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Sample records for site-specific dna damage

  1. Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

    PubMed

    Grandchamp, Nicolas; Altémir, Dorothée; Philippe, Stéphanie; Ursulet, Suzanna; Pilet, Héloïse; Serre, Marie-Claude; Lenain, Aude; Serguera, Che; Mallet, Jacques; Sarkis, Chamsy

    2014-01-01

    Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable. PMID:24956106

  2. Viral induction of site-specific chromosome damage.

    PubMed

    Fortunato, Elizabeth A; Spector, Deborah H

    2003-01-01

    The advent of advanced cell culture and cytogenetics techniques in the 1950s opened a new avenue for research on the pathogenic interactions between animal viruses and their hosts. Studies of many viruses revealed their ability to nonspecifically induce cytogenetic damage to their host cell's chromosomes. However, only three viruses, the oncogenic adenoviruses, herpes simplex virus (HSV) and human cytomegalovirus (HCMV), have been found to cause non-random, site-specific chromosomal damage. Adenovirus (Ad) type 12 induces fragility at four distinct loci (RNU1, RNU2, RN5S and PSU1) in many different types of human cells. A common feature of these loci is that they contain a repeated array of transcriptionally active genes encoding small structural RNAs. Site-specific induction of breaks also requires the virally encoded E1B protein of M(r) 55000 and the C-terminus of the cellular p53 protein. Analysis of the induction of damage by HSV and HCMV necessitates consideration of several factors, including the strain of virus used, the timing of infection, the type of cell used, and the multiplicity of infection. Both HSV strains 1 and 2 are cytotoxic, although the former seems to be more proficient at inducing damage. At early times post infection, HSV induces breaks and specific uncoiling of the centromeres of chromosomes 1, 9 and 16. This is followed at later times by a more complete severing of all of the chromosomes, termed pulverisation. Damage by HSV requires viral entry and de novo viral protein synthesis, with immediate early viral proteins responsible for the induction of breaks and uncoiling and early gene products (most likely nucleases) involved in the extensive pulverisation seen later. HCMV has been studied primarily in permissive human fibroblasts. Its ability to induce specific damage in chromosome 1 at two loci, 1q21 and 1q42, was only recently revealed as the cells must be in S-phase when they are infected for the breaks to be observed. In contrast to

  3. Site-specifically modified oligodeoxyribonucleotides as templates for Escherichia coli DNA polymerase I.

    PubMed Central

    O'Connor, D; Stöhrer, G

    1985-01-01

    Oligodeoxyribonucleotides with site-specific modifications have been used as substrates for Escherichia coli DNA polymerase I holoenzyme and Klenow fragment. Modifications included the bulky guanine-8-aminofluorene adduct and a guanine oxidation product resembling the product of photosensitized DNA oxidation. By a combination of primers and "nick-mers", conditions of single-strand-directed DNA synthesis and nick-translation could be created. Our results show that the polymerase can bypass both types of lesions. Bypass occurs on a single-stranded template but is facilitated on a nicked, double-stranded template. Only purines, with guanine more favored than adenine, are incorporated across both lesions. Hesitation during bypass could not be detected. The results indicate that site-specifically modified oligonucleotides can be sensitive probes for the action of polymerases on damaged templates. They also suggest a function for polymerase I, in its nick-translation capacity, during DNA repair and mutagenesis. Images PMID:3887400

  4. STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

    PubMed Central

    Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

    2009-01-01

    Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

  5. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.

    PubMed Central

    McClelland, M; Nelson, M; Raschke, E

    1994-01-01

    Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes. PMID:7937074

  6. Targeting and Processing of Site-specific DNA Interstrand Crosslinks

    PubMed Central

    Vasquez, Karen M.

    2010-01-01

    DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, and thus ICL-inducing agents such as cyclophosphamide, melphalan, cisplatin, psoralen and mitomycin C have been used clinically as anti-cancer drugs for decades. ICLs can also be formed endogenously as a consequence of cellular metabolic processes. ICL-inducing agents continue to be among the most effective chemotherapeutic treatments for many cancers; however, treatment with these agents can lead to secondary malignancies, in part due to mutagenic processing of the DNA lesions. The mechanisms of ICL repair have been characterized more thoroughly in bacteria and yeast than in mammalian cells. Thus, a better understanding the molecular mechanisms of ICL processing offers the potential to improve the efficacy of these drugs in cancer therapy. In mammalian cells it is thought that ICLs are repaired by the coordination of proteins from several pathways, including nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR), translesion synthesis (TLS), and proteins involved in Fanconi anemia (FA). In this review, we focus on the potential functions of NER, MMR, and HR proteins in the repair of and response to ICLs in human cells and in mice. We will also discuss a unique approach, using psoralen covalently linked to triplex-forming oligonucleotides to direct ICLs to specific sites in the mammalian genome. PMID:20196133

  7. Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication.

    PubMed

    Yeo, Jung Eun; Wickramaratne, Susith; Khatwani, Santoshkumar; Wang, Yen-Chih; Vervacke, Jeffrey; Distefano, Mark D; Tretyakova, Natalia Y

    2014-08-15

    DNA-protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA-protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts. PMID:24918113

  8. Site-specific control of silica mineralization on DNA using a designed peptide.

    PubMed

    Ozaki, Makoto; Nagai, Kazuma; Nishiyama, Hiroto; Tsuruoka, Takaaki; Fujii, Satoshi; Endoh, Tamaki; Imai, Takahito; Tomizaki, Kin-Ya; Usui, Kenji

    2016-03-01

    We developed a site-specific method for precipitating inorganic compounds using organic compounds, DNA, and designed peptides with peptide nucleic acids (PNAs). Such a system for site-specific precipitation represents a powerful tool for use in nanobiochemistry and materials chemistry. PMID:26690695

  9. Germinal transmission of site-specific excised genomic DNA by the bacterial ParA resolvase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome engineering is an essential tool in research and product development. Behind some of the recent advances in plant gene transfer is the development of site-specific recombination systems that enable the precise manipulation of DNA, e.g. the deletion, integration or translocation of DNA. DNA ...

  10. CRISPR Outsourcing: Commissioning IHF for Site-Specific Integration of Foreign DNA at the CRISPR Array.

    PubMed

    Wei, Yunzhou; Terns, Michael P

    2016-06-16

    In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA. PMID:27315553

  11. Mapping site-specific endonuclease binding to DNA by direct imaging with AFM

    SciTech Connect

    Allison, D.P.; Thundat, T.; Doktycz, M.J.; Kerper, P.S.; Warmack, R.J.; Modrich, P.; Isfort, R.J.

    1995-12-31

    Physical mapping of DNA can be accomplished by direct AFM imaging of site specific proteins bound to DNA molecules. Using Gln-111, a mutant of EcoRI endonuclease with a specific affinity for EcoRI sites 1,000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 10{sup 4}, the authors demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS{sup +}) or two (pMP{sup 32}) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin gold prior to imaging. Image enhancement coupled with improvements in the preparation techniques for imaging large DNA molecules, such as lambda DNA (47 kb), has the potential to contribute to direct AFM restriction mapping of cosmid-sized genomic DNAs.

  12. Synthesis of DNA Oligodeoxynucleotides Containing Site-Specific 1,3-Butadiene- Deoxyadenosine Lesions

    PubMed Central

    Wickramaratne, Susith; Seiler, Christopher L.

    2016-01-01

    Post-oligomerization synthesis is a useful technique for preparing site-specifically modified DNA oligomers. This approach involves site-specific incorporation of inherently reactive halogenated nucleobases into DNA strands using standard solid phase synthesis, followed by post-oligomerization nucleophilic aromatic substitution (SNAr) reactions with carcinogen-derived synthons. In these reactions, the inherent reactivities of DNA and carcinogen-derived species are reversed: the modified DNA nucleobase acts as an electrophile, while the carcinogen-derived species acts as a nucleophile. In the present protocol, we describe the use of the post-oligomerization approach to prepare DNA strands containing site- and stereospecific N6-adenine and N1, N6-adenine adducts induced by epoxide metabolites of the known human and animal carcinogen, 1,3-butadiene (BD). The resulting oligomers containing site specific, structurally defined DNA adducts can be used in structural and biological studies to reveal the roles of specific BD adducts in carcinogenesis and mutagenesis. PMID:26344227

  13. Synthesis of Site-Specific DNA–Protein Conjugates and Their Effects on DNA Replication

    PubMed Central

    2015-01-01

    DNA–protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA–protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts. PMID:24918113

  14. Site-specifically arraying small molecules or proteins on DNA using an expanded genetic alphabet.

    PubMed

    Li, Zhengtao; Lavergne, Thomas; Malyshev, Denis A; Zimmermann, Jörg; Adhikary, Ramkrishna; Dhami, Kirandeep; Ordoukhanian, Phillip; Sun, Zhelin; Xiang, Jie; Romesberg, Floyd E

    2013-10-11

    A class of replicable unnatural DNA base pairs formed between d5SICS and either dMMO2, dDMO, or dNaM were developed. To explore the use of these pairs to produce site-specifically labeled DNA, the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase-mediated replication, and subsequent site-specific modification of the amplified DNA by Click chemistry is reported. With the d5SICS scaffold a propynyl ether linker is accommodated better than its aliphatic analogue, but not as well as the protected propargyl amine linker explored previously. It was also found that with the dMMO2 and dDMO analogues, the dMMO2 position para to the glycosidic linkage is best suited for linker attachment and that although aliphatic and ether-based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogues, a variety of them were used to site-selectively attach a biotin tag to the amplified DNA. Finally, we use d5SICS(CO) -dNaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps "evolution" of nanomaterials. PMID:24026962

  15. Site-specifically arraying small molecules or proteins on DNA using an expanded genetic alphabet

    PubMed Central

    Zimmermann, Jörg; Adhikary, Ramkrishna; Dhami, Kirandeep; Ordoukhanian, Phillip; Sun, Zhelin; Xiang, Jie; Romesberg, Floyd E.

    2014-01-01

    We have developed a class of replicable unnatural DNA base pairs formed between d5SICS and either dMMO2, dDMO, or dNaM. To explore the use of these pairs to produce site-specifically labeled DNA, we report the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase-mediated replication, and subsequent site-specific modification of the amplified DNA via Click chemistry. We find that with the d5SICS scaffold, a propynyl ether linker is accommodated better than its aliphatic analog, but not as well as the protected propargyl amine linker explored previously. We also find that with the dMMO2 and dDMO analogs, the dMMO2 position para to the glycosidic linkage is best suited for linker attachment, and that while aliphatic and ether-based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogs, a variety of them are used to site-selectively attach a biotin tag to the amplified DNA. Finally, we use d5SICSCO-dNaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps “evolution” of nanomaterials. PMID:24026962

  16. The Energetic Contribution of Induced Electrostatic Asymmetry to DNA Bending by a Site-Specific Protein

    PubMed Central

    Hancock, Stephen P.; Hiller, David A.; Perona, John J.; Jen-Jacobson, Linda

    2012-01-01

    DNA bending can be promoted by reducing the net negative electrostatic potential around phosphates on one face of the DNA, such that electrostatic repulsion among phosphates on the opposite face drives bending toward the less negative surface. To provide the first assessment of the energetic contribution to DNA bending when electrostatic asymmetry is induced by a site-specific DNA binding protein, we manipulated the electrostatics in the EcoRV endonuclease-DNA complex by mutation of cationic sidechains that contact DNA phosphates and/or by replacing a selected phosphate in each strand with uncharged methylphosphonate. Reducing the net negative charge at two symmetrically located phosphates on the concave DNA face contributes −2.3 to −0.9 kcal/mol (depending on position) to complex formation. In contrast, reducing negative charge on the opposing convex face produces a penalty of +1.3 kcal/mol. Förster resonance energy transfer experiments show that the extent of axial DNA bending (about 50°) is little affected in the modified complexes, implying that modification affects the energetic cost but not the extent of DNA bending. Kinetic studies show that favorable effects of induced electrostatic asymmetry on equilibrium binding derive primarily from a reduced rate of complex dissociation, suggesting stabilization of the specific complex between protein and markedly bent DNA. A smaller increase in the association rate may suggest that the DNA in the initial encounter complex is mildly bent. The data imply that protein-induced electrostatic asymmetry makes a significant contribution to DNA bending, but is not itself sufficient to drive full bending in the specific EcoRV-DNA complex. PMID:21167173

  17. Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases

    PubMed Central

    Krishnakumar, Radha; Grose, Carissa; Haft, Daniel H.; Zaveri, Jayshree; Alperovich, Nina; Gibson, Daniel G.; Merryman, Chuck; Glass, John I.

    2014-01-01

    Toward achieving rapid and large scale genome modification directly in a target organism, we have developed a new genome engineering strategy that uses a combination of bioinformatics aided design, large synthetic DNA and site-specific recombinases. Using Cre recombinase we swapped a target 126-kb segment of the Escherichia coli genome with a 72-kb synthetic DNA cassette, thereby effectively eliminating over 54 kb of genomic DNA from three non-contiguous regions in a single recombination event. We observed complete replacement of the native sequence with the modified synthetic sequence through the action of the Cre recombinase and no competition from homologous recombination. Because of the versatility and high-efficiency of the Cre-lox system, this method can be used in any organism where this system is functional as well as adapted to use with other highly precise genome engineering systems. Compared to present-day iterative approaches in genome engineering, we anticipate this method will greatly speed up the creation of reduced, modularized and optimized genomes through the integration of deletion analyses data, transcriptomics, synthetic biology and site-specific recombination. PMID:24914053

  18. Isolation and characterization of site-specific DNA-methyltransferases from Bacillus coagulans K.

    PubMed

    Svadbina, I V; Zelinskaya, N V; Kovalevskaya, N P; Zheleznaya, L A; Matvienko, N I

    2004-03-01

    Two site-specific DNA methyltransferases, M.BcoKIA and M.BcoKIB, were isolated from the thermophilic strain Bacillus coagulans K. Each of the methylases protects the recognition site 5'-CTCTTC-3'/5'-GAAGAG-3' from cleavage with the cognate restriction endonuclease BcoKI. It is shown that M.BcoKIB is an N6-adenine specific methylase and M.BcoKIA is an N4-cytosine specific methylase. According to bisulfite mapping, M.BcoKIA methylates the first cytosine in the sequence 5'-CTCTTC-3'. PMID:15061697

  19. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

    PubMed Central

    McDonald, James I.; Celik, Hamza; Rois, Lisa E.; Fishberger, Gregory; Fowler, Tolison; Rees, Ryan; Kramer, Ashley; Martens, Andrew; Edwards, John R.

    2016-01-01

    ABSTRACT Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate. PMID:27170255

  20. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation.

    PubMed

    McDonald, James I; Celik, Hamza; Rois, Lisa E; Fishberger, Gregory; Fowler, Tolison; Rees, Ryan; Kramer, Ashley; Martens, Andrew; Edwards, John R; Challen, Grant A

    2016-01-01

    Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate. PMID:27170255

  1. Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency

    PubMed Central

    Nishigaki, Yutaka; Martí, Ramon; Copeland, William C.; Hirano, Michio

    2003-01-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5′-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations. PMID:12813027

  2. Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency.

    PubMed

    Nishigaki, Yutaka; Martí, Ramon; Copeland, William C; Hirano, Michio

    2003-06-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5'-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations. PMID:12813027

  3. Site-specific DNA excision in transgenic rice with a cell-permeable cre recombinase.

    PubMed

    Cao, Ming-Xia; Huang, Jian-Qiu; Yao, Quan-Hong; Liu, Sheng-Jun; Wang, Cheng-Long; Wei, Zhi-Ming

    2006-01-01

    The removal of selected marker genes from transgenic plants is necessary to address biosafety concerns and to carry out further experiments with transgenic organisms. In the present study, the 12-amino-acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF)-4 was used as a carrier to deliver enzymatically active Cre proteins into living plant cells, and to produce a site-specific DNA excision in transgenic rice plants. The process, which made cells permeable to Cre recombinase-mediated DNA recombination, circumvented the need to express Cre under spatiotemporal control and was proved to be a simple and efficient system to achieve marker-free transgenic plants. The ultimate aim of the present study is to develop commercial rice cultivars free from selected marker genes to hasten public acceptance of transgenic crops. PMID:16382182

  4. ParA resolvase catalyzes site-specific excision of DNA from the Arabidopsis genome.

    PubMed

    Thomson, James G; Yau, Yuan-Yeu; Blanvillain, Robert; Nunes, Wylla M; Chiniquy, Dawn; Thilmony, Roger; Ow, David W

    2009-04-01

    The small serine resolvase ParA from bacterial plasmids RK2 and RP4 catalyzes the recombination of two identical 133 bp recombination sites known as MRS. Previously, we reported that ParA is active in the fission yeast Schizosaccharomyces pombe. In this work, the parA recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis lines harboring a chromosomally integrated MRS-flanked target. The ParA recombinase excised the MRS-flanked DNA and the excision event was detected in subsequent generations in the absence of ParA, indicating germinal transmission of the excision event. The precise site-specific deletion by the ParA recombination system in planta demonstrates that the ParA recombinase can be used to remove transgenic DNA, such as selectable markers or other introduced transgenes that are no longer desired in the final product. PMID:18704739

  5. DNA origami based visualization system for studying site-specific recombination events.

    PubMed

    Suzuki, Yuki; Endo, Masayuki; Katsuda, Yousuke; Ou, Keiyu; Hidaka, Kumi; Sugiyama, Hiroshi

    2014-01-01

    Site-specific recombination involves reciprocal exchange between defined DNA sites. The reaction initiates from the formation of a recombinase-DNA synaptic complex, in which two recombination sites arrange in an appropriate configuration. However, there is incomplete information about how the topological state of the substrate influences the synapsis and outcome of the reaction. Here, we show that Cre-mediated recombination can be regulated by controlling the orientation and topology of the loxP substrate in a DNA frame nanoscaffold. High-speed atomic force microscopy analyses revealed that the loxP-containing substrate strands in the antiparallel orientation can be recombined only through formation of synaptic complexes. By tethering Holliday junction (HJ) intermediates to DNA frames in different connection patterns and using them as a starting substrate, we found that the topological state of the HJ intermediates dictates the outcome of the resolution. Our approach should provide a new platform for structural-functional studies of various DNA targeting enzymes, especially which require formation of synaptic complexes. PMID:24328161

  6. Site-Specific Surface Functionalization of Gold Nanorods Using DNA Origami Clamps.

    PubMed

    Shen, Chenqi; Lan, Xiang; Lu, Xuxing; Meyer, Travis A; Ni, Weihai; Ke, Yonggang; Wang, Qiangbin

    2016-02-17

    Precise control over surface functionalities of nanomaterials offers great opportunities for fabricating complex functional nanoarchitectures but still remains challenging. In this work, we successfully developed a novel strategy to modify a gold nanorod (AuNR) with specific surface recognition sites using a DNA origami clamp. AuNRs were encapsulated by the DNA origami through hybridization of single-stranded DNA on the AuNRs and complementary capture strands inside the clamp. Another set of capture strands on the outside of the clamp create the specific recognition sites on the AuNR surface. By means of this strategy, AuNRs were site-specifically modified with gold nanoparticles at the top, middle, and bottom of the surface, respectively, to construct a series of well-defined heterostructures with controlled "chemical valence". Our study greatly expands the utility of DNA origami as a tool for building complex nanoarchitectures and represents a new approach for precise tailoring of nanomaterial surfaces. PMID:26824749

  7. Site-specific inter-strand cross-links of DNA duplexes

    PubMed Central

    Ye, Miao; Guillaume, Johan; Liu, Yu; Sha, Ruojie; Wang, Risheng

    2013-01-01

    We report the development of technology that allows inter-strand coupling across various positions within one turn of DNA. Four 2′-modified nucleotides were synthesized as protected phosphoramidites and incorporated into DNA oligonucleotides. The modified nucleotides contain either 5-atom or 16-atom linker components, with either amine or carboxylic acid functional groups at their termini, forming 10 or 32 atom (11 or 33 bond) linkages. Chemical coupling of the amine and carboxylate groups in designed strands resulted in the formation of an amide bond. Coupling efficiency as a function of trajectory distance between the individual linker components was examined. For those nucleotides capable of forming inter-strand cross-links (ICLs), coupling yields were found to depend on temperature, distance, and linker length, enabling several approaches that can control regioselective linkage. In the most favorable cases, the coupling yields are quantitative. Spectroscopic measurements of strands that were chemically cross-linked indicate that the global structure of the DNA duplex does not appear to be distorted from the B form after coupling. Thermal denaturing profiles of those strands were shifted to somewhat higher temperatures than those of their respective control duplexes. Thus, the robust amide ICLs formed by this approach are site-specific, do not destabilize the rest of the duplex, and only minimally perturb the secondary structure. PMID:23894693

  8. Melanoplus sanguinipes entomopoxvirus DNA topoisomerase: site-specific DNA transesterification and effects of 5'-bridging phosphorothiolates.

    PubMed

    Krogh, B O; Cheng, C; Burgin, A; Shuman, S

    1999-11-25

    Melanoplus sanguinipes entomopoxvirus (MsEPV) encodes a 328 amino acid polypeptide related to the type I topoisomerases of six other genera of vertebrate and insect poxviruses. The gene encoding MsEPV topoisomerase was expressed in bacteria, and the recombinant protein was purified by ion-exchange chromatography and glycerol gradient sedimentation. MsEPV topoisomerase, a monomeric protein, catalyzed the relaxation of supercoiled plasmid DNA at approximately 0.6 supercoils/s. Like other poxvirus topoisomerases, the MsEPV enzyme formed a covalent adduct with duplex DNA at the target sequence CCCTT downward arrow. The kinetic and equilibrium parameters of the DNA transesterification reaction of MsEPV topoisomerase were k(cl) = 0.3 s(-1) and K(cl) = 0.25. The introduction of a 5'-bridging phosphorothiolate at the scissile phosphate increased the cleavage equilibrium constant from 0.25 to >/=30. Similar phosphorothiolate effects were observed with vaccinia topoisomerase. Kinetic analysis of single-turnover cleavage and religation reactions established that the altered equilibrium was the result of a approximately 10(-4) decrement in the rate of topoisomerase-catalyzed attack of 5'-SH DNA on the DNA-(3'-phosphotyrosyl)-enzyme intermediate. 5'-bridging phosphorothiolates at the scissile phosphate and other positions within the CCCTT element had no significant effect on k(cl). PMID:10562506

  9. Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity

    SciTech Connect

    Joshi, Rakesh; Ho, Kwok Ki; Tenney, Kristen; Chen, Jui-Hui; Golden, Barbara L.; Gimble, Frederick S.

    2013-09-18

    Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G{sub +4} base pair for the wild-type A:T{sub +4} base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T{sub +4} were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T{sub +4} or the C:G{sub +4} base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G{sub +4} recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T{sub +4} target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G{sub +4} target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed -36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G{sub +4} substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.

  10. DNA Damage Response

    PubMed Central

    Giglia-Mari, Giuseppina; Zotter, Angelika; Vermeulen, Wim

    2011-01-01

    Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required. PMID:20980439

  11. Site-specific AGE modifications in the ECM: a role for glyoxal in protein damage in diabetes

    PubMed Central

    Voziyan, Paul; Brown, Kyle L.; Chetyrkin, Sergei; Hudson, Billy

    2014-01-01

    Non-enzymatic modification of proteins in hyperglycemia is a major proposed mechanism of diabetic complications. Specifically, advanced glycation end products (AGEs) derived from hyperglycemia-induced reactive carbonyl species (RCS) can have pathogenic consequences when they target functionally critical protein residues. Modification of a small number of these critical residues, often undetectable by the methodologies relying on measurements of total AGE levels, can cause significant functional damage. Therefore, detection of specific sites of protein damage in diabetes is central to understanding molecular basis of diabetic complications and for identification of biomarkers which are mechanistically linked to the disease. The current paradigm of RCS-derived protein damage places the major focus on methylglyoxal (MGO), an intermediate of cellular glycolysis. We propose that glyoxal (GO) is a major contributor to extracellular matrix (ECM) damage in diabetes. Here, we review the current knowledge and provide new data about GO-derived site-specific ECM modification in experimental diabetes. PMID:23492568

  12. A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation.

    PubMed

    Arakawa, Toshiya; Ohta, Tohru; Abiko, Yoshihiro; Okayama, Miki; Mizoguchi, Itaru; Takuma, Taishin

    2011-09-15

    DNA methylation is an important epigenetic modification that leads to a wide variety of biological functions, including transcription, growth and development, and diseases associated with altered gene expression such as cancers. However, tools to insert site-specific methylation into DNA for analyzing epigenetic functions are limited. Here we describe a novel polymerase chain reaction (PCR)-based approach to provide site-specific DNA methylation at any site, including CpG or CpNpG islands. This method is simple and versatile, and it consists of four steps to construct the DNA methylation vector: (I) design and synthesis of methylated primers, (II) PCR amplification, (III) isolation of single-stranded DNA, and (IV) annealing and ligation of isolated single-stranded DNAs. First we produced and validated a linear green fluorescence protein (GFP) vector by this method. Next we applied this method to introduce methyl groups into the promoter of the cyclooxygenase-2 (COX-2) gene and found that site-specific DNA methylation at the CRE element significantly altered COX-2 gene expression. These results demonstrate that this PCR-based approach is useful for the analysis of biological functions that depend on DNA methylation. PMID:21669180

  13. Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs.

    PubMed

    Gagné, Jean-Philippe; Ethier, Chantal; Defoy, Daniel; Bourassa, Sylvie; Langelier, Marie-France; Riccio, Amanda A; Pascal, John M; Moon, Kyung-Mee; Foster, Leonard J; Ning, Zhibin; Figeys, Daniel; Droit, Arnaud; Poirier, Guy G

    2015-06-01

    An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process. PMID:25800440

  14. DNA damage tolerance.

    PubMed

    Branzei, Dana; Psakhye, Ivan

    2016-06-01

    Accurate chromosomal DNA replication is fundamental for optimal cellular function and genome integrity. Replication perturbations activate DNA damage tolerance pathways, which are crucial to complete genome duplication as well as to prevent formation of deleterious double strand breaks. Cells use two general strategies to tolerate lesions: recombination to a homologous template, and trans-lesion synthesis with specialized polymerases. While key players of these processes have been outlined, much less is known on their choreography and regulation. Recent advances have uncovered principles by which DNA damage tolerance is regulated locally and temporally - in relation to replication timing and cell cycle stage -, and are beginning to elucidate the DNA dynamics that mediate lesion tolerance and influence chromosome structure during replication. PMID:27060551

  15. Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.

    PubMed

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Shirai, Makoto

    2016-06-01

    The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of β-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited β-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes. PMID:26870903

  16. Vascular nanomedicine: Site specific delivery of elastin stabilizing therapeutics to damaged arteries

    NASA Astrophysics Data System (ADS)

    Sinha, Aditi

    improved resistance to elastolytic digestion. We further show that the same polyphenols interact with monomeric tropoelastin released by the vascular cells and dramatically increasing their self-assembly in-vitro. In addition, we demonstrate the elastogenic ability of these polyphenols in aiding the crosslinking of tropoelastin released by aneurysmal cells converting it into mature elastin. Finally, we developed a nanoparticle system functionalized with elastin antibody on the surface that, upon systemic delivery, can recognize and bind to sites of damaged elastin in the aorta. We are able to show that this nanoparticle system works in representative animal models for MAC and AAA. These nanoparticles demonstrated spatial and functional specificity for degraded elastin. In conclusion, our work is focused on understanding the role of elastin degradation in vascular calcification and aortic aneurysms. We tested approaches to halt elastin degradation and to regenerate elastin in arteries so that homeostasis can be achieved.

  17. Mutagenesis by site-specific arylamine adducts in plasmid DNA: Enhancing replication of the adducted strand alters mutation frequency

    SciTech Connect

    Reid, T.M.; Lee, Meisie; King, C.M. )

    1990-07-03

    Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by >7-fold to 2.9% and of the AAF adduct by >12-fold to 0.75%. The AF adduct produced primarily single-base deletions in the absence of uracil but only base substitutions in the uracil-containing constructs. The AAF adduct produced mutations only in the uracil-containing DNA, which included both frame shifts and base substitutions. Mutations produced by both adducts were SOS dependent.

  18. Theory on the mechanism of site-specific DNA-protein interactions in the presence of traps.

    PubMed

    Niranjani, G; Murugan, R

    2016-01-01

    The speed of site-specific binding of transcription factor (TFs) proteins with genomic DNA seems to be strongly retarded by the randomly occurring sequence traps. Traps are those DNA sequences sharing significant similarity with the original specific binding sites (SBSs). It is an intriguing question how the naturally occurring TFs and their SBSs are designed to manage the retarding effects of such randomly occurring traps. We develop a simple random walk model on the site-specific binding of TFs with genomic DNA in the presence of sequence traps. Our dynamical model predicts that (a) the retarding effects of traps will be minimum when the traps are arranged around the SBS such that there is a negative correlation between the binding strength of TFs with traps and the distance of traps from the SBS and (b) the retarding effects of sequence traps can be appeased by the condensed conformational state of DNA. Our computational analysis results on the distribution of sequence traps around the putative binding sites of various TFs in mouse and human genome clearly agree well the theoretical predictions. We propose that the distribution of traps can be used as an additional metric to efficiently identify the SBSs of TFs on genomic DNA. PMID:27434174

  19. Phasing in the presence of severe site-specific radiation damage through dose-dependent modelling of heavy atoms.

    PubMed

    Schiltz, M; Dumas, P; Ennifar, E; Flensburg, C; Paciorek, W; Vonrhein, C; Bricogne, G

    2004-06-01

    The case of a brominated RNA crystal structure determination in which standard three-wavelength MAD phasing was unsuccessful because of fast X-ray-induced debromination was reinvestigated [Ennifar et al. (2002), Acta Cryst. D58, 1262-1268]. It was found that if the data are kept unmerged and if a dose-stamp is associated with each reflection measurement, dose-dependent occupancies can be refined for the Br atoms. Such a parametrization has been implemented in the macromolecular phasing program SHARP. Refining such dose-dependent occupancies on an unmerged data set gave a dramatic improvement, even for SAD phases from only the first wavelength (peak), and resulted in a good electron-density map after solvent flattening. The adverse effect of radiation damage has been turned into a beneficial one. The crucial difference is made by the use of unmerged data: phasing power is generated through the intensity differences of symmetry-related reflections recorded at different doses, i.e. corresponding to different states of the X-ray-induced debromination. This approach should prove useful in all situations of experimental phasing where site-specific radiation damage occurs unavoidably and undesirably and not only in cases in which radiation damage is purposely being created in order to demonstrate its potential usefulness. PMID:15159561

  20. Method for performing site-specific affinity fractionation for use in DNA sequencing

    DOEpatents

    Mirzabekov, A.D.; Lysov, Y.P.; Dubley, S.A.

    1999-05-18

    A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between the cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting the extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to the extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from the array. 14 figs.

  1. Method for performing site-specific affinity fractionation for use in DNA sequencing

    DOEpatents

    Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

    1999-01-01

    A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

  2. Ribonucleotide triggered DNA damage and RNA-DNA damage responses

    PubMed Central

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage. PMID:25692233

  3. Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities

    PubMed Central

    Berger, Michael F.; Philippakis, Anthony A.; Qureshi, Aaron M.; He, Fangxue S.; Estep, Preston W.; Bulyk, Martha L.

    2015-01-01

    Transcription factors (TFs) regulate the expression of genes involved in myriad cellular processes through sequence-specific interactions with DNA. In order to predict DNA regulatory elements and the TFs targeting them with greater accuracy, detailed knowledge of the binding preferences of TFs is needed. Protein binding microarray (PBM) technology permits rapid, high-throughput characterization of the in vitro DNA binding specificities of proteins1. Here, we present a novel, maximally compact, synthetic DNA sequence design that represents all possible DNA sequence variants of a given length k (i.e., all “k-mers”) on a single, universal microarray. We constructed such all k-mer microarrays covering all 10 base pair (bp) binding sites by converting high-density single-stranded oligonucleotide arrays to double-stranded DNA arrays. Using these microarrays, we comprehensively determined the binding specificities over a full range of affinities for five TFs of diverse structural classes from yeast, worm, mouse, and human. Importantly, the unbiased coverage of all k-mers permits an interrogation of binding site preferences, including nucleotide interdependencies, at unprecedented resolution. PMID:16998473

  4. Site-Specific DNA Structural and Dynamic Features Revealed by Nucleotide-Independent Nitroxide Probes

    SciTech Connect

    Popova, Anna; Kalai, Tamas; Hideg, Kalman; Qin, Peter Z.

    2009-09-15

    In site-directed spin labeling, a covalently attached nitroxide probe containing a chemically inert unpaired electron is utilized to obtain information on the local environment of the parent macromolecule. Studies presented here examine the feasibility of probing local DNA structural and dynamic features using a class of nitroxide probes that are linked to chemically substituted phosphorothioate positions at the DNA backbone. Two members of this family, designated as R5 and R5a, were attached to eight different sites of a dodecameric DNA duplex without severely perturbing the native B-form conformation. Measured X-band electron paramagnetic resonance (EPR) spectra, which report on nitroxide rotational motions, were found to vary depending on the location of the label (e.g., duplex center vs termini) and the surrounding DNA sequence. This indicates that R5 and R5a can provide information on the DNA local environment at the level of an individual nucleotide. As these probes can be attached to arbitrary nucleotides within a nucleic acid sequence, they may provide a means to “scan” a given DNA molecule in order to interrogate its local structural and dynamic features.

  5. Eubacterial SpoVG Homologs Constitute a New Family of Site-Specific DNA-Binding Proteins

    PubMed Central

    Jutras, Brandon L.; Chenail, Alicia M.; Rowland, Christi L.; Carroll, Dustin; Miller, M. Clarke; Bykowski, Tomasz; Stevenson, Brian

    2013-01-01

    A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG α-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent β-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins. PMID:23818957

  6. Theory of site-specific interactions of the combinatorial transcription factors with DNA

    NASA Astrophysics Data System (ADS)

    Murugan, R.

    2010-05-01

    We derive a functional relationship between the mean first passage time associated with the concurrent binding of multiple transcription factors (TFs) at their respective combinatorial cis-regulatory module sites (CRMs) and the number n of TFs involved in the regulation of the initiation of transcription of a gene of interest. Our results suggest that the overall search time τs that is required by the n TFs to locate their CRMs which are all located on the same DNA chain scales with n as τs~nα where α ~ (2/5). When the jump size k that is associated with the dynamics of all the n TFs along DNA is higher than that of the critical jump size kc that scales with the size of DNA N as kc ~ N2/3, we observe a similar power law scaling relationship and also the exponent α. When k < kc, α shows a strong dependence on both n and k. Apparently there is a critical number of combinatorial TFs nc ~ 20 that is required to efficiently regulate the initiation of transcription of a given gene below which (2/5) < α < 1 and beyond which α > 1. These results seem to be independent of the initial distances between the TFs and their corresponding CRMs and also suggest that the maximum number of TFs involved in a given combinatorial regulation of the initiation of transcription of a gene of interest seems to be restricted by the degree of condensation of the genomic DNA. The optimum number mopt of roadblock protein molecules per genome at which the search time associated with these n TFs to locate their binding sites is a minimum seems to scale as mopt~Lnα/2 where L is the sliding length of TFs whose maximum value seems to be such that L <= 104 bps for the E. coli bacterial genome.

  7. Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

    PubMed

    Smidt, M P; Russchen, B; Snippe, L; Wijnholds, J; Ab, G

    1995-07-11

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. PMID:7630716

  8. Site-specific hydrolysis and alcoholysis of human immunodeficiency virus DNA termini mediated by the viral integrase protein.

    PubMed Central

    Vink, C; Yeheskiely, E; van der Marel, G A; van Boom, J H; Plasterk, R H

    1991-01-01

    Before integration of the human immunodeficiency virus (HIV) DNA, two nucleotides are removed from the 3' ends of the viral DNA by the integrase (IN) protein. We studied the chemistry of this reaction, and found that IN mediates site-specific hydrolysis of a phosphodiester bond, resulting in release of a dinucleotide. A class of alcohols (including glycerol, 1,2-propanediol, but not 1,3-propanediol) can also act as nucleophile in this reaction, and likewise the alcoholic amino acids L-serine and L-threonine can be covalently linked to the dinucleotide. No evidence was found for a covalent linkage between the IN protein and this dinucleotide, suggesting that IN directs a single nucleophilic attack of water at the specific phosphodiester bond. Images PMID:1662361

  9. Subunit composition determines E2F DNA-binding site specificity.

    PubMed Central

    Tao, Y; Kassatly, R F; Cress, W D; Horowitz, J M

    1997-01-01

    The product of the retinoblastoma (Rb) susceptibility gene, Rb-1, regulates the activity of a wide variety of transcription factors, such as E2F, in a cell cycle-dependent fashion. E2F is a heterodimeric transcription factor composed of two subunits each encoded by one of two related gene families, denoted E2F and DP. Five E2F genes, E2F-1 through E2F-5, and two DP genes, DP-1 and DP-2, have been isolated from mammals, and heterodimeric complexes of these proteins are expressed in most, if not all, vertebrate cells. It is not yet clear whether E2F/DP complexes regulate overlapping and/or specific cellular genes. Moreover, little is known about whether Rb regulates all or a subset of E2F-dependent genes. Using recombinant E2F, DP, and Rb proteins prepared in baculovirus-infected cells and a repetitive immunoprecipitation-PCR procedure (CASTing), we have identified consensus DNA-binding sites for E2F-1/DP-1, E2F-1/DP-2, E2F-4/DP-1, and E2F-4/DP-2 complexes as well as an Rb/E2F-1/DP-1 trimeric complex. Our data indicate that (i) E2F, DP, and Rb proteins each influence the selection of E2F-binding sites; (ii) E2F sites differ with respect to their intrinsic DNA-bending properties; (iii) E2F/DP complexes induce distinct degrees of DNA bending; and (iv) complex-specific E2F sites selected in vitro function distinctly as regulators of cell cycle-dependent transcription in vivo. These data indicate that the specific sequence of an E2F site may determine its role in transcriptional regulation and suggest that Rb/E2F complexes may regulate subsets of E2F-dependent cellular genes. PMID:9372931

  10. Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA.

    PubMed

    Karow, Marisa; Chavez, Christopher L; Farruggio, Alfonso P; Geisinger, Jonathan M; Keravala, Annahita; Jung, W Edward; Lan, Feng; Wu, Joseph C; Chen-Tsai, Yanru; Calos, Michele P

    2011-11-01

    Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting. PMID:21898697

  11. Drought-induced site-specific DNA methylation and its association with drought tolerance in rice (Oryza sativa L.)

    PubMed Central

    Wang, Wen-Sheng; Pan, Ya-Jiao; Zhao, Xiu-Qin; Dwivedi, D.; Zhu, Ling-Hua; Ali, J.; Fu, Bin-Ying; Li, Zhi-Kang

    2011-01-01

    An indica pyramiding line, DK151, and its recurrent parent, IR64, were evaluated under drought stress and non-stress conditions for three consecutive seasons. DK151 showed significantly improved tolerance to drought. The DNA methylation changes in DK151 and IR64 under drought stress and subsequent recovery were assessed using methylation-sensitive amplified polymorphism analysis. Our results indicate that drought-induced genome-wide DNA methylation changes accounted for ∼12.1% of the total site-specific methylation differences in the rice genome. This drought-induced DNA methylation pattern showed three interesting properties. The most important one was its genotypic specificity reflected by large differences in the detected DNA methylation/demethylation sites between DK151 and IR64, which result from introgressed genomic fragments in DK151. Second, most drought-induced methylation/demethylation sites were of two major types distinguished by their reversibility, including 70% of the sites at which drought-induced epigenetic changes were reversed to their original status after recovery, and 29% of sites at which the drought-induced DNA demethylation/methylation changes remain even after recovery. Third, the drought-induced DNA methylation alteration showed a significant level of developmental and tissue specificity. Together, these properties are expected to have contributed greatly to rice response and adaptation to drought stress. Thus, induced epigenetic changes in rice genome can be considered as a very important regulatory mechanism for rice plants to adapt to drought and possibly other environmental stresses. PMID:21193578

  12. Mitochondrial DNA Damage and Diseases

    PubMed Central

    Singh, Gyanesh; Pachouri, U C; Khaidem, Devika Chanu; Kundu, Aman; Chopra, Chirag; Singh, Pushplata

    2015-01-01

    Various endogenous and environmental factors can cause mitochondrial DNA (mtDNA) damage.  One of the reasons for enhanced mtDNA damage could be its proximity to the source of oxidants, and lack of histone-like protective proteins. Moreover, mitochondria contain inadequate DNA repair pathways, and, diminished DNA repair capacity may be one of the factors responsible for high mutation frequency of the mtDNA. mtDNA damage might cause impaired mitochondrial function, and, unrepaired mtDNA damage has been frequently linked with several diseases. Exploration of mitochondrial perspective of diseases might lead to a better understanding of several diseases, and will certainly open new avenues for detection, cure, and prevention of ailments.

  13. Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo.

    PubMed

    You, Changjun; Wang, Yinsheng

    2015-09-01

    Aberrant transcription induced by DNA damage may confer risk for the development of cancer and other human diseases. Traditional methods for measuring lesion-induced transcriptional alterations often involve extensive colony screening and DNA sequencing procedures. Here we describe a protocol for the quantitative assessment of the effects of DNA lesions on the efficiency and fidelity of transcription in vitro and in mammalian cells. The method is also amenable to investigating the influence of specific DNA repair proteins on the biological response toward DNA damage during transcription by manipulating their gene expression. Specifically, we present detailed, step-by-step procedures, including DNA template preparation, in vitro and in vivo transcription, RNA purification, reverse-transcription PCR (RT-PCR) and restriction digestion of RT-PCR products. Analyses of restriction fragments of interest are performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and polyacrylamide gel electrophoresis (PAGE). The entire procedure described in this protocol can be completed in 15-20 d. PMID:26292071

  14. Amino acid sequence homology between Piv, an essential protein in site-specific DNA inversion in Moraxella lacunata, and transposases of an unusual family of insertion elements.

    PubMed Central

    Lenich, A G; Glasgow, A C

    1994-01-01

    Deletion analysis of the subcloned DNA inversion region of Moraxella lacunata indicates that Piv is the only M. lacunata-encoded factor required for site-specific inversion of the tfpQ/tfpI pilin segment. The predicted amino acid sequence of Piv shows significant homology solely with the transposases/integrases of a family of insertion sequence elements, suggesting that Piv is a novel site-specific recombinase. Images PMID:8021196

  15. TFIIIB subunit locations on U6 gene promoter DNA mapped by site-specific protein-DNA photo-cross-linking.

    PubMed

    Kang, Jin Joo; Kang, Yoon Soon; Stumph, William E

    2016-05-01

    RNA polymerase III-transcribed U6 snRNA genes have gene-external promoters that contain TATA boxes. U6 TATA sequences are bound by TFIIIB that in Drosophila contains the three subunits TBP, Brf1, and Bdp1. The overall structure of TFIIIB is still not well understood. We have therefore studied the mode of TFIIIB binding to DNA by site-specific protein-DNA photo-cross-linking. The results indicate that a portion of Brf1 is sandwiched between Bdp1 and TBP upstream of the TATA box. Furthermore, Bdp1 traverses the DNA under the N-terminal stirrup of TBP to interact with the DNA (and very likely Brf1) downstream of the TATA sequence. PMID:27112515

  16. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

    PubMed Central

    Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  17. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  18. DNA damage checkpoints in mammals.

    PubMed

    Niida, Hiroyuki; Nakanishi, Makoto

    2006-01-01

    DNA damage is a common event and probably leads to mutation or deletion within chromosomal DNA, which may cause cancer or premature aging. DNA damage induces several cellular responses including DNA repair, checkpoint activity and the triggering of apoptotic pathways. DNA damage checkpoints are associated with biochemical pathways that end delay or arrest of cell-cycle progression. These checkpoints engage damage sensor proteins, such as the Rad9-Rad1-Hus1 (9-1-1) complex, and the Rad17-RFC complex, in the detection of DNA damage and transduction of signals to ATM, ATR, Chk1 and Chk2 kinases. Chk1 and Chk2 kinases regulate Cdc25, Wee1 and p53 that ultimately inactivate cyclin-dependent kinases (Cdks) which inhibit cell-cycle progression. In this review, we discuss the molecular mechanisms by which DNA damage is recognized by sensor proteins and signals are transmitted to Cdks. We classify the genes involved in checkpoint signaling into four categories, namely sensors, mediators, transducers and effectors, although their proteins have the broad activity, and thus this classification is for convenience and is not definitive. PMID:16314342

  19. Deciphering the DNA Damage Response.

    PubMed

    Haber, James E

    2015-09-10

    This year's Albert Lasker Basic Medical Research Award honors Evelyn Witkin and Stephen J. Elledge, two pioneers in elucidating the DNA damage response, whose contributions span more than 40 years. PMID:26359974

  20. Optical detection of DNA damage

    NASA Astrophysics Data System (ADS)

    Rogers, Kim R.; Apostol, A.; Cembrano, J.

    1999-02-01

    A rapid and sensitive fluorescence assay for oxidative damage to calf thymus DNA is reported. A decrease in the transition temperature for strand separation resulted from exposure of the DNA to the reactive decomposition products of 3- morpholinosydnonimine (SIN-1) (i.e., nitric oxide, superoxide, peroxynitrite, hydrogen peroxide, and hydroxyl radicals). A decrease in melting temperature of 12 degrees Celsius was indicative of oxidative damage including single strand chain breaks. Double stranded (ds) and single stranded (ss) forms of DNA were determined using the indicator dyes ethidium bromide and PicoGreen. The change in DNA 'melting' curves was dependant on the concentration of SIN-1 and was most pronounced at 75 degrees Celsius. This chemically induced damage was significantly inhibited by sodium citrate, tris(hydroxymethyl)aminomethane (Tris), and diethylenetriaminepentaacetic acid (DTPA), but was unaffected by superoxide dismutase (SOD), catalase, ethylenediamine tetraacietic acid (EDTA), or deferoxamine. Lowest observable effect level for SIN-1-induced damage was 200 (mu) M.

  1. Replicating Damaged DNA in Eukaryotes

    PubMed Central

    Chatterjee, Nimrat; Siede, Wolfram

    2013-01-01

    DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail. PMID:24296172

  2. DNA damage in neurodegenerative diseases.

    PubMed

    Coppedè, Fabio; Migliore, Lucia

    2015-06-01

    Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis, which represent three of the most common neurodegenerative pathologies in humans. PMID:26255941

  3. Sperm DNA oxidative damage and DNA adducts.

    PubMed

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm

  4. Modulation of charge transport across double-stranded DNA by the site-specific incorporation of copper bis-phenanthroline complexes.

    PubMed

    Dauphin-Ducharme, Philippe; Rosati, Fiora; Greschner, Andrea; De Bruijn, A Dowine; Salvatore, Danielle; Toader, Violeta; Lau, Kai Lin; Mauzeroll, Janine; Sleiman, Hanadi

    2015-02-10

    The site-specific incorporation of transition-metal complexes within DNA duplexes, followed by their immobilization on a gold surface, was studied by electrochemistry to characterize their ability to mediate charge. Cyclic voltammetry, square-wave voltammetry, and control experiments were carried out on fully matched and mismatched DNA strands that are mono- or bis-labeled with transition-metal complexes. These experiments are all consistent with the ability of the metal centers to act as a redox probe that is well coupled to the DNA π-stack, allowing DNA-mediated charge transport. PMID:25590949

  5. Types and Consequences of DNA Damage

    EPA Science Inventory

    This review provides a concise overview of the types of DNA damage and the molecular mechanisms by which a cell senses DNA damage, repairs the damage, converts the damage into a mutation, or dies as a consequence of unrepaired DNA damage. Such information is important in consid...

  6. Site-specific deletion in cauliflower mosaic virus DNA: possible involvement of RNA splicing and reverse transcription

    PubMed Central

    Hirochika, Hirohiko; Takatsuji, Hiroshi; Ubasawa, Aiko; Ikeda, Joh-E

    1985-01-01

    A frequent site-specific deletion was observed in the life cycle of cauliflower mosaic virus (S strain). Analysis of the sequence around the deletion site and the parental sequence implied that the deletion was promoted at sequences similar to the donor and acceptor consensus sequences of RNA splicing, designated as the deletion donor and acceptor sequences, respectively. To elucidate the mechanism of this site-specific deletion, point mutations were introduced into the deletion donor sequence (GT to GG or GA transversion). Deletion at the original deletion donor site did not occur in these mutants, instead, new (cryptic) donor sites were activated. All of these activated cryptic sites had sequences similar to the splicing consensus sequence. In all cases except one, the original deletion acceptor site was used. These results can be most readily explained by postulating that the site-specific deletion occurs by reverse transcription of spliced viral RNA. This frequent site-specific deletion was not observed in other strains. For a virus which replicates by reverse transcription, a mechanism to regulate the rate of splicing is required to ensure the intactness of the viral genome. We discuss the possibility that the S strain has a mutation in this regulatory mechanism. ImagesFig. 1.Fig. 3.Fig. 5.Fig. 7. PMID:16453624

  7. DNA Damage and Pulmonary Hypertension

    PubMed Central

    Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

    2016-01-01

    Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. PMID:27338373

  8. DNA Damage and Pulmonary Hypertension.

    PubMed

    Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

    2016-01-01

    Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. PMID:27338373

  9. DNA Damage and Repair in Vascular Disease.

    PubMed

    Uryga, Anna; Gray, Kelly; Bennett, Martin

    2016-01-01

    DNA damage affecting both genomic and mitochondrial DNA is present in a variety of both inherited and acquired vascular diseases. Multiple cell types show persistent DNA damage and a range of lesions. In turn, DNA damage activates a variety of DNA repair mechanisms, many of which are activated in vascular disease. Such DNA repair mechanisms either stall the cell cycle to allow repair to occur or trigger apoptosis or cell senescence to prevent propagation of damaged DNA. Recent evidence has indicated that DNA damage occurs early, is progressive, and is sufficient to impair function of cells composing the vascular wall. The consequences of persistent genomic and mitochondrial DNA damage, including inflammation, cell senescence, and apoptosis, are present in vascular disease. DNA damage can thus directly cause vascular disease, opening up new possibilities for both prevention and treatment. We review the evidence for and the causes, types, and consequences of DNA damage in vascular disease. PMID:26442438

  10. Mutations in the Dimer Interface of Dihydrolipoamide Dehydrogenase Promote Site-specific Oxidative Damages in Yeast and Human Cells*

    PubMed Central

    Vaubel, Rachael A.; Rustin, Pierre; Isaya, Grazia

    2011-01-01

    Dihydrolipoamide dehydrogenase (DLD) is a multifunctional protein well characterized as the E3 component of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes. Previously, conditions predicted to destabilize the DLD dimer revealed that DLD could also function as a diaphorase and serine protease. However, the relevance of these cryptic activities remained undefined. We analyzed human DLD mutations linked to strikingly different clinical phenotypes, including E340K, D444V, R447G, and R460G in the dimer interface domain that are responsible for severe multisystem disorders of infancy and G194C in the NAD+-binding domain that is typically associated with milder presentations. In vitro, all of these mutations decreased to various degrees dihydrolipoamide dehydrogenase activity, whereas dimer interface mutations also enhanced proteolytic and/or diaphorase activity. Human DLD proteins carrying each individual mutation complemented fully the respiratory-deficient phenotype of yeast cells lacking endogenous DLD even when residual dihydrolipoamide dehydrogenase activity was as low as 21% of controls. However, under elevated oxidative stress, expression of DLD proteins with dimer interface mutations greatly accelerated the loss of respiratory function, resulting from enhanced oxidative damage to the lipoic acid cofactor of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase and other mitochondrial targets. This effect was not observed with the G194C mutation or a mutation that disrupts the proteolytic active site of DLD. As in yeast, lipoic acid cofactor was damaged in human D444V-homozygous fibroblasts after exposure to oxidative stress. We conclude that the cryptic activities of DLD promote oxidative damage to neighboring molecules and thus contribute to the clinical severity of DLD mutations. PMID:21930696

  11. Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase.

    PubMed

    De Paepe, Annelies; De Buck, Sylvie; Nolf, Jonah; Van Lerberge, Els; Depicker, Ann

    2013-07-01

    Random T-DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T-DNA copies, the risk of mutating the host genome and the difficulty of stacking well-defined traits. Therefore, recombination systems have been proposed to integrate the T-DNA at a pre-selected site in the host genome. Here, we demonstrate the capacity of the ϕC31 integrase (INT) for efficient targeted T-DNA integration. Moreover, we show that the iterative site-specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre-selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T-DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site-specific integration (SSI) of this T-DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium-mediated integration, allowing the serial integration of transgenic DNA sequences in plants. PMID:23574114

  12. Method for assaying clustered DNA damages

    DOEpatents

    Sutherland, Betsy M.

    2004-09-07

    Disclosed is a method for detecting and quantifying clustered damages in DNA. In this method, a first aliquot of the DNA to be tested for clustered damages with one or more lesion-specific cleaving reagents under conditions appropriate for cleavage of the DNA to produce single-strand nicks in the DNA at sites of damage lesions. The number average molecular length (Ln) of double stranded DNA is then quantitatively determined for the treated DNA. The number average molecular length (Ln) of double stranded DNA is also quantitatively determined for a second, untreated aliquot of the DNA. The frequency of clustered damages (.PHI..sub.c) in the DNA is then calculated.

  13. Insights into the architecture and stoichiometry of Escherichia coli PepA*DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy.

    PubMed

    Minh, Phu Nguyen Le; Devroede, Neel; Massant, Jan; Maes, Dominique; Charlier, Daniel

    2009-04-01

    Multifunctional Aminopeptidase A (PepA) from Escherichia coli is involved in the control of two distinct DNA transaction processes: transcriptional repression of the carAB operon, encoding carbamoyl phosphate synthase and site-specific resolution of ColE1-type plasmid multimers. Both processes require communication at a distance along a DNA molecule and PepA is the major structural component of the nucleoprotein complexes that underlie this communication. Atomic Force Microscopy was used to analyze the architecture of PepA.carAB and PepA.cer site complexes. Contour length measurements, bending angle analyses and volume determinations demonstrate that the carP1 operator is foreshortened by approximately 235 bp through wrapping around one PepA hexamer. The highly deformed part of the operator extends from slightly upstream of the -35 hexamer of the carP1 promoter to just downstream of the IHF-binding site, and comprises the binding sites for the PurR and RutR transcriptional regulators. This extreme remodeling of the carP1 control region provides a straightforward explanation for the strict requirement of PepA in the establishment of pyrimidine and purine-specific repression of carAB transcription. We further provide a direct physical proof that PepA is able to synapse two cer sites in direct repeat in a large interwrapped nucleoprotein complex, likely comprising two PepA hexamers. PMID:19136463

  14. Insights into the architecture and stoichiometry of Escherichia coli PepA•DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy

    PubMed Central

    Minh, Phu Nguyen Le; Devroede, Neel; Massant, Jan; Maes, Dominique; Charlier, Daniel

    2009-01-01

    Multifunctional Aminopeptidase A (PepA) from Escherichia coli is involved in the control of two distinct DNA transaction processes: transcriptional repression of the carAB operon, encoding carbamoyl phosphate synthase and site-specific resolution of ColE1-type plasmid multimers. Both processes require communication at a distance along a DNA molecule and PepA is the major structural component of the nucleoprotein complexes that underlie this communication. Atomic Force Microscopy was used to analyze the architecture of PepA·carAB and PepA·cer site complexes. Contour length measurements, bending angle analyses and volume determinations demonstrate that the carP1 operator is foreshortened by ∼235 bp through wrapping around one PepA hexamer. The highly deformed part of the operator extends from slightly upstream of the –35 hexamer of the carP1 promoter to just downstream of the IHF-binding site, and comprises the binding sites for the PurR and RutR transcriptional regulators. This extreme remodeling of the carP1 control region provides a straightforward explanation for the strict requirement of PepA in the establishment of pyrimidine and purine-specific repression of carAB transcription. We further provide a direct physical proof that PepA is able to synapse two cer sites in direct repeat in a large interwrapped nucleoprotein complex, likely comprising two PepA hexamers. PMID:19136463

  15. Targeting DNA damage response in cancer therapy

    PubMed Central

    Hosoya, Noriko; Miyagawa, Kiyoshi

    2014-01-01

    Cancer chemotherapy and radiotherapy are designed to kill cancer cells mostly by inducing DNA damage. DNA damage is normally recognized and repaired by the intrinsic DNA damage response machinery. If the damaged lesions are successfully repaired, the cells will survive. In order to specifically and effectively kill cancer cells by therapies that induce DNA damage, it is important to take advantage of specific abnormalities in the DNA damage response machinery that are present in cancer cells but not in normal cells. Such properties of cancer cells can provide biomarkers or targets for sensitization. For example, defects or upregulation of the specific pathways that recognize or repair specific types of DNA damage can serve as biomarkers of favorable or poor response to therapies that induce such types of DNA damage. Inhibition of a DNA damage response pathway may enhance the therapeutic effects in combination with the DNA-damaging agents. Moreover, it may also be useful as a monotherapy when it achieves synthetic lethality, in which inhibition of a complementary DNA damage response pathway selectively kills cancer cells that have a defect in a particular DNA repair pathway. The most striking application of this strategy is the treatment of cancers deficient in homologous recombination by poly(ADP-ribose) polymerase inhibitors. In this review, we describe the impact of targeting the cancer-specific aberrations in the DNA damage response by explaining how these treatment strategies are currently being evaluated in preclinical or clinical trials. PMID:24484288

  16. DNA damage checkpoint, damage repair, and genome stability.

    PubMed

    Liu, Wei-Feng; Yu, Shan-Shan; Chen, Guan-Jun; Li, Yue-Zhong

    2006-05-01

    Genomic DNA is under constant attack from both endogenous and exogenous sources of DNA damaging agents. Without proper care, the ensuing DNA damages would lead to alteration of genomic structure thus affecting the faithful transmission of genetic information. During the process of evolution, organisms have acquired a series of mechanisms responding to and repairing DNA damage, thus assuring the maintenance of genome stability and faithful transmission of genetic information. DNA damage checkpoint is one such important mechanism by which, in the face of DNA damage, a cell can respond to amplified damage signals, either by actively halting the cell cycle until it ensures that critical processes such as DNA replication or mitosis are complete or by initiating apoptosis as a last resort. Over the last decade, complex hierarchical interactions between the key components like ATM/ATR in the checkpoint pathway and various other mediators, effectors including DNA damage repair proteins have begun to emerge. In the meantime, an intimate relationship between mechanisms of damage checkpoint pathway, DNA damage repair, and genome stability was also uncovered. Reviewed herein are the recent findings on both the mechanisms of activation of checkpoint pathways and their coordination with DNA damage repair machinery as well as their effect on genomic integrity. PMID:16722332

  17. Miniaturized reaction vessel system, method for performing site-specific biochemical reactions and affinity fractionation for use in DNA sequencing

    DOEpatents

    Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

    2000-01-01

    A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

  18. Site-specific DNA cleavage by artificial zinc finger-type nuclease with cerium-binding peptide

    SciTech Connect

    Nakatsukasa, Takako; Shiraishi, Yasuhisa; Negi, Shigeru; Imanishi, Miki; Futaki, Shiroh; Sugiura, Yukio . E-mail: sugiura@scl.kyoto-u.ac.jp

    2005-04-29

    The addition of a new function to native proteins is one of the most attractive protein-based designs. In this study, we have converted a C{sub 2}H{sub 2}-type zinc finger as a DNA-binding motif into a novel zinc finger-type nuclease by connecting two distinct zinc finger proteins (Sp1 and GLI) with a functional linker possessing DNA cleavage activity. As a DNA cleavage domain, we chose an analogue of the metal-binding loop (12 amino acid residues), peptide P1, which has been reported to exhibit a strong binding affinity for a lanthanide ion and DNA cleavage ability in the presence of Ce(IV). Our newly designed nucleases, Sp1(P1)GLI and Sp1(P1G)GLI, can strongly bind to a lanthanide ion and show a unique DNA cleavage pattern, in which certain positions between the two DNA-binding sites are specifically cleaved. The present result provides useful information for expanding the design strategy for artificial nucleases.

  19. Site specific cleavage of thetaX-174 replicative form DNA after modification by N-acetoxy-N-2-acetylaminofluorene

    SciTech Connect

    Bases, R.; Mendez, F.; Mendez, L.

    1983-01-01

    Three kinds of structural disturbances were found in an 88 base pair (bp) fragment of thetaX-174 DNA after exposure to N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF). Frequent strand scissions at two specific guanine sites on the 5' /sup 32/P-end-labeled fragment were identified by base sequence analysis. Scissions at these two sites were induced at neutral pH and they were not increased by treatment with apurinic endonuclease. They are an immediate consequence of N-Aco-AAF action and are not primarily apurinic sites. Alkali treatment with 1 M piperidine at 90/sup 0/C induced strand scissions at every guanine, demonstrating adduct slices, depurination and strand scissions. Adducted DNA was sensitive to single-strand specific nuclease digestion, suggesting unwound DNA. These studies indicate the prediliction of N-Aco-AAF for certain DNA sites and they suggest three kinds of DNA modifications which can be expected after adduction by this carcinogen. Some of the sites may be premutational carcinogen-induced DNA structural modifications.

  20. Direct and site-specific quantification of RNA 2'-O-methylation by PCR with an engineered DNA polymerase.

    PubMed

    Aschenbrenner, Joos; Marx, Andreas

    2016-05-01

    Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR. PMID:27016740

  1. Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase

    PubMed Central

    Aschenbrenner, Joos; Marx, Andreas

    2016-01-01

    Methylation of the 2′-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2′-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2′-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2′-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR. PMID:27016740

  2. Repetitive, marker-free, site-specific integration as a novel tool for multiple chromosomal integration of DNA.

    PubMed

    Petersen, Kia Vest; Martinussen, Jan; Jensen, Peter Ruhdal; Solem, Christian

    2013-06-01

    We present a tool for repetitive, marker-free, site-specific integration in Lactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusion of a minimal bacterial attachment site (attB(min)), two mutated loxP sequences (lox66 and lox71) allowing for removal of undesirable vector elements (antibiotic resistance marker), and a counterselection marker (oroP) for selection of loxP recombination on the pKV6 vector. When transformed into L. lactis expressing the phage TP901-1 integrase, pKV6 integrates with high frequency into the chromosome, where it is flanked by attL and attR hybrid attachment sites. After expression of Cre recombinase from a plasmid that is not able to replicate in L. lactis, loxP recombinants can be selected for by using 5-fluoroorotic acid. The introduced attB(min) site can subsequently be used for a second round of integration. To examine if attP recombination was specific to the attB site, integration was performed in strains containing the attB, attL, and attR sites or the attL and attR sites only. Only attP-attB recombination was observed when all three sites were present. In the absence of the attB site, a low frequency of attP-attL recombination was observed. To demonstrate the functionality of the system, the xylose utilization genes (xylABR and xylT) from L. lactis strain KF147 were integrated into the chromosome of L. lactis strain MG1363 in two steps. PMID:23542630

  3. Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways.

    PubMed

    Qi, Yiping; Zhang, Yong; Zhang, Feng; Baller, Joshua A; Cleland, Spencer C; Ryu, Yungil; Starker, Colby G; Voytas, Daniel F

    2013-03-01

    Improved methods for engineering sequence-specific nucleases, including zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs), have made it possible to precisely modify plant genomes. However, the success of genome modification is largely dependent on the intrinsic activity of the engineered nucleases. In this study, we sought to enhance ZFN-mediated targeted mutagenesis and gene targeting (GT) in Arabidopsis by manipulating DNA repair pathways. Using a ZFN that creates a double-strand break (DSB) at the endogenous ADH1 locus, we analyzed repair outcomes in the absence of DNA repair proteins such as KU70 and LIG4 (both involved in classic nonhomologous end-joining, NHEJ) and SMC6B (involved in sister-chromatid-based homologous recombination, HR). We achieved a fivefold to 16-fold enhancement in HR-based GT in a ku70 mutant and a threefold to fourfold enhancement in GT in the lig4 mutant. Although the NHEJ mutagenesis frequency was not significantly changed in ku70 or lig4, DNA repair was shifted to microhomology-dependent alternative NHEJ. As a result, mutations in both ku70 and lig4 were predominantly large deletions, which facilitates easy screening for mutations by PCR. Interestingly, NHEJ mutagenesis and GT at the ADH1 locus were enhanced by sixfold to eightfold and threefold to fourfold, respectively, in a smc6b mutant. The increase in NHEJ-mediated mutagenesis by loss of SMC6B was further confirmed using ZFNs that target two other Arabidopsis genes, namely, TT4 and MPK8. Considering that components of DNA repair pathways are highly conserved across species, mutations in DNA repair genes likely provide a universal strategy for harnessing repair pathways to achieve desired targeted genome modifications. PMID:23282329

  4. A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells

    PubMed Central

    Zhang, Ya; Huang, Liang; Fu, Haiqing; Smith, Owen K.; Lin, Chii Mei; Utani, Koichi; Rao, Mishal; Reinhold, William C.; Redon, Christophe E.; Ryan, Michael; Kim, RyangGuk; You, Yang; Hanna, Harlington; Boisclair, Yves; Long, Qiaoming; Aladjem, Mirit I.

    2016-01-01

    Mammalian chromosome replication starts from distinct sites; however, the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. Here we identify a replication-initiation determinant (RepID) protein that binds a subset of replication-initiation sites. A large fraction of RepID-binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from RepID-bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID-depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication-initiation events. These observations are consistent with a model, suggesting that RepID facilitates replication initiation at a distinct group of human replication origins. PMID:27272143

  5. A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells.

    PubMed

    Zhang, Ya; Huang, Liang; Fu, Haiqing; Smith, Owen K; Lin, Chii Mei; Utani, Koichi; Rao, Mishal; Reinhold, William C; Redon, Christophe E; Ryan, Michael; Kim, RyangGuk; You, Yang; Hanna, Harlington; Boisclair, Yves; Long, Qiaoming; Aladjem, Mirit I

    2016-01-01

    Mammalian chromosome replication starts from distinct sites; however, the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. Here we identify a replication-initiation determinant (RepID) protein that binds a subset of replication-initiation sites. A large fraction of RepID-binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from RepID-bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID-depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication-initiation events. These observations are consistent with a model, suggesting that RepID facilitates replication initiation at a distinct group of human replication origins. PMID:27272143

  6. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage

    SciTech Connect

    Yuan,Y.; Pei, Y.; Ma, J.; Kuryavyi, V.; Zhadina, M.; Meister, G.; Chen, H.; Dauter, Z.; Tuschi, T.; Patel, D.

    2005-01-01

    Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in 'slicer' activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

  7. The N-terminal part of the E.coli DNA binding protein FIS is essential for stimulating site-specific DNA inversion but is not required for specific DNA binding.

    PubMed Central

    Koch, C; Ninnemann, O; Fuss, H; Kahmann, R

    1991-01-01

    FIS protein is involved in several different cellular processes stimulating site-specific recombination in phages Mu and lambda as well as transcription of stable RNA operons in E.coli. We have performed a mutational analysis of fis and provide genetic and biochemical evidence that a truncated version of FIS lacking the N-terminal region is sufficient for specific DNA binding and for stimulating lambda excision. These mutants also retain their ability to autoregulate fis gene expression. Such mutant proteins, however, cannot stimulate the enhancer dependent DNA inversion reaction. Images PMID:1834996

  8. Site-specific DNA binding of the Schizosaccharomyces pombe origin recognition complex is determined by the Orc4 subunit.

    PubMed

    Kong, D; DePamphilis, M L

    2001-12-01

    The mechanism by which origin recognition complexes (ORCs) identify replication origins was investigated using purified Orc proteins from Schizosaccharomyces pombe. Orc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand but were devoid of either alternating A and T residues or GC-rich sequences. Addition of a complex consisting of Orc1, -2, -3, -5, and -6 proteins (ORC-5) altered neither Orc4p binding to origin DNA nor Orc4p protection of specific sequences. ORC-5 alone bound weakly and nonspecifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit. PMID:11689699

  9. MicroRNA response to DNA damage

    PubMed Central

    Wan, Guohui; Mathur, Rohit; Hu, Xiaoxiao; Zhang, Xinna; Lu, Xiongbin

    2011-01-01

    Faithful transmission of genetic material in eukaryotic cells requires not only accurate DNA replication and chromosome distribution, but also the ability to sense and repair spontaneous and induced DNA damage. To maintain genomic integrity, cells undergo a DNA damage response using a complex network of signaling pathways, composed of coordinate sensors, transducers and effectors in cell cycle arrest, apoptosis and DNA repair. Emerging evidence has suggested that microRNAs (miRNAs) play a critical role in regulation of DNA damage response. Here, we discuss the recent findings on how miRNAs interact with the canonical DNA damage response and how miRNA expression is regulated after DNA damage. PMID:21741842

  10. Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo

    PubMed Central

    McNeer, Nicole A.; Schleifman, Erica B.; Cuthbert, Amy; Brehm, Michael; Jackson, Andrew; Cheng, Christopher; Anandalingam, Kavitha; Kumar, Priti; Shultz, Leonard D.; Greiner, Dale L.

    2013-01-01

    In vivodelivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD-scid IL2rγnull mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA, and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen, and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the β-globin gene using nanoparticles carrying β-globin-targeted PNAs/DNAs, demonstrating this method’s versatility. In vivo testing in an EGFP- β-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo. PMID:23076379

  11. Cellular responses to environmental DNA damage

    SciTech Connect

    Not Available

    1994-08-01

    This volume contains the proceedings of the conference entitled Cellular Responses to Environmental DNA Damage held in Banff,Alberta December 1--6, 1991. The conference addresses various aspects of DNA repair in sessions titled DNA repair; Basic Mechanisms; Lesions; Systems; Inducible Responses; Mutagenesis; Human Population Response Heterogeneity; Intragenomic DNA Repair Heterogeneity; DNA Repair Gene Cloning; Aging; Human Genetic Disease; and Carcinogenesis. Individual papers are represented as abstracts of about one page in length.

  12. DNA Damage Signals and Space Radiation Risk

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.

    2011-01-01

    Space radiation is comprised of high-energy and charge (HZE) nuclei and protons. The initial DNA damage from HZE nuclei is qualitatively different from X-rays or gamma rays due to the clustering of damage sites which increases their complexity. Clustering of DNA damage occurs on several scales. First there is clustering of single strand breaks (SSB), double strand breaks (DSB), and base damage within a few to several hundred base pairs (bp). A second form of damage clustering occurs on the scale of a few kbp where several DSB?s may be induced by single HZE nuclei. These forms of damage clusters do not occur at low to moderate doses of X-rays or gamma rays thus presenting new challenges to DNA repair systems. We review current knowledge of differences that occur in DNA repair pathways for different types of radiation and possible relationships to mutations, chromosomal aberrations and cancer risks.

  13. DNA Damage in Major Psychiatric Diseases.

    PubMed

    Raza, Muhammad Ummear; Tufan, Turan; Wang, Yan; Hill, Christopher; Zhu, Meng-Yang

    2016-08-01

    Human cells are exposed to exogenous insults and continuous production of different metabolites. These insults and unwanted metabolic products might interfere with the stability of genomic DNA. Recently, many studies have demonstrated that different psychiatric disorders show substantially high levels of oxidative DNA damage in the brain accompanied with morphological and functional alterations. It reveals that damaged genomic DNA may contribute to the pathophysiology of these mental illnesses. In this article, we review the roles of oxidative damage and reduced antioxidant ability in some vastly studied psychiatric disorders and emphasize the inclusion of treatment options involving DNA repair. In addition, while most currently used antidepressants are based on the manipulation of the neurotransmitter regulation in managing different mental abnormalities, they are able to prevent or reverse neurotoxin-induced DNA damage. Therefore, it may be plausible to target on genomic DNA alterations for psychiatric therapies, which is of pivotal importance for future antipsychiatric drug development. PMID:27126805

  14. Persistent damage induces mitochondrial DNA degradation

    PubMed Central

    Shokolenko, Inna N.; Wilson, Glenn L.; Alexeyev, Mikhail F.

    2013-01-01

    Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by six hours after induction of mutant uracil-N-glycosylase and by twelve hours after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and “foaming” of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence

  15. Site-specific cleavage/packaging of herpes simplex virus DNA and the selective maturation of nucleocapsids containing full-length viral DNA

    PubMed Central

    Vlazny, Donald A.; Kwong, Ann; Frenkel, Niza

    1982-01-01

    Defective genomes present in serially passaged herpes simplex virus (HSV) stocks have been shown to consist of tandemly arranged repeat units containing limited sets of the standard virus DNA sequences. Invariably, the HSV defective genomes terminate with the right (S component) terminus of HSV DNA. Because the oligomeric forms can arise from a single repeat unit, it has been concluded that the defective genomes arise by a rolling circle mechanism of replication. We now report on our studies of defective genomes packaged in viral capsids accumulating in the nuclei and in mature virions (enveloped capsids) translocated into the cytoplasm of cells infected with serially passaged virus. These studies have revealed that, upon electrophoresis in agarose gels, the defective genomes prepared from cytoplasmic virions comigrated with nondefective standard virus DNA (Mr 100 × 106). In contrast, DNA prepared from capsids accumulating in nuclei consisted of both full-length defective virus DNA molecules and smaller DNA molecules of discrete sizes, ranging in Mr from 5.5 to 100 × 106. These smaller DNA species were shown to consist of different integral numbers (from 1 to approximately 18) of defective genome repeat units and to terminate with sequences corresponding to the right terminal sequences of HSV DNA. We conclude on the basis of these studies that (i) sequences from the right end of standard virus DNA contain a recognition signal for the cleavage and packaging of concatemeric viral DNA, (ii) the sequence-specific cleavage is either a prerequisite for or occurs during the entry of viral DNA into capsid structures, and (iii) DNA molecules significantly shorter than full-length standard viral DNA can become encapsidated within nuclear capsids provided they contain the cleavage/packaging signal. However, capsids containing DNA molecules significantly shorter than standard virus DNA are not translocated into the cytoplasm. Images PMID:6280181

  16. Ubiquitylation, neddylation and the DNA damage response

    PubMed Central

    Brown, Jessica S.; Jackson, Stephen P.

    2015-01-01

    Failure of accurate DNA damage sensing and repair mechanisms manifests as a variety of human diseases, including neurodegenerative disorders, immunodeficiency, infertility and cancer. The accuracy and efficiency of DNA damage detection and repair, collectively termed the DNA damage response (DDR), requires the recruitment and subsequent post-translational modification (PTM) of a complex network of proteins. Ubiquitin and the ubiquitin-like protein (UBL) SUMO have established roles in regulating the cellular response to DNA double-strand breaks (DSBs). A role for other UBLs, such as NEDD8, is also now emerging. This article provides an overview of the DDR, discusses our current understanding of the process and function of PTM by ubiquitin and NEDD8, and reviews the literature surrounding the role of ubiquitylation and neddylation in DNA repair processes, focusing particularly on DNA DSB repair. PMID:25833379

  17. Identification of barriers to rotation of DNA segments in yeast from the topology of DNA rings excised by an inducible site-specific recombinase.

    PubMed Central

    Gartenberg, M R; Wang, J C

    1993-01-01

    Controlled excision of DNA segments to yield intracellular DNA rings of well-defined sequences was utilized to study the determinants of transcriptional supercoiling of closed circular DNA in the yeast Saccharomyces cerevisiae. In delta top1 top2ts strains of S. cerevisiae expressing Escherichia coli DNA topoisomerase I, accumulation of positive supercoils in intracellular DNA normally occurs upon thermal inactivation of DNA topoisomerase II because of the simultaneous generation of positively and negatively supercoiled domains by transcription and the preferential relaxation of the latter by the bacterial enzyme. Positive supercoil accumulation in DNA rings is shown to depend on the presence of specific sequence elements; one likely cause of this dependence is that the persistence of oppositely supercoiled domains in an intracellular DNA ring requires the presence of barriers to rotation of the DNA segments connecting the domains. Analysis of the S. cerevisiae 2-microns plasmid partition system by this approach suggests that the plasmid-encoded REP1 and REP2 proteins are involved in forming such a barrier in DNA containing the REP3 sequence. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8248138

  18. Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

    PubMed Central

    Nicolás, A L; Young, C S

    1994-01-01

    Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell. Images PMID:8264584

  19. PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

    PubMed

    Gonzalez-Hunt, Claudia P; Rooney, John P; Ryde, Ian T; Anbalagan, Charumathi; Joglekar, Rashmi; Meyer, Joel N

    2016-01-01

    Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  20. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage

    PubMed Central

    Gonzalez-Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi

    2016-01-01

    Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  1. Triplex-Induced DNA Damage Response

    PubMed Central

    Rogers, Faye A.; Tiwari, Meetu Kaushik

    2013-01-01

    Cellular DNA damage response is critical to preserving genomic integrity following exposure to genotoxic stress. A complex series of networks and signaling pathways become activated after DNA damage and trigger the appropriate cellular response, including cell cycle arrest, DNA repair, and apoptosis. The response elicited is dependent upon the type and extent of damage sustained, with the ultimate goal of preventing propagation of the damaged DNA. A major focus of our studies is to determine the cellular pathways involved in processing damage induced by altered helical structures, specifically triplexes. Our lab has demonstrated that the TFIIH factor XPD occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. We have shown that XPD co-localizes with γH2AX, and its presence is required for the phosphorylation of H2AX tyrosine142, which stimulates the signaling pathway to recruit pro-apoptotic factors to the damage site. Herein, we examine the cellular pathways activated in response to triplex formation and discuss our finding that suggests that XPD-dependent apoptosis plays a role in preserving genomic integrity in the presence of excessive structurally induced DNA damage. PMID:24348211

  2. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.

    PubMed

    Langbein, L; Heid, H W; Moll, I; Franke, W W

    1993-12-01

    /plantar epidermis was found positive. By in situ hybridization and immunolocalization we further showed that CK 9 is only expressed in the suprabasal cell layers of this special epidermal tissue. We discuss the molecular properties of CK 9 and its cell type- and body site-specific expression in relation to the special differentiation of palmar/plantar epidermis and to diseases specific for this body site. PMID:7507869

  3. Age to survive: DNA damage and aging.

    PubMed

    Schumacher, Björn; Garinis, George A; Hoeijmakers, Jan H J

    2008-02-01

    Aging represents the progressive functional decline and increased mortality risk common to nearly all metazoans. Recent findings experimentally link DNA damage and organismal aging: longevity-regulating genetic pathways respond to the accumulation of DNA damage and other stress conditions and conversely influence the rate of damage accumulation and its impact for cancer and aging. This novel insight has emerged from studies on human progeroid diseases and mouse models that have deficient DNA repair pathways. Here we discuss a unified concept of an evolutionarily conserved 'survival' response that shifts the organism's resources from growth to maintenance as an adaptation to stresses, such as starvation and DNA damage. This shift protects the organism from cancer and promotes healthy aging. PMID:18192065

  4. Attenuation of drug-stimulated topoisomerase II-DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIalpha.

    PubMed Central

    Aoyama, M; Grabowski, D R; Dubyak, G R; Constantinou, A I; Rybicki, L A; Bukowski, R M; Ganapathi, M K; Hickson, I D; Ganapathi, R

    1998-01-01

    that lead to the site-specific hypophosphorylation of topo IIalpha are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons. PMID:9841887

  5. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  6. The RNA Splicing Response to DNA Damage

    PubMed Central

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  7. Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

    PubMed Central

    Adams, Vicki; Bantwal, Radhika; Stevenson, Lauren; Cheung, Jackie K.; Awad, Milena M.; Nicholson, Joel; Carter, Glen P.; Mackin, Kate E.; Rood, Julian I.

    2014-01-01

    TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia. PMID:24682304

  8. DNA damage responses in mammalian oocytes.

    PubMed

    Collins, Josie K; Jones, Keith T

    2016-07-01

    DNA damage acquired during meiosis can lead to infertility and miscarriage. Hence, it should be important for an oocyte to be able to detect and respond to such events in order to make a healthy egg. Here, the strategies taken by oocytes during their stages of growth to respond to DNA damaging events are reviewed. In particular, recent evidence of a novel pathway in fully grown oocytes helps prevent the formation of mature eggs with DNA damage. It has been found that fully grown germinal vesicle stage oocytes that have been DNA damaged do not arrest at this point in meiosis, but instead undergo meiotic resumption and stall during the first meiotic division. The Spindle Assembly Checkpoint, which is a well-known mitotic pathway employed by somatic cells to monitor chromosome attachment to spindle microtubules, appears to be utilised by oocytes also to respond to DNA damage. As such maturing oocytes are arrested at metaphase I due to an active Spindle Assembly Checkpoint. This is surprising given this checkpoint has been previously studied in oocytes and considered to be weak and ineffectual because of its poor ability to be activated in response to microtubule attachment errors. Therefore, the involvement of the Spindle Assembly Checkpoint in DNA damage responses of mature oocytes during meiosis I uncovers a novel second function for this ubiquitous cellular checkpoint. PMID:27069010

  9. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    SciTech Connect

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  10. Historical perspective on the DNA damage response.

    PubMed

    Hanawalt, Philip C

    2015-12-01

    The DNA damage response (DDR) has been broadly defined as a complex network of cellular pathways that cooperate to sense and repair lesions in DNA. Multiple types of DNA damage, some natural DNA sequences, nucleotide pool deficiencies and collisions with transcription complexes can cause replication arrest to elicit the DDR. However, in practice, the term DDR as applied to eukaryotic/mammalian cells often refers more specifically to pathways involving the activation of the ATM (ataxia-telangiectasia mutated) and ATR (ATM-Rad3-related) kinases in response to double-strand breaks or arrested replication forks, respectively. Nevertheless, there are distinct responses to particular types of DNA damage that do not involve ATM or ATR. In addition, some of the aberrations that cause replication arrest and elicit the DDR cannot be categorized as direct DNA damage. These include nucleotide pool deficiencies, nucleotide sequences that can adopt non-canonical DNA structures, and collisions between replication forks and transcription complexes. The response to these aberrations can be called the genomic stress response (GSR), a term that is meant to encompass the sensing of all types of DNA aberrations together with the mechanisms involved in coping with them. In addition to fully functional cells, the consequences of processing genomic aberrations may include mutagenesis, genomic rearrangements and lethality. PMID:26507443

  11. DNA damage may drive nucleosomal reorganization to facilitate damage detection

    NASA Astrophysics Data System (ADS)

    LeGresley, Sarah E.; Wilt, Jamie; Antonik, Matthew

    2014-03-01

    One issue in genome maintenance is how DNA repair proteins find lesions at rates that seem to exceed diffusion-limited search rates. We propose a phenomenon where DNA damage induces nucleosomal rearrangements which move lesions to potential rendezvous points in the chromatin structure. These rendezvous points are the dyad and the linker DNA between histones, positions in the chromatin which are more likely to be accessible by repair proteins engaged in a random search. The feasibility of this mechanism is tested by considering the statistical mechanics of DNA containing a single lesion wrapped onto the nucleosome. We consider lesions which make the DNA either more flexible or more rigid by modeling the lesion as either a decrease or an increase in the bending energy. We include this energy in a partition function model of nucleosome breathing. Our results indicate that the steady state for a breathing nucleosome will most likely position the lesion at the dyad or in the linker, depending on the energy of the lesion. A role for DNA binding proteins and chromatin remodelers is suggested based on their ability to alter the mechanical properties of the DNA and DNA-histone binding, respectively. We speculate that these positions around the nucleosome potentially serve as rendezvous points where DNA lesions may be encountered by repair proteins which may be sterically hindered from searching the rest of the nucleosomal DNA. The strength of the repositioning is strongly dependent on the structural details of the DNA lesion and the wrapping and breathing of the nucleosome. A more sophisticated evaluation of this proposed mechanism will require detailed information about breathing dynamics, the structure of partially wrapped nucleosomes, and the structural properties of damaged DNA.

  12. DNA damage induction of ribonucleotide reductase.

    PubMed Central

    Elledge, S J; Davis, R W

    1989-01-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidase activity in yeast strains containing the RNR2-lacZ fusion was inducible in response to DNA-damaging agents (UV light, 4-nitroquinoline-1-oxide [4-NQO], and methyl methanesulfonate [MMS]) and agents that block DNA replication (hydroxyurea [HU] and methotrexate) but not heat shock. When MATa cells were arrested in G1 by alpha-factor, RNR2 mRNA was still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. In addition, RNR2 induction was not blocked by the presence of cycloheximide and is therefore likely to be independent of protein synthesis. A mutation, rnr2-314, was found to confer hypersensitivity to HU and increased sensitivity to MMS. In rnr2-314 mutant strains, the DNA damage stress response was found to be partially constitutive as well as hypersensitive to induction by HU but not MMS. The induction properties of RNR2 were examined in a rad4-2 mutant background; in this genetic background, RNR2 was hypersensitive to induction by 4-NQO but not MMS. Induction of the RNR2-lacZ fusion in a RAD(+) strain in response to 4-NQO was not enhanced by the presence of an equal number of rad4-2 cells that lacked the fusion, implying that the DNA damage stress response in cell autonomous. Images PMID:2513480

  13. Chromatin Remodeling, DNA Damage Repair and Aging

    PubMed Central

    Liu, Baohua; Yip, Raymond KH; Zhou, Zhongjun

    2012-01-01

    Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging. PMID:23633913

  14. Interactions between lac repressor protein and site-specific bromodeoxyuridine-substituted operator DNA. Ultraviolet footprinting and protein-DNA cross-link formation

    SciTech Connect

    Wick, K.L.; Matthews, K.S. )

    1991-04-05

    Specific contacts between the lac repressor and operator have been explored using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40-base pair operator provides substrate for ultraviolet irradiation; upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed 13 unique sites of inducer-sensitive protection along the operator sequence using this method compared to complete substitution with BrdU; differences were observed at two positions for singly substituted versus completely substituted DNAs. The ability of these photosensitive DNAs to form short range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified. At one site, cross-linking without protection from strand scission was observed; this result suggests an unusual mechanism of strand scission and/or cross-linking at this site. Comparison of the UV protection results and the cross-linking data show that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts.

  15. Site-specific photochemistry

    NASA Astrophysics Data System (ADS)

    Koplitz, Brent D.; Brum, Jeffrey L.; Deshmukh, Subhash; Xu, Xiaodong; Wang, Zhongrui; Yen, Yu-Fong

    1992-04-01

    We present results on `site-specific' H-atom production in photolysis experiments conducted under collisionless conditions. H and D atoms are used as labels to investigate the site(s) at which C-H (or C-D) bond cleavage occurs in a variety of haloalkane systems. Experiments using two photolysis lasers clearly indicate that photon absorption by an intermediate, presumably an alkyl radical, is important in many of the systems studied. The site(s) (e.g., (alpha) , (beta) , or (gamma) ) at which C-H (or C-D) bond cleavage occurs is dependent not only on the nature of the molecule, but also on the photolysis wavelength. As a diagnostic tool, H- and D-atom Doppler spectroscopy allows us to gain insight into the energetics associated with the various dissociation processes. Our overall aim is to gain a further understanding of the photolysis properties of a variety of simple molecules and their associated radicals.

  16. Epigenome Maintenance in Response to DNA Damage.

    PubMed

    Dabin, Juliette; Fortuny, Anna; Polo, Sophie E

    2016-06-01

    Organism viability relies on the stable maintenance of specific chromatin landscapes, established during development, that shape cell functions and identities by driving distinct gene expression programs. Yet epigenome maintenance is challenged during transcription, replication, and repair of DNA damage, all of which elicit dynamic changes in chromatin organization. Here, we review recent advances that have shed light on the specialized mechanisms contributing to the restoration of epigenome structure and function after DNA damage in the mammalian cell nucleus. By drawing a parallel with epigenome maintenance during replication, we explore emerging concepts and highlight open issues in this rapidly growing field. In particular, we present our current knowledge of molecular players that support the coordinated maintenance of genome and epigenome integrity in response to DNA damage, and we highlight how nuclear organization impacts genome stability. Finally, we discuss possible functional implications of epigenome plasticity in response to genotoxic stress. PMID:27259203

  17. Molecular mechanisms involved in initiation of the DNA damage response

    PubMed Central

    Barnum, Kevin J; O’Connell, Matthew J

    2015-01-01

    DNA is subject to a wide variety of damage. In order to maintain genomic integrity, cells must respond to this damage by activating repair and cell cycle checkpoint pathways. The initiating events in the DNA damage response entail recognition of the lesion and the assembly of DNA damage response complexes at the DNA. Here, we review what is known about these processes for various DNA damage pathways. PMID:27308403

  18. The RNA Response to DNA Damage.

    PubMed

    Giono, Luciana E; Nieto Moreno, Nicolás; Cambindo Botto, Adrián E; Dujardin, Gwendal; Muñoz, Manuel J; Kornblihtt, Alberto R

    2016-06-19

    Multicellular organisms must ensure genome integrity to prevent accumulation of mutations, cell death, and cancer. The DNA damage response (DDR) is a complex network that senses, signals, and executes multiple programs including DNA repair, cell cycle arrest, senescence, and apoptosis. This entails regulation of a variety of cellular processes: DNA replication and transcription, RNA processing, mRNA translation and turnover, and post-translational modification, degradation, and relocalization of proteins. Accumulated evidence over the past decades has shown that RNAs and RNA metabolism are both regulators and regulated actors of the DDR. This review aims to present a comprehensive overview of the current knowledge on the many interactions between the DNA damage and RNA fields. PMID:26979557

  19. Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

    PubMed Central

    Friedman, Joshua I.; Stivers, James T.

    2010-01-01

    A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location. PMID:20469926

  20. UV damage in DNA promotes nucleosome unwrapping.

    PubMed

    Duan, Ming-Rui; Smerdon, Michael J

    2010-08-20

    The association of DNA with histones in chromatin impedes DNA repair enzymes from accessing DNA lesions. Nucleosomes exist in a dynamic equilibrium in which portions of the DNA molecule spontaneously unwrap, transiently exposing buried DNA sites. Thus, nucleosome dynamics in certain regions of chromatin may provide the exposure time and space needed for efficient repair of buried DNA lesions. We have used FRET and restriction enzyme accessibility to study nucleosome dynamics following DNA damage by UV radiation. We find that FRET efficiency is reduced in a dose-dependent manner, showing that the presence of UV photoproducts enhances spontaneous unwrapping of DNA from histones. Furthermore, this UV-induced shift in unwrapping dynamics is associated with increased restriction enzyme accessibility of histone-bound DNA after UV treatment. Surprisingly, the increased unwrapping dynamics is even observed in nucleosome core particles containing a single UV lesion at a specific site. These results highlight the potential for increased "intrinsic exposure" of nucleosome-associated DNA lesions in chromatin to repair proteins. PMID:20562439

  1. Oxidation of DNA: damage to nucleobases.

    PubMed

    Kanvah, Sriram; Joseph, Joshy; Schuster, Gary B; Barnett, Robert N; Cleveland, Charles L; Landman, Uzi

    2010-02-16

    All organisms store the information necessary to maintain life in their DNA. Any process that damages DNA, causing a loss or corruption of that information, jeopardizes the viability of the organism. One-electron oxidation is such a process. In this Account, we address three of the central features of one-electron oxidation of DNA: (i) the migration of the radical cation away from the site of its formation; (ii) the electronic and structural factors that determine the nucleobases at which irreversible reactions most readily occur; (iii) the mechanism of reaction for nucleobase radical cations. The loss of an electron (ionization) from DNA generates an electron "hole" (a radical cation), located most often on its nucleobases, that migrates reversibly through duplex DNA by hopping until it is trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In aqueous solution, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counterions to the phosphate groups (typically Na(+)) play an important role in facilitating both hopping and the eventual reaction of the radical cation with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA, comprising the four common DNA nucleobases G, C, A, and T, reaction occurs most commonly at a guanine, resulting in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step, primarily by a tandem process. The oxidative damage of DNA is a complex process, influenced by charge transport and reactions that are controlled by a combination of enthalpic, entropic, steric, and

  2. Autophosphorylation and Pin1 binding coordinate DNA damage-induced HIPK2 activation and cell death.

    PubMed

    Bitomsky, Nadja; Conrad, Elisa; Moritz, Christian; Polonio-Vallon, Tilman; Sombroek, Dirk; Schultheiss, Kathrin; Glas, Carolina; Greiner, Vera; Herbel, Christoph; Mantovani, Fiamma; del Sal, Giannino; Peri, Francesca; Hofmann, Thomas G

    2013-11-01

    Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1-HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis. PMID:24145406

  3. FIBER OPTIC BIOSENSOR FOR DNA DAMAGE

    EPA Science Inventory

    This paper describes a fiber optic biosensor for the rapid and sensitive detection of radiation-induced or chemically-induced oxidative DNA damage. The assay is based on the hybridization and temperature-induced dissociation (melting curves) of synthetic oligonucleotides. The...

  4. DNA Damage and Aging Around the Clock.

    PubMed

    Gutierrez-Martinez, Paula; Rossi, Derrick J; Beerman, Isabel

    2016-08-01

    The hematopoietic system undergoes many changes during aging, but the causes and molecular mechanisms behind these changes are not well understood. Wang et al. have recently implicated a circadian rhythm gene, Per2, as playing a role in the DNA damage response and in the expression of lymphoid genes in aged hematopoietic stem cells. PMID:27345866

  5. Oxidative DNA Damage and Nucleotide Excision Repair

    PubMed Central

    Melis, Joost P.M.; Luijten, Mirjam

    2013-01-01

    Abstract Significance: Oxidative DNA damage is repaired by multiple, overlapping DNA repair pathways. Accumulating evidence supports the hypothesis that nucleotide excision repair (NER), besides base excision repair (BER), is also involved in neutralizing oxidative DNA damage. Recent Advances: NER includes two distinct sub-pathways: transcription-coupled NER (TC-NER) and global genome repair (GG-NER). The CSA and CSB proteins initiate the onset of TC-NER. Recent findings show that not only CSB, but also CSA is involved in the repair of oxidative DNA lesions, in the nucleus as well as in mitochondria. The XPG protein is also of importance for the removal of oxidative DNA lesions, as it may enhance the initial step of BER. Substantial evidence exists that support a role for XPC in NER and BER. XPC deficiency not only results in decreased repair of oxidative lesions, but has also been linked to disturbed redox homeostasis. Critical Issues: The role of NER proteins in the regulation of the cellular response to oxidative (mitochondrial and nuclear) DNA damage may be the underlying mechanism of the pathology of accelerated aging in Cockayne syndrome patients, a driving force for internal cancer development in XP-A and XP-C patients, and a contributor to the mixed exhibited phenotypes of XP-G patients. Future Directions: Accumulating evidence indicates that DNA repair factors can be involved in multiple DNA repair pathways. However, the distinct detailed mechanism and consequences of these additional functions remain to be elucidated and can possibly shine a light on clinically related issues. Antioxid. Redox Signal. 18, 2409–2419. PMID:23216312

  6. Maternal diabetes triggers DNA damage and DNA damage response in neurulation stage embryos through oxidative stress.

    PubMed

    Dong, Daoyin; Yu, Jingwen; Wu, Yanqing; Fu, Noah; Villela, Natalia Arias; Yang, Peixin

    2015-11-13

    DNA damage and DNA damage response (DDR) in neurulation stage embryos under maternal diabetes conditions are not well understood. The purpose of this study was to investigate whether maternal diabetes and high glucose in vitro induce DNA damage and DDR in the developing embryo through oxidative stress. In vivo experiments were conducted by mating superoxide dismutase 1 (SOD1) transgenic male mice with wild-type (WT) female mice with or without diabetes. Embryonic day 8.75 (E8.75) embryos were tested for the DNA damage markers, phosphorylated histone H2A.X (p-H2A.X) and DDR signaling intermediates, including phosphorylated checkpoint 1 (p-Chk1), phosphorylated checkpoint 2 (p-Chk2), and p53. Levels of the same DNA damage markers and DDR signaling intermediates were also determined in the mouse C17.2 neural stem cell line. Maternal diabetes and high glucose in vitro significantly increased the levels of p-H2A.X. Levels of p-Chk1, p-Chk2, and p53, were elevated under both maternal diabetic and high glucose conditions. SOD1 overexpression blocked maternal diabetes-induced DNA damage and DDR in vivo. Tempol, a SOD1 mimetic, diminished high glucose-induced DNA damage and DDR in vitro. In conclusion, maternal diabetes and high glucose in vitro induce DNA damage and activates DDR through oxidative stress, which may contribute to the pathogenesis of diabetes-associated embryopathy. PMID:26427872

  7. Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells.

    PubMed

    Izhar, Lior; Ziv, Omer; Cohen, Isadora S; Geacintov, Nicholas E; Livneh, Zvi

    2013-04-16

    DNA lesions can block replication forks and lead to the formation of single-stranded gaps. These replication complications are mitigated by DNA damage tolerance mechanisms, which prevent deleterious outcomes such as cell death, genomic instability, and carcinogenesis. The two main tolerance strategies are translesion DNA synthesis (TLS), in which low-fidelity DNA polymerases bypass the blocking lesion, and homology-dependent repair (HDR; postreplication repair), which is based on the homologous sister chromatid. Here we describe a unique high-resolution method for the simultaneous analysis of TLS and HDR across defined DNA lesions in mammalian genomes. The method is based on insertion of plasmids carrying defined site-specific DNA lesions into mammalian chromosomes, using phage integrase-mediated integration. Using this method we show that mammalian cells use HDR to tolerate DNA damage in their genome. Moreover, analysis of the tolerance of the UV light-induced 6-4 photoproduct, the tobacco smoke-induced benzo[a]pyrene-guanine adduct, and an artificial trimethylene insert shows that each of these three lesions is tolerated by both TLS and HDR. We also determined the specificity of nucleotide insertion opposite these lesions during TLS in human genomes. This unique method will be useful in elucidating the mechanism of DNA damage tolerance in mammalian chromosomes and their connection to pathological processes such as carcinogenesis. PMID:23530190

  8. Oxidative DNA damage accumulation in gastric carcinogenesis

    PubMed Central

    Farinati, F; Cardin, R; Degan, P; Rugge, M; Di, M; Bonvicini, P; Naccarato, R

    1998-01-01

    Background—Gastric carcinogenesis is a multifactorial, multistep process, in which chronic inflammation plays a major role. 
Aims—In order to ascertain whether free radical mediated oxidative DNA damage is involved in such a process, concentrations of 8-hydroxydeoxyguanosine (8OHdG), a mutagenic/carcinogenic adduct, and thiobarbituric acid reactive substances (TBARS), as an indirect measure of free radical mediated damage, were determined in biopsy specimens from patients undergoing endoscopy. 
Patients—Eighty eight patients were divided into histological subgroups as follows: 27 with chronic non-atrophic gastritis, 41 with atrophic gastritis, six with gastric cancer, and 14 unaffected controls. 
Methods—Intestinal metaplasia, Helicobacter pylori infection, and disease activity were semiquantitatively scored. 8OHdG concentrations were assessed by HPLC with electrochemical detection, and TBARS concentrations were fluorimetrically assayed. 
Results—8OHdG concentrations (mean number of adducts/105 dG residues) were significantly higher in chronic atrophic gastritis (p=0.0009). Significantly higher concentrations were also detected in the presence of severe disease activity (p=0.02), intestinal metaplasia (p=0.035), and H pylori infection (p=0.001). TBARS concentrations were also higher in atrophic gastritis, though not significantly so. In a multiple logistic regression analysis, 8OHdG concentrations correlated best with the presence and severity of H pylori infection (r=0.53, p=0.002). 
Conclusions—Chronic gastritis is characterised by the accumulation of oxidative DNA damage with mutagenic and carcinogenic potential. H pylori infection is the major determinant for DNA adduct formation. 

 Keywords: free radicals; oxidative DNA damage; gastric carcinogenesis; precancerous changes; peroxidative damage PMID:9577340

  9. DNA damage checkpoint recovery and cancer development

    SciTech Connect

    Wang, Haiyong; Zhang, Xiaoshan; Teng, Lisong; Legerski, Randy J.

    2015-06-10

    Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observed to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint

  10. DNA damage tolerance by recombination: Molecular pathways and DNA structures.

    PubMed

    Branzei, Dana; Szakal, Barnabas

    2016-08-01

    Replication perturbations activate DNA damage tolerance (DDT) pathways, which are crucial to promote replication completion and to prevent fork breakage, a leading cause of genome instability. One mode of DDT uses translesion synthesis polymerases, which however can also introduce mutations. The other DDT mode involves recombination-mediated mechanisms, which are generally accurate. DDT occurs prevalently postreplicatively, but in certain situations homologous recombination is needed to restart forks. Fork reversal can function to stabilize stalled forks, but may also promote error-prone outcome when used for fork restart. Recent years have witnessed important advances in our understanding of the mechanisms and DNA structures that mediate recombination-mediated damage-bypass and highlighted principles that regulate DDT pathway choice locally and temporally. In this review we summarize the current knowledge and paradoxes on recombination-mediated DDT pathways and their workings, discuss how the intermediate DNA structures may influence genome integrity, and outline key open questions for future research. PMID:27236213

  11. Heat Stress-Induced DNA Damage

    PubMed Central

    Kantidze, O.L.; Velichko, A.K.; Luzhin, A.V.; Razin, S.V.

    2016-01-01

    Although the heat-stress response has been extensively studied for decades, very little is known about its effects on nucleic acids and nucleic acid-associated processes. This is due to the fact that the research has focused on the study of heat shock proteins and factors (HSPs and HSFs), their involvement in the regulation of transcription, protein homeostasis, etc. Recently, there has been some progress in the study of heat stress effects on DNA integrity. In this review, we summarize and discuss well-known and potential mechanisms of formation of various heat stress-induced DNA damage. PMID:27437141

  12. DNA Damage, Homology-Directed Repair, and DNA Methylation

    PubMed Central

    Angrisano, Tiziana; Morano, Annalisa; Lee, Bongyong; Pardo, Alba Di; Messina, Samantha; Iuliano, Rodolfo; Fusco, Alfredo; Santillo, Maria R; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Avvedimento, Enrico V

    2007-01-01

    To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments. PMID:17616978

  13. O6-alkylguanine-DNA transferase (SNAP) as capture module for site-specific covalent bioconjugation of targeting protein on nanoparticles

    NASA Astrophysics Data System (ADS)

    Mazzucchelli, Serena; Colombo, Miriam; Galbiati, Elisabetta; Corsi, Fabio; Montenegro, Josè M.; Parak, Wolfgang J.; Prosperi, Davide

    2013-02-01

    A bimodular genetic fusion comprising a delivery module (scFv) and a capture module (SNAP) is proposed as a novel strategy for the biologically mediated site-specific covalent conjugation of targeting proteins to nanoparticles. ScFv800E6, an scFv mutant selective for HER2 antigen overexpressed in breast cancer cells was chosen as targeting ligand. The fusion protein SNAP-scFv was irreversibly immobilized on magnetofluorescent nanoparticles through the recognition between SNAP module and pegylated O6-alkylguanine derivative. The targeting efficiency of the resulting nanoparticle against HER2-positive breast cancer cells was assessed by flow cytometry and immunofluorescence.

  14. Mitochondrial DNA damage and efficiency of ATP biosynthesis: mathematical model.

    PubMed

    Beregovskaya, N; Maiboroda, R

    1995-01-21

    The role of mitochondrial DNA (mtDNA) damage in ageing processes and in malignant transformation of a cell is discussed. A mathematical model of the mtDNA population in a cell and in tissue is constructed. The model describes the effects of mtDNA damages accumulated during ageing and some features of malignant transformation and regeneration. PMID:7891454

  15. DNA-damaging autoantibodies and cancer: the lupus butterfly theory.

    PubMed

    Noble, Philip W; Bernatsky, Sasha; Clarke, Ann E; Isenberg, David A; Ramsey-Goldman, Rosalind; Hansen, James E

    2016-07-01

    Autoantibodies reactive against host DNA are detectable in the circulation of most people with systemic lupus erythematosus (SLE). The long-held view that antibodies cannot penetrate live cells has been disproved. A subset of lupus autoantibodies penetrate cells, translocate to nuclei, and inhibit DNA repair or directly damages DNA. The result of these effects depends on the microenvironment and genetic traits of the cell. Some DNA-damaging antibodies alone have little impact on normal cells, but in the presence of other conditions, such as pre-existing DNA-repair defects, can become highly toxic. These findings raise new questions about autoimmunity and DNA damage, and reveal opportunities for new targeted therapies against malignancies particularly vulnerable to DNA damage. In this Perspectives article, we review the known associations between SLE, DNA damage and cancer, and propose a theory for the effects of DNA-damaging autoantibodies on SLE pathophysiology and cancer risk. PMID:27009542

  16. Mitochondrial DNA damage induced autophagy, cell death, and disease

    PubMed Central

    Van Houten, Bennett; Hunter, Senyene E.; Meyer, Joel N.

    2016-01-01

    Mammalian mitochondria contain multiple small genomes. While these organelles have efficient base excision removal of oxidative DNA lesions and alkylation damage, many DNA repair systems that work on nuclear DNA damage are not active in mitochondria. What is the fate of DNA damage in the mitochondria that cannot be repaired or that overwhelms the repair system? Some forms of mitochondrial DNA damage can apparently trigger mitochondrial DNA destruction, either via direct degradation or through specific forms of autophagy, such as mitophagy. However, accumulation of certain types of mitochondrial damage, in the absence of DNA ligase III (Lig3) or exonuclease G (EXOG), enzymes required for repair, can directly trigger cell death. This review examines the cellular effects of persistent damage to mitochondrial genomes and discusses the very different cell fates that occur in response to different kinds of damage. PMID:26709760

  17. Direct Detection and Sequencing of Damaged DNA Bases

    PubMed Central

    2011-01-01

    Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications. PMID:22185597

  18. Influenza infection induces host DNA damage and dynamic DNA damage responses during tissue regeneration

    PubMed Central

    Li, Na; Parrish, Marcus; Chan, Tze Khee; Yin, Lu; Rai, Prashant; Yoshiyuki, Yamada; Abolhassani, Nona; Tan, Kong Bing; Kiraly, Orsolya; Chow, Vincent TK; Engelward, Bevin P.

    2016-01-01

    Influenza viruses account for significant morbidity worldwide. Inflammatory responses, including excessive generation of reactive oxygen and nitrogen species (RONS), mediate lung injury in severe Influenza infections. However, the molecular basis of inflammation-induced lung damage is not fully understood. Here, we studied influenza H1N1 infected cells in vitro, as well as H1N1 infected mice, and we monitored molecular and cellular responses over the course of two weeks in vivo. We show that influenza induces DNA damage both when cells are directly exposed to virus in vitro (measured using the comet assay) and also when cells are exposed to virus in vivo (estimated via γH2AX foci). We show that DNA damage, as well as responses to DNA damage, persist in vivo until long after virus has been cleared, at times when there are inflammation associated RONS (measured by xanthine oxidase activity and oxidative products). The frequency of lung epithelial and immune cells with increased γH2AX foci is elevated in vivo, especially for dividing cells (Ki-67 positive) exposed to oxidative stress during tissue regeneration. Additionally, we observed a significant increase in apoptotic cells as well as increased levels of DSB repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA both in vitro and in vivo, and that DNA damage responses are activated, raising the possibility that DNA repair capacity may be a determining factor for tissue recovery and disease outcome. PMID:25809161

  19. Genetic and non-genetic influences during pregnancy on infant global and site specific DNA methylation: role for folate gene variants and vitamin B12.

    PubMed

    McKay, Jill A; Groom, Alexandra; Potter, Catherine; Coneyworth, Lisa J; Ford, Dianne; Mathers, John C; Relton, Caroline L

    2012-01-01

    Inter-individual variation in patterns of DNA methylation at birth can be explained by the influence of environmental, genetic and stochastic factors. This study investigates the genetic and non-genetic determinants of variation in DNA methylation in human infants. Given its central role in provision of methyl groups for DNA methylation, this study focuses on aspects of folate metabolism. Global (LUMA) and gene specific (IGF2, ZNT5, IGFBP3) DNA methylation were quantified in 430 infants by Pyrosequencing®. Seven polymorphisms in 6 genes (MTHFR, MTRR, FOLH1, CβS, RFC1, SHMT) involved in folate absorption and metabolism were analysed in DNA from both infants and mothers. Red blood cell folate and serum vitamin B(12) concentrations were measured as indices of vitamin status. Relationships between DNA methylation patterns and several covariates viz. sex, gestation length, maternal and infant red cell folate, maternal and infant serum vitamin B(12), maternal age, smoking and genotype were tested. Length of gestation correlated positively with IGF2 methylation (rho = 0.11, p = 0.032) and inversely with ZNT5 methylation (rho = -0.13, p = 0.017). Methylation of the IGFBP3 locus correlated inversely with infant vitamin B(12) concentration (rho = -0.16, p = 0.007), whilst global DNA methylation correlated inversely with maternal vitamin B(12) concentrations (rho = 0.18, p = 0.044). Analysis of common genetic variants in folate pathway genes highlighted several associations including infant MTRR 66G>A genotype with DNA methylation (χ(2) = 8.82, p = 0.003) and maternal MTHFR 677C>T genotype with IGF2 methylation (χ(2) = 2.77, p = 0.006). These data support the hypothesis that both environmental and genetic factors involved in one-carbon metabolism influence DNA methylation in infants. Specifically, the findings highlight the importance of vitamin B(12) status, infant MTRR genotype and maternal MTHFR genotype, all of which may influence the supply of methyl groups for DNA

  20. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities

    PubMed Central

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K.

    2014-01-01

    Loss of WRN function causes Werner Syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN’s DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor hydroxyurea. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency. PMID:24965941

  1. Yap1: a DNA damage responder in Saccharomyces cerevisiae.

    PubMed

    Rowe, Lori A; Degtyareva, Natalya; Doetsch, Paul W

    2012-04-01

    Activation of signaling pathways in response to genotoxic stress is crucial for cells to properly repair DNA damage. In response to DNA damage, intracellular levels of reactive oxygen species increase. One important function of such a response could be to initiate signal transduction processes. We have employed the model eukaryote Saccharomyces cerevisiae to delineate DNA damage sensing mechanisms. We report a novel, unanticipated role for the transcription factor Yap1 as a DNA damage responder, providing direct evidence that reactive oxygen species are an important component of the DNA damage signaling process. Our findings reveal an epistatic link between Yap1 and the DNA base excision repair pathway. Corruption of the Yap1-mediated DNA damage response influences cell survival and genomic stability in response to exposure to genotoxic agents. PMID:22433435

  2. DNA Damage Response and Immune Defense: Links and Mechanisms

    PubMed Central

    Nakad, Rania; Schumacher, Björn

    2016-01-01

    DNA damage plays a causal role in numerous human pathologies including cancer, premature aging, and chronic inflammatory conditions. In response to genotoxic insults, the DNA damage response (DDR) orchestrates DNA damage checkpoint activation and facilitates the removal of DNA lesions. The DDR can also arouse the immune system by for example inducing the expression of antimicrobial peptides as well as ligands for receptors found on immune cells. The activation of immune signaling is triggered by different components of the DDR including DNA damage sensors, transducer kinases, and effectors. In this review, we describe recent advances on the understanding of the role of DDR in activating immune signaling. We highlight evidence gained into (i) which molecular and cellular pathways of DDR activate immune signaling, (ii) how DNA damage drives chronic inflammation, and (iii) how chronic inflammation causes DNA damage and pathology in humans. PMID:27555866

  3. Nucleotide Salvage Deficiencies, DNA Damage and Neurodegeneration

    PubMed Central

    Fasullo, Michael; Endres, Lauren

    2015-01-01

    Nucleotide balance is critically important not only in replicating cells but also in quiescent cells. This is especially true in the nervous system, where there is a high demand for adenosine triphosphate (ATP) produced from mitochondria. Mitochondria are particularly prone to oxidative stress-associated DNA damage because nucleotide imbalance can lead to mitochondrial depletion due to low replication fidelity. Failure to maintain nucleotide balance due to genetic defects can result in infantile death; however there is great variability in clinical presentation for particular diseases. This review compares genetic diseases that result from defects in specific nucleotide salvage enzymes and a signaling kinase that activates nucleotide salvage after DNA damage exposure. These diseases include Lesch-Nyhan syndrome, mitochondrial depletion syndromes, and ataxia telangiectasia. Although treatment options are available to palliate symptoms of these diseases, there is no cure. The conclusions drawn from this review include the critical role of guanine nucleotides in preventing neurodegeneration, the limitations of animals as disease models, and the need to further understand nucleotide imbalances in treatment regimens. Such knowledge will hopefully guide future studies into clinical therapies for genetic diseases. PMID:25923076

  4. Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression

    PubMed Central

    Cline, Susan D.

    2012-01-01

    How mitochondria process DNA damage and whether a change in the steady-state level of mitochondrial DNA damage (mtDNA) contributes to mitochondrial dysfunction are questions that fuel burgeoning areas of research into aging and disease pathogenesis. Over the past decade, researchers have identified and measured various forms of endogenous and environmental mtDNA damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to contain the full range of DNA repair mechanisms that operate in the nucleus, although mtDNA contains types of damage that are targets of each nuclear DNA repair pathway. The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome. Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA polymerase, or by inducing transcriptional mutagenesis or premature transcript termination. This review summarizes our current knowledge of mtDNA damage, its repair, and its effects on mtDNA integrity and gene expression. PMID:22728831

  5. Impact of Alternative DNA Structures on DNA Damage, DNA Repair, and Genetic Instability

    PubMed Central

    Wang, Guliang; Vasquez, Karen M.

    2014-01-01

    Repetitive genomic sequences can adopt a number of alternative DNA structures that differ from the canonical B-form duplex (i.e. non-B DNA). These non-B DNA-forming sequences have been shown to have many important biological functions related to DNA metabolic processes; for example, they may have regulatory roles in DNA transcription and replication. In addition to these regulatory functions, non-B DNA can stimulate genetic instability in the presence or absence of DNA damage, via replication-dependent and/or replication-independent pathways. This review focuses on the interactions of non-B DNA conformations with DNA repair proteins and how these interactions impact genetic instability. PMID:24767258

  6. Cellular commitment to reentry into the cell cycle after stalled DNA is determined by site-specific phosphorylation of Chk1 and PTEN

    PubMed Central

    Martin, Sarah A.; Ouchi, Toru

    2016-01-01

    In this study, we show that depletion of Chk1 by small interfering RNA (siRNA) results in failure of reentry to the cell cycle after DNA replication has been stalled by exposure to hydroxyurea (HU). Casein kinase II (CKII) is degraded in these cells in a proteasome-dependent manner, resulting in decreased phosphorylation and PTEN levels. We show that phosphorylation of Chk1 at Ser317 but not at Ser345 is required for phosphorylation of PTEN at Thr383 by CKII, making cell cycle reentry after HU treatment possible. Like Chk1 depletion, loss of PTEN due to siRNA is followed by inability to return to the cell cycle following HU. In Chk1-siRNA cells, reintroduction of wild-type PTEN but not PTEN T383A restores the ability of the cell to reenter the G2-M phase of the cell cycle after stalled DNA replication. We conclude that, in response to stalled DNA replication, Chk1 is phosphorylated at Ser317 by ATR resulting in stabilization of CKII, which in turn leads to phosphorylation of PTEN at Thr383. PMID:18723495

  7. Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river

    USGS Publications Warehouse

    Karouna-Renier, Natalie K.; White, Carl; Perkins, Christopher R.; Schmerfeld, John J.; Yates, David

    2014-01-01

    Historical discharges of Hg into the South River near the town of Waynesboro, VA, USA, have resulted in persistently elevated Hg concentrations in sediment, surface water, ground water, soil, and wildlife downstream of the discharge site. In the present study, we examined mercury (Hg) levels in in little brown bats (Myotis lucifugus) from this location and assessed the utility of a non-destructively collected tissue sample (wing punch) for determining mitochondrial DNA (mtDNA) damage in Hg exposed bats. Bats captured 1 and 3 km from the South River, exhibited significantly higher levels of total Hg (THg) in blood and fur than those from the reference location. We compared levels of mtDNA damage using real-time quantitative PCR (qPCR) analysis of two distinct regions of mtDNA. Genotoxicity is among the many known toxic effects of Hg, resulting from direct interactions with DNA or from oxidative damage. Because it lacks many of the protective protein structures and repair mechanisms associated with nuclear DNA, mtDNA is more sensitive to the effects of genotoxic chemicals and therefore may be a useful biomarker in chronically exposed organisms. Significantly higher levels of damage were observed in both regions of mtDNA in bats captured 3 km from the river than in controls. However, levels of mtDNA damage exhibited weak correlations with fur and blood THg levels, suggesting that other factors may play a role in the site-specific differences.

  8. Decoupling of DNA damage response signaling from DNA damages underlies temozolomide resistance in glioblastoma cells☆

    PubMed Central

    Cui, Bo; Johnson, Stewart P.; Bullock, Nancy; Ali-Osman, Francis; Bigner, Darell D.; Friedman, Henry S.

    2010-01-01

    Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor in adults. Current therapy includes surgery, radiation and chemotherapy with temozolomide (TMZ). Major determinants of clinical response to TMZ include methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter and mismatch repair (MMR) status. Though the MGMT promoter is methylated in 45% of cases, for the first nine months of follow-up, TMZ does not change survival outcome. Furthermore, MMR deficiency makes little contribution to clinical resistance, suggesting that there exist unrecognized mechanisms of resistance. We generated paired GBM cell lines whose resistance was attributed to neither MGMT nor MMR. We show that, responding to TMZ, these cells exhibit a decoupling of DNA damage response (DDR) from ongoing DNA damages. They display methylation-resistant synthesis in which ongoing DNA synthesis is not inhibited. They are also defective in the activation of the S and G2 phase checkpoint. DDR proteins ATM, Chk2, MDC1, NBS1 and gammaH2AX also fail to form discrete foci. These results demonstrate that failure of DDR may play an active role in chemoresistance to TMZ. DNA damages by TMZ are repaired by MMR proteins in a futile, reiterative process, which activates DDR signaling network that ultimately leads to the onset of cell death. GBM cells may survive genetic insults in the absence of DDR. We anticipate that our findings will lead to more studies that seek to further define the role of DDR in ultimately determining the fate of a tumor cell in response to TMZ and other DNA methylators. PMID:23554659

  9. Nanoparticles for Site Specific Genome Editing

    NASA Astrophysics Data System (ADS)

    McNeer, Nicole Ali

    Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 by "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in treatment or cure of inherited disorders of the blood such as beta-thalassemia. Gene editing in HSPCs and differentiated T cells could help combat HIV/AIDs by modifying receptors, such as CCR5, necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. In vivo gene editing could also provide novel treatment for systemic monogenic disorders such as cystic fibrosis, an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane receptor. Here, we have engineered biodegradable nanoparticles to deliver oligonucleotides for site-specific genome editing of disease-relevant genes in human cells, with high efficiency, low toxicity, and editing of clinically relevant cell types. We designed nanoparticles to edit the human beta-globin and CCR5 genes in hematopoietic cells. We show that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can delivery PNA and donor DNA for site-specific gene modification in human hematopoietic cells in vitro and in vivo in NOD-scid IL2rgammanull mice. Nanoparticles delivered by tail vein localized to hematopoietic compartments in the spleen and bone marrow of humanized mice, resulting in modification of the beta-globin and CCR5 genes. Modification frequencies ranged from 0.005 to 20% of cells depending on the organ and cell type, without detectable toxicity. This project developed highly versatile methods for delivery of therapeutics to hematolymphoid cells and hematopoietic stem cells, and will help to

  10. 8-Oxoguanine DNA glycosylase-1 augments pro-inflammatory gene expression by facilitating the recruitment of site-specific transcription factors

    PubMed Central

    Ba, Xueqing; Bacsi, Attila; Luo, Jixian; Aguilera-Aguirre, Leopoldo; Zeng, Xianlu; Radak, Zsolt; Brasier, Allan R; Boldogh, Istvan

    2014-01-01

    Among the insidious DNA base lesions, 8-oxo-7,8-dihydroguanine (8-oxoG) is one of the most abundant, a lesion that arises through the attack by reactive oxygen species on guanine, especially when located in cis-regulatory elements. 8-oxoG is repaired by the 8-oxoguanine glycosylase 1 (OGG1)-initiated DNA base excision repair (BER) pathway. Here we investigated whether 8-oxoG repair by OGG1 in promoter regions is compatible with a prompt gene expression and a host innate immune response. For this purpose, we utilized a mouse model of airway inflammation, supplemented with cell cultures, chromatin immunoprecipitation, siRNA knockdown, real-time PCR, Comet and reporter transcription assays. Our data show that exposure of cells to tumor necrosis factor alpha (TNF-α) altered cellular redox, increased the 8-oxoG level in DNA, recruited OGG1 to promoter sequences and transiently inhibited BER of 8-oxoG. Promoter-associated OGG1 then enhanced NF-êB/RelA binding to cis-elements and facilitated recruitment of Specificity Protein 1 (SP1), transcription initiation factor II-D (TFIID), and phospho-RNA polymerase II, resulting in the rapid expression of chemokines/cytokines and inflammatory cell accumulation in mouse airways. siRNA depletion of OGG1 or prevention of guanine oxidation significantly decreased TNF-α-induced inflammatory responses. Together, these results show that non-productive binding of OGG1 to 8-oxoG in promoter sequences could be an epigenetic mechanism to modulate gene expression for a prompt innate immune response. PMID:24489103

  11. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  12. The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons

    SciTech Connect

    Xiong, Y.; Eickbush, T.H.

    1988-01-01

    Two types of insertion elements, R1 and R2 (previously called type I and type II), are known to interrupt the 28S ribosomal genes of several insect species. In the silkmoth, Bombyx mori, each element occupies approximately 10% of the estimated 240 ribosomal DNA units, while at most only a few copies are located outside the ribosomal DNA units. The authors present here the complete nucleotide sequence of an R1 insertion from B. mori (R1Bm). This 5.1-kilobase element contains two overlapping open reading frames (ORFs) which together occupy 88% of its length. ORF1 is 461 amino acids in length and exhibits characteristics of retroviral gag genes. ORF2 is 1,051 amino acids in length and contains homology to reverse transcriptase-like enzymes. The analysis of 3' and 5' ends of independent isolates from the ribosomal locus supports the suggestion that R1 is still functioning as a transposable element. The precise location of the element within the genome implies that its transposition must occur with remarkable insertion sequence specificity. Comparison of the deduced amino acid sequences from six retrotransposons, R1 and R2 of B. mori, I factor and F element of Drosophila melanogaster, L1 of Mus domesticus, and Ingi of Trypanosoma brucei, reveals a relatively high level of sequence homology in the reverse transcriptase region. Like R1, these elements lack long terminal repeats. The authors therefore named this class of related elements the non-long-terminal-repeat (non-LTR) retrotransposons.

  13. Maintenance of the DNA-Damage Checkpoint Requires DNA-Damage-Induced Mediator Protein Oligomerization

    PubMed Central

    Usui, Takehiko; Foster, Steven S.; Petrini, John H.J.

    2010-01-01

    SUMMARY Oligomeric assembly of Brca1 C-terminal (BRCT) domain-containing mediator proteins occurs at sites of DNA damage. However, the functional significance and regulation of such assemblies are not well understood. In this study, we defined the molecular mechanism of DNA-damage-induced oligomerization of the S. cerevisiae BRCT protein Rad9. Our data suggest that Rad9’s tandem BRCT domain mediates Rad9 oligomerization via its interaction with its own Mec1/Tel1-phosphorylated SQ/TQ cluster domain (SCD). Rad53 activation is unaffected by mutations that impair Rad9 oligomerization, but checkpoint maintenance is lost, indicating that oligomerization is required to sustain checkpoint signaling. Once activated, Rad53 phosphorylates the Rad9 BRCT domain, which attenuates the BRCT-SCD interaction. Failure to phosphorylate the Rad9 BRCT results in cytologically visible Rad9 foci. This suggests a feedback loop wherein Rad53 activity and Rad9 oligomerization are regulated to tune the DNA-damage response. PMID:19187758

  14. Transposon-specified site-specific recombination.

    PubMed Central

    Kitts, P; Symington, L; Burke, M; Reed, R; Sherratt, D

    1982-01-01

    Cointegrate DNA molecules containing two copies of a transposable element appear to be intermediates in the transposition process. These structures are resolved by site-specific recombination to yield the normal end products of transposition. The transposable element gamma delta (Tn1000) synthesizes a product interchangeable with the Tn1/3tnpR protein in promoting Tn1/3 site-specific recombination. These data support the hypothesis that cointegrates containing directly repeated copies of Tn1/3 are obligatory intermediates in interreplicon transposition of Tn1/3. In addition, we show here that the reaction is independent of the element-encoded tnpA gene product. Tn501, which specifies mercury resistance, also produces cointegrates as intermediates in interreplicon transposition. The appearance of Tn501-specified recombination activity that can act on these cointegrates requires growth of cells in the presence of Hg2+. Images PMID:6275390

  15. Oxidative DNA damage stalls the human mitochondrial replisome

    PubMed Central

    Stojkovič, Gorazd; Makarova, Alena V.; Wanrooij, Paulina H.; Forslund, Josefin; Burgers, Peter M.; Wanrooij, Sjoerd

    2016-01-01

    Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase γ holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage, and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism. PMID:27364318

  16. DNA damage and neurotoxicity of chronic alcohol abuse

    PubMed Central

    Kruman, Inna I; Henderson, George I; Bergeson, Susan E

    2013-01-01

    Chronic alcohol abuse results in a variety of pathological effects including damage to the brain. The causes of alcohol-induced brain pathology are presently unclear. Several mechanisms of pathogenicity of chronic alcoholism have been proposed, including accumulation of DNA damage in the absence of repair, resulting in genomic instability and death of neurons. Genomic instability is a unified genetic mechanism leading to a variety of neurodegenerative disorders. Ethanol also likely interacts with various metabolic pathways, including one-carbon metabolism (OCM). OCM is critical for the synthesis of DNA precursors, essential for DNA repair, and as a methyl donor for various methylation events, including DNA methylation. Both DNA repair and DNA methylation are critical for maintaining genomic stability. In this review, we outline the role of DNA damage and DNA repair dysfunction in chronic