAS cells. The results from flow cytometric assay indicated that Gyp-induced cytotoxic effects led to a decrease in the percentage of viable SAS cells. The results from comet assay revealed that the incubation of SAS cells with Gyp led to a longer DNA migration smear (comet tail) when compared with control and this effect was dose-dependent. The results from real-time PCR analysis indicated that treatment of SAS cells with 180 mug/ml of Gyp for 24 h led to a decrease in 14-3-3sigma, DNA-dependent serine/threonine protein kinase (DNAPK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression. These observations may explain the cell death caused by Gyp in SAS cells. Taken together, Gyp induced DNA damage and inhibited DNA repair-associated gene expressions in human oral cancer SAS cells in vitro.

  19. Frequency distribution of DNA repair genes ERCC1 and ERCC2 polymorphisms in South Indian healthy population.

    PubMed

    Rao, Katiboina Srinivasa; SureshKumar, Srinivasamurthy; Umamaheswaran, Gurusamy; Paul, Abialbon; Dubashi, Biswajit; Gunaseelan, Karunanithi; Dkhar, Steven Aibor

    2014-09-01

    DNA repair genes are crucial in maintaining the integrity of the whole genome. Single nucleotide polymorphisms (SNPs) in DNA repair genes have been attributed to the development of various cancers. SNPs of DNA repair genes (ERCC1 and ERCC2) have been implicated in the causation of various cancers as well as inter-individual variability in the therapeutic outcomes of platinum based therapy. Thus establishing the frequency of these functional SNPs in the healthy population is of significance. The present study was aimed to establish the allele and genotype frequencies of ERCC1 (19007C>T, rs11615; 8092C>A, rs3212986) and ERCC2 (Asp312Asn, rs1799793) genes in South Indian healthy population and to compare the data from HapMap populations. The study population consisted of 128 healthy South Indian unrelated individuals of either sex aged between 18 and 60 years. Standard phenol-chloroform method was used to extract DNA from peripheral leukocytes. The genotype of DNA repair gene polymorphisms was determined by quantitative real-time polymerase chain reaction using TaqMan genotyping assay. The observed frequency of the studied polymorphisms followed Hardy-Weinberg equilibrium (p>0.05). The frequencies of the minor alleles of the SNPs rs11615 (T), rs3212986 (A) and rs1799793 (A) were 43.8%, 29.3% and 35.6%, respectively. Gender-based analysis showed no significant difference in the frequency pattern. The observed allele and genotype frequencies showed significant ethnic difference between South Indians and other HapMap populations. This is the first study to provide the normative frequency data of allele and genotype distribution of three SNPs of ERCC1 and ERCC2 in South Indian healthy population. It might be useful in future genotype-phenotype association studies, especially for predicting the efficacy and adverse events of platinum based drugs. PMID:25155628

  20. Contribution of DNA double-strand break repair gene XRCC3 genotypes to oral cancer susceptibility in Taiwan.

    PubMed

    Tsai, Chia-Wen; Chang, Wen-Shin; Liu, Juhn-Cherng; Tsai, Ming-Hsui; Lin, Cheng-Chieh; Bau, Da-Tian

    2014-06-01

    The DNA repair gene X-ray repair cross complementing protein 3 (XRCC3) is thought to play a major role in double-strand break repair and in maintaining genomic stability. Very possibly, defective double-strand break repair of cells can lead to carcinogenesis. Therefore, a case-control study was performed to reveal the contribution of XRCC3 genotypes to individual oral cancer susceptibility. In this hospital-based research, the association of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotypes with oral cancer risk in a Taiwanese population was investigated. In total, 788 patients with oral cancer and 956 age- and gender-matched healthy controls were genotyped. The results showed that there was significant differential distribution among oral cancer and controls in the genotypic (p=0.001428) and allelic (p=0.0013) frequencies of XRCC3 rs861539. As for the other polymorphisms, there was no difference between case and control groups. In gene-lifestyle interaction analysis, we have provided the first evidence showing that there is an obvious joint effect of XRCC3 rs861539 genotype with individual areca chewing habits on oral cancer risk. In conclusion, the T allele of XRCC3 rs861539, which has an interaction with areca chewing habit in oral carcinogenesis, may be an early marker for oral cancer in Taiwanese.

  1. Clinical features and mismatch repair gene mutation screening in Chinese patients with hereditary nonpolyposis colorectal carcinoma

    PubMed Central

    Liu, Shan-Run; Zhao, Bo; Wang, Zhen-Jun; Wan, Yuan-Lian; Huang, Yan-Ting

    2004-01-01

    AIM: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominantly-inherited cancer-susceptibility syndrome that confers an increased risk for colorectal cancer and a variety of other tumors at a young age. It has been associated with germline mutations in five mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, hPMS2, and hMSH6/GTBP). The great majority of germline mutations were found in hMSH2 and hMLH1. The purpose of this study was to analyze the clinical features of Chinese HNPCC patients and to screen hMSH2 and hMLH1 gene mutations. METHODS: Twenty-eight independent Chinese families were collected, of which 15 met Amsterdam criteria I and 13 met the Japanese clinical diagnosis criteria. The data were recorded including sex, site of colorectal cancer (CRC), age of diagnosis, history of synchronous and/or metachronous CRC, instance of extracolonic cancers, and histopathology of tumors. Peripheral blood samples were collected from all pedigrees after formal written consents were signed. PCR and denaturing high-performance liquid chromatography (DHPLC) were used to screen the coding regions of hMSH2 and hMLH1 genes. The samples showing abnormal DHPLC profiles were sequenced by a 377 DNA sequencer. RESULTS: One hundred and seventy malignant neoplasms were found in one hundred and twenty-six patients (multiple cancer in twenty-three), including one hundred and twenty-seven CRCs, fifteen gastric, seven endometrial, and five esophageal cancers. Seventy-seven point eight percent of the patients had CRCs, sharing the features of early occurrence (average age of onset, 45.9 years) and of the right-sided predominance reported in the literature. In Chinese HNPCC patients, gastric cancer occurred more frequently, accounting for 11.9% of all cancers patients and ranking second in the spectrum of HNPCC predisposing cancers. Synchronous CRCs occurred less frequently, only accounting for 3.1% of the total CRCs. Twenty percent of the colorectal patients had

  2. Role of donor and host cells in muscle-derived stem cell-mediated bone repair: differentiation vs. paracrine effects

    PubMed Central

    Gao, Xueqin; Usas, Arvydas; Proto, Jonathan D.; Lu, Aiping; Cummins, James H.; Proctor, Alexander; Chen, Chien-Wen; Huard, Johnny

    2014-01-01

    Murine muscle-derived stem cells (MDSCs) have been shown capable of regenerating bone in a critical size calvarial defect model when transduced with BMP 2 or 4; however, the contribution of the donor cells and their interactions with the host cells during the bone healing process have not been fully elucidated. To address this question, C57/BL/6J mice were divided into MDSC/BMP4/GFP, MDSC/GFP, and scaffold groups. After transplanting MDSCs into the critical-size calvarial defects created in normal mice, we found that mice transplanted with BMP4GFP-transduced MDSCs healed the bone defect in 4 wk, while the control groups (MDSC-GFP and scaffold) demonstrated no bone healing. The newly formed trabecular bone displayed similar biomechanical properties as the native bone, and the donor cells directly participated in endochondral bone formation via their differentiation into chondrocytes, osteoblasts, and osteocytes via the BMP4-pSMAD5 and COX-2-PGE2 signaling pathways. In contrast to the scaffold group, the MDSC groups attracted more inflammatory cells initially and incurred faster inflammation resolution, enhanced angiogenesis, and suppressed initial immune responses in the host mice. MDSCs were shown to attract macrophages via the secretion of monocyte chemotactic protein 1 and promote endothelial cell proliferation by secreting multiple growth factors. Our findings indicated that BMP4GFP-transduced MDSCs not only regenerated bone by direct differentiation, but also positively influenced the host cells to coordinate and promote bone tissue repair through paracrine effects.—Gao, X., Usas, A., Proto, J. D., Lu, A., Cummins, J. H., Proctor, A., Chen, C.-W., Huard, J. Role of donor and host cells in muscle-derived stem cell-mediated bone repair: differentiation vs. paracrine effects. PMID:24843069

  3. Phosphorylation of Sae2 Mediates Forkhead-associated (FHA) Domain-specific Interaction and Regulates Its DNA Repair Function.

    PubMed

    Liang, Jason; Suhandynata, Raymond T; Zhou, Huilin

    2015-04-24

    Saccharomyces cerevisiae Sae2 and its ortholog CtIP in higher eukaryotes have a conserved role in the initial processing of DNA lesions and influencing their subsequent repair pathways. Sae2 is phosphorylated by the ATR/ATM family kinases Mec1 and Tel1 in response to DNA damage. Among the Mec1/Tel1 consensus phosphorylation sites of Sae2, we found that mutations of Thr-90 and Thr-279 of Sae2 into alanine caused a persistent Rad53 activation in response to a transient DNA damage, similar to the loss of Sae2. To gain insight into the function of this phosphorylation of Sae2, we performed a quantitative proteomics analysis to identify its associated proteins. We found that phosphorylation of Thr-90 of Sae2 mediates its interaction with Rad53, Dun1, Xrs2, Dma1, and Dma2, whereas Rad53 and Dun1 additionally interact with phosphorylated Thr-279 of Sae2. Mutations of the ligand-binding residues of Forkhead-associated (FHA) domains of Rad53, Dun1, Xrs2, Dma1, and Dma2 abolished their interactions with Sae2, revealing the involvement of FHA-specific interactions. Mutations of Thr-90 and Thr-279 of Sae2 caused a synergistic defect when combined with sgs1Δ and exo1Δ and elevated gross chromosomal rearrangements. Likewise, mutations of RAD53 and DUN1 caused a synthetic growth defect with sgs1Δ and elevated gross chromosomal rearrangements. These findings suggest that threonine-specific phosphorylation of Sae2 by Mec1 and Tel1 contributes to DNA repair and genome maintenance via its interactions with Rad53 and Dun1.

  4. AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

    PubMed

    Yu, Hongwei; Fischer, Gregory; Hogan, Quinn H

    2016-01-01

    Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.

  5. Studies of DEAE-dextran-mediated gene transfer.

    PubMed

    Yang, Y W; Yang, J C

    1997-02-01

    DEAE-dextran-mediated gene transfer was studied for the introduction of pSV2neo DNA into Fisher-rat 3T3 (FR3T3) cells. Zeta (zeta) potentials of the DEAE-dextran-DNA complexes and FR3T3 cells were found to be dependent on the concentration of DEAE-dextran in the medium. The maximum transfection efficiency occurred at a DEAE-dextran/DNA ratio of 50:1 or thereabouts. The interaction between DNA and cells is determined by the adsorption process. The results obtained, along with the correlation between the kinetic adsorption behaviour of 3H-labelled DNA and the transfection efficiency, indicated that adsorption of DEAE-dextran-DNA complexes to the negatively charged cell surfaces, due to electrostatic and dispersion attraction, plays the decisive role in determining the DNA transfection efficiencies.

  6. Control of pollen-mediated gene flow in transgenic trees.

    PubMed

    Zhang, Chunsheng; Norris-Caneda, Kim H; Rottmann, William H; Gulledge, Jon E; Chang, Shujun; Kwan, Brian Yow-Hui; Thomas, Anita M; Mandel, Lydia C; Kothera, Ronald T; Victor, Aditi D; Pearson, Leslie; Hinchee, Maud A W

    2012-08-01

    Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.

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

    PubMed Central

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

    2016-01-01

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

  8. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

    SciTech Connect

    Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; Juhasz, Szilvia; Morocz, Monika; Gali, Himabindu; Dhe-Paganon, Sirano; Venclovas, Česlovas; Haracska, Lajos

    2015-09-08

    Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.

  9. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

    PubMed Central

    Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; Juhasz, Szilvia; Morocz, Monika; Gali, Himabindu; Dhe-Paganon, Sirano; Venclovas, Česlovas; Haracska, Lajos

    2015-01-01

    Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. In more general terms, we suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal. PMID:26350214

  10. SNMIB/Apollo protects leading-strand telomeres against NHEJ-mediated repair.

    PubMed

    Lam, Yung C; Akhter, Shamima; Gu, Peili; Ye, Jing; Poulet, Anaïs; Giraud-Panis, Marie-Josèphe; Bailey, Susan M; Gilson, Eric; Legerski, Randy J; Chang, Sandy

    2010-07-01

    Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo's conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/Apollo(-/-)ATM(-/-) MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5'-3' exonuclease function of SNM1B/Apollo in the generation of 3' single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway. PMID:20551906

  11. SNMIB/Apollo protects leading-strand telomeres against NHEJ-mediated repair.

    PubMed

    Lam, Yung C; Akhter, Shamima; Gu, Peili; Ye, Jing; Poulet, Anaïs; Giraud-Panis, Marie-Josèphe; Bailey, Susan M; Gilson, Eric; Legerski, Randy J; Chang, Sandy

    2010-07-01

    Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo's conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/Apollo(-/-)ATM(-/-) MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5'-3' exonuclease function of SNM1B/Apollo in the generation of 3' single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway.

  12. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

    DOE PAGES

    Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; Juhasz, Szilvia; Morocz, Monika; Gali, Himabindu; Dhe-Paganon, Sirano; Venclovas, Česlovas; Haracska, Lajos

    2015-09-08

    Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteinsmore » retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.« less

  13. Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

    PubMed Central

    Harris, P V; Mazina, O M; Leonhardt, E A; Case, R B; Boyd, J B; Burtis, K C

    1996-01-01

    Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms. PMID:8816490

  14. Percutaneous repair of iatrogenic subclavian artery injury by suture-mediated closure device

    PubMed Central

    Chivate, Rahul S; Kulkarni, Suyash S; Shetty, Nitin S; Polnaya, Ashwin M; Gala, Kunal B; Patel, Paresh G

    2016-01-01

    Central venous catheterization through internal jugular vein is done routinely in intensive care units. It is generally safe, more so when the procedure is performed under ultrasound guidance. However, there could be inadvertent puncture of other vessels in the neck when the procedure is not performed under real-time sonographic guidance. Closure of this vessel opening can pose a challenge if it is an artery, in a location difficult to compress, and is further complicated by deranged coagulation profile. Here, we discuss the removal of an inadvertently placed catheter from subclavian artery with closure of arteriotomy percutaneously using arterial suture-mediated closure device. PMID:27413277

  15. Percutaneous repair of iatrogenic subclavian artery injury by suture-mediated closure device.

    PubMed

    Chivate, Rahul S; Kulkarni, Suyash S; Shetty, Nitin S; Polnaya, Ashwin M; Gala, Kunal B; Patel, Paresh G

    2016-01-01

    Central venous catheterization through internal jugular vein is done routinely in intensive care units. It is generally safe, more so when the procedure is performed under ultrasound guidance. However, there could be inadvertent puncture of other vessels in the neck when the procedure is not performed under real-time sonographic guidance. Closure of this vessel opening can pose a challenge if it is an artery, in a location difficult to compress, and is further complicated by deranged coagulation profile. Here, we discuss the removal of an inadvertently placed catheter from subclavian artery with closure of arteriotomy percutaneously using arterial suture-mediated closure device. PMID:27413277

  16. Functional repair of human donor lungs by IL-10 gene therapy.

    PubMed

    Cypel, Marcelo; Liu, Mingyao; Rubacha, Matt; Yeung, Jonathan C; Hirayama, Shin; Anraku, Masaki; Sato, Masaaki; Medin, Jeffrey; Davidson, Beverly L; de Perrot, Marc; Waddell, Thomas K; Slutsky, Arthur S; Keshavjee, Shaf

    2009-10-28

    More than 80% of potential donor lungs are injured during brain death of the donor and from complications experienced in the intensive care unit, and therefore cannot be used for transplantation. These lungs show inflammation and disruption of the alveolar-capillary barrier, leading to poor gas exchange. Although the number of patients in need of lung transplantation is increasing, the number of donors is static. We investigated the potential to use gene therapy with an adenoviral vector encoding human interleukin-10 (AdhIL-10) to repair injured donor lungs ex vivo before transplantation. IL-10 is an anti-inflammatory cytokine that mainly exerts its suppressive functions by the inactivation of antigen-presenting cells with consequent inhibition of proinflammatory cytokine secretion. In pigs, AdhIL-10-treated lungs exhibited attenuated inflammation and improved function after transplantation. Lungs from 10 human multiorgan donors that had suffered brain death were determined to be clinically unsuitable for transplantation. They were then maintained for 12 hours at body temperature in an ex vivo lung perfusion system with or without intra-airway delivery of AdhIL-10 gene therapy. AdhIL-10-treated lungs showed significant improvement in function (arterial oxygen pressure and pulmonary vascular resistance) when compared to controls, a favorable shift from proinflammatory to anti-inflammatory cytokine expression, and recovery of alveolar-blood barrier integrity. Thus, treatment of injured human donor lungs with the cytokine IL-10 can improve lung function, potentially rendering injured lungs suitable for transplantation into patients. PMID:20368171

  17. Risk of colorectal cancer for people with a mutation in both a MUTYH and a DNA mismatch repair gene.

    PubMed

    Win, Aung Ko; Reece, Jeanette C; Buchanan, Daniel D; Clendenning, Mark; Young, Joanne P; Cleary, Sean P; Kim, Hyeja; Cotterchio, Michelle; Dowty, James G; MacInnis, Robert J; Tucker, Katherine M; Winship, Ingrid M; Macrae, Finlay A; Burnett, Terrilea; Le Marchand, Loïc; Casey, Graham; Haile, Robert W; Newcomb, Polly A; Thibodeau, Stephen N; Lindor, Noralane M; Hopper, John L; Gallinger, Steven; Jenkins, Mark A

    2015-12-01

    The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understandin g the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95% confidence interval (CI) 9.19-50.1; p < 0.001], but not different from that for carriers of a MMR gene mutation alone (HR 1.94, 95% CI 0.63-5.99; p = 0.25). Within the limited power of this study, there was no evidence that a monoallelic MUTYH gene mutation confers additional risk of colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative.

  18. Nucleotide Excision Repair Gene Polymorphisms, Meat Intake and Colon Cancer Risk

    PubMed Central

    Steck, Susan E.; Butler, Lesley M.; Keku, Temitope; Antwi, Samuel; Galanko, Joseph; Sandler, Robert S.; Hu, Jennifer J.

    2014-01-01

    Purpose Much of the DNA damage from colon cancer-related carcinogens, including heterocyclic amines (HCA) and polycyclic aromatic hydrocarbons (PAH) from red meat cooked at high temperature, are repaired by the nucleotide excision repair (NER) pathway. Thus, we examined whether NER non-synonymous single nucleotide polymorphisms (nsSNPs) modified the association between red meat intake and colon cancer risk. Methods The study consists of 244 African-American and 311 white colon cancer cases and population-based controls (331 African Americans and 544 whites) recruited from 33 counties in North Carolina from 1996 to 2000. Information collected by food frequency questionnaire on meat intake and preparation methods were used to estimate HCA and benzo(a)pyrene (BaP, a PAH) intake. We tested 7 nsSNPs in 5 NER genes: XPC A499V and K939Q, XPD D312N and K751Q, XPF R415Q, XPG D1104H, and RAD23B A249V. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated using unconditional logistic regression. Results Among African Americans, we observed a statistically significant positive association between colon cancer risk and XPC 499 AV+VV genotype (OR=1.7, 95% CI: 1.1, 2.7, AA as referent), and an inverse association with XPC 939 QQ (OR=0.3, 95%CI: 0.2, 0.8, KK as referent). These associations were not observed among whites. For both races combined, there was interaction between the XPC 939 genotype, well-done red meat intake and colon cancer risk (OR=1.5, 95% CI=1.0, 2.2 for high well-done red meat and KK genotype as compared to low well-done red meat and KK genotype, pinteraction =0.05). Conclusions Our data suggest that NER nsSNPs are associated with colon cancer risk and may modify the association between well-done red meat intake and colon cancer risk. PMID:24607854

  19. Expression of genes responsible for the repair of mispaired bases of the DNA (MLH1) in invasive ductal breast carcinoma.

    PubMed

    Milanović, Rudolf; Stanec, Sanda; Stanec, Mladen; Korusić, Andelko; Husedzinović, Ino; Razumović, Jasminka Jakić

    2013-09-01

    Breast cancer is a heterogeneous group of diseases determined and distinguished by cellular type, gene expression and clinical signs and symptoms. Identification of histological and biological markers is of great value in predicting the progression of tumor growth and anticipating the expected response to various treatment options. Due to a high degree of cell proliferation in breast tumors and high genetic instability of these tumors, as a consequence of defective DNA repair mechanisms, chemotherapy as a treatment option often renders very successful results. During our scientific research we wanted to determine the involvement of the genetic polymorphisms of DNA mismatch repair system (MLH1 gene) and the subsequent development of breast carcinoma. This study included 108 patients who were surgically treated for invasive breast cancer at the Department of Plastic, Reconstructive and Aesthetic Surgery, University Hospital "Dubrava". The expression of the MLH1 gene was determined by immunohistochemical methods. The results showed that 82.9% of tumor cells expressed the MLH1 gene. Analysis of survival rate for patients with invasive ductal breast cancer showed a statistically significant (p = 0.043) correlation with the expression of MLH1 genes. The overall five year survival rate of our patients was 78.7%. These results indicate that there is a possible involvement of MLH1 gene in the progression and development of breast cancer.

  20. Occupational solvent exposure, genetic variation of DNA repair genes, and the risk of non-Hodgkin's lymphoma.

    PubMed

    Jiao, Jie; Zheng, Tongzhang; Lan, Qing; Chen, Yingtai; Deng, Qian; Bi, Xiaofeng; Kim, Christopher; Holford, Theodore; Leaderer, Brian; Boyle, Peter; Ba, Yue; Xia, Zhaolin; Chanock, Stephen J; Rothman, Nathaniel; Zhang, Yawei

    2012-11-01

    The main objective of this study was to test the hypothesis that genetic variations in DNA repair genes may modify the association between occupational exposure to solvents and the risk of non-Hodgkin's lymphoma (NHL). A population-based case-control study was conducted on Connecticut women including 518 histologically confirmed incident NHL cases and 597 controls. Unconditional logistic regression models were used to estimate the odds ratios and effect modification from the 30 single nucleotide polymorphisms in 16 DNA repair genes of the association between solvent exposure and the risk of NHL overall and subtypes. Single nucleotide polymorphisms in MGMT (rs12917) and NBS1 (rs1805794) significantly modified the association between exposure to chlorinated solvents and the risk of NHL (Pfor interaction=0.0003 and 0.0048, respectively). After stratification by major NHL histological subtypes, MGMT (rs12917) modified the association between chlorinated solvents and the risk of diffuse large B-cell lymphoma (Pfor interaction=0.0027) and follicular lymphoma (Pfor interaction=0.0024). A significant interaction was also observed between occupational exposure to benzene and BRCA2 (rs144848) for NHL overall (Pfor interaction=0.0042). Our study results suggest that genetic variations in DNA repair genes modify the association between occupational exposure to solvents and the risk of NHL. PMID:22430443

  1. Polymorphisms in DNA repair genes, smoking, and bladder cancer risk: findings from the International Consortium of Bladder Cancer

    PubMed Central

    Stern, Mariana C.; Lin, Jie; Figueroa, Jonine D.; Kelsey, Karl T.; Kiltie, Anne E.; Yuan, Jian-Min; Matullo, Giuseppe; Fletcher, Tony; Benhamou, Simone; Taylor, Jack A.; Placidi, Donatella; Zhang, Zuo-Feng; Steineck, Gunnar; Rothman, Nathaniel; Kogevinas, Manolis; Silverman, Debra; Malats, Nuria; Chanock, Stephen; Wu, Xifeng; Karagas, Margaret R.; Andrew, Angeline S.; Nelson, Heather H.; Bishop, D. Timothy; Sak, Sei Chung; Choudhury, Ananya; Barrett, Jennifer H; Elliot, Faye; Corral, Román; Joshi, Amit D.; Gago-Dominguez, Manuela; Cortessis, Victoria K.; Xiang, Yong-Bing; Vineis, Paolo; Sacerdote, Carlotta; Guarrera, Simonetta; Polidoro, Silvia; Allione, Alessandra; Gurzau, Eugen; Koppova, Kvetoslava; Kumar, Rajiv; Rudnai, Peter; Porru, Stefano; Carta, Angela; Campagna, Marcello; Arici, Cecilia; Park, SungShim Lani; Garcia-Closas, Montserrat

    2009-01-01

    Tobacco smoking is the most important and well-established bladder cancer risk factor, and a rich source of chemical carcinogens and reactive oxygen species that can induce damage to DNA in urothelial cells. Therefore, common variation in DNA repair genes might modify bladder cancer risk. In this study we present results from meta- and pooled analyses conducted as part of the International Consortium of Bladder Cancer. We included data on 10 single nucleotide polymorphisms corresponding to 7 DNA repair genes from 13 studies. Pooled- and meta-analyses included 5,282 cases and 5,954 controls of non-Latino white origin. We found evidence for weak but consistent associations with ERCC2 D312N (rs1799793) (per allele OR = 1.10; 95% CI = 1.01–1.19; p = 0.021), NBN E185Q (rs1805794) (per allele OR = 1.09; 95% CI = 1.01–1.18; p = 0.028), and XPC A499V (rs2228000) (per allele OR = 1.10; 95% CI = 1.00–1.21, p = 0.044). The association with NBN E185Q was limited to ever smokers (interaction p = 0.002), and was strongest for the highest levels of smoking dose and smoking duration. Overall, our study provides the strongest evidence to date for a role of common variants in DNA repair genes in bladder carcinogenesis. PMID:19706757

  2. Efficient TALEN-mediated gene knockout in livestock.

    PubMed

    Carlson, Daniel F; Tan, Wenfang; Lillico, Simon G; Stverakova, Dana; Proudfoot, Chris; Christian, Michelle; Voytas, Daniel F; Long, Charles R; Whitelaw, C Bruce A; Fahrenkrug, Scott C

    2012-10-23

    Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.

  3. Induction of AhR-Mediated Gene Transcription by Coffee

    PubMed Central

    Ishikawa, Toshio; Takahashi, Satoshi; Morita, Koji; Okinaga, Hiroko; Teramoto, Tamio

    2014-01-01

    Background Aryl hydrocarbon receptor (AhR) is classically known to be activated by xenobiotics such as dioxins and polycyclic aromatic hydrocarbons (PAHs). Although it has been reported that PAHs are contained in roasted coffee beans, in general coffee beverages are not considered to be AhR activators. We tested whether exposure to coffee would activate AhR in cultured cells. Methods HepG2 cells stably expressing an AhR-responsive reporter gene were treated with coffee samples. Also, expression of CYP1A1, an endogenous AhR-responsive gene, was quantitated by RT-PCR and Western blotting in HepG2, Caco-2, and MCF-7 cells, after treatment with coffee. In order to obtain sensitive and reproducible results, all the experiments were performed with the cells placed in either phosphate-buffered saline (PBS) or pure serum, instead of routinely-used culture medium, whose intrinsic AhR-stimulating activity turned out to be so strong as to interfere with the analyses. Results All the coffee samples tested robustly stimulated AhR-mediated transcription in the reporter gene assays. Of note, to what extent coffee and other AhR agonists activated AhR was different, depending on whether the experiments were done in PBS or serum. CYP1A1 mRNA was induced by coffee, in HepG2, Caco-2, and MCF-7 cells placed in either PBS or serum. CYP1A1 protein expression, which was not detected in these cells incubated in PBS, was also increased by coffee in cells placed in serum. Conclusions By using culture medium-free experimental settings, we have shown that coffee is a strong AhR activator. Our observation may help elucidate as-yet-unrecognized effects of coffee on human health. PMID:25007155

  4. Expression of nuclear factor, erythroid 2-like 2-mediated genes differentiates tuberculosis.

    PubMed

    Qian, Zhongqing; Lv, Jingzhu; Kelly, Gabriel T; Wang, Hongtao; Zhang, Xiaojie; Gu, Wanjun; Yin, Xiaofeng; Wang, Ting; Zhou, Tong

    2016-07-01

    During infection and host defense, nuclear factor, erythroid 2-like 2 (Nrf2) dependent signaling is an efficient antioxidant defensive mechanism used by host cells to control the destructive effects of reactive oxygen species. This allows for effective defense responses against microbes while minimizing oxidative injury to the host cell itself. As a central regulator of antioxidant genes, Nrf2 has gained great attention in its pivotal role in infection, especially in tuberculosis (TB), the top infectious disease killer worldwide. To elucidate the genes potentially regulated by Nrf2 in TB, we conducted a meta-analysis on published gene expression datasets. Firstly, we compared the global gene expression profiles between control and Nrf2-deficient human cells. The differentially expressed genes were deemed as "Nrf2-mediated genes". Next, the whole blood gene expression pattern of TB patients was compared with that of healthy controls, pneumonia patients, and lung cancer patients. We found that the genes deregulated in TB significantly overlap with the Nrf2-mediated genes. Based on the intersection of Nrf2-mediated and TB-regulated genes, we identified an Nrf2-mediated 17-gene signature, which reflects a cluster of gene ontology terms highly related to TB physiology. We demonstrated that the 17-gene signature can be used to distinguish TB patients from healthy controls and patients with latent TB infection, pneumonia, or lung cancer. Also, the Nrf2-mediated gene signature can be used as an indicator of the anti-TB therapeutic response. More importantly, we confirmed that the predictive power of the Nrf2-mediated 17-gene signature is significantly better than the random gene sets selected from the human transcriptome. Also, the 17-gene signature performs even better than the random gene signatures selected from TB-associated genes. Our study confirms the central role of Nrf2 in TB pathogenesis and provides a novel and useful diagnostic method to differentiate TB

  5. Gene Expression by PBMC in Primary Sclerosing Cholangitis: Evidence for Dysregulation of Immune Mediated Genes

    PubMed Central

    Aoki, Christopher A.; Dawson, Kevin; Kenny, Thomas P.; Gershwin, M. Eric; Bowlus, Christopher L.

    2006-01-01

    Primary sclerosing cholangitis (PSC) is a chronic disease of the bile ducts characterized by an inflammatory infiltrate and obliterative fibrosis. The precise role of the immune system in the pathogenesis of PSC remains unknown. We used RNA microarray analysis to identify immune-related genes and pathways that are differentially expressed in PSC. Messenger RNA (mRNA) from peripheral blood mononuclear cells (PBMC) was isolated from both patients with PSC and age and sex matched healthy controls. Samples from 5 PSC patients and 5 controls were analyzed by microarray and based upon rigorous statistical analysis of the data, relevant genes were chosen for confirmation by RT-PCR in 10 PSC patients and 10 controls. Using unsupervised hierarchical clustering, gene expression in PSC was statistically different from our control population. Interestingly, genes within the IL-2 receptor beta, IL-6 and MAP Kinase pathways were found to be differently expressed in patients with PSC compared to controls. Further, individual genes, TNF-α induced protein 6 (TNFaip6) and membrane-spanning 4-domains, subfamily A (ms4a) were found to be upregulated in PSC while similar to Mothers against decapentaplegic homolog 5 (SMAD 5) was downregulated. In conclusion, several immune-related pathways and genes were differentially expressed in PSC compared to control patients, giving further evidence that this disease is systemic and immune-mediated. PMID:17162367

  6. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    SciTech Connect

    Cho, Eun-Ah; Juhnn, Yong-Sung

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  7. I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.

    PubMed

    Ouedraogo, Jean Paul; Arentshorst, Mark; Nikolaev, Igor; Barends, Sharief; Ram, Arthur F J

    2015-12-01

    Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.

  8. A unique horizontal gene transfer event has provided the octocoral mitochondrial genome with an active mismatch repair gene that has potential for an unusual self-contained function

    PubMed Central

    2011-01-01

    Background The mitochondrial genome of the Octocorallia has several characteristics atypical for metazoans, including a novel gene suggested to function in DNA repair. This mtMutS gene is favored for octocoral molecular systematics, due to its high information content. Several hypotheses concerning the origins of mtMutS have been proposed, and remain equivocal, although current weight of support is for a horizontal gene transfer from either an epsilonproteobacterium or a large DNA virus. Here we present new and compelling evidence on the evolutionary origin of mtMutS, and provide the very first data on its activity, functional capacity and stability within the octocoral mitochondrial genome. Results The mtMutS gene has the expected conserved amino acids, protein domains and predicted tertiary protein structure. Phylogenetic analysis indicates that mtMutS is not a member of the MSH family and therefore not of eukaryotic origin. MtMutS clusters closely with representatives of the MutS7 lineage; further support for this relationship derives from the sharing of a C-terminal endonuclease domain that confers a self-contained mismatch repair function. Gene expression analyses confirm that mtMutS is actively transcribed in octocorals. Rates of mitochondrial gene evolution in mtMutS-containing octocorals are lower than in their hexacoral sister-group, which lacks the gene, although paradoxically the mtMutS gene itself has higher rates of mutation than other octocoral mitochondrial genes. Conclusions The octocoral mtMutS gene is active and codes for a protein with all the necessary components for DNA mismatch repair. A lower rate of mitochondrial evolution, and the presence of a nicking endonuclease domain, both indirectly support a theory of self-sufficient DNA mismatch repair within the octocoral mitochondrion. The ancestral affinity of mtMutS to non-eukaryotic MutS7 provides compelling support for an origin by horizontal gene transfer. The immediate vector of transmission

  9. Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord

    PubMed Central

    Wu, Min-fei; Zhang, Shu-quan; Gu, Rui; Liu, Jia-bei; Li, Ye; Zhu, Qing-san

    2015-01-01

    The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pcDNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco's modified Eagle's medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1–4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the

  10. Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord.

    PubMed

    Wu, Min-Fei; Zhang, Shu-Quan; Gu, Rui; Liu, Jia-Bei; Li, Ye; Zhu, Qing-San

    2015-09-01

    The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pcDNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco's modified Eagle's medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1-4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the

  11. Chromatin-modifying agents for epigenetic reprogramming and endogenous neural stem cell-mediated repair in stroke.

    PubMed

    Qureshi, Irfan A; Mehler, Mark F

    2011-03-01

    The recent explosion of interest in epigenetics and chromatin biology has made a significant impact on our understanding of the pathophysiology of cerebral ischemia and led to the identification of new treatment strategies for stroke, such as those that employ histone deacetylase inhibitors. These are key advances; however, the rapid pace of discovery in chromatin biology and innovation in the development of chromatin-modifying agents implies there are emerging classes of drugs that may also have potential benefits in stroke. Herein, we discuss how various chromatin regulatory factors and their recently identified inhibitors may serve as drug targets and therapeutic agents for stroke, respectively. These factors primarily include members of the repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor macromolecular complex, polycomb group (PcG) proteins, and associated chromatin remodeling factors, which have been linked to the pathophysiology of cerebral ischemia. Further, we suggest that, because of the key roles played by REST, PcG proteins and other chromatin remodeling factors in neural stem and progenitor cell (NSPC) biology, chromatin-modifying agents can be utilized not only to mitigate ischemic injury directly but also potentially to promote endogenous NSPC-mediated brain repair mechanisms. PMID:24014083

  12. Chromatin-Modifying Agents for Epigenetic Reprogramming and Endogenous Neural Stem Cell-Mediated Repair in Stroke

    PubMed Central

    Qureshi, Irfan A.; Mehler, Mark F.

    2013-01-01

    The recent explosion of interest in epigenetics and chromatin biology has made a significant impact on our understanding of the pathophysiology of cerebral ischemia and led to the identification of new treatment strategies for stroke, such as those that employ histone deacetylase inhibitors. These are key advances; however, the rapid pace of discovery in chromatin biology and innovation in the development of chromatin-modifying agents implies there are emerging classes of drugs that may also have potential benefits in stroke. Herein, we discuss how various chromatin regulatory factors and their recently identified inhibitors may serve as drug targets and therapeutic agents for stroke, respectively. These factors primarily include members of the repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor macromolecular complex, polycomb group (PcG) proteins, and associated chromatin remodeling factors, which have been linked to the pathophysiology of cerebral ischemia. Further, we suggest that, because of the key roles played by REST, PcG proteins and other chromatin remodeling factors in neural stem and progenitor cell (NSPC) biology, chromatin-modifying agents can be utilized not only to mitigate ischemic injury directly but also potentially to promote endogenous NSPC-mediated brain repair mechanisms. PMID:24014083

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

    SciTech Connect

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

    1989-07-01

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

  14. RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae.

    PubMed

    Ivanov, E L; Haber, J E

    1995-04-01

    HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in DSB-induced recombination and repair.

  15. Large-scale genomic analyses link reproductive ageing to hypothalamic signaling, breast cancer susceptibility and BRCA1-mediated DNA repair

    PubMed Central

    Lunetta, Kathryn L.; Pervjakova, Natalia; Chasman, Daniel I.; Stolk, Lisette; Finucane, Hilary K.; Sulem, Patrick; Bulik-Sullivan, Brendan; Esko, Tõnu; Johnson, Andrew D.; Elks, Cathy E.; Franceschini, Nora; He, Chunyan; Altmaier, Elisabeth; Brody, Jennifer A.; Franke, Lude L.; Huffman, Jennifer E.; Keller, Margaux F.; McArdle, Patrick F.; Nutile, Teresa; Porcu, Eleonora; Robino, Antonietta; Rose, Lynda M.; Schick, Ursula M.; Smith, Jennifer A.; Teumer, Alexander; Traglia, Michela; Vuckovic, Dragana; Yao, Jie; Zhao, Wei; Albrecht, Eva; Amin, Najaf; Corre, Tanguy; Hottenga, Jouke-Jan; Mangino, Massimo; Smith, Albert V.; Tanaka, Toshiko; Abecasis, Goncalo; Andrulis, Irene L.; Anton-Culver, Hoda; Antoniou, Antonis C.; Arndt, Volker; Arnold, Alice M.; Barbieri, Caterina; Beckmann, Matthias W.; Beeghly-Fadiel, Alicia; Benitez, Javier; Bernstein, Leslie; Bielinski, Suzette J.; Blomqvist, Carl; Boerwinkle, Eric; Bogdanova, Natalia V.; Bojesen, Stig E.; Bolla, Manjeet K.; Borresen-Dale, Anne-Lise; Boutin, Thibaud S; Brauch, Hiltrud; Brenner, Hermann; Brüning, Thomas; Burwinkel, Barbara; Campbell, Archie; Campbell, Harry; Chanock, Stephen J.; Chapman, J. Ross; Chen, Yii-Der Ida; Chenevix-Trench, Georgia; Couch, Fergus J.; Coviello, Andrea D.; Cox, Angela; Czene, Kamila; Darabi, Hatef; De Vivo, Immaculata; Demerath, Ellen W.; Dennis, Joe; Devilee, Peter; Dörk, Thilo; dos-Santos-Silva, Isabel; Dunning, Alison M.; Eicher, John D.; Fasching, Peter A.; Faul, Jessica D.; Figueroa, Jonine; Flesch-Janys, Dieter; Gandin, Ilaria; Garcia, Melissa E.; García-Closas, Montserrat; Giles, Graham G.; Girotto, Giorgia G.; Goldberg, Mark S.; González-Neira, Anna; Goodarzi, Mark O.; Grove, Megan L.; Gudbjartsson, Daniel F.; Guénel, Pascal; Guo, Xiuqing; Haiman, Christopher A.; Hall, Per; Hamann, Ute; Henderson, Brian E.; Hocking, Lynne J.; Hofman, Albert; Homuth, Georg; Hooning, Maartje J.; Hopper, John L.; Hu, Frank B.; Huang, Jinyan; Humphreys, Keith; Hunter, David J.; Jakubowska, Anna; Jones, Samuel E.; Kabisch, Maria; Karasik, David; Knight, Julia A.; Kolcic, Ivana; Kooperberg, Charles; Kosma, Veli-Matti; Kriebel, Jennifer; Kristensen, Vessela; Lambrechts, Diether; Langenberg, Claudia; Li, Jingmei; Li, Xin; Lindström, Sara; Liu, Yongmei; Luan, Jian’an; Lubinski, Jan; Mägi, Reedik; Mannermaa, Arto; Manz, Judith; Margolin, Sara; Marten, Jonathan; Martin, Nicholas G.; Masciullo, Corrado; Meindl, Alfons; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L.; Müller-Nurasyid, Martina; Nalls, Michael; Neale, Ben M.; Nevanlinna, Heli; Neven, Patrick; Newman, Anne B.; Nordestgaard, Børge G.; Olson, Janet E.; Padmanabhan, Sandosh; Peterlongo, Paolo; Peters, Ulrike; Petersmann, Astrid; Peto, Julian; Pharoah, Paul D.P.; Pirastu, Nicola N.; Pirie, Ailith; Pistis, Giorgio; Polasek, Ozren; Porteous, David; Psaty, Bruce M.; Pylkäs, Katri; Radice, Paolo; Raffel, Leslie J.; Rivadeneira, Fernando; Rudan, Igor; Rudolph, Anja; Ruggiero, Daniela; Sala, Cinzia F.; Sanna, Serena; Sawyer, Elinor J.; Schlessinger, David; Schmidt, Marjanka K.; Schmidt, Frank; Schmutzler, Rita K.; Schoemaker, Minouk J.; Scott, Robert A.; Seynaeve, Caroline M.; Simard, Jacques; Sorice, Rossella; Southey, Melissa C.; Stöckl, Doris; Strauch, Konstantin; Swerdlow, Anthony; Taylor, Kent D.; Thorsteinsdottir, Unnur; Toland, Amanda E.; Tomlinson, Ian; Truong, Thérèse; Tryggvadottir, Laufey; Turner, Stephen T.; Vozzi, Diego; Wang, Qin; Wellons, Melissa; Willemsen, Gonneke; Wilson, James F.; Winqvist, Robert; Wolffenbuttel, Bruce B.H.R.; Wright, Alan F.; Yannoukakos, Drakoulis; Zemunik, Tatijana; Zheng, Wei; Zygmunt, Marek; Bergmann, Sven; Boomsma, Dorret I.; Buring, Julie E.; Ferrucci, Luigi; Montgomery, Grant W.; Gudnason, Vilmundur; Spector, Tim D.; van Duijn, Cornelia M; Alizadeh, Behrooz Z.; Ciullo, Marina; Crisponi, Laura; Easton, Douglas F.; Gasparini, Paolo P.; Gieger, Christian; Harris, Tamara B.; Hayward, Caroline; Kardia, Sharon L.R.; Kraft, Peter; McKnight, Barbara; Metspalu, Andres; Morrison, Alanna C.; Reiner, Alex P.; Ridker, Paul M.; Rotter, Jerome I.; Toniolo, Daniela; Uitterlinden, André G.; Ulivi, Sheila; Völzke, Henry; Wareham, Nicholas J.; Weir, David R.; Yerges-Armstrong, Laura M.; Price, Alkes L.; Stefansson, Kari; Visser, Jenny A.; Ong, Ken K.; Chang-Claude, Jenny; Murabito, Joanne M.; Perry, John R.B.; Murray, Anna

    2015-01-01

    Menopause timing has a substantial impact on infertility and risk of disease, including breast cancer, but the underlying mechanisms are poorly understood. We report a dual strategy in ~70,000 women to identify common and low-frequency protein-coding variation associated with age at natural menopause (ANM). We identified 44 regions with common variants, including two harbouring additional rare missense alleles of large effect. We found enrichment of signals in/near genes involved in delayed puberty, highlighting the first molecular links between the onset and end of reproductive lifespan. Pathway analyses revealed a major association with DNA damage-response (DDR) genes, including the first common coding variant in BRCA1 associated with any complex trait. Mendelian randomisation analyses supported a causal effect of later ANM on breast cancer risk (~6% risk increase per-year, P=3×10−14), likely mediated by prolonged sex hormone exposure, rather than DDR mechanisms. PMID:26414677

  16. Large-scale genomic analyses link reproductive aging to hypothalamic signaling, breast cancer susceptibility and BRCA1-mediated DNA repair.

    PubMed

    Day, Felix R; Ruth, Katherine S; Thompson, Deborah J; Lunetta, Kathryn L; Pervjakova, Natalia; Chasman, Daniel I; Stolk, Lisette; Finucane, Hilary K; Sulem, Patrick; Bulik-Sullivan, Brendan; Esko, Tõnu; Johnson, Andrew D; Elks, Cathy E; Franceschini, Nora; He, Chunyan; Altmaier, Elisabeth; Brody, Jennifer A; Franke, Lude L; Huffman, Jennifer E; Keller, Margaux F; McArdle, Patrick F; Nutile, Teresa; Porcu, Eleonora; Robino, Antonietta; Rose, Lynda M; Schick, Ursula M; Smith, Jennifer A; Teumer, Alexander; Traglia, Michela; Vuckovic, Dragana; Yao, Jie; Zhao, Wei; Albrecht, Eva; Amin, Najaf; Corre, Tanguy; Hottenga, Jouke-Jan; Mangino, Massimo; Smith, Albert V; Tanaka, Toshiko; Abecasis, Gonçalo R; Andrulis, Irene L; Anton-Culver, Hoda; Antoniou, Antonis C; Arndt, Volker; Arnold, Alice M; Barbieri, Caterina; Beckmann, Matthias W; Beeghly-Fadiel, Alicia; Benitez, Javier; Bernstein, Leslie; Bielinski, Suzette J; Blomqvist, Carl; Boerwinkle, Eric; Bogdanova, Natalia V; Bojesen, Stig E; Bolla, Manjeet K; Borresen-Dale, Anne-Lise; Boutin, Thibaud S; Brauch, Hiltrud; Brenner, Hermann; Brüning, Thomas; Burwinkel, Barbara; Campbell, Archie; Campbell, Harry; Chanock, Stephen J; Chapman, J Ross; Chen, Yii-Der Ida; Chenevix-Trench, Georgia; Couch, Fergus J; Coviello, Andrea D; Cox, Angela; Czene, Kamila; Darabi, Hatef; De Vivo, Immaculata; Demerath, Ellen W; Dennis, Joe; Devilee, Peter; Dörk, Thilo; Dos-Santos-Silva, Isabel; Dunning, Alison M; Eicher, John D; Fasching, Peter A; Faul, Jessica D; Figueroa, Jonine; Flesch-Janys, Dieter; Gandin, Ilaria; Garcia, Melissa E; García-Closas, Montserrat; Giles, Graham G; Girotto, Giorgia G; Goldberg, Mark S; González-Neira, Anna; Goodarzi, Mark O; Grove, Megan L; Gudbjartsson, Daniel F; Guénel, Pascal; Guo, Xiuqing; Haiman, Christopher A; Hall, Per; Hamann, Ute; Henderson, Brian E; Hocking, Lynne J; Hofman, Albert; Homuth, Georg; Hooning, Maartje J; Hopper, John L; Hu, Frank B; Huang, Jinyan; Humphreys, Keith; Hunter, David J; Jakubowska, Anna; Jones, Samuel E; Kabisch, Maria; Karasik, David; Knight, Julia A; Kolcic, Ivana; Kooperberg, Charles; Kosma, Veli-Matti; Kriebel, Jennifer; Kristensen, Vessela; Lambrechts, Diether; Langenberg, Claudia; Li, Jingmei; Li, Xin; Lindström, Sara; Liu, Yongmei; Luan, Jian'an; Lubinski, Jan; Mägi, Reedik; Mannermaa, Arto; Manz, Judith; Margolin, Sara; Marten, Jonathan; Martin, Nicholas G; Masciullo, Corrado; Meindl, Alfons; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L; Müller-Nurasyid, Martina; Nalls, Michael; Neale, Benjamin M; Nevanlinna, Heli; Neven, Patrick; Newman, Anne B; Nordestgaard, Børge G; Olson, Janet E; Padmanabhan, Sandosh; Peterlongo, Paolo; Peters, Ulrike; Petersmann, Astrid; Peto, Julian; Pharoah, Paul D P; Pirastu, Nicola N; Pirie, Ailith; Pistis, Giorgio; Polasek, Ozren; Porteous, David; Psaty, Bruce M; Pylkäs, Katri; Radice, Paolo; Raffel, Leslie J; Rivadeneira, Fernando; Rudan, Igor; Rudolph, Anja; Ruggiero, Daniela; Sala, Cinzia F; Sanna, Serena; Sawyer, Elinor J; Schlessinger, David; Schmidt, Marjanka K; Schmidt, Frank; Schmutzler, Rita K; Schoemaker, Minouk J; Scott, Robert A; Seynaeve, Caroline M; Simard, Jacques; Sorice, Rossella; Southey, Melissa C; Stöckl, Doris; Strauch, Konstantin; Swerdlow, Anthony; Taylor, Kent D; Thorsteinsdottir, Unnur; Toland, Amanda E; Tomlinson, Ian; Truong, Thérèse; Tryggvadottir, Laufey; Turner, Stephen T; Vozzi, Diego; Wang, Qin; Wellons, Melissa; Willemsen, Gonneke; Wilson, James F; Winqvist, Robert; Wolffenbuttel, Bruce B H R; Wright, Alan F; Yannoukakos, Drakoulis; Zemunik, Tatijana; Zheng, Wei; Zygmunt, Marek; Bergmann, Sven; Boomsma, Dorret I; Buring, Julie E; Ferrucci, Luigi; Montgomery, Grant W; Gudnason, Vilmundur; Spector, Tim D; van Duijn, Cornelia M; Alizadeh, Behrooz Z; Ciullo, Marina; Crisponi, Laura; Easton, Douglas F; Gasparini, Paolo P; Gieger, Christian; Harris, Tamara B; Hayward, Caroline; Kardia, Sharon L R; Kraft, Peter; McKnight, Barbara; Metspalu, Andres; Morrison, Alanna C; Reiner, Alex P; Ridker, Paul M; Rotter, Jerome I; Toniolo, Daniela; Uitterlinden, André G; Ulivi, Sheila; Völzke, Henry; Wareham, Nicholas J; Weir, David R; Yerges-Armstrong, Laura M; Price, Alkes L; Stefansson, Kari; Visser, Jenny A; Ong, Ken K; Chang-Claude, Jenny; Murabito, Joanne M; Perry, John R B; Murray, Anna

    2015-11-01

    Menopause timing has a substantial impact on infertility and risk of disease, including breast cancer, but the underlying mechanisms are poorly understood. We report a dual strategy in ∼70,000 women to identify common and low-frequency protein-coding variation associated with age at natural menopause (ANM). We identified 44 regions with common variants, including two regions harboring additional rare missense alleles of large effect. We found enrichment of signals in or near genes involved in delayed puberty, highlighting the first molecular links between the onset and end of reproductive lifespan. Pathway analyses identified major association with DNA damage response (DDR) genes, including the first common coding variant in BRCA1 associated with any complex trait. Mendelian randomization analyses supported a causal effect of later ANM on breast cancer risk (∼6% increase in risk per year; P = 3 × 10(-14)), likely mediated by prolonged sex hormone exposure rather than DDR mechanisms. PMID:26414677

  17. Large-scale genomic analyses link reproductive aging to hypothalamic signaling, breast cancer susceptibility and BRCA1-mediated DNA repair.

    PubMed

    Day, Felix R; Ruth, Katherine S; Thompson, Deborah J; Lunetta, Kathryn L; Pervjakova, Natalia; Chasman, Daniel I; Stolk, Lisette; Finucane, Hilary K; Sulem, Patrick; Bulik-Sullivan, Brendan; Esko, Tõnu; Johnson, Andrew D; Elks, Cathy E; Franceschini, Nora; He, Chunyan; Altmaier, Elisabeth; Brody, Jennifer A; Franke, Lude L; Huffman, Jennifer E; Keller, Margaux F; McArdle, Patrick F; Nutile, Teresa; Porcu, Eleonora; Robino, Antonietta; Rose, Lynda M; Schick, Ursula M; Smith, Jennifer A; Teumer, Alexander; Traglia, Michela; Vuckovic, Dragana; Yao, Jie; Zhao, Wei; Albrecht, Eva; Amin, Najaf; Corre, Tanguy; Hottenga, Jouke-Jan; Mangino, Massimo; Smith, Albert V; Tanaka, Toshiko; Abecasis, Gonçalo R; Andrulis, Irene L; Anton-Culver, Hoda; Antoniou, Antonis C; Arndt, Volker; Arnold, Alice M; Barbieri, Caterina; Beckmann, Matthias W; Beeghly-Fadiel, Alicia; Benitez, Javier; Bernstein, Leslie; Bielinski, Suzette J; Blomqvist, Carl; Boerwinkle, Eric; Bogdanova, Natalia V; Bojesen, Stig E; Bolla, Manjeet K; Borresen-Dale, Anne-Lise; Boutin, Thibaud S; Brauch, Hiltrud; Brenner, Hermann; Brüning, Thomas; Burwinkel, Barbara; Campbell, Archie; Campbell, Harry; Chanock, Stephen J; Chapman, J Ross; Chen, Yii-Der Ida; Chenevix-Trench, Georgia; Couch, Fergus J; Coviello, Andrea D; Cox, Angela; Czene, Kamila; Darabi, Hatef; De Vivo, Immaculata; Demerath, Ellen W; Dennis, Joe; Devilee, Peter; Dörk, Thilo; Dos-Santos-Silva, Isabel; Dunning, Alison M; Eicher, John D; Fasching, Peter A; Faul, Jessica D; Figueroa, Jonine; Flesch-Janys, Dieter; Gandin, Ilaria; Garcia, Melissa E; García-Closas, Montserrat; Giles, Graham G; Girotto, Giorgia G; Goldberg, Mark S; González-Neira, Anna; Goodarzi, Mark O; Grove, Megan L; Gudbjartsson, Daniel F; Guénel, Pascal; Guo, Xiuqing; Haiman, Christopher A; Hall, Per; Hamann, Ute; Henderson, Brian E; Hocking, Lynne J; Hofman, Albert; Homuth, Georg; Hooning, Maartje J; Hopper, John L; Hu, Frank B; Huang, Jinyan; Humphreys, Keith; Hunter, David J; Jakubowska, Anna; Jones, Samuel E; Kabisch, Maria; Karasik, David; Knight, Julia A; Kolcic, Ivana; Kooperberg, Charles; Kosma, Veli-Matti; Kriebel, Jennifer; Kristensen, Vessela; Lambrechts, Diether; Langenberg, Claudia; Li, Jingmei; Li, Xin; Lindström, Sara; Liu, Yongmei; Luan, Jian'an; Lubinski, Jan; Mägi, Reedik; Mannermaa, Arto; Manz, Judith; Margolin, Sara; Marten, Jonathan; Martin, Nicholas G; Masciullo, Corrado; Meindl, Alfons; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L; Müller-Nurasyid, Martina; Nalls, Michael; Neale, Benjamin M; Nevanlinna, Heli; Neven, Patrick; Newman, Anne B; Nordestgaard, Børge G; Olson, Janet E; Padmanabhan, Sandosh; Peterlongo, Paolo; Peters, Ulrike; Petersmann, Astrid; Peto, Julian; Pharoah, Paul D P; Pirastu, Nicola N; Pirie, Ailith; Pistis, Giorgio; Polasek, Ozren; Porteous, David; Psaty, Bruce M; Pylkäs, Katri; Radice, Paolo; Raffel, Leslie J; Rivadeneira, Fernando; Rudan, Igor; Rudolph, Anja; Ruggiero, Daniela; Sala, Cinzia F; Sanna, Serena; Sawyer, Elinor J; Schlessinger, David; Schmidt, Marjanka K; Schmidt, Frank; Schmutzler, Rita K; Schoemaker, Minouk J; Scott, Robert A; Seynaeve, Caroline M; Simard, Jacques; Sorice, Rossella; Southey, Melissa C; Stöckl, Doris; Strauch, Konstantin; Swerdlow, Anthony; Taylor, Kent D; Thorsteinsdottir, Unnur; Toland, Amanda E; Tomlinson, Ian; Truong, Thérèse; Tryggvadottir, Laufey; Turner, Stephen T; Vozzi, Diego; Wang, Qin; Wellons, Melissa; Willemsen, Gonneke; Wilson, James F; Winqvist, Robert; Wolffenbuttel, Bruce B H R; Wright, Alan F; Yannoukakos, Drakoulis; Zemunik, Tatijana; Zheng, Wei; Zygmunt, Marek; Bergmann, Sven; Boomsma, Dorret I; Buring, Julie E; Ferrucci, Luigi; Montgomery, Grant W; Gudnason, Vilmundur; Spector, Tim D; van Duijn, Cornelia M; Alizadeh, Behrooz Z; Ciullo, Marina; Crisponi, Laura; Easton, Douglas F; Gasparini, Paolo P; Gieger, Christian; Harris, Tamara B; Hayward, Caroline; Kardia, Sharon L R; Kraft, Peter; McKnight, Barbara; Metspalu, Andres; Morrison, Alanna C; Reiner, Alex P; Ridker, Paul M; Rotter, Jerome I; Toniolo, Daniela; Uitterlinden, André G; Ulivi, Sheila; Völzke, Henry; Wareham, Nicholas J; Weir, David R; Yerges-Armstrong, Laura M; Price, Alkes L; Stefansson, Kari; Visser, Jenny A; Ong, Ken K; Chang-Claude, Jenny; Murabito, Joanne M; Perry, John R B; Murray, Anna

    2015-11-01

    Menopause timing has a substantial impact on infertility and risk of disease, including breast cancer, but the underlying mechanisms are poorly understood. We report a dual strategy in ∼70,000 women to identify common and low-frequency protein-coding variation associated with age at natural menopause (ANM). We identified 44 regions with common variants, including two regions harboring additional rare missense alleles of large effect. We found enrichment of signals in or near genes involved in delayed puberty, highlighting the first molecular links between the onset and end of reproductive lifespan. Pathway analyses identified major association with DNA damage response (DDR) genes, including the first common coding variant in BRCA1 associated with any complex trait. Mendelian randomization analyses supported a causal effect of later ANM on breast cancer risk (∼6% increase in risk per year; P = 3 × 10(-14)), likely mediated by prolonged sex hormone exposure rather than DDR mechanisms.

  18. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.

    PubMed

    Gantz, Valentino M; Jasinskiene, Nijole; Tatarenkova, Olga; Fazekas, Aniko; Macias, Vanessa M; Bier, Ethan; James, Anthony A

    2015-12-01

    Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda. PMID:26598698

  19. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.

    PubMed

    Gantz, Valentino M; Jasinskiene, Nijole; Tatarenkova, Olga; Fazekas, Aniko; Macias, Vanessa M; Bier, Ethan; James, Anthony A

    2015-12-01

    Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.

  20. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi

    PubMed Central

    Gantz, Valentino M.; Tatarenkova, Olga; Fazekas, Aniko; Macias, Vanessa M.; Bier, Ethan; James, Anthony A.

    2015-01-01

    Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda. PMID:26598698

  1. Ultrasound-induced hyperthermia for the spatio-temporal control of gene expression in bone repair

    NASA Astrophysics Data System (ADS)

    Wilson, Christopher; Padilla, Frédéric; Zhang, Man; Vilaboa, Nuria; Kripfgans, Oliver; Fowlkes, Brian; Franceschi, Renny

    2012-10-01

    Spatial and temporal control over the expression of growth/differentiation factors is of great interest for regeneration of bone, but technologies capable of providing tight and active control over gene expression remain elusive. We propose the use of focused ultrasound for the targeted activation of heat shock-sensitive expression systems in engineered bone. We report in vitro results with cells that express firefly luciferase (fLuc) under the control of a heat shock protein promoter. Cells were embedded in fibrin scaffolds and exposed to focused ultrasound, using a custom 3.3MHz transducer (focal length 4", f-number 1.33", focal dimension 1.2mm lateral FWHM) in CW mode for 2-20 minutes at intensities ISPTA=120-440 W/cm2. The kinetics of ultrasound-mediated activation of the cells was compared with that of strictly thermal activation. Bioluminescence imaging revealed fLuc expression in an area ≥2.5mm in diameter at the position of the ultrasound focus, and the diameter and intensity of the signal increased with the amplitude of the acoustic energy. We also found that ultrasound activated fLuc expression with substantially shorter exposures than thermal activation. Our results demonstrate the potential for focused ultrasound to selectively activate the expression of a gene of interest in an engineered tissue and suggest that focused ultrasound activates the heat shock pathway by a combination of thermal and non-thermal mechanisms.

  2. Association of base excision repair gene polymorphisms with ESRD risk in a Chinese population.

    PubMed

    Cai, Zhenming; Chen, Huimei; Tao, Jing; Guo, Wenwen; Liu, Xiufang; Zheng, Bixia; Sun, Wei; Wang, Yaping

    2012-01-01

    The base excision repair (BER) pathway, containing OGG1, MTH1 and MUTYH, is a major protector from oxidative DNA damage in humans, while 8-oxoguanine (8-OHdG), an index of DNA oxidation, is increased in maintenance hemodialysis (HD) patients. Four polymorphisms of BER genes, OGG1 c.977C > G (rs1052133), MTH1 c.247G > A (rs4866), MUTYH c.972G > C (rs3219489), and AluYb8MUTYH (rs10527342), were examined in 337 HD patients and 404 healthy controls. And the 8-OHdG levels in leukocyte DNA were examined in 116 HD patients. The distribution of MUTYH c.972 GG or AluYb8MUTYH differed between the two groups and was associated with a moderately increased risk for end-stage renal disease (ESRD) (P = 0.013 and 0.034, resp.). The average 8-OHdG/10(6) dG value was significantly higher in patients with the OGG1 c.977G, MUTYH c.972G or AluYb8MUTYH alleles (P < 0.001 via ANOVA). Further analysis showed that combination of MUTYH c.972GG with OGG1 c.977GG or AluYb8MUTYH increased both the risk for ESRD and leukocyte DNA 8-OHdG levels in HD patients. Our study showed that MUTYH c.972GG, AluYb8MUTYH, and combination of OGG1 c.977GG increased the risk for ESRD development in China and suggested that DNA oxidative damage might be involved in such process.

  3. The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

    PubMed

    Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

    2016-07-01

    The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. PMID:27246098

  4. Preferential repair of cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription.

    PubMed Central

    Sweder, K S; Hanawalt, P C

    1992-01-01

    While preferential repair of the transcribed strands within active genes has been demonstrated in organisms as diverse as humans and Escherichia coli, it has not previously been shown to occur in chromosomal genes in the yeast Saccharomyces cerevisiae. We found that repair of cyclobutane pyrimidine dimers in the transcribed strand of the expressed RPB2 gene in the chromosome of a repair-proficient strain is much more rapid than that in the nontranscribed strand. Furthermore, a copy of the RPB2 gene borne on a centromeric ARS1 plasmid showed the same strand bias in repair. To investigate the relation of this strand bias to transcription, we studied repair in a yeast strain with the temperature-sensitive mutation, rpb1-1, in the largest subunit of RNA polymerase II. When exponentially growing rpb1-1 cells are shifted to the nonpermissive temperature, they rapidly cease mRNA synthesis. At the permissive temperature, both rpb1-1 and the wild-type, parental cells exhibited rapid, proficient repair in the transcribed strand of chromosomal and plasmid-borne copies of the RPB2 gene. At the nonpermissive temperature, the rate of repair in the transcribed strand in rpb1-1 cells was reduced to that in the nontranscribed strand. These findings establish the dependence of strand bias in repair on transcription by RNA polymerase II in the chromosomes and in plasmids, and they validate the use of plasmids for analysis of the relation of repair to transcription in yeast. Images PMID:1438266

  5. RNAi mediated Tiam1 gene knockdown inhibits invasion of retinoblastoma.

    PubMed

    Subramanian, Nithya; Navaneethakrishnan, Saranya; Biswas, Jyotirmay; Kanwar, Rupinder K; Kanwar, Jagat R; Krishnakumar, Subramanian

    2013-01-01

    T lymphoma invasion and metastasis protein (Tiam1) is up-regulated in variety of cancers and its expression level is related to metastatic potential of the type of cancer. Earlier, Tiam1 was shown to be overexpressed in retinoblastoma (RB) and we hypothesized that it was involved in invasiveness of RB. This was tested by silencing Tiam1 in RB cell lines (Y79 and Weri-Rb1) using siRNA pool, targeting different regions of Tiam1 mRNA. The cDNA microarray of Tiam1 silenced cells showed gene regulations altered by Tiam1 were predominantly on the actin cytoskeleton interacting proteins, apoptotic initiators and tumorogenic potential targets. The silenced phenotype resulted in decreased growth and increased apoptosis with non-invasive characteristics. Transfection of full length and N-terminal truncated construct (C1199) clearly revealed membrane localization of Tiam1 and not in the case of C580 construct. F-actin staining showed the interaction of Tiam1 with actin in the membrane edges that leads to ruffling, and also imparts varying invasive potential to the cell. The results obtained from our study show for the first time that Tiam1 modulates the cell invasion, mediated by actin cytoskeleton remodeling in RB. PMID:23950931

  6. Retinoic acid-mediated gene expression in transgenic reporter zebrafish.

    PubMed

    Perz-Edwards, A; Hardison, N L; Linney, E

    2001-01-01

    Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

  7. Deficiency in Nucleotide Excision Repair Family Gene Activity, Especially ERCC3, Is Associated with Non-Pigmented Hair Fiber Growth

    PubMed Central

    Yu, Mei; Bell, Robert H.; Ho, Maggie M.; Leung, Gigi; Haegert, Anne; Carr, Nicholas; Shapiro, Jerry; McElwee, Kevin J.

    2012-01-01

    We conducted a microarray study to discover gene expression patterns associated with a lack of melanogenesis in non-pigmented hair follicles (HF) by microarray. Pigmented and non-pigmented HFs were collected and micro-dissected into the hair bulb (HB) and the upper hair sheaths (HS) including the bulge region. In comparison to pigmented HS and HBs, nucleotide excision repair (NER) family genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH exhibited statistically significantly lower expression in non- pigmented HS and HBs. Quantitative PCR verified microarray data and identified ERCC3 as highly differentially expressed. Immunohistochemistry confirmed ERCC3 expression in HF melanocytes. A reduction in ERCC3 by siRNA interference in human melanocytes in vitro reduced their tyrosinase production ability. Our results suggest that loss of NER gene function is associated with a loss of melanin production capacity. This may be due to reduced gene transcription and/or reduced DNA repair in melanocytes which may eventually lead to cell death. These results provide novel information with regard to melanogenesis and its regulation. PMID:22615732

  8. Restoration of Chinese hamster cell radiation resistance by the human repair gene ERCC-5 and progress in molecular cloning of this gene

    SciTech Connect

    Strniste, G.F.; Chen, D.J.; deBruin, D.; McCoy, L.S.; Luke, J.A.; Mudgett, J.S.; Nickols, J.W.; Okinaka, R.T.; Tesmer, J.G.; MacInnes, M.A.

    1988-01-01

    The uv-sensitive Chinese hamster cell uv-135 is being used to identify and isolate the human gene, ERCC-5, which corrects nucleotide excision repair in this incision-defective mutant. A cosmid library, constructed from a 3/sup 0/ transformant of uv-135, has been screened for transfected gpt and human Alu family sequences. An ordered physical map of overlapping positives cosmids has been determined. Molecular evidence suggests a region of this map of <40 Kbp contains the ERCC-5 gene. 10 refs., 2 figs.

  9. NPM-ALK mediates phosphorylation of MSH2 at tyrosine 238, creating a functional deficiency in MSH2 and the loss of mismatch repair.

    PubMed

    Bone, K M; Wang, P; Wu, F; Wu, C; Li, L; Bacani, J T; Andrew, S E; Lai, R

    2015-05-15

    The vast majority of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ALCL) tumors express the characteristic oncogenic fusion protein NPM-ALK, which mediates tumorigenesis by exerting its constitutive tyrosine kinase activity on various substrates. We recently identified MSH2, a protein central to DNA mismatch repair (MMR), as a novel binding partner and phosphorylation substrate of NPM-ALK. Here, using liquid chromatography-mass spectrometry, we report for the first time that MSH2 is phosphorylated by NPM-ALK at a specific residue, tyrosine 238. Using GP293 cells transfected with NPM-ALK, we confirmed that the MSH2(Y238F) mutant is not tyrosine phosphorylated. Furthermore, transfection of MSH2(Y238F) into these cells substantially decreased the tyrosine phosphorylation of endogenous MSH2. Importantly, gene transfection of MSH2(Y238F) abrogated the binding of NPM-ALK with endogenous MSH2, re-established the dimerization of MSH2:MSH6 and restored the sensitivity to DNA mismatch-inducing drugs, indicative of MMR return. Parallel findings were observed in two ALK+ALCL cell lines, Karpas 299 and SUP-M2. In addition, we found that enforced expression of MSH2(Y238F) into ALK+ALCL cells alone was sufficient to induce spontaneous apoptosis. In conclusion, our findings have identified NPM-ALK-induced phosphorylation of MSH2 at Y238 as a crucial event in suppressing MMR. Our studies have provided novel insights into the mechanism by which oncogenic tyrosine kinases disrupt MMR.

  10. TaMSH7: A cereal mismatch repair gene that affects fertility in transgenic barley (Hordeum vulgare L.)

    PubMed Central

    Lloyd, Andrew H; Milligan, Andrew S; Langridge, Peter; Able, Jason A

    2007-01-01

    Background Chromosome pairing, recombination and DNA repair are essential processes during meiosis in sexually reproducing organisms. Investigating the bread wheat (Triticum aestivum L.) Ph2 (Pairing homoeologous) locus has identified numerous candidate genes that may have a role in controlling such processes, including TaMSH7, a plant specific member of the DNA mismatch repair family. Results Sequencing of the three MSH7 genes, located on the short arms of wheat chromosomes 3A, 3B and 3D, has revealed no significant sequence divergence at the amino acid level suggesting conservation of function across the homoeogroups. Functional analysis of MSH7 through the use of RNAi loss-of-function transgenics was undertaken in diploid barley (Hordeum vulgare L.). Quantitative real-time PCR revealed several T0 lines with reduced MSH7 expression. Positive segregants from two T1 lines studied in detail showed reduced MSH7 expression when compared to transformed controls and null segregants. Expression of MSH6, another member of the mismatch repair family which is most closely related to the MSH7 gene, was not significantly reduced in these lines. In both T1 lines, reduced seed set in positive segregants was observed. Conclusion Results presented here indicate, for the first time, a distinct functional role for MSH7 in vivo and show that expression of this gene is necessary for wild-type levels of fertility. These observations suggest that MSH7 has an important function during meiosis and as such remains a candidate for Ph2. PMID:18096080

  11. Analysis of gene expression dynamics revealed delayed and abnormal epidermal repair process in aged compared to young skin.

    PubMed

    Sextius, Peggy; Marionnet, Claire; Tacheau, Charlotte; Bon, François-Xavier; Bastien, Philippe; Mauviel, Alain; Bernard, Bruno A; Bernerd, Françoise; Dubertret, Louis

    2015-05-01

    With aging, epidermal homeostasis and barrier function are disrupted. In a previous study, we analyzed the transcriptomic response of young skin epidermis after stratum corneum removal, and obtained a global kinetic view of the molecular processes involved in barrier function recovery. In the present study, the same analysis was performed in aged skin in order to better understand the defects which occur with aging. Thirty healthy male volunteers (67 ± 4 years old) were involved. Tape-strippings were carried out on the inner face of one forearm, the other unstripped forearm serving as control. At 2, 6, 18, 30 and 72 h after stripping, TEWL measurements were taken, and epidermis samples were collected. Total RNA was extracted and analyzed using DermArray(®) cDNA microarrays. The results highlighted that barrier function recovery and overall kinetics of gene expression were delayed following stripping in aged skin. Indeed, the TEWL measurements showed that barrier recovery in the young group appeared to be dramatically significant during the overall kinetics, while there were no significant evolution in the aged group until 30 h. Moreover, gene expression analysis revealed that the number of modulated genes following tape stripping increased as a function of time and reached a peak at 6 h after tape stripping in young skin, while it was at 30 h in aged skin, showing that cellular activity linked to the repair process may be engaged earlier in young epidermis than in aged epidermis. A total of 370 genes were modulated in the young group. In the aged group, 382 genes were modulated, whose 184 were also modulated in the young group. Only eight genes that were modulated in both groups were significantly differently modulated. The characterization of these genes into 15 functional families helped to draw a scenario for the aging process affecting epidermal repair capacity. PMID:25740152

  12. Identification of the class I genes of the mouse major histocompatibility complex by DNA-mediated gene transfer.

    PubMed

    Goodenow, R S; McMillan, M; Nicolson, M; Sher, B T; Eakle, K; Davidson, N; Hood, L

    1982-11-18

    DNA-mediated gene transfer was used to identify cloned class I genes from the major histocompatibility complex of the BALB/c mouse. Three genes encoding the transplantation antigens H-2 Kd, Dd and Ld were identified as well as genes encoding the Qa-2,3 and two TL differentiation antigens. As many as 10 putative novel class I genes were detected by the association of their gene products with beta 2-microglobulin. Alloantiserum prepared to one of the novel antigens was used to demonstrate the expression of the previously undetected antigen on spleen cells of various inbred, congeneic, and recombinant congeneic strains of mice. PMID:6815535

  13. A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy

    SciTech Connect

    Weeda, G.; Donker, I.; Vermeulen, W.

    1997-02-01

    Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and nails, mental retardation, impaired sexual development, and ichthyosis. Photosensitivity has been reported in {approximately}50% of the cases, but no skin cancer is associated with TTD. Virtually all photosensitive TTD patients have a deficiency in the nucleotide excision repair (NER) of UV-induced DNA damage that is indistinguishable from that of xeroderma pigmentosum (XP) complementation group D (XP-D) patients. DNA repair defects in XP-D are associated with two additional, quite different diseases; XP, a sun-sensitive and cancer-prone repair disorder, and Cockayne syndrome (CS), a photosensitive condition characterized by physical and mental retardation and wizened facial appearance. One photosensitive TTD case constitutes a new repair-deficient complementation group, TTD-A. Remarkably, both TTD-A and XP-D defects are associated with subunits of TFIIH, a basal transcription factor with a second function in DNA repair. Thus, mutations in TFIIH components may, on top of a repair defect, also cause transcriptional insufficiency, which may explain part of the non-XP clinical features of TTD. To date, three patients with the remarkable conjunction of XP and CS but not TM have been assigned to XP complementation group B (XP-B). Here we present the characterization of the NER defect in two mild TTD patients (TTD6VI and TTD4VI) and confirm the assignment to X-PB. The causative mutation was found to be a single base substitution resulting in a missense mutation (T119P) in a region of the XPB protein. These findings define a third TTD complementation group, extend the clinical heterogeneity associated with XP-B, stress the exclusive relationship between TTD and mutations in subunits of repair/transcription factor TFIIH, and strongly support the concept of {open_quotes}transcription syndromes.{close_quotes} 46 refs., 6 figs., 2 tabs.

  14. Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

    PubMed Central

    Carrasco, Begoña; Yadav, Tribhuwan; Serrano, Ester; Alonso, Juan C.

    2015-01-01

    Genetic data have revealed that the absence of Bacillus subtilis RecO and one of the end-processing avenues (AddAB or RecJ) renders cells as sensitive to DNA damaging agents as the null recA, suggesting that both end-resection pathways require RecO for recombination. RecA, in the rATP·Mg2+ bound form (RecA·ATP), is inactive to catalyze DNA recombination between linear double-stranded (ds) DNA and naked complementary circular single-stranded (ss) DNA. We showed that RecA·ATP could not nucleate and/or polymerize on SsbA·ssDNA or SsbB·ssDNA complexes. RecA·ATP nucleates and polymerizes on RecO·ssDNA·SsbA complexes more efficiently than on RecO·ssDNA·SsbB complexes. Limiting SsbA concentrations were sufficient to stimulate RecA·ATP assembly on the RecO·ssDNA·SsbB complexes. RecO and SsbA are necessary and sufficient to ‘activate’ RecA·ATP to catalyze DNA strand exchange, whereas the AddAB complex, RecO alone or in concert with SsbB was not sufficient. In presence of AddAB, RecO and SsbA are still necessary for efficient RecA·ATP-mediated three-strand exchange recombination. Based on genetic and biochemical data, we proposed that SsbA and RecO (or SsbA, RecO and RecR in vivo) are crucial for RecA activation for both, AddAB and RecJ–RecQ (RecS) recombinational repair pathways. PMID:26001966

  15. Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair.

    PubMed

    Carrasco, Begoña; Yadav, Tribhuwan; Serrano, Ester; Alonso, Juan C

    2015-07-13

    Genetic data have revealed that the absence of Bacillus subtilis RecO and one of the end-processing avenues (AddAB or RecJ) renders cells as sensitive to DNA damaging agents as the null recA, suggesting that both end-resection pathways require RecO for recombination. RecA, in the rATP·Mg(2+) bound form (RecA·ATP), is inactive to catalyze DNA recombination between linear double-stranded (ds) DNA and naked complementary circular single-stranded (ss) DNA. We showed that RecA·ATP could not nucleate and/or polymerize on SsbA·ssDNA or SsbB·ssDNA complexes. RecA·ATP nucleates and polymerizes on RecO·ssDNA·SsbA complexes more efficiently than on RecO·ssDNA·SsbB complexes. Limiting SsbA concentrations were sufficient to stimulate RecA·ATP assembly on the RecO·ssDNA·SsbB complexes. RecO and SsbA are necessary and sufficient to 'activate' RecA·ATP to catalyze DNA strand exchange, whereas the AddAB complex, RecO alone or in concert with SsbB was not sufficient. In presence of AddAB, RecO and SsbA are still necessary for efficient RecA·ATP-mediated three-strand exchange recombination. Based on genetic and biochemical data, we proposed that SsbA and RecO (or SsbA, RecO and RecR in vivo) are crucial for RecA activation for both, AddAB and RecJ-RecQ (RecS) recombinational repair pathways. PMID:26001966

  16. ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle

    PubMed Central

    Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A.

    2016-01-01

    Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation. PMID:27442013

  17. ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

    PubMed

    Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A

    2016-01-01

    Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation. PMID:27442013

  18. Magnetic nanoparticle-mediated gene transfer to oligodendrocyte precursor cell transplant populations is enhanced by magnetofection strategies.

    PubMed

    Jenkins, Stuart I; Pickard, Mark R; Granger, Nicolas; Chari, Divya M

    2011-08-23

    This study has tested the feasibility of using physical delivery methods, employing static and oscillating field "magnetofection" techniques, to enhance magnetic nanoparticle-mediated gene transfer to rat oligodendrocyte precursor cells derived for transplantation therapies. These cells are a major transplant population to mediate repair of damage as occurs in spinal cord injury and neurological diseases such as multiple sclerosis. We show for the first time that magnetic nanoparticles mediate effective transfer of reporter and therapeutic genes to oligodendrocyte precursors; transfection efficacy was significantly enhanced by applied static or oscillating magnetic fields, the latter using an oscillating array employing high-gradient NdFeB magnets. The effects of oscillating fields were frequency-dependent, with 4 Hz yielding optimal results. Transfection efficacies obtained using magnetofection methods were highly competitive with or better than current widely used nonviral transfection methods (e.g., electroporation and lipofection) with the additional critical advantage of high cell viability. No adverse effects were found on the cells' ability to divide or give rise to their daughter cells, the oligodendrocytes-key properties that underpin their regeneration-promoting effects. The transplantation potential of transfected cells was tested in three-dimensional tissue engineering models utilizing brain slices as the host tissue; modified transplanted cells were found to migrate, divide, give rise to daughter cells, and integrate within host tissue, further evidencing the safety of the protocols used. Our findings strongly support the concept that magnetic nanoparticle vectors in conjunction with state-of-the-art magnetofection strategies provide a technically simple and effective alternative to current methods for gene transfer to oligodendrocyte precursor cells.

  19. Smoking and selected DNA repair gene polymorphisms in controls: Systematic review and meta-analysis

    PubMed Central

    Hodgson, M. Elizabeth; Poole, Charles; Olshan, Andrew F.; North, Kari E.; Zeng, Donglin; Millikan, Robert C.

    2010-01-01

    Background When the case-only study design is used to estimate statistical interaction between genetic (G) and environmental (E) exposures, G and E must be independent in the underlying population, or the case-only estimate of interaction (COR) will be biased. Few studies have examined the occurrence of G-E association in published control group data. Methods To examine the assumption of G-E independence in empirical data, we conducted a systematic review and meta-analysis of G-E associations in controls for frequently investigated DNA repair genes (XRCC1 Arg399Gln, Arg194Trp, or Arg280His, XPD Lys751Gln, and Asp312Asn, and XRCC3 Thr241Met) and smoking (ever/never smoking, current/not current smoker, smoking duration, smoking intensity and pack-years). Results Across the 55 included studies, SNP-smoking associations in controls (ORz) were not reliably at the null value of 1.0 for any SNP-smoking combinations. Two G-E combinations were too heterogeneous for summary estimates: XRCC1 399 and ever-never smoking (N=21), and XPD 751 and pack-years (N=12). ORz ranges for these combinations were: [ORz (95% confidence interval (CI)] 0.7 (0.4, 1.2) – 1.9 (1.2, 2.8) and 0.8 (0.5, 1.3) – 2.3 (0.8, 6.1), respectively). Estimates for studies considered homogeneous (Cochran’s Q p-value <0.10) varied 2- to 5-fold. No study characteristics were identified that could explain heterogeneity. Conclusions We recommend the independence assumption be evaluated in the population underlying any potential case-only study, rather than in a proxy control group(s) or pooled controls. Impact These results suggest that G-E association in controls may be population-specific. Increased access to control data would improve evaluation of the independence assumption. PMID:20935063

  20. Mismatch repair gene defects in sporadic colorectal cancer enhance immune surveillance

    PubMed Central

    Canal, Fabio; Scarpa, Melania; Basato, Silvia; Erroi, Francesca; Fiorot, Alain; Dall'Agnese, Lucia; Pozza, Anna; Porzionato, Andrea; Castagliuolo, Ignazio; Dei Tos, Angelo P.; Bassi, Nicolò; Castoro, Carlo

    2015-01-01

    Background There is evidence that colorectal cancers (CRC) with DNA mismatch repair deficiency (MMR-D) are associated with a better prognosis than the generality of large bowel malignancies. Since an active immune surveillance process has been demonstrated to influence CRC outcome, we investigated whether MMR-D can enhance the immune response in CRC. Patients and Methods A group of 113 consecutive patients operated for CRC (42 stage I or II and 71 with stage III or IV) was retrospectively analyzed. The expression of MMR genes (MSH2, MLH1, MSH6 and PSM2) and co-stimulatory molecule CD80 was assessed by tissue microarray immunohistochemistry. In addition, tumor infiltrating mononuclear cells (TIMC) and T cell subpopulations (CD4, CD8, T-bet and FoxP-3) were quantified. The effect of specific siRNA (siMSH2, siMLH1, siMSH6 and siPSM2) transfection in HT29 on CD80 expression was quantified by flow cytometry. Non parametric statistics and survival analysis were used. Results Patients with MMR-D showed a higher T-bet/CD4 ratio (p = 0.02), a higher rate of CD80 expression and CD8 lymphocyte infiltration compared to those with no MMR-D. Moreover, in the MMR-D group, the Treg marker FoxP-3 was not expressed (p = 0.05). MMR-D patients with stage I or II and T-bet expression had a significant better survival (p = 0.009). Silencing of MSH2, MLH1 and MSH6, but not PSM2, significantly increased the rate of CD80+ HT29 cells (p = 0.007, p = 0.023 and p = 0.015, respectively). Conclusions CRC with MMR-D showed a higher CD80 expression, and CD8+ and Th1 T-cell infiltration. In vitro silencing of MSH2, MLH1 and MSH6 significantly increased CD80+ cell rate. These results suggest an enhanced immune surveillance mechanism in presence of MMR-D. PMID:26496037

  1. Transcriptome analyses and virus induced gene silencing identify genes in the Rpp4-mediated Asian soybean rust resistance pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rpp4 (Resistance to Phakopsora pachyrhizi 4) confers resistance to P. pachyrhizi, the causal agent of Asian soybean rust (ASR). By combining expression profiling and virus induced gene silencing (VIGS), we are developing a genetic framework for Rpp4-mediated resistance. We measured gene expression i...

  2. Purkinje Cell Degeneration in pcd Mice Reveals Large Scale Chromatin Reorganization and Gene Silencing Linked to Defective DNA Repair*

    PubMed Central

    Baltanás, Fernando C.; Casafont, Iñigo; Lafarga, Vanesa; Weruaga, Eduardo; Alonso, José R.; Berciano, María T.; Lafarga, Miguel

    2011-01-01

    DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death. PMID:21700704

  3. Beryllium chloride-induced oxidative DNA damage and alteration in the expression patterns of DNA repair-related genes.

    PubMed

    Attia, Sabry M; Harisa, Gamaleldin I; Hassan, Memy H; Bakheet, Saleh A

    2013-09-01

    Beryllium metal has physical properties that make its use essential for very specific applications, such as medical diagnostics, nuclear/fusion reactors and aerospace applications. Because of the widespread human exposure to beryllium metals and the discrepancy of the genotoxic results in the reported literature, detail assessments of the genetic damage of beryllium are warranted. Mice exposed to beryllium chloride at an oral dose of 23mg/kg for seven consecutive days exhibited a significant increase in the level of DNA-strand breaking and micronuclei formation as detected by a bone marrow standard comet assay and micronucleus test. Whereas slight beryllium chloride-induced oxidative DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in considerable increases in oxidative DNA damage after the 11.5 and 23mg/kg/day treatment as detected by enzyme-modified comet assays. Increased 8-hydroxydeoxyguanosine was also directly correlated with increased bone marrow micronuclei formation and DNA strand breaks, which further confirm the involvement of oxidative stress in the induction of bone marrow genetic damage after exposure to beryllium chloride. Gene expression analysis on the bone marrow cells from beryllium chloride-exposed mice showed significant alterations in genes associated with DNA damage repair. Therefore, beryllium chloride may cause genetic damage to bone marrow cells due to the oxidative stress and the induced unrepaired DNA damage is probably due to the down-regulation in the expression of DNA repair genes, which may lead to genotoxicity and eventually cause carcinogenicity.

  4. Isolation and Characterization of Two Saccharomyces Cerevisiae Genes Encoding Homologs of the Bacterial Hexa and Muts Mismatch Repair Proteins

    PubMed Central

    Reenan, R. A.; Kolodner, R. D.

    1992-01-01

    Homologs of the Escherichia coli (mutL, S and uvrD) and Streptococcus pneumoniae (hexA, B) genes involved in mismatch repair are known in several distantly related organisms. Degenerate oligonucleotide primers based on conserved regions of E. coli MutS protein and its homologs from Salmonella typhimurium, S. pneumoniae and human were used in the polymerase chain reaction (PCR) to amplify and clone mutS/hexA homologs from Saccharomyces cerevisiae. Two DNA sequences were amplified whose deduced amino acid sequences both shared a high degree of homology with MutS. These sequences were then used to clone the full-length genes from a yeast genomic library. Sequence analysis of the two MSH genes (MSH = mutS homolog), MSH1 and MSH2, revealed open reading frames of 2877 bp and 2898 bp. The deduced amino acid sequences predict polypeptides of 109.3 kD and 109.1 kD, respectively. The overall amino acid sequence identity with the E. coli MutS protein is 28.6% for MSH1 and 25.2% for MSH2. Features previously found to be shared by MutS homologs, such as the nucleotide binding site and the helix-turn-helix DNA binding motif as well as other highly conserved regions whose function remain unknown, were also found in the two yeast homologs. Evidence presented in this and a companion study suggest that MSH1 is involved in repair of mitochondrial DNA and that MSH2 is involved in nuclear DNA repair. PMID:1459447

  5. Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells

    PubMed Central

    Truong, Lan N.; Li, Yongjiang; Shi, Linda Z.; Hwang, Patty Yi-Hwa; He, Jing; Wang, Hailong; Razavian, Niema; Berns, Michael W.; Wu, Xiaohua

    2013-01-01

    Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ—even with very limited end resection—requires cyclin-dependent kinase activities and increases significantly when cells enter S phase. We also showed that MMEJ shares the initial end resection step with homologous recombination (HR) by requiring meiotic recombination 11 homolog A (Mre11) nuclease activity, which is needed for subsequent recruitment of Bloom syndrome protein (BLM) and exonuclease 1 (Exo1) to DNA double-strand breaks (DSBs) to promote extended end resection and HR. MMEJ does not require S139-phosphorylated histone H2AX (γ-H2AX), suggesting that initial end resection likely occurs at DSB ends. Using a MMEJ and HR competition repair substrate, we demonstrated that MMEJ with short end resection is used in mammalian cells at the level of 10–20% of HR when both HR and nonhomologous end joining are available. Furthermore, MMEJ is used to repair DSBs generated at collapsed replication forks. These studies suggest that MMEJ not only is a backup repair pathway in mammalian cells, but also has important physiological roles in repairing DSBs to maintain cell viability, especially under genomic stress. PMID:23610439

  6. Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori.

    PubMed

    Michielse, C B; Arentshorst, M; Ram, A F J; van den Hondel, C A M J J

    2005-01-01

    In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement.

  7. The Fanconi Anaemia Components UBE2T and FANCM Are Functionally Linked to Nucleotide Excision Repair

    PubMed Central

    Kelsall, Ian R.; Langenick, Judith; MacKay, Craig; Patel, Ketan J.; Alpi, Arno F.

    2012-01-01

    The many proteins that function in the Fanconi anaemia (FA) monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes – UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair. PMID:22615860

  8. The sensitivity of Cockayne's syndrome cells to DNA-damaging agents is not due to defective transcription-coupled repair of active genes.

    PubMed

    van Oosterwijk, M F; Versteeg, A; Filon, R; van Zeeland, A A; Mullenders, L H

    1996-08-01

    Two of the hallmarks of Cockayne's syndrome (CS) are the hypersensitivity of cells to UV light and the lack of recovery of the ability to synthesize RNA following exposure of cells to UV light, in spite of the normal repair capacity at the overall genome level. The prolonged repressed RNA synthesis has been attributed to a defect in transcription-coupled repair, resulting in slow removal of DNA lesions from the transcribed strand of active genes. This model predicts that the sensitivity of CS cells to another DNA-damaging agent, i.e., the UV-mimetic agent N-acetoxy-2-acetylaminofluorene (NA-AAF), should also be associated with a lack of resumption of RNA synthesis and defective transcription-coupled repair of NA-AAF-induced DNA adducts. We tested this by measuring the rate of excision of DNA adducts in the adenosine deaminase gene of primary normal human fibroblasts and two CS (complementation group A and B) fibroblast strains. High-performance liquid chromatography analysis of DNA adducts revealed that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the main adduct induced by NA-AAF in both normal and CS cells. No differences were found between normal and CS cells with respect to induction of this lesion either at the level of the genome overall or at the gene level. Moreover, repair of dG-C8-AF in the active adenosine deaminase gene occurred at similar rates and without strand specificity in normal and CS cells, indicating that transcription-coupled repair does not contribute significantly to repair of dG-C8-AF in active genes. Yet CS cells are threefold more sensitive to NA-AAF than are normal cells and are unable to recover the ability to synthesize RNA. Our data rule out defective transcription-coupled repair as the cause of the increased sensitivity of CS cells to DNA-damaging agents and suggest that the cellular sensitivity and the prolonged repressed RNA synthesis are primarily due to a transcription defect. We hypothesize that upon treatment of cells

  9. Impaired Cytogenetic Damage Repair and Cell Cycle Regulation in Response to Ionizing Radiation in Human Fibroblast Cells with Individual Knock-down of 25 Genes

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish; Jeevarajan, Antony; Pierson, Duane; 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 demonstrated that genes with upregulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. In our present study, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yield of MN and/or CA formation were significantly increased by suppressed expression of 5 genes that included Ku70 in the DSB repair pathway; XPA in the NER pathway; RPA1 in the MMR pathway; RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes including MRE11A, RAD51 in the DSB pathway, and SESN1 and SUMO1 showed significant inhibition of cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, p21 and MLH1 expression resulted in both enhanced cell cycle progression and significantly higher yield of cytogenetic damage, indicating the involvement of these gene products in both cell cycle control and DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

  10. Evaluation of adipose-derived stem cells for tissue-engineered muscle repair construct-mediated repair of a murine model of volumetric muscle loss injury.

    PubMed

    Kesireddy, Venu

    2016-01-01

    Volumetric muscle loss (VML) can occur from congenital defects, muscle wasting diseases, civilian or military injuries, and as a result of surgical removal of muscle tissue (eg, cancer), all of which can lead to irrevocable functional and cosmetic defects. Current tissue engineering strategies to repair VML often employ muscle-derived progenitor cells (MDCs) as one component. However, there are some inherent limitations in their in vitro culture expansion. Thus, this study explores the potential of adipose-derived stem cells (ADSCs) as an alternative cell source to MDCs for tissue engineering of skeletal muscle. A reproducible VML injury model in murine latissimus dorsi muscle was used to evaluate tissue-engineered muscle repair (TEMR) constructs incorporating MDCs or ADSCs. Importantly, histological analysis revealed that ADSC-seeded constructs displayed regeneration potential that was comparable to those seeded with MDCs 2 months postrepair. Furthermore, morphological analysis of retrieved constructs demonstrated signs of neotissue formation, including cell fusion, fiber formation, and scaffold remodeling. Immunohistochemistry demonstrated positive staining for both structural and functional proteins. Positive staining for vascular structures indicated the potential for long-term neotissue survival and integration with existing musculature. Qualitative observation of lentivirus-Cherry-labeled donor cells by immunohistochemistry indicates that participation of ADSCs in new hybrid myofiber formation incorporating donor cells was relatively low, compared to donor MDCs. However, ADSCs appear to participate in vascularization. In summary, I have demonstrated that TEMR constructs generated with ADSCs displayed skeletal muscle regeneration potential comparable to TEMR-MDC constructs as previously reported. PMID:27114706

  11. Evaluation of adipose-derived stem cells for tissue-engineered muscle repair construct-mediated repair of a murine model of volumetric muscle loss injury

    PubMed Central

    Kesireddy, Venu

    2016-01-01

    Volumetric muscle loss (VML) can occur from congenital defects, muscle wasting diseases, civilian or military injuries, and as a result of surgical removal of muscle tissue (eg, cancer), all of which can lead to irrevocable functional and cosmetic defects. Current tissue engineering strategies to repair VML often employ muscle-derived progenitor cells (MDCs) as one component. However, there are some inherent limitations in their in vitro culture expansion. Thus, this study explores the potential of adipose-derived stem cells (ADSCs) as an alternative cell source to MDCs for tissue engineering of skeletal muscle. A reproducible VML injury model in murine latissimus dorsi muscle was used to evaluate tissue-engineered muscle repair (TEMR) constructs incorporating MDCs or ADSCs. Importantly, histological analysis revealed that ADSC-seeded constructs displayed regeneration potential that was comparable to those seeded with MDCs 2 months postrepair. Furthermore, morphological analysis of retrieved constructs demonstrated signs of neotissue formation, including cell fusion, fiber formation, and scaffold remodeling. Immunohistochemistry demonstrated positive staining for both structural and functional proteins. Positive staining for vascular structures indicated the potential for long-term neotissue survival and integration with existing musculature. Qualitative observation of lentivirus-Cherry-labeled donor cells by immunohistochemistry indicates that participation of ADSCs in new hybrid myofiber formation incorporating donor cells was relatively low, compared to donor MDCs. However, ADSCs appear to participate in vascularization. In summary, I have demonstrated that TEMR constructs generated with ADSCs displayed skeletal muscle regeneration potential comparable to TEMR–MDC constructs as previously reported. PMID:27114706

  12. Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms.

    PubMed

    Kostyrko, Kaja; Mermod, Nicolas

    2016-04-01

    DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms. Transfection of these assay vectors in Chinese hamster ovary (CHO) cells and characterization of the repaired DNA sequences indicated that while simple MMEJ is able to mediate relatively efficient DSB repair if longer microhomologies are present, the majority of DSBs were repaired using the highly error-prone SD-MMEJ pathway. To validate the involvement of DNA synthesis in the repair process, siRNA knock-down of different genes proposed to play a role in MMEJ were performed, revealing that the knock-down of DNA polymerase θ inhibited DNA end resection and repair through simple MMEJ, thus favoring the other repair pathway. Overall, we conclude that this approach provides a convenient assay to study MMEJ-related DNA repair pathways.

  13. Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

    PubMed Central

    Sitaram, A; Plitas, G; Wang, W; Scicchitano, D A

    1997-01-01

    Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair. PMID:9001209

  14. POLYMORPHISMS IN THE DNA NUCLEOTIDE EXCISION REPAIR GENES AND LUNG CANCER RISK IN XUAN WEI, CHINA

    EPA Science Inventory

    The lung cancer mortality rate in Xuan Wei County, China is among the highest in the country and has been etiologically attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NE...

  15. Multiple Rad52-Mediated Homology-Directed Repair Mechanisms Are Required to Prevent Telomere Attrition-Induced Senescence in Saccharomyces cerevisiae

    PubMed Central

    2016-01-01

    Most human somatic cells express insufficient levels of telomerase, which can result in telomere shortening and eventually senescence, both of which are hallmarks of ageing. Homology-directed repair (HDR) is important for maintaining proper telomere function in yeast and mammals. In Saccharomyces cerevisiae, Rad52 is required for almost all HDR mechanisms, and telomerase-null cells senesce faster in the absence of Rad52. However, its role in preventing accelerated senescence has been unclear. In this study, we make use of rad52 separation-of-function mutants to find that multiple Rad52-mediated HDR mechanisms are required to delay senescence, including break-induced replication and sister chromatid recombination. In addition, we show that misregulation of histone 3 lysine 56 acetylation, which is known to be defective in sister chromatid recombination, also causes accelerated senescence. We propose a model where Rad52 is needed to repair telomere attrition-induced replication stress. PMID:27428329

  16. Repair of benzo[a]pyrene diol epoxide-DNA adducts in the DHFR gene of a human embryonic kidney cell line

    SciTech Connect

    Schild, L.J.; Baird, W.M.; Smith, C.A.

    1997-10-01

    Benzo[a]pyrene (BaP) can be metabolized in cells to (+) anti-BaP-7,8-diol-9,10-epoxide (BPDE), an ultimate carcinogen which binds extensively to dG in DNA. Chen et al. found that BPDE adducts were removed by transcription coupled repair in the HPRT gene of human fibroblast cultures. We examined repair of BPDE adducts in the amplified DHFR gene of a human fibroblast cultures. We examined repair of BPDE adducts in the amplified DHFR gene of a human embryonic kidney cell line, 293c18 mtx-r, using a laser cleavage-Southern analysis technique. Attempts to determine whether BPDE adducts were repaired by transcription coupled repair were very toxic to the cells and little total repair was observed. Cells treated with 1.5 {mu}M BPDE contained 26 pmol BPDE/mg DNA after 1 hr and 19 pmol BPDE/mg DNA after 24 hr. To determine if the toxicity of the BPDE affected repair in these cells, cultures were treated with 0.5 {mu}M and 1 {mu}M BPDE. Postlabeling and HPLC analysis of total adducts demonstrated that the 1{mu}M treatment resulted in 18 pmol BPDE/mg DNA at 1 hr and this was reduced to 3 pmol BPDE/mg DNA by 48 hr. Laser induced cleavage of KpnI-restricted DNA demonstrated breakdown of a 20 kb DHFR fragment. Analysis of the repair of BPDE-DNA adducts in the transcribed and non-transcribed strands of the DHFR gene are in progress.

  17. Defective DNA repair increases susceptibility to senescence through extension of Chk1-mediated G2 checkpoint activation

    PubMed Central

    Johmura, Yoshikazu; Yamashita, Emiri; Shimada, Midori; Nakanishi, Keiko; Nakanishi, Makoto

    2016-01-01

    Susceptibility to senescence caused by defective DNA repair is a major hallmark of progeroid syndrome patients, but molecular mechanisms of how defective DNA repair predisposes to senescence are largely unknown. We demonstrate here that suppression of DNA repair pathways extends the duration of Chk1-dependent G2 checkpoint activation and sensitizes cells to senescence through enhancement of mitosis skipping. Extension of G2 checkpoint activation by introduction of the TopBP1 activation domain and the nondegradable mutant of Claspin sensitizes cells to senescence. In contrast, a shortening of G2 checkpoint activation by expression of SIRT6 or depletion of OTUB2 reduces susceptibility to senescence. Fibroblasts from progeroid syndromes tested shows a correlation between an extension of G2 checkpoint activation and an increase in the susceptibility to senescence. These results suggest that extension of G2 checkpoint activation caused by defective DNA repair is critical for senescence predisposition in progeroid syndrome patients. PMID:27507734

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

    PubMed Central

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

    2014-01-01

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

  19. Decreased DNA repair gene XRCC1 expression is associated with radiotherapy-induced acute side effects in breast cancer patients.

    PubMed

    Batar, Bahadir; Guven, Gulgun; Eroz, Seda; Bese, Nuran Senel; Guven, Mehmet

    2016-05-10

    DNA repair plays a critical role in response to ionizing radiation (IR) and developing of radiotherapy induced normal tissue reactions. In our study, we investigated the association of radiotherapy related acute side effects, with X-ray repair cross complementing group 1 (XRCC1) and Poly (ADP-ribose) polymerase 1 (PARP1) DNA repair gene expression levels, their changes in protein expression and DNA damage levels in breast cancer patients. The study included 40 women with newly diagnosed breast cancer; an experimental case group (n=20) with acute side effects and the control group (n=20) without side effects. For gene and protein expression analysis, lymphocytes were cultured for 72 h and followed by in vitro 2 Gray (Gy) gamma-irradiation. For detection of DNA damage levels, lymphocytes were irradiated with in vitro 2 Gy gamma-rays and followed by incubation for 72 h. XRCC1 mRNA and protein expression levels were significantly higher in controls than in experimental cases (P=0.020). In terms of DNA damage levels, an increased frequency of micronucleus (MN) was observed in experimental cases versus controls, but this association was not significant (P=0.206). We also observed a significant negative correlation between MN frequency and XRCC1 protein levels in experimental (r=-0.469, P=0.037) vs control (r=-0.734, P<0.001). Our results suggested that decreased XRCC1 expression levels might be associated with the increased risk of therapeutic IR-related acute side effects in patients with breast cancer.

  20. Decreased DNA repair gene XRCC1 expression is associated with radiotherapy-induced acute side effects in breast cancer patients.

    PubMed

    Batar, Bahadir; Guven, Gulgun; Eroz, Seda; Bese, Nuran Senel; Guven, Mehmet

    2016-05-10

    DNA repair plays a critical role in response to ionizing radiation (IR) and developing of radiotherapy induced normal tissue reactions. In our study, we investigated the association of radiotherapy related acute side effects, with X-ray repair cross complementing group 1 (XRCC1) and Poly (ADP-ribose) polymerase 1 (PARP1) DNA repair gene expression levels, their changes in protein expression and DNA damage levels in breast cancer patients. The study included 40 women with newly diagnosed breast cancer; an experimental case group (n=20) with acute side effects and the control group (n=20) without side effects. For gene and protein expression analysis, lymphocytes were cultured for 72 h and followed by in vitro 2 Gray (Gy) gamma-irradiation. For detection of DNA damage levels, lymphocytes were irradiated with in vitro 2 Gy gamma-rays and followed by incubation for 72 h. XRCC1 mRNA and protein expression levels were significantly higher in controls than in experimental cases (P=0.020). In terms of DNA damage levels, an increased frequency of micronucleus (MN) was observed in experimental cases versus controls, but this association was not significant (P=0.206). We also observed a significant negative correlation between MN frequency and XRCC1 protein levels in experimental (r=-0.469, P=0.037) vs control (r=-0.734, P<0.001). Our results suggested that decreased XRCC1 expression levels might be associated with the increased risk of therapeutic IR-related acute side effects in patients with breast cancer. PMID:26826460

  1. Neurotrophic factor small-molecule mimetics mediated neuroregeneration and synaptic repair: emerging therapeutic modality for Alzheimer's disease.

    PubMed

    Kazim, Syed Faraz; Iqbal, Khalid

    2016-01-01

    Alzheimer's disease (AD) is an incurable and debilitating chronic progressive neurodegenerative disorder which is the leading cause of dementia worldwide. AD is a heterogeneous and multifactorial disorder, histopathologically characterized by the presence of amyloid β (Aβ) plaques and neurofibrillary tangles composed of Aβ peptides and abnormally hyperphosphorylated tau protein, respectively. Independent of the various etiopathogenic mechanisms, neurodegeneration is a final common outcome of AD neuropathology. Synaptic loss is a better correlate of cognitive impairment in AD than Aβ or tau pathologies. Thus a highly promising therapeutic strategy for AD is to shift the balance from neurodegeneration to neuroregeneration and synaptic repair. Neurotrophic factors, by virtue of their neurogenic and neurotrophic activities, have potential for the treatment of AD. However, the clinical therapeutic usage of recombinant neurotrophic factors is limited because of the insurmountable hurdles of unfavorable pharmacokinetic properties, poor blood-brain barrier (BBB) permeability, and severe adverse effects. Neurotrophic factor small-molecule mimetics, in this context, represent a potential strategy to overcome these short comings, and have shown promise in preclinical studies. Neurotrophic factor small-molecule mimetics have been the focus of intense research in recent years for AD drug development. Here, we review the relevant literature regarding the therapeutic beneficial effect of neurotrophic factors in AD, and then discuss the recent status of research regarding the neurotrophic factor small-molecule mimetics as therapeutic candidates for AD. Lastly, we summarize the preclinical studies with a ciliary neurotrophic factor (CNTF) small-molecule peptide mimetic, Peptide 021 (P021). P021 is a neurogenic and neurotrophic compound which enhances dentate gyrus neurogenesis and memory processes via inhibiting leukemia inhibitory factor (LIF) signaling pathway and increasing

  2. Regulated expression of the Saccharomyces cerevisiae DNA repair gene RAD7 in response to DNA damage and during sporulation.

    PubMed

    Jones, J S; Prakash, L; Prakash, S

    1990-06-11

    The RAD7 gene of Saccharomyces cerevisiae affects the proficiency of excision repair of DNA damaged by UV light. Here, we report our studies on the regulation of the RAD7 gene in response to UV irradiation and during sporulation. RAD7 transcript levels increased 6-fold within 40 min of exposure of cells to 37 J/m2 of UV light. Higher UV doses also elicited rapid increases in the level of RAD7 mRNA. RAD7 mRNA levels increased in sporulating MATa/MAT alpha diploid cells, but not in the asporogenous MATa/MATa strain exposed to sporulation conditions. The increase in RAD7 mRNA level in MATa/MAT alpha cells was 15-fold after 6 h and 9-fold after 7 h in sporulation medium; thereafter, RAD7 mRNA levels declined. Periodic transcription of RAD7 during sporulation suggests a role for RAD7 in this process.

  3. Diversification of the Primary Antibody Repertoire by AID-Mediated Gene Conversion.

    PubMed

    Lanning, Dennis K; Knight, Katherine L

    2015-01-01

    Gene conversion, mediated by activation-induced cytidine deaminase (AID), has been found to contribute to generation of the primary antibody repertoire in several vertebrate species. Generation of the primary antibody repertoire by gene conversion of immunoglobulin (Ig) genes occurs primarily in gut-associated lymphoid tissues (GALT) and is best described in chicken and rabbit. Here, we discuss current knowledge of the mechanism of gene conversion as well as the contribution of the microbiota in promoting gene conversion of Ig genes. Finally, we propose that the antibody diversification strategy used in GALT species, such as chicken and rabbit, is conserved in a subset of human and mouse B cells.

  4. Analysis of aberrant methylation in DNA repair genes during malignant transformation of human bronchial epithelial cells induced by cadmium.

    PubMed

    Zhou, Zhi-heng; Lei, Yi-xiong; Wang, Cai-xia

    2012-02-01

    Cadmium (Cd) and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood yet. Aberrant methylation was investigated in order to obtain insight into the DNA repair-related epigenetic mechanisms underlying CdCl(2)-induced malignant transformation of human bronchial epithelial cells (16HBE). Gene expression and DNA methylation were assessed in untreated control cells; 5th, 15th, and 35th passage of CdCl2-treated cells and tumorigenic cells (TCs) from nude mice by using high-performance liquid chromatography, real-time PCR, Western blot analysis, and methylation-specific PCR assay. During Cd-induced malignant transformation, global DNA methylation progressively increased and was associated with the overexpression of the DNA methyltransferase genes DNMT1 and DNMT3a but not DNMT3b. Expression of both the messenger RNA and proteins of the DNA repair genes (hMSH2, ERCC1, XRCC1, and hOGG1) progressively reduced and DNA damage increased with Cd-induced transformation. The promoter regions of hMSH2, ERCC1, XRCC1, and hOGG1 were heavily methylated in the 35th passage transformed cells and the TCs. The DNA demethylating agent 5-aza-2'-deoxycytidine could reverse the Cd-induced global DNA hypermethylation, DNMT hyperactivity, and the silencing of hMSH2, ERCC1, XRCC1, and hOGG1 in a time-dependent manner. The results indicate that DNMT1 and DNMT3a overexpression can result in global DNA hypermethylation and silencing of the hMSH2, ERCC1, XRCC1, and hOGG1 genes. They may partly explain the epigenetic mechanisms underlying the carcinogenesis due to Cd.

  5. Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene.

    PubMed Central

    Xiao, W; Derfler, B; Chen, J; Samson, L

    1991-01-01

    We previously identified and characterized biochemically an O6-methylguanine (O6MeG) DNA repair methyltransferase (MTase) in the yeast Saccharomyces cerevisiae and showed that it recognizes both O6MeG and O4-methylthymine (O4MeT) in vitro. Here we characterize the cloned S. cerevisiae O6MeG DNA MTase gene (MGT1) and determine its in vivo role in protecting yeast from DNA alkylation damage. We isolated a yeast DNA fragment that suppressed alkylation-induced killing and mutation in Escherichia coli ada ogt MTase deficient mutants and produced in these cells a protein similar to the yeast MTase. The cloned yeast fragment was mapped to chromosome IV and DNA sequencing identified an open reading frame, designated MGT1, which encodes a 188 amino acid protein with a molecular weight of 21,500 daltons. An 88 amino acid stretch of the MGT1 protein displays remarkable homology with four bacterial MTases and the human DNA MTase. S.cerevisiae mutants bearing an insertion in the MGT1 gene lacked DNA MTase activity and were very sensitive to alkylation induced killing and mutation. MGT1 transcript levels are not increased in response to DNA alkylation damage, nor is the MGT1 MTase involved in the regulation of the yeast 3-methyladenine DNA glycosylase gene (MAG). Expression of the MGT1 gene in E.coli prevented the induction by alkylating agents of both G:C to A:T and A:T to G:C transition mutations indicating that this eukaryotic MTase repairs both O6MeG and O4MeT in vivo. Images PMID:2065659

  6. The yellow fluorescent protein (YFP-H) mouse reveals neuroprotection as a novel mechanism underlying chondroitinase ABC-mediated repair after spinal cord injury.

    PubMed

    Carter, Lucy M; Starkey, Michelle L; Akrimi, Sonia F; Davies, Meirion; McMahon, Stephen B; Bradbury, Elizabeth J

    2008-12-24

    Chondroitinase ABC (ChABC) represents a promising therapeutic strategy for the treatment of spinal cord injury due to its potent effects on restoring function to spinal-injured adult mammals. However, there is limited mechanistic insight as to the underlying effects of ChABC treatment, where the effects are mediated, and which signaling pathways are involved in ChABC-mediated repair. Here we use a transgenic (YFP-H) mouse to demonstrate that cortical layer V projection neurons undergo severe atrophy 4 weeks after thoracic dorsal column injury and that ChABC is neuroprotective for these neurons after ICV infusion. ChABC also prevented cell atrophy after localized delivery to the spinal cord, suggesting a possible retrograde neuroprotective effect mediated at the injury site. Furthermore, neuroprotection of corticospinal cell somata coincided with increased axonal sprouting in the spinal cord. In addition, Western blot analysis of a number of kinases important in survival and growth signaling revealed a significant increase in phosphorylated ERK1 at the spinal injury site after in vivo ChABC treatment, indicating that activated ERK may play a role in downstream repair processes after ChABC treatment. Total forms of PKC and AKT were also elevated, indicating that modification of the glial scar by ChABC promotes long-lasting signaling changes at the lesion site. Thus, using the YFP-H mouse as a novel tool to study degenerative changes and repair after spinal cord injury we demonstrate, for the first time, that ChABC treatment regulates multiple signaling cascades at the injury site and exerts protective effects on axotomized corticospinal projection neurons.

  7. Frequency of mutations in mismatch repair genes in a population-based study of women with ovarian cancer

    PubMed Central

    Pal, T; Akbari, M R; Sun, P; Lee, J-H; Fulp, J; Thompson, Z; Coppola, D; Nicosia, S; Sellers, T A; McLaughlin, J; Risch, H A; Rosen, B; Shaw, P; Schildkraut, J; Narod, S A

    2012-01-01

    Background: Mutations in genes for hereditary non-polyposis colorectal cancer (HNPCC) in ovarian cancer patients remains poorly defined. We sought to estimate the frequency and characteristics of HNPCC gene mutations in a population-based sample of women with epithelial ovarian cancer. Methods: The analysis included 1893 women with epithelial ovarian cancer ascertained from three population-based studies. Full-germline DNA sequencing of the coding regions was performed on three HNPCC genes, MLH1, MSH2 and MSH6. Collection of demographic, clinical and family history information was attempted in all women. Results: Nine clearly pathogenic mutations were identified, including five in MSH6, two each in MLH1 and MSH2. In addition, 28 unique predicted pathogenic missense variants were identified in 55 patients. Pathogenic mutation carriers had an earlier mean age at diagnosis of ovarian cancer, overrepresentation of cancers with non-serous histologies and a higher number of relatives with HNPCC-related cancers. Conclusions: Our findings suggest that fewer than 1% of women with ovarian cancer harbour a germline mutation in the HNPCC genes, with overrepresentation of MSH6 mutations. This represents a lower-range estimate due to the large number of predicted pathogenic variants in which pathogenicity could not definitively be determined. Identification of mismatch repair gene mutations has the potential to impact screening and treatment decisions in these women. PMID:23047549

  8. Mismatch Repair in Schizosaccharomyces Pombe Requires the Mutl Homologous Gene Pms1: Molecular Cloning and Functional Analysis

    PubMed Central

    Schar, P.; Baur, M.; Schneider, C.; Kohli, J.

    1997-01-01

    Homologues of the bacterial mutS and mutL genes involved in DNA mismatch repair have been found in organisms from bacteria to humans. Here, we describe the structure and function of a newly identified Schizosaccharomyces pombe gene that encodes a predicted amino acid sequence of 794 residues with a high degree of homology to MutL related proteins. On the basis of its closer relationship to the eukaryotic ``PMS'' genes than to the ``MLH'' genes, we have designated the S. pombe homologue pms1. Disruption of the pms1 gene causes a significant increase of spontaneous mutagenesis as documented by reversion rate measurements. Tetrad analyses of crosses homozygous for the pms1 mutation reveal a reduction of spore viability from >92% to 80% associated with a low proportion (~50%) of meioses producing four viable spores and a significant, allele-dependent increase of the level of post-meiotic segregation of genetic marker allele pairs. The mutant phenotypes are consistent with a general function of pms1 in correction of mismatched base pairs arising as a consequence of DNA polymerase errors during DNA synthesis, or of hybrid DNA formation between homologous but not perfectly complementary DNA strands during meiotic recombination. PMID:9258673

  9. Heterogeneity of excision repair cross-complementation group 1 gene expression in non-small-cell lung cancer patients

    PubMed Central

    SMIRNOV, SERHEY; PASHKEVICH, ANASTASIYA; LIUNDYSHEVA, VALERIYA; BABENKO, ANDREY; SMOLYAKOVA, RAISA

    2015-01-01

    Excision repair cross-complementation group 1 (ERCC1) gene expression analysis is currently used widely in the molecular diagnosis of cancer. According to numerous studies, ERCC1 gene expression correlates with overall survival and effectiveness of chemotherapy with platinum agents. However, the degree of this correlation differs among various studies, with certain authors reporting a complete lack of such a correlation. These contradictions may be attributed to a number of factors, including the heterogeneity of the tumor tissue. In this study, we attempted to assess the degree of genetic heterogeneity exhibited by tissue samples obtained from non-small-cell lung cancer (NSCLC) through the expression of the ERCC1 gene. This study included 25 samples of tumor tissue from patients with a morphologically confirmed NSCLC diagnosis. A total of three randomized sections of each specimen were used. The ERCC1 gene expression was assessed by quantitative polymerase chain reaction (qPCR) in the TaqMan format. When planning the experiment and analysis of qPCR data, the MIQE guidelines were taken into consideration. We established that the coefficient of variation of the relative level of ERCC1 gene expression in the majority of the samples exceeded 33% (P<0.05), indicating the significant heterogeneity of the sample. We also demonstrated that the degree of heterogeneity of the tumor tissue is largely dependent on disease stage. PMID:25469300

  10. MicroRNA regulation of DNA repair gene expression in hypoxic stress.

    PubMed

    Crosby, Meredith E; Kulshreshtha, Ritu; Ivan, Mircea; Glazer, Peter M

    2009-02-01

    Genetic instability is a hallmark of cancer; the hypoxic tumor microenvironment has been implicated as a cause of this phenomenon. MicroRNAs (miR) are small nonprotein coding RNAs that can regulate various cellular pathways. We report here that two miRs, miR-210 and miR-373, are up-regulated in a hypoxia-inducible factor-1alpha-dependent manner in hypoxic cells. Bioinformatics analyses suggested that these miRs could regulate factors implicated in DNA repair pathways. Forced expression of miR-210 was found to suppress the levels of RAD52, which is a key factor in homology-dependent repair (HDR); the forced expression of miR-373 led to a reduction in the nucleotide excision repair (NER) protein, RAD23B, as well as in RAD52. Consistent with these results, both RAD52 and RAD23B were found to be down-regulated in hypoxia, but in both cases, the hypoxia-induced down-regulation could be partially reversed by antisense inhibition of miR-210 and miR-373. Importantly, luciferase reporter assays indicated that miR-210 is capable of interacting with the 3' untranslated region (UTR) of RAD52 and that miR-373 can act on the 3' UTR of RAD23B. These results indicate that hypoxia-inducible miR-210 and miR-373 play roles in modulating the expression levels of key proteins involved in the HDR and NER pathways, providing new mechanistic insight into the effect of hypoxia on DNA repair and genetic instability in cancer.

  11. Genetic variants in DNA repair genes as potential predictive markers for oxaliplatin chemotherapy in colorectal cancer.

    PubMed

    Kap, E J; Seibold, P; Richter, S; Scherer, D; Habermann, N; Balavarca, Y; Jansen, L; Becker, N; Pfütze, K; Popanda, O; Hoffmeister, M; Ulrich, A; Benner, A; Ulrich, C M; Burwinkel, B; Brenner, H; Chang-Claude, J

    2015-12-01

    Oxaliplatin-based chemotherapy exerts its effects through generating DNA damage. Hence, genetic variants in DNA repair pathways could modulate treatment response. We used a prospective cohort of 623 colorectal cancer patients with stage II-IV disease treated with adjuvant/palliative chemotherapy to comprehensively investigate 1727 genetic variants in the DNA repair pathways as potential predictive markers for oxaliplatin treatment. Single nucleotide polymorphisms (SNP) associations with overall survival and recurrence-free survival were assessed using a Cox regression model. Pathway analysis was performed using the gamma method. Patients carrying variant alleles of rs3783819 (MNAT1) and rs1043953 (XPC) experienced a longer overall survival after treatment with oxaliplatin than patients who did not carry the variant allele, while the opposite association was found in patients who were not treated with oxaliplatin (false discovery rate-adjusted P-values for heterogeneity 0.0047 and 0.0237, respectively). The nucleotide excision repair (NER) pathway was found to be most likely associated with overall survival in patients who received oxaliplatin (P-value=0.002). Our data show that genetic variants in the NER pathway are potentially predictive of treatment response to oxaliplatin.

  12. CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells.

    PubMed

    Zhu, Li; Mon, Hiroaki; Xu, Jian; Lee, Jae Man; Kusakabe, Takahiro

    2015-01-01

    Gene targeting can be achieved by precise genetic modifications through homology-directed repair (HDR) after DNA breaks introduced by genome editing tools such as CRISPR/Cas9 system. The most common form of HDR is homologous recombination (HR). Binding to the DNA breaks by HR factors is thought to compete with non-homologous end joining (NHEJ), an alternative DNA repair pathway. Here, we knocked out the factors in NHEJ by CRISPR/Cas9 system in silkworm cells, so that increased the activities of HR up to 7-fold. Also efficient HR-mediated genome editing events occurred between the chromosomal BmTUDOR-SN gene and donor DNA sequences with an EGFP gene in the middle of two homologous arms for the target gene. Utilizing the NHEJ-deficient silkworm cells, we found that homologous arms as short as 100 bp in donor DNA could be designed to perform precise genome editing. These studies should greatly accelerate investigations into genome editing of silkworm. PMID:26657947

  13. Polymorphisms in DNA repair genes as risk factors for asbestos-related malignant mesothelioma in a general population study.

    PubMed

    Dianzani, I; Gibello, L; Biava, A; Giordano, M; Bertolotti, M; Betti, M; Ferrante, D; Guarrera, S; Betta, G P; Mirabelli, D; Matullo, G; Magnani, C

    2006-07-25

    Differences in response to carcinogenic agents are due to the allelic variants of the genes that control it. Key genes are those involved in the repair of the DNA damage caused by such agents. This paper describes the results of a case-control epidemiological study designed to determine the genotypes of four of these genes in persons exposed to a single genotoxic factor, i.e. asbestos, who had or had not developed malignant mesothelioma (MM). Our working hypothesis was that an imperfect DNA repair, as revealed by subtle polymorphic variants, could reduce protection against the chronic DNA insult provoked by asbestos and eventually result in mutagenesis and cancer. Seven variants (i.e. XRCC1-R399Q-NCBI SNP, XRCC1-R194W, XRCC3-T241M, XRCC3-IVS6-14, XPD-K751Q, XPD-D312N, OGG1-S326C) were investigated in 81 patients and 110 age and sex-matched controls, all residents at Casale Monferrato, a Piedmontese town highly exposed to asbestos pollution. Unconditional multivariable logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). When considered as a categorical variable, XRCC1-399Q showed an increased OR both in heterozygotes (OR=2.08; 95% CI=1.00-4.33) and homozygotes (2.38; 95% CI=0.82-6.94), although individual ORs were not significant. When it was considered as a continuous variable OR was significant (OR=1.68; 95% CI: 1.02-2.75). When genotypes were divided into "non-risk" and "risk" genotypes, i.e. those thought to be associated with increased risk in the light of the functional significance of the variants, XRCC1-399Q (Q homozygotes+Q/R heterozygotes versus R homozygotes) had an OR=2.147 (95% CI: 1.08-4.28), whereas that of XRCC3-241T (T homozygotes+M/T heterozygotes versus M homozygotes) was 4.09 (95% CI: 1.26-13.21) and that of OGG1-326C was increased, though not significantly. None of the haplotypes showed a significantly different frequency between patients and controls. This is the first report of an association between

  14. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    SciTech Connect

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo

    2013-07-19

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways.

  15. Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells

    PubMed Central

    Zhu, Shenyin; Yan, Yu; Zhu, Yi; Li, Min; Wang, Zhigang; Xu, Ronald X.

    2015-01-01

    Ultrasound-targeted microbubble destruction (UTMD) technique can be potentially used for non-viral delivery of gene therapy. Targeting wild-type p53 (wtp53) tumor suppressor gene may provide a clinically promising treatment for patients with ovarian cancer. However, UTMD mediated gene therapy typically uses non-targeted microbubbles with suboptimal gene transfection efficiency. We synthesized a targeted microbubble agent for UTMD mediated wtp53 gene therapy in ovarian cancer cells. Lipid micro-bubbles were conjugated with a Luteinizing Hormone–Releasing Hormone analog (LHRHa) via an avidin– biotin linkage to target the ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFP-N1-wtp53 plasmid. Upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm2) for 30 s, the wtp53 gene was transfected to the ovarian cancer cells. The transfection efficiency was (43.90 ± 6.19)%. The expression of wtp53 mRNA after transfection was (97.08 ± 12.18)%. The cell apoptosis rate after gene therapy was (39.67 ± 5.95)%. In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher transfection efficiency and higher cell apoptosis rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene transfection efficiency in ovarian cancer cells. PMID:22841613

  16. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants. PMID:23821951

  17. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  18. UV-enhanced reactivation of a UV-damaged reporter gene suggests transcription-coupled repair is UV-inducible in human cells.

    PubMed

    Francis, M A; Rainbow, A J

    1999-01-01

    The genetic disorders xeroderma pigmentosum (XP) and Cockayne syndrome (CS) exhibit deficiencies in the repair of UV-induced DNA damage. CS fibroblasts retain proficient nucleotide excision repair (NER) of inactive (or bulk) DNA, but are deficient in the transcription-coupled repair (TCR) of active genes. In contrast, XP complementation group C (XP-C) fibroblasts retain proficient TCR, but are deficient in bulk DNA repair. The remaining NER-deficient XP groups exhibit deficiencies in both repair pathways. Ad5HCMVsp1lacZ is a recombinant adenovirus vector that is unable to replicate in human fibroblasts, but can efficiently infect and express the beta-galactosidase reporter gene in these cells. We have examined the host cell reactivation (HCR) of beta-galactosidase activity for UV-irradiated Ad5HCMVsp1lacZ in non-irradiated and UV-irradiated normal, XP-B, XP-C, XP-D, XP-F, XP-G, CS-A and CS-B fibroblasts. HCR of beta-galactosidase activity for UV-irradiated Ad5HCMVsp1lacZ was reduced in non-irradiated cells from each of the repair-deficient groups examined (including XP-C) relative to that in non-irradiated normal cells. Prior irradiation of cells with low UV fluences resulted in an enhancement of HCR for normal and XP-C strains, but not for the remaining XP and CS strains. HCR of the UV-damaged reporter gene in UV-irradiated XP and CS strains was similar to measurements of TCR reported previously for these cells. These results suggest that UV treatment results in an induced repair of UV-damaged DNA in the transcribed strand of an active gene in XP-C and normal cells through an enhancement of TCR or a mechanism which involves the TCR pathway. PMID:9934845

  19. Mutations Affecting Ty-Mediated Expression of the HIS4 Gene of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Winston, Fred; Chaleff, Deborah T.; Valent, Barbara; Fink, Gerald R.

    1984-01-01

    We have identified mutations in seven unlinked genes (SPT genes) that affect the phenotypes of Ty and δ insertion mutations in the 5' noncoding region of the HIS4 gene of S. cerevisiae. Spt mutants were selected for suppression of his4-912δ, a solo δ derivative of Ty912. Other Ty and δ insertions at HIS4 are suppressed by mutations in some but not all of the SPT genes. Only spt4 suppresses a non-Ty insertion at HIS4. In addition to their effects on Ty and δ insertions, mutations in several SPT genes show defects in general cellular functions—mating. DNA repair and growth. PMID:6329902

  20. Repair of endonuclease-induced double-strand breaks in Saccharomyces cerevisiae: essential role for genes associated with nonhomologous end-joining.

    PubMed Central

    Lewis, L K; Westmoreland, J W; Resnick, M A

    1999-01-01

    Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous end-joining (NHEJ) is not well characterized in the yeast Saccharomyces cerevisiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Galactose-induced expression of EcoRI endonuclease in rad50, mre11, or xrs2 mutants, which are deficient in plasmid DSB end-joining and some forms of recombination, resulted in G2 arrest and rapid cell killing. Endonuclease synthesis also produced moderate cell killing in sir4 strains. In contrast, EcoRI caused prolonged cell-cycle arrest of recombination-defective rad51, rad52, rad54, rad55, and rad57 mutants, but cells remained viable. Cell-cycle progression was inhibited in excision repair-defective rad1 mutants, but not in rad2 cells, indicating a role for Rad1 processing of the DSB ends. Phenotypic responses of additional mutants, including exo1, srs2, rad5, and rdh54 strains, suggest roles in recombinational repair, but not in NHEJ. Interestingly, the rapid cell killing in haploid rad50 and mre11 strains was largely eliminated in diploids, suggesting that the cohesive-ended DSBs could be efficiently repaired by homologous recombination throughout the cell cycle in the diploid mutants. These results demonstrate essential but separable roles for NHEJ pathway genes in the repair of chromosomal DSBs that are structurally similar to those occurring during cellular development. PMID:10430580

  1. Calmodulin Mediates DNA Repair Pathways Involving H2AX in Response to Low-Dose Radiation Exposure of RAW 264.7 Macrophages

    SciTech Connect

    Smallwood, Heather S.; Lopez Ferrer, Daniel; Eberlein, P. Elis; Watson, David J.; Squier, Thomas C.

    2009-02-05

    Understanding the molecular mechanisms that modulate macrophage radioresistance is necessary for the development of effective radiation therapies, as tumor-associated macrophages promote both angiogenesis and matrix remodeling that, in turn, enhance metastasis. In this respect, we have identified a dose-dependent increase in the abundance of the calcium regulatory protein calmodulin (CaM) in RAW 264.7 macrophages upon irradiation. CaM overexpression results in increased macrophage survival following radiation exposure, acting to diminish the sensitivity to low-dose exposures. Increases in CaM abundance also result in an increase in the number of phosphorylated histone H2AX protein complexes associated with DNA repair following macrophage irradiation, with no change in the extent of double-stranded DNA damage. In comparison, when NFκB-dependent pathways are inhibited, through the expression of a dominant-negative IκB construct, there is no significant increase in phosphorylated H2AX upon irradiation. These results indicate that the molecular basis for the up-regulation of histone H2AX mediated DNA-repair pathways is not the result of nonspecific NFκB-dependent pathways or a specific threshold of DNA damage. Rather, increases in CaM abundance act to minimize the low-dose hypersensitivity to radiation to enhance macrophage radioresistance through processes that include the upregulation of DNA repair pathways involving histone protein H2AX phosphorylation.

  2. RNAi-mediated gene knockdown and in vivo diuresis assay in adult female Aedes aegypti mosquitoes.

    PubMed

    Drake, Lisa L; Price, David P; Aguirre, Sarah E; Hansen, Immo A

    2012-07-14

    This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis. The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

  3. eIF4AII is dispensable for miRNA-mediated gene silencing

    PubMed Central

    Galicia-Vázquez, Gabriela; Chu, Jennifer; Pelletier, Jerry

    2015-01-01

    MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression through partial complementary base-pairing to the 3′ untranslated region (UTR) of target mRNAs. Inhibition of translation initiation has been identified as an early event of miRNA-mediated gene repression, but the underlying mechanistic details of this process are not well understood. Recently, eukaryotic initiation factor (eIF) 4AII was identified as a critical modulator of miRNA activity with depletion of this factor alleviating miRNA-mediated gene repression. Using the CRISPR/Cas9-editing system, we generated a novel cell line in which expression of eIF4AII was eliminated. The absence of eIF4AII does not affect cell viability, proliferation, or global mRNA translation. Importantly, we show that eIF4AII is dispensable for miRNA-mediated gene silencing. PMID:26286746

  4. DNA repair genes implicated in triple negative familial non-BRCA1/2 breast cancer predisposition

    PubMed Central

    Ollier, Marie; Radosevic-Robin, Nina; Kwiatkowski, Fabrice; Ponelle, Flora; Viala, Sandrine; Privat, Maud; Uhrhammer, Nancy; Bernard-Gallon, Dominique; Penault-Llorca, Frédérique; Bignon, Yves-Jean; Bidet, Yannick

    2015-01-01

    Among breast cancers, 10 to 15% of cases would be due to hereditary risk. In these familial cases, mutations in BRCA1 and BRCA2 are found in only 15% to 20%, meaning that new susceptibility genes remain to be found. Triple-negative breast cancers represent 15% of all breast cancers, and are generally aggressive tumours without targeted therapies available. Our hypothesis is that some patients with triple negative breast cancer could share a genetic susceptibility different from other types of breast cancers. We screened 36 candidate genes, using pyrosequencing, in all the 50 triple negative breast cancer patients with familial history of cancer but no BRCA1 or BRCA2 mutation of a population of 3000 families who had consulted for a familial breast cancer between 2005 and 2013. Any mutations were also sequenced in available relatives of cases. Protein expression and loss of heterozygosity were explored in tumours. Seven deleterious mutations in 6 different genes (RAD51D, MRE11A, CHEK2, MLH1, MSH6, PALB2) were observed in one patient each, except the RAD51D mutation found in two cases. Loss of heterozygosity in the tumour was found for 2 of the 7 mutations. Protein expression was absent in tumour tissue for 5 mutations. Taking into consideration a specific subtype of tumour has revealed susceptibility genes, most of them in the homologous recombination DNA repair pathway. This may provide new possibilities for targeted therapies, along with better screening and care of patients. PMID:26328243

  5. Polysaccharides of Aloe vera induce MMP-3 and TIMP-2 gene expression during the skin wound repair of rat.

    PubMed

    Tabandeh, Mohammad Reza; Oryan, Ahmad; Mohammadalipour, Adel

    2014-04-01

    Polysaccharides are the main macromolecules of Aloe vera gel but no data about their effect on extracellular matrix (ECM) elements are available. Here, mannose rich Aloe vera polysaccharides (AVP) with molecular weight between 50 and 250 kDa were isolated and characterized. Open cutaneous wounds on the back of 45 rats (control and treated) were daily treated with 25mg (n=15) and 50 mg (n=15) AVP for 30 days. The levels of MMP-3 and TIMP-2 gene expression were analyzed using real time PCR. The levels of n-acetyl glucosamine (NAGA), n-acetyl galactosamine (NAGLA) and collagen contents were also measured using standard biochemical methods. Faster wound closure was observed at day 15 post wounding in AVP treated animals in comparison with untreated group. At day 10 post wounding, AVP inhibited MMP-3 gene expression, while afterwards MMP-3 gene expression was upregulated. AVP enhanced TIMP-2 gene expression, collagen, NAGLA and NAGA synthesis in relation to untreated wounds. Our results suggest that AVP has positive effects on the regulation of ECM factor synthesis, which open up new perspectives for the wound repair activity of Aloe vera polysaccharide at molecular level.

  6. DNA repair genes implicated in triple negative familial non-BRCA1/2 breast cancer predisposition.

    PubMed

    Ollier, Marie; Radosevic-Robin, Nina; Kwiatkowski, Fabrice; Ponelle, Flora; Viala, Sandrine; Privat, Maud; Uhrhammer, Nancy; Bernard-Gallon, Dominique; Penault-Llorca, Frédérique; Bignon, Yves-Jean; Bidet, Yannick

    2015-01-01

    Among breast cancers, 10 to 15% of cases would be due to hereditary risk. In these familial cases, mutations in BRCA1 and BRCA2 are found in only 15% to 20%, meaning that new susceptibility genes remain to be found. Triple-negative breast cancers represent 15% of all breast cancers, and are generally aggressive tumours without targeted therapies available. Our hypothesis is that some patients with triple negative breast cancer could share a genetic susceptibility different from other types of breast cancers. We screened 36 candidate genes, using pyrosequencing, in all the 50 triple negative breast cancer patients with familial history of cancer but no BRCA1 or BRCA2 mutation of a population of 3000 families who had consulted for a familial breast cancer between 2005 and 2013. Any mutations were also sequenced in available relatives of cases. Protein expression and loss of heterozygosity were explored in tumours. Seven deleterious mutations in 6 different genes (RAD51D, MRE11A, CHEK2, MLH1, MSH6, PALB2) were observed in one patient each, except the RAD51D mutation found in two cases. Loss of heterozygosity in the tumour was found for 2 of the 7 mutations. Protein expression was absent in tumour tissue for 5 mutations. Taking into consideration a specific subtype of tumour has revealed susceptibility genes, most of them in the homologous recombination DNA repair pathway. This may provide new possibilities for targeted therapies, along with better screening and care of patients. PMID:26328243

  7. Base excision repair genes and risk of lung cancer among San Francisco Bay Area Latinos and African-Americans

    PubMed Central

    Chang, Jeffrey S.; Wrensch, Margaret R.; Hansen, Helen M.; Sison, Jennette D.; Aldrich, Melinda C.; Quesenberry, Charles P.; Seldin, Michael F.; Kelsey, Karl T.; Wiencke, John K.

    2009-01-01

    Base excision repair (BER) is the primary DNA damage repair mechanism for repairing small base lesions resulting from oxidation and alkylation damage. This study examines the association between 24 single-nucleotide polymorphisms (SNPs) belonging to five BER genes (XRCC1, APEX1, PARP1, MUTYH and OGG1) and lung cancer among Latinos (113 cases and 299 controls) and African-Americans (255 cases and 280 controls). The goal was to evaluate the differences in genetic contribution to lung cancer risk by ethnic groups. Analyses of individual SNPs and haplotypes were performed using unconditional logistic regressions adjusted for age, sex and genetic ancestry. Four SNPs among Latinos and one SNP among African-Americans were significantly (P < 0.05) associated with either risk of all lung cancer or non-small cell lung cancer (NSCLC). However, only the association between XRCC1 Arg399Gln (rs25487) and NSCLC among Latinos (odds ratio associated with every copy of Gln = 1.52; 95% confidence interval: 1.01–2.28) had a false-positive report probability of <0.5. Arg399Gln is a SNP with some functional evidence and has been shown previously to be an important SNP associated with lung cancer, mostly for Asians. Since the analyses were adjusted for genetic ancestry, the observed association between Arg399Gln and NSCLC among Latinos is unlikely to be confounded by population stratification; however, this result needs to be confirmed by additional studies among the Latino population. This study suggests that there are genetic differences in the association between BER pathway and lung cancer between Latinos and African-Americans. PMID:19029194

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

    Park, E; Guzder, S N; Koken, M H; Jaspers-Dekker, I; Weeda, G; Hoeijmakers, J H; Prakash, S; Prakash, L

    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(r) 95,356). The RAD25 (SSL2)- and XPBC-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 UV 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 Lys392 residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD25 (SSL2) ATPase/DNA helicase activity in viability. PMID:1333609

  10. Macrophage mediated PCI enhanced gene-directed enzyme prodrug therapy

    NASA Astrophysics Data System (ADS)

    Christie, Catherine E.; Zamora, Genesis; Kwon, Young J.; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2015-03-01

    Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

  11. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    PubMed

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  12. Fur-Mediated Activation of Gene Transcription in the Human Pathogen Neisseria gonorrhoeae

    PubMed Central

    Yu, Chunxiao

    2012-01-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  13. Baculovirus-mediated gene delivery and RNAi applications.

    PubMed

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-04-22

    Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism.

  14. Baculovirus-mediated Gene Delivery and RNAi Applications

    PubMed Central

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-01-01

    Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism. PMID:25912715

  15. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm‑2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  16. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm-2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  17. Proton Irradiation Sensitizes Radioresistant Non-small Cell Lung Cancer Cells by Modulating Epidermal Growth Factor Receptor-mediated DNA Repair.

    PubMed

    Park, Hyung Ju; Oh, Jeong Su; Chang, Jong Wook; Hwang, Sang-Gu; Kim, Jae-Sung

    2016-01-01

    Although proton radiotherapy is effective in treating various types of cancer, little is known on the biological responses triggered by proton irradiation. In the present study, we investigated protein profiles following proton irradiation of non-small cell lung cancer (NSCLC) cells and defined the role of proton-induced epidermal growth factor receptor (EGFR) expression in NSCLC cells. We found that proton irradiation more effectively sensitized NSCLC cells than gamma irradiation did. The expression profiles of radiosensitive and radioresistant NSCLC cells following proton and gamma irradiation were examined using antibody arrays. With regard to proteins, expression of EGFR was the most highly induced by proton irradiation. In addition, we found that EGFR inhibition with gefinitib significantly increased the radiosensitivity of NSCLC cells, and that increased radiosensitivity due to gefinitib was mediated by the suppression of DNA repair in radioresistant NSCLC cells. Thus, our data provide the first evidence that proton irradiation sensitizes radioresistant NSCLC cancer cells by modulating EGFR-mediated DNA repair.

  18. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    SciTech Connect

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  19. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

    PubMed

    Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

  20. Transcript length mediates developmental timing of gene expression across Drosophila.

    PubMed

    Artieri, Carlo G; Fraser, Hunter B

    2014-11-01

    The time required to transcribe genes with long primary transcripts may limit their ability to be expressed in cells with short mitotic cycles, a phenomenon termed intron delay. As such short cycles are a hallmark of the earliest stages of insect development, we tested the impact of intron delay on the Drosophila developmental transcriptome. We find that long zygotically expressed genes show substantial delay in expression relative to their shorter counterparts, which is not observed for maternally deposited transcripts. Patterns of RNA-seq coverage along transcripts show that this delay is consistent with their inability to completely transcribe long transcripts, but not with transcriptional initiation-based regulatory control. We further show that highly expressed zygotic genes maintain compact transcribed regions across the Drosophila phylogeny, allowing conservation of embryonic expression patterns. We propose that the physical constraints of intron delay affect patterns of expression and the evolution of gene structure of a substantial portion of the Drosophila transcriptome.

  1. Transcription mediated insulation and interference direct gene cluster expression switches.

    PubMed

    Nguyen, Tania; Fischl, Harry; Howe, Françoise S; Woloszczuk, Ronja; Serra Barros, Ana; Xu, Zhenyu; Brown, David; Murray, Struan C; Haenni, Simon; Halstead, James M; O'Connor, Leigh; Shipkovenska, Gergana; Steinmetz, Lars M; Mellor, Jane

    2014-11-19

    In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change.

  2. Transcription mediated insulation and interference direct gene cluster expression switches

    PubMed Central

    Nguyen, Tania; Brown, David; Murray, Struan C; Haenni, Simon; Halstead, James M; O'Connor, Leigh; Shipkovenska, Gergana; Steinmetz, Lars M; Mellor, Jane

    2014-01-01

    In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change. DOI: http://dx.doi.org/10.7554/eLife.03635.001 PMID:25407679

  3. Nanoparticle-mediated delivery of suicide genes in cancer therapy.

    PubMed

    Vago, Riccardo; Collico, Veronica; Zuppone, Stefania; Prosperi, Davide; Colombo, Miriam

    2016-09-01

    Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo, this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy.

  4. Gene expression profiling of the androgen independent prostate cancer cells demonstrates complex mechanisms mediating resistance to docetaxel

    PubMed Central

    Desarnaud, Frank; Geck, Peter; Parkin, Christopher; Carpinito, Gino

    2011-01-01

    The molecular mechanisms conferring resistance to docetaxel in prostate cancer patients remain partially understood. We generated docetaxel resistant derivatives of the androgen independent prostate cancer cell lines PC-3 and DU-145. Docetaxel rapidly induces DU-145 cell death via apoptosis and the drug resistant cells were produced by periodically exposing proliferating DU-145 cultures to small doses of docetaxel. In PC-3 cells docetaxel induces delayed cell death via mitotic catastrophe evident by profound multinucleation and formation of giant cells. Mononucleated progeny of the giant PC-3 cells shows significant resistance to docetaxel. Gene expression profiling of these docetaxel resistant PC-3 cells revealed sets of docetaxel inducible and constitutively expressed genes associated with major cancer pathways. A contradictory overlap with DU-145 docetaxel resistant cells was also found. Analyses suggested significant changes associated with apoptotic function, DNA repair, cell growth, survival and proliferation, metabolism, maintenance of cytoskeleton and extracellular matrix formation. These cellular processes often contribute to drug resistance and our study identified a set of genes managing this phenotype. Additional analyses of the drug resistant PC-3 cells using shRNA constructs determined direct relevance of Cyclin G2 to docetaxel resistance as well as prevention of multinucleation, whereas the knockdown of upregulated CYP1B1 showed no effect on either of these processes. Downregulated GBP1 was explored by ectopic overexpression and even though GBP1 has a potential to mediate resistance to docetaxel, it was not utilized in PC-3 cells. The results suggest complex combination of gene expression pattern changes that enables resistance to docetaxel while preventing death via multinucleation. PMID:21057205

  5. Evolutionary conservation of excision repair in Schizosaccharomyces pombe: evidence for a family of sequences related to the Saccharomyces cerevisiae RAD2 gene.

    PubMed Central

    Carr, A M; Sheldrick, K S; Murray, J M; al-Harithy, R; Watts, F Z; Lehmann, A R

    1993-01-01

    Cells mutated at the rad13 locus in the fission yeast, Schizosaccharomyces pombe are deficient in excision-repair of UV damage. We have cloned the S.pombe rad13 gene by its ability to complement the UV sensitivity of a rad13 mutant. The gene is not essential for cell proliferation. Sequence analysis of the cloned gene revealed an open reading-frame of 1113 amino acids with structural homology to the RAD2 gene of the distantly related Saccharomyces cerevisiae. The sequence similarity is confined to three domains, two close to the N-terminus of the encoded protein, the third being close to the C-terminus. The central region of about 500 amino acids shows little similarity between the two organisms. The first and third domains are also found in a related yet distinct pair of homologous S.pombe/S.cerevisiae DNA repair genes (rad2/YKL510), which have only a very short region between these two conserved domains. Using the polymerase chain reaction with degenerate primers, we have isolated fragments from a gene homologous to rad13/RAD2 from Aspergillus nidulans. These findings define new functional domains involved in excision-repair, as well as identifying a conserved family of genes related to RAD2. Images PMID:8464724

  6. Male infertility, genetic analysis of the DAZ genes on the human Y chromosome and genetic analysis of DNA repair.

    PubMed

    Fox, M S; Reijo Pera, R A

    2001-11-26

    Many genes that are required for fertility have been identified in model organisms (). Mutations in these genes cause infertility due to defects in development of the germ cell lineage, but the organism is otherwise healthy. Although human reproduction is undoubtedly as complex as that of other organisms, very few fertility loci have been mapped (). This is in spite of the prevalence of human infertility, the lack of effective treatments to remedy germ cell defects, and the cost to couples and society of assisted reproductive techniques. Fifteen percent of couples are infertile and half of all cases can be traced to the male partner. Aside from defects in sperm production, most infertile men are otherwise healthy. This review is divided into two distinct parts to discuss work that: (i) led to the identification of several genes on the Y chromosome that likely function in sperm production; and (ii) implicates DNA repair in male infertility via increased frequency of mutations in DNA from men with meiotic arrest. PMID:11694340

  7. LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

    PubMed Central

    van Ravesteyn, Thomas W.; Dekker, Marleen; Fish, Alexander; Sixma, Titia K.; Wolters, Astrid; Dekker, Rob J.; te Riele, Hein P. J.

    2016-01-01

    Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype. PMID:26951689

  8. Electroporation-mediated gene transfer directly to the swine heart.

    PubMed

    Hargrave, B; Downey, H; Strange, R; Murray, L; Cinnamond, C; Lundberg, C; Israel, A; Chen, Y-J; Marshall, W; Heller, R

    2013-02-01

    In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using three different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the electrocardiogram were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on the anterior wall of the left ventricle were treated. Animals were killed 48 h after injection and electroporation and gene expression was determined. Results were compared with sites in the heart that received plasmid injection but no electric pulses or were not treated. Gene expression was higher in all electroporated sites when compared with injection only sites demonstrating the robustness of this approach. Our results provide evidence that in vivo electroporation can be a safe and effective non-viral method for delivering genes to the heart, in vivo.

  9. Challenges and complexities in estimating both the functional impact and the disease risk associated with the extensive genetic variation in human DNA repair genes.

    PubMed

    Mohrenweiser, Harvey W; Wilson, David M; Jones, Irene M

    2003-05-15

    Individual risk and the population incidence of disease result from the interaction of genetic susceptibility and exposure. DNA repair is an example of a cellular process where genetic variation in families with extreme predisposition is documented to be associated with high disease likelihood, including syndromes of premature aging and cancer. Although the identification and characterization of new genes or variants in cancer families continues to be important, the focus of this paper is the current status of efforts to define the impact of polymorphic amino acid substitutions in DNA repair genes on individual and population cancer risk. There is increasing evidence that mild reductions in DNA repair capacity, assumed to be the consequence of common genetic variation, affect cancer predisposition. The extensive variation being found in the coding regions of DNA repair genes and the large number of genes in each of the major repair pathways results in complex genotypes with potential to impact cancer risk in the general population. The implications of this complexity for molecular epidemiology studies, as well as concepts that may make these challenges more manageable, are discussed. The concepts include both experimental and computational approaches that could be employed to develop predictors of disease susceptibility based on DNA repair genotype, focusing initially on studies to assess functional impact on individual proteins and pathways and then on molecular epidemiology studies to assess exposure-dependent health risk. In closing, we raise some of the non-technical challenges to the utilization of the full richness of the genetic variation to reduce disease occurrence and ultimately improve health care. PMID:12714187

  10. Mediator Kinase Inhibition Further Activates Super-Enhancer Associated Genes in AML

    PubMed Central

    Nitulescu, Ioana I.; Tangpeerachaikul, Anupong; Poss, Zachary C.; Da Silva, Diogo H.; Caruso, Brittany T.; Arefolov, Alexander; Fadeyi, Olugbeminiyi; Christie, Amanda L.; Du, Karrie; Banka, Deepti; Schneider, Elisabeth V.; Jestel, Anja; Zou, Ge; Si, Chong; Ebmeier, Christopher C.; Bronson, Roderick T.; Krivtsov, Andrei V.; Myers, Andrew G.; Kohl, Nancy E.; Kung, Andrew L.; Armstrong, Scott A.; Lemieux, Madeleine E.; Taatjes, Dylan J.; Shair, Matthew D.

    2015-01-01

    Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML. PMID:26416749

  11. Is repetitive wounding and bone marrow-derived stem cell mediated-repair an etiology of lung cancer development and dissemination?

    PubMed

    Haura, Eric B

    2006-01-01

    The prevailing view of lung cancer is multi-step progression of normal cells into cancer cells through gain of function oncogenes coupled with loss of tumor suppressor genes. This progression of genetic damage ultimately results in the hallmarks of cancer. This theory has strong support from studies finding genetic damage in early stage preneoplastic lesions in lung epithelial cells from current or former smokers. This paper discusses an alternate theory that lung cancer is a bone marrow stem cell derived disease. Chronic cigarette smoking results in lung inflammation and epithelial damage that activates a chronic wound repair program. Recent studies have demonstrated that ability of bone marrow derived stem cells to respond to epithelial wounding and contribute to epithelial repair. The identification of cancer stem cells that are distinct from the bulk tumor cells through their ability of self-renewal may suggest that such cells are important in the development of lung cancer. The evidence supporting the hypothesis along with its implications are discussed. Confirmation of the hypothesis would suggest that the transition time from a normal cell to overt cancer cell may be much shorter than that based on the multi-step cancer progression model. Additionally, if wounding in other organs is a beacon that attracts bone marrow derived tumor cells, efforts to ameliorate areas of epithelial injury and compensatory wounding may block bone marrow derived tumor cell homing, aberrant repair, and metastasis. Finally, a bone marrow derived lung cancer stem cell would require stem cell poisons for cure.

  12. slo K+ channel gene regulation mediates rapid drug tolerance

    NASA Astrophysics Data System (ADS)

    Ghezzi, Alfredo; Al-Hasan, Yazan M.; Larios, Leo E.; Bohm, Rudolf A.; Atkinson, Nigel S.

    2004-12-01

    Changes in neural activity caused by exposure to drugs may trigger homeostatic mechanisms that attempt to restore normal neural excitability. In Drosophila, a single sedation with the anesthetic benzyl alcohol changes the expression of the slo K+ channel gene and induces rapid drug tolerance. We demonstrate linkage between these two phenomena by using a mutation and a transgene. A mutation that eliminates slo expression prevents tolerance, whereas expression from an inducible slo transgene mimics tolerance in naïve animals. The behavioral response to benzyl alcohol can be separated into an initial phase of hyperkinesis and a subsequent phase of sedation. The hyperkinetic phase causes a drop in slo gene expression and makes animals more sensitive to benzyl alcohol. It is the sedative phase that stimulates slo gene expression and induces tolerance. We demonstrate that the expression level of slo is a predictor of drug sensitivity. drug abuse | potassium channel | transcription regulation

  13. Alterations of DNA repair genes in the NCI-60 cell lines and their predictive value for anticancer drug activity

    PubMed Central

    Sousa, Fabricio G.; Matuo, Renata; Tang, Sai-Wen; Rajapakse, Vinodh N.; Luna, Augustin; Sander, Chris; Varma, Sudhir; Simon, Paul H.G.; Doroshow, James H.; Reinhold, William C.; Pommier, Yves

    2015-01-01

    Loss of function of DNA repair (DNAR) genes is associated with genomic instability and cancer predisposition; it also makes cancer cells reliant on a reduced set of DNAR pathways to resist DNA-targeted therapy, which remains the core of the anticancer armamentarium. Because the landscape of DNAR defects across numerous types of cancers and its relation with drug activity have not been systematically examined, we took advantage of the unique drug and genomic databases of the US National Cancer Institute cancer cell lines (the NCI-60) to characterize 260 DNAR genes with respect to deleterious mutations and expression down-regulation; 169 genes exhibited a total of 549 function-affecting alterations, with 39 of them scoring as putative knockouts across 31 cell lines. Those mutations were compared to tumor samples from 12 studies of The Cancer Genome Atlas (TCGA) and The Cancer Cell Line Encyclopedia (CCLE). Based on this compendium of alterations, we determined which DNAR genomic alterations predicted drug response for 20,195 compounds present in the NCI-60 drug database. Among 242 DNA damaging agents, 202 showed associations with at least one DNAR genomic signature. In addition to SLFN11, the Fanconi anemia-scaffolding gene SLX4 (FANCP/BTBD12) stood out among the genes most significantly related with DNA synthesis and topoisomerase inhibitors. Depletion and complementation experiments validated the causal relationship between SLX4 defects and sensitivity to raltitrexed and cytarabine in addition to camptothecin. Therefore, we propose new rational uses for existing anticancer drugs based on a comprehensive analysis of DNAR genomic parameters. PMID:25758781

  14. Alterations of DNA repair genes in the NCI-60 cell lines and their predictive value for anticancer drug activity.

    PubMed

    Sousa, Fabricio G; Matuo, Renata; Tang, Sai-Wen; Rajapakse, Vinodh N; Luna, Augustin; Sander, Chris; Varma, Sudhir; Simon, Paul H G; Doroshow, James H; Reinhold, William C; Pommier, Yves

    2015-04-01

    Loss of function of DNA repair (DNAR) genes is associated with genomic instability and cancer predisposition; it also makes cancer cells reliant on a reduced set of DNAR pathways to resist DNA-targeted therapy, which remains the core of the anticancer armamentarium. Because the landscape of DNAR defects across numerous types of cancers and its relation with drug activity have not been systematically examined, we took advantage of the unique drug and genomic databases of the US National Cancer Institute cancer cell lines (the NCI-60) to characterize 260 DNAR genes with respect to deleterious mutations and expression down-regulation; 169 genes exhibited a total of 549 function-affecting alterations, with 39 of them scoring as putative knockouts across 31 cell lines. Those mutations were compared to tumor samples from 12 studies of The Cancer Genome Atlas (TCGA) and The Cancer Cell Line Encyclopedia (CCLE). Based on this compendium of alterations, we determined which DNAR genomic alterations predicted drug response for 20,195 compounds present in the NCI-60 drug database. Among 242 DNA damaging agents, 202 showed associations with at least one DNAR genomic signature. In addition to SLFN11, the Fanconi anemia-scaffolding gene SLX4 (FANCP/BTBD12) stood out among the genes most significantly related with DNA synthesis and topoisomerase inhibitors. Depletion and complementation experiments validated the causal relationship between SLX4 defects and sensitivity to raltitrexed and cytarabine in addition to camptothecin. Therefore, we propose new rational uses for existing anticancer drugs based on a comprehensive analysis of DNAR genomic parameters.

  15. Nanoparticle-mediated delivery of suicide genes in cancer therapy.

    PubMed

    Vago, Riccardo; Collico, Veronica; Zuppone, Stefania; Prosperi, Davide; Colombo, Miriam

    2016-09-01

    Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo, this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy. PMID:27436147

  16. Symposium overview: genetic polymorphisms in DNA repair and cancer risk.

    PubMed

    Hu, J J; Mohrenweiser, H W; Bell, D A; Leadon, S A; Miller, M S

    2002-11-15

    A symposium, Genetic Polymorphisms in DNA Repair and Cancer Risk, was presented at the 40th Annual Meeting of the Society of Toxicology, held in San Francisco, California, in March 2001. A brief report of the symposium was published (Kaiser, Science 292, 837-838, 2001). Molecular epidemiological studies have shown that polymorphic variants of genes involved in the metabolism and repair of carcinogens can act as cancer susceptibility genes. These variants of drug metabolic and DNA-repair enzymes either increase the activation of chemical carcinogens or decrease the cells' ability to detoxify/repair mutagenic damages. Although on an individual basis these variant alleles may only slightly change catalytic activity and increase cancer risk, their polymorphic frequency in the human population may contribute to a high proportion of cancer cases. Studies conducted over the past few years have identified variant alleles for a number of DNA-repair genes, some of which have been shown to change DNA-repair capacity. Identifying these genotypic alterations in DNA-repair enzymes and their association with cancer may help to elucidate the mechanisms of cancer etiology and to predict both disease risk and response to cancer therapy, since most antineoplastic treatments mediate their effects through DNA damage.

  17. LONG DISTANCE POLLEN-MEDIATED GENE FLOW FROM CREEPING BENTGRASS

    EPA Science Inventory

    Researchers from USEPA WED have measured gene flow from experimental fields of Roundup? herbicide resistant genetically modified (GM) creeping bentgrass a grass used primarily on golf courses, to compatible non-crop relatives. Using a sampling design based on the estimated time ...

  18. Transient gene expression mediated by integrase-defective retroviral vectors.

    PubMed

    Yu, Seung Shin; Dan, Kazuyuki; Chono, Hideto; Chatani, Emi; Mineno, Junichi; Kato, Ikunoshin

    2008-04-18

    Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

  19. RNAi-Mediated Inactivation of Autophagy Genes in Caenorhabditis elegans.

    PubMed

    Palmisano, Nicholas J; Meléndez, Alicia

    2016-02-01

    RNA interference (RNAi) is a process that results in the sequence-specific silencing of endogenous mRNA through the introduction of double-stranded RNA (dsRNA). In the nematode Caenorhabditis elegans, RNA inactivation can be used at any specific developmental stage or during adulthood to inhibit a given target gene. Investigators can take advantage of the fact that, in C. elegans, RNAi is unusual in that it is systemic, meaning that dsRNA can spread throughout the animal and can affect virtually all tissues except neurons. Here, we describe a protocol for the most common method to achieve RNAi in C. elegans, which is to feed them bacteria that express dsRNA complementary to a specific target gene. This method has various advantages, including the availability of libraries that essentially cover the whole genome, the ability to treat animals at any developmental stage, and that it is relatively cost effective. We also discuss how RNAi specific to autophagy genes has proven to be an excellent method to study the role of these genes in autophagy, as well as other cellular and developmental processes, while also highlighting the caveats that must be applied. PMID:26832686

  20. Myelin repair and functional recovery mediated by neural cell transplantation in a mouse model of multiple sclerosis

    PubMed Central

    Bai, Lianhua; Hecker, Jordan; Kerstetter, Amber; Miller, Robert H.

    2014-01-01

    Cellular therapies are becoming a major focus for the treatment of demyelinating diseases such as multiple sclerosis (MS), therefore it is important to identify the most effective cell types that promote myelin repair. Several components contribute to the relative benefits of specific cell types including the overall efficacy of the cell therapy, the reproducibility of treatment, the mechanisms of action of distinct cell types and the ease of isolation and generation of therapeutic populations. A range of distinct cell populations promote functional recovery in animal models of MS including neural stem cells and mesenchymal stem cells derived from different tissues. Each of these cell populations has advantages and disadvantages and likely works through distinct mechanisms. The relevance of such mechanisms to myelin repair in the adult central nervous system is unclear since the therapeutic cells are generally derived from developing animals. Here we describe the isolation and characterization of a population of neural cells from the adult spinal cord that are characterized by the expression of the cell surface glycoprotein NG2. In functional studies, injection of adult NG2+ cells into mice with ongoing MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) enhanced remyelination in the CNS while the number of CD3+ T cells in areas of spinal cord demyelination was reduced approximately three-fold. In vivo studies indicated that in EAE, NG2+ cells stimulated endogenous repair while in vitro they responded to signals in areas of induced inflammation by differentiating into oligodendrocytes. These results suggested that adult NG2+ cells represent a useful cell population for promoting neural repair in a variety of different conditions including demyelinating diseases such as MS. PMID:23471865

  1. The Cellular Response to Oxidatively Induced DNA Damage and Polymorphism of Some DNA Repair Genes Associated with Clinicopathological Features of Bladder Cancer.

    PubMed

    Savina, Nataliya V; Nikitchenko, Nataliya V; Kuzhir, Tatyana D; Rolevich, Alexander I; Krasny, Sergei A; Goncharova, Roza I

    2016-01-01

    Genome instability and impaired DNA repair are hallmarks of carcinogenesis. The study was aimed at evaluating the DNA damage response in H2O2-treated lymphocytes using the alkaline comet assay in bladder cancer (BC) patients as compared to clinically healthy controls, elderly persons, and individuals with chronic inflammations. Polymorphism in DNA repair genes involved in nucleotide excision repair (NER) and base excision repair (BER) was studied using the PCR-RFLP method in the Belarusian population to elucidate the possible association of their variations with both bladder cancer risk and clinicopathological features of tumors. The increased level of H2O2-induced DNA damage and a higher proportion of individuals sensitive to oxidative stress were found among BC patients as compared to other groups under study. Heterozygosity in the XPD gene (codon 751) increased cancer risk: OR (95% CI) = 1.36 (1.03-1.81), p = 0.031. The frequency of the XPD 312Asn allele was significantly higher in T ≥ 2 high grade than in T ≥ 2 low grade tumors (p = 0.036); the ERCC6 1097Val/Val genotype was strongly associated with muscle-invasive tumors. Combinations of homozygous wild type alleles occurred with the increased frequency in patients with non-muscle-invasive tumors suggesting that the maintenance of normal DNA repair activity may prevent cancer progression.

  2. The Cellular Response to Oxidatively Induced DNA Damage and Polymorphism of Some DNA Repair Genes Associated with Clinicopathological Features of Bladder Cancer

    PubMed Central

    Savina, Nataliya V.; Nikitchenko, Nataliya V.; Kuzhir, Tatyana D.; Rolevich, Alexander I.; Krasny, Sergei A.; Goncharova, Roza I.

    2016-01-01

    Genome instability and impaired DNA repair are hallmarks of carcinogenesis. The study was aimed at evaluating the DNA damage response in H2O2-treated lymphocytes using the alkaline comet assay in bladder cancer (BC) patients as compared to clinically healthy controls, elderly persons, and individuals with chronic inflammations. Polymorphism in DNA repair genes involved in nucleotide excision repair (NER) and base excision repair (BER) was studied using the PCR-RFLP method in the Belarusian population to elucidate the possible association of their variations with both bladder cancer risk and clinicopathological features of tumors. The increased level of H2O2-induced DNA damage and a higher proportion of individuals sensitive to oxidative stress were found among BC patients as compared to other groups under study. Heterozygosity in the XPD gene (codon 751) increased cancer risk: OR (95% CI) = 1.36 (1.03–1.81), p = 0.031. The frequency of the XPD 312Asn allele was significantly higher in T ≥ 2 high grade than in T ≥ 2 low grade tumors (p = 0.036); the ERCC6 1097Val/Val genotype was strongly associated with muscle-invasive tumors. Combinations of homozygous wild type alleles occurred with the increased frequency in patients with non-muscle-invasive tumors suggesting that the maintenance of normal DNA repair activity may prevent cancer progression. PMID:26649138

  3. Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line

    SciTech Connect

    Wu Mei; Zhang Zunzhen Che Wangjun

    2008-05-01

    Bleomycin (BLM) has been found to induce 8-oxoguanine and DNA strand breaks through producing oxidative free radicals, thereby leading to cell cycle arrest, apoptosis and cell death. Cellular DNA damage repair mechanisms such as single strand DNA break repair/base excision repair (BER) are responsible for removing bleomycin-induced DNA damage, therefore confer chemotherapeutic resistance to bleomycin. In this study, we have investigated if down-regulation of human 8-oxoguanine DNA glycosylase (hOGG1), an important BER enzyme, could alter cellular sensitivity to bleomycin, thereby reducing chemotherapeutic resistance in human tumor cell. A human lung cancer cell line with hOGG1 deficiency (A549-R) was created by ribozyme gene knockdown technique. Bleomycin cellular sensitivity and DNA/chromosomal damages were examined using MTT, colony forming assay, comet assay as well as micronucleus assay. We demonstrated that hOGG1 gene knockdown enhanced bleomycin cytotoxicity and reduced the ability of colony formation of the lung cancer cell lines. We further demonstrated that bleomycin-induced DNA strand breaks resulted in an increase of micronucleus rate. hOGG1 deficiency significantly reduced DNA damage repair capacity of the lung cancer cell lines. Our results indicated that hOGG1 deficiency allowed the accumulation of bleomycin-induced DNA damage and chromosomal breaks by compromising DNA damage repair capacity, thereby increasing cellular sensitivity to bleomycin.

  4. A Study of Molecular Signals Deregulating Mismatch Repair Genes in Prostate Cancer Compared to Benign Prostatic Hyperplasia

    PubMed Central

    Basu, Sanmitra; Majumder, Subhadipa; Bhowal, Ankur; Ghosh, Alip; Naskar, Sukla; Nandy, Sumit; Mukherjee, Subhabrata; Sinha, Rajan Kumar; Basu, Keya; Karmakar, Dilip; Banerjee, Soma; Sengupta, Sanghamitra

    2015-01-01

    Prostate cancer is one of the leading causes of mortality among aging males. There is an unmet requirement of clinically useful biomarkers for early detection of prostate cancer to reduce the liabilities of overtreatment and accompanying morbidity. The present population-based study investigates the factors disrupting expression of multiple functionally related genes of DNA mismatch repair pathway in prostate cancer patients to identify molecular attributes distinguishing adenocarcinoma from benign hyperplasia of prostate. Gene expression was compared between tissue samples from prostate cancer and benign prostatic hyperplasia using real-time-PCR, western blot and immunohistochemistry. Assessment of genotypes of seven single-nucleotide-polymorphisms of three MMR genes was conducted using PCR-coupled RFLP and sequencing. Promoter methylation was interrogated by methylation-specific-PCR and bisulfite-sequencing. Interaction between microRNAs and MMR genes was verified by 3'UTR-based dual luciferase assays. Concurrent reduction of three MMR genes namely hMLH1, hMSH6 and hMSH2 (34-85%, P<0.05) was observed in prostate cancer tissues. hMSH6 polymorphism rs1800932(Pro92Pro) conferred a borderline protection in cancer patients (OR = 0.33, 95% CI = 0.15-0.75). Relative transcript level of hMLH1 was inversely related (r = -0.59, P<0.05) with methylation quotient of its promoter which showed a significantly higher methylation density (P = 0.008, Z = -2.649) in cancer patients. hsa-miR-155, hsa-miR-141 and hsa-miR-21 gene expressions were significantly elevated (66-85%, P<0.05) in tumor specimens and negatively correlated (r = -0.602 to -0.527, P<0.05) with that of MMR genes. hsa-miR-155 & hsa-miR-141 and hsa-miR-155 & hsa-miR-21 were demonstrated to bind to their putative seed sequences in hMLH1 and hMSH6 3’UTRs respectively. Relatively higher expression of DNA methyl-transferases (DNMT1 and DNMT3b) and HIF-1α genes (34-50%, P<0.05) were also detected in tumor tissues

  5. Stromal cell-derived factor-1 mediates changes of bone marrow stem cells during the bone repair process.

    PubMed

    Okada, Kiyotaka; Kawao, Naoyuki; Yano, Masato; Tamura, Yukinori; Kurashimo, Shinzi; Okumoto, Katsumi; Kojima, Kotarou; Kaji, Hiroshi

    2016-01-01

    Osteoblasts, osteoclasts, chondrocytes, and macrophages that participate in the bone repair process are derived from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the roles of these stem cells during the repair of injured bone tissue are still unclear. In the present study, we examined the effects of bone defect on HSCs and MSCs in bone marrow and spleen in 75 mice and its mechanism. We analyzed the HSC and MSC populations in these tissues of a mouse with femoral bone damage by using flow cytometry. The number of HSCs in the bone marrow of mice with damaged femurs was significantly lower than the number of these cells in the bone marrow of the contralateral intact femurs on day 2 after injury. Meanwhile, the number of MSCs in the bone marrow of mice with damaged femurs was significantly higher than that of the contralateral femurs. Both intraperitoneal administration of AMD3100, a C-X-C chemokine receptor 4 (CXCR4) antagonist, and local treatment with an anti-stromal cell-derived factor-1 (SDF-1) antibody blunted the observed decrease in HSC and increase in MSC populations within the bone marrow of injured femurs. In conclusion, the present study revealed that there is a concurrent decrease and increase in the numbers of HSCs and MSCs, respectively, in the bone marrow during repair of mouse femoral bone damage. Furthermore, the SDF-1/CXCR4 system was implicated as contributing to the changes in these stem cell populations upon bone injury.

  6. Polymerase chain reaction-mediated gene synthesis: synthesis of a gene coding for isozyme c of horseradish peroxidase.

    PubMed Central

    Jayaraman, K; Fingar, S A; Shah, J; Fyles, J

    1991-01-01

    The synthesis of a gene coding for horseradish peroxidase (HRP, isozyme c; EC 1.11.1.7) is described using a polymerase chain reaction (PCR)-mediated gene synthesis approach developed in our laboratory. In this approach, all the oligonucleotides making up the gene are ligated in a single step by using the two outer oligonucleotides as PCR primers and the crude ligation mixture as the target. The PCR facilitates synthesis and purification of the gene simultaneously. The gene for HRP was synthesized by ligating all 40 oligonucleotides in a single step followed by PCR amplification. The gene was also synthesized from its fragments by using an overlap extension method similar to the procedure as described [Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. (1989) Gene 77, 61-68]. A method for combining different DNA fragments, in-frame, by using the PCR was also developed and used to synthesize the HRP gene from its gene fragments. This method is applicable to the synthesis of even larger genes and to combine any DNA fragments in-frame. After the synthesis, preliminary characterization of the HRP gene was also carried out by the PCR to confirm the arrangement of oligonucleotides in the gene. This was done by carrying out the PCR with several sets of primers along the gene and comparing the product sizes with the expected sizes. The gene and the fragments generated by PCR were cloned in Escherichia coli and the sequence was confirmed by manual and automated DNA sequencing. Images PMID:1851991

  7. RNAi Pathway Genes Are Resistant to Small RNA Mediated Gene Silencing in the Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Pompey, Justine M.; Morf, Laura; Singh, Upinder

    2014-01-01

    The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a “trigger” to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing. PMID:25198343

  8. RNAi pathway genes are resistant to small RNA mediated gene silencing in the protozoan parasite Entamoeba histolytica.

    PubMed

    Pompey, Justine M; Morf, Laura; Singh, Upinder

    2014-01-01

    The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a "trigger" to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing.

  9. Repair of uv damaged DNA: Genes and proteins of yeast and human. Progress report, November 1, 1991--April 15, 1992

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  10. The Saccharomyces cerevisiae RAD7 and RAD16 genes are required for inducible excision of endonuclease III sensitive-sites, yet are not needed for the repair of these lesions following a single UV dose.

    PubMed

    Scott, A D; Waters, R

    1997-01-31

    The RAD7 and RAD16 genes of Saccharomyces cerevisiae have roles in the repair of UV induced CPDs in nontranscribed genes [1], and in the repair of CPDs in the nontranscribed strand of transcribed genes [2]. Previously, we identified an inducible component to nucleotide excision repair (NER), which is absent in a rad16 delta strain [3]. We have examined the repair of UV induced endonuclease III sensitive-sites (EIIISS), and have shown repair of these lesions to proceed by NER but their removal from nontranscribed regions is independent of RAD7 and RAD16. Furthermore, EIIISS are repaired with equal efficiency from both transcribed and nontranscribed genes [4]. In order to dissect the roles of RAD7 and RAD16 in the above processes we examined the repair of EIIISS in the MAT alpha and HML alpha loci, which are, respectively, transcriptionally active and inactive in alpha haploid cells. These loci have elevated levels of these lesions after UV (in genomic DNA EIIISS constitute about 10% of total lesions, whereas CPDs are about 70% of total lesions). We have shown that excision of UV induced EIIISS is enhanced following a prior UV irradiation. No enhancement of repair was detected in either the rad7 delta or the rad16 delta mutant. The fact that RAD7 and RAD16 are not required for the repair of EIIISS per se yet are required for the enhanced excision of these lesions from MAT alpha and HML alpha suggests two possibilities. These genes have two roles in NER, namely in the repair of CPDs from nontranscribed sequences, and in enhancing NER itself regardless of whether these genes' products are required for the excision of the specific lesion being repaired. In the latter case, the induction of RAD7 and RAD16 may increase the turnover of complexes stalled in nontranscribed DNA so as to increase the availability of NER proteins for the repair of CPDs and EIIISS in all regions of the genome.

  11. Immunoglobulin genes undergo legitimate repair in human B cells not only after cis- but also frequent trans-class switch recombination.

    PubMed

    Laffleur, B; Bardet, S M; Garot, A; Brousse, M; Baylet, A; Cogné, M

    2014-01-01

    Immunoglobulin (Ig) genes specifically recruit activation-induced deaminase (AID) for 'on-target' DNA deamination, initiating either variable (V) region somatic hypermutation, or double-strand break intermediates of class switch recombination (CSR). Such breaks overwhelmingly undergo legitimate intra-Ig repair rather than rare illegitimate and potentially oncogenic junctions outside of Ig loci. We show that in human B cells, legitimate synapsis and repair efficiently join Ig genes whether physically linked on one chromosome or located apart on both alleles. This indicates mechanisms faithfully recognizing and/or pairing loci with homology in structure and accessibility, thus licensing interchromosomal trans-CSR junctions while usually preventing illegitimate interchromosomal recombination with AID off-target genes. Physical linkage of IgH genes in cis on the same allele just increases the likelihood of legitimate repair by another fourfold. The strongest force driving CSR might thus be recognition of legitimate target genes. Formation of IgH intra-allelic loops along this process would then constitute a consequence rather than a pre-requisite of this gene-pairing process.

  12. CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation

    PubMed Central

    Tsui, Shanli; Wang, Jie; Wang, Ling; Dai, Wei; Lu, Luo

    2016-01-01

    Background The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. Methods Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. Results Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. Conclusion Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate

  13. Double-strand break repair and colorectal cancer: gene variants within 3′ UTRs and microRNAs binding as modulators of cancer risk and clinical outcome

    PubMed Central

    Naccarati, Alessio; Rosa, Fabio; Vymetalkova, Veronika; Barone, Elisa; Jiraskova, Katerina; Di Gaetano, Cornelia; Novotny, Jan; Levy, Miroslav; Vodickova, Ludmila; Gemignani, Federica; Buchler, Tomas; Landi, Stefano

    2016-01-01

    Genetic variations in 3′ untranslated regions of target genes may affect microRNA binding, resulting in differential protein expression. microRNAs regulate DNA repair, and single-nucleotide polymorphisms in miRNA binding sites (miRSNPs) may account for interindividual differences in the DNA repair capacity. Our hypothesis is that miRSNPs in relevant DNA repair genes may ultimately affect cancer susceptibility and impact prognosis. In the present study, we analysed the association of polymorphisms in predicted microRNA target sites of double-strand breaks (DSBs) repair genes with colorectal cancer (CRC) risk and clinical outcome. Twenty-one miRSNPs in non-homologous end-joining and homologous recombination pathways were assessed in 1111 cases and 1469 controls. The variant CC genotype of rs2155209 in MRE11A was strongly associated with decreased cancer risk when compared with the other genotypes (OR 0.54, 95% CI 0.38–0.76, p = 0.0004). A reduced expression of the reporter gene was observed for the C allele of this polymorphism by in vitro assay, suggesting a more efficient interaction with potentially binding miRNAs. In colon cancer patients, the rs2155209 CC genotype was associated with shorter survival while the TT genotype of RAD52 rs11226 with longer survival when both compared with their respective more frequent genotypes (HR 1.63, 95% CI 1.06-2.51, p = 0.03 HR 0.60, 95% CI 0.41–0.89, p = 0.01, respectively). miRSNPs in DSB repair genes involved in the maintenance of genomic stability may have a role on CRC susceptibility and clinical outcome. PMID:26735576

  14. Genetic variants in DNA double-strand break repair genes and risk of salivary gland carcinoma: a case-control study.

    PubMed

    Xu, Li; Tang, Hongwei; El-Naggar, Adel K; Wei, Peng; Sturgis, Erich M

    2015-01-01

    DNA double strand break (DSB) repair is the primary defense mechanism against ionizing radiation-induced DNA damage. Ionizing radiation is the only established risk factor for salivary gland carcinoma (SGC). We hypothesized that genetic variants in DSB repair genes contribute to individual variation in susceptibility to SGC. To test this hypothesis, we conducted a case-control study in which we analyzed 415 single nucleotide polymorphisms (SNPs) in 45 DSB repair genes in 352 SGC cases and 598 controls. Multivariate logistic regression analysis was performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Rs3748522 in RAD52 and rs13180356 in XRCC4 were significantly associated with SGC after Bonferroni adjustment; ORs (95% CIs) for the variant alleles of these SNPs were 1.71 (1.40-2.09, P = 1.70 × 10(-7)) and 0.58 (0.45-0.74, P = 2.00 × 10(-5)) respectively. The genetic effects were modulated by histological subtype. The association of RAD52-rs3748522 with SGC was strongest for mucoepidermoid carcinoma (OR = 2.21, 95% CI: 1.55-3.15, P = 1.25 × 10(-5), n = 74), and the association of XRCC4-rs13180356 with SGC was strongest for adenoid cystic carcinoma (OR = 0.60, 95% CI: 0.42-0.87, P = 6.91 × 10(-3), n = 123). Gene-level association analysis revealed one gene, PRKDC, with a marginally significant association with SGC risk in non-Hispanic whites. To our knowledge, this study is the first to comprehensively evaluate the genetic effect of DSB repair genes on SGC risk. Our results indicate that genetic variants in the DSB repair pathways contribute to inter-individual differences in susceptibility to SGC and show that the impact of genetic variants differs by histological subtype. Independent studies are warranted to confirm these findings. PMID:26035306

  15. Sleeping Beauty-Mediated Drug Resistance Gene Transfer in Human Hematopoietic Progenitor Cells

    PubMed Central

    Hyland, Kendra A.; Olson, Erik R.; McIvor, R. Scott

    2015-01-01

    The Sleeping Beauty (SB) transposon system can insert sequences into mammalian chromosomes, supporting long-term expression of both reporter and therapeutic genes. Hematopoietic progenitor cells (HPCs) are an ideal therapeutic gene transfer target as they are used in therapy for a variety of hematologic and metabolic conditions. As successful SB-mediated gene transfer into human CD34+ HPCs has been reported by several laboratories, we sought to extend these studies to the introduction of a therapeutic gene conferring resistance to methotrexate (MTX), potentially providing a chemoprotective effect after engraftment. SB-mediated transposition of hematopoietic progenitors, using a transposon encoding an L22Y variant dihydrofolate reductase fused to green fluorescent protein, conferred resistance to methotrexate and dipyridamole, a nucleoside transport inhibitor that tightens MTX selection conditions, as assessed by in vitro hematopoietic colony formation. Transposition of individual transgenes was confirmed by sequence analysis of transposon–chromosome junctions recovered by linear amplification-mediated PCR. These studies demonstrate the potential of SB-mediated transposition of HPCs for expression of drug resistance genes for selective and chemoprotective applications. PMID:26176276

  16. Sleeping Beauty-Mediated Drug Resistance Gene Transfer in Human Hematopoietic Progenitor Cells.

    PubMed

    Hyland, Kendra A; Olson, Erik R; McIvor, R Scott

    2015-10-01

    The Sleeping Beauty (SB) transposon system can insert sequences into mammalian chromosomes, supporting long-term expression of both reporter and therapeutic genes. Hematopoietic progenitor cells (HPCs) are an ideal therapeutic gene transfer target as they are used in therapy for a variety of hematologic and metabolic conditions. As successful SB-mediated gene transfer into human CD34(+) HPCs has been reported by several laboratories, we sought to extend these studies to the introduction of a therapeutic gene conferring resistance to methotrexate (MTX), potentially providing a chemoprotective effect after engraftment. SB-mediated transposition of hematopoietic progenitors, using a transposon encoding an L22Y variant dihydrofolate reductase fused to green fluorescent protein, conferred resistance to methotrexate and dipyridamole, a nucleoside transport inhibitor that tightens MTX selection conditions, as assessed by in vitro hematopoietic colony formation. Transposition of individual transgenes was confirmed by sequence analysis of transposon-chromosome junctions recovered by linear amplification-mediated PCR. These studies demonstrate the potential of SB-mediated transposition of HPCs for expression of drug resistance genes for selective and chemoprotective applications.

  17. Lactase gene promoter fragments mediate differential spatial and temporal expression patterns in transgenic mice.

    PubMed

    Wang, Zhi; Maravelias, Charalambos; Sibley, Eric

    2006-04-01

    Lactase gene expression is spatiotemporally regulated during mammalian gut development. We hypothesize that distinct DNA control regions specify appropriate spatial and temporal patterning of lactase gene expression. In order to define regions of the lactase promoter involved in mediating intestine-specific and spatiotemporal restricted expression, transgenic mice harboring 100 bp, 1.3- and 2.0- kb fragments of the 5' flanking region of the rat lactase gene cloned upstream of a luciferase reporter were characterized. The 100-bp lactase promoter-reporter transgenic mouse line expressed maximal luciferase activity in the intestine with a posterior shift in spatial restriction and ectopic expression in the stomach and lung. The temporal pattern of expression mediated by the 1.3-kb promoter?reporter transgene increases with postnatal maturation in contrast with the postnatal decline mediated by the 2.0-kb promoter-reporter transgene and the endogenous lactase gene. The differential transgene expression patterns mediated by the lactase promoter fragments suggests that intestine-specific spatial and temporal control elements reside in distinct regions of the DNA sequences upstream of the lactase gene transcription start-site.

  18. Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

    SciTech Connect

    Yamaguchi, Kazuki; Feril, Loreto B.; Tachibana, Katsuro; Takahashi, Akira; Matsuo, Miki; Endo, Hitomi; Harada, Yoshimi; Nakayama, Juichiro

    2011-07-22

    Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  19. Negative regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12.

    PubMed Central

    Tsui, H C; Feng, G; Winkler, M E

    1997-01-01

    The MutS, MutL, and MutH proteins play major roles in several DNA repair pathways. We previously reported that the cellular amounts of MutS and MutH decreased by as much as 10-fold in stationary-phase cultures. Consequently, we tested whether the amounts of MutS, MutL, and MutH were regulated by two global regulators, RpoS (sigma38) and Hfq (HF-I [putative RNA chaperone]), which are involved in stationary-phase transition. We report here that mutations in hfq and rpoS reversed the stationary-phase down-regulation of the amounts of MutS and MutH. hfq regulation of the amount of MutS in stationary-phase cultures was mediated by RpoS-dependent and -independent mechanisms, whereas hfq regulation of the amount of MutH was mediated only through RpoS. Consistent with this interpretation, the amount of MutS but not MutH was regulated by Hfq, but not RpoS, in exponentially growing cells. The amount of MutL remained unchanged in rpoS, hfq-1, and rpoS+, hfq+ strains in exponentially growing and stationary-phase cultures and served as a control. The beta-galactosidase activities of single-copy mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally in exponentially growing cultures. RNase T2 protection assays revealed increased amounts of mutS transcript that are attributed to increased mutS transcript stability in hfq-1 mutants. Lack of Hfq also increased the amounts and stabilities of transcripts initiated from P(miaA) and P1hfqHS, two of the promoters for hfq, suggesting autoregulation, but did not change the half-life of bulk mRNA. These results suggest that the amounts of MutS and MutH may be adjusted in cells subjected to different stress conditions by an RpoS-dependent mechanism. In addition, Hfq directly or indirectly regulates several genes, including mutS, hfq, and miaA, by an RpoS-independent mechanism that destabilizes transcripts. PMID:9393714

  20. AAV-mediated gene targeting methods for human cells

    PubMed Central

    Khan, Iram F; Hirata, Roli K; Russell, David W

    2013-01-01

    Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10−5 to 10−2 per infected cell. these targeting frequencies are 1–4 logs higher than those obtained by conventional transfection or electroporation approaches. a wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by southern blots. this protocol (from vector design through a single round of targeting and screening) can be completed in ~10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks. PMID:21455185

  1. Ultrasound mediated delivery of drugs and genes to solid tumors

    PubMed Central

    Frenkel, Victor

    2008-01-01

    It has long been shown that therapeutic ultrasound can be used effectively to ablate solid tumors, and a variety of cancers are presently being treated in the clinic using these types of ultrasound exposures. There is, however, an ever-increasing body of preclinical literature that demonstrates how ultrasound energy can also be used non-destructively for increasing the efficacy of drugs and genes for improving cancer treatment. In this review, a summary of the most important ultrasound mechanisms will be given with a detailed description of how each one can be employed for a variety of applications. This includes the manner by which acoustic energy deposition can be used to create changes in tissue permeability for enhancing the delivery of conventional agents, as well as for deploying and activating drugs and genes via specially tailored vehicles and formulations. PMID:18474406

  2. Repressor-mediated tissue-specific gene expression in plants

    DOEpatents

    Meagher, Richard B.; Balish, Rebecca S.; Tehryung, Kim; McKinney, Elizabeth C.

    2009-02-17

    Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

  3. Identifying genes that mediate anthracyline toxicity in immune cells.

    PubMed

    Frick, Amber; Suzuki, Oscar T; Benton, Cristina; Parks, Bethany; Fedoriw, Yuri; Richards, Kristy L; Thomas, Russell S; Wiltshire, Tim

    2015-01-01

    The role of the immune system in response to chemotherapeutic agents remains elusive. The interpatient variability observed in immune and chemotherapeutic cytotoxic responses is likely, at least in part, due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at identifying genes underlying these chemotherapeutic cytotoxic effects on immune cells. Using genome-wide association studies (GWAS), we identified four genome-wide significant quantitative trait loci (QTL) that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. Of particular interest, a locus on chromosome 16 was significantly associated with cell viability following idarubicin administration (p = 5.01 × 10(-8)). Within this QTL lies App, which encodes amyloid beta precursor protein. Comparison of dose-response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those of C57BL/6J control mice (p < 0.05). In conclusion, the cellular screening approach coupled with GWAS led to the identification and subsequent validation of a gene involved in T-cell viability after idarubicin treatment. Previous studies have suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. Further investigations should include performing mechanistic studies, validating additional genes from the GWAS, including Ppfia1 and Ppfibp1, and ultimately translating the findings to in vivo and human studies.

  4. Mismatch repair gene defects contribute to the genetic basis of double primary cancers of the colorectum and endometrium.

    PubMed

    Millar, A L; Pal, T; Madlensky, L; Sherman, C; Temple, L; Mitri, A; Cheng, H; Marcus, V; Gallinger, S; Redston, M; Bapat, B; Narod, S

    1999-05-01

    Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome caused by germline defects of mismatch repair (MMR) genes. Endometrial cancer is the most common extracolonic neoplasm in HNPCC and is the primary clinical manifestation of the syndrome in some families. The cumulative incidence of endometrial cancer among HNPCC mutation carriers is high, estimated to be from 22 to 43%. We hypothesized that women with double primary cancers of the colorectum and endometrium are likely to be members of HNPCC families. In order to determine how frequently HNPCC manifests in the context of double primary cancers, we examined alterations of two MMR genes, hMSH2 and hMLH1, in 40 unrelated women affected with double primary cancers. These cases were identified using hospital-based and population-based cancer registries in Ontario, Canada. MMR gene mutations were screened by single-strand conformation polymorphism analysis and confirmed by direct sequencing. Eighteen percent (seven of 40) were found to harbor mutations of one of the two MMR genes. Analysis of colorectal and/or endometrial tumors of mutation-negative probands found microsatellite instability in seven of 20 cases. Six of seven mutation-positive probands had strong family histories suggestive of HNPCC. First degree relatives of mutation-positive probands had a very high relative risk (RR) of colorectal cancer (RR = 8.1, CI 3. 5-15.9) and endometrial cancer (RR = 23.8, CI 6.4-61.0). The relative risk of mutation-negative cases was 2.8 (CI 1.7-4.5) for colorectal cancer and 5.4 (CI 2.0-11.7) for endometrial cancer. We recommend that all double primary patients with cancers at these sites should have a genetic evaluation, including molecular analysis for HNPCC where appropriate. PMID:10196371

  5. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    SciTech Connect

    Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J

    1995-04-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

  6. Laminin mediates tissue-specific gene expression in mammary epithelia

    PubMed Central

    1995-01-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta- casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain. PMID:7730398

  7. Recent developments with the human repair genes ERCC2, ERCC4, and XRCC1

    SciTech Connect

    Thompson, L.H.; Caldecott, K.W.; Brookman, K.W.; Weber, C.A.; Salazar, E.S.; Takayama, K.; Fornace, A.J.

    1992-11-06

    ERCC2 was first identified as a gene on human chromosome 19 that complemented the UV sensitivity of CHO UV5 cells in somatic cell hybrids. Subsequent studies localized ERCC2 to the same chromosomal region (19q13.2--13.3) as the ERCC1 gene and showed that the two genes were less than 250 kb apart. Cloning of ERCC2 was accomplished by transfection of genomic DNA into UV5 cells and rescue of the gene from a secondary transformant. Recovery of the gene was aide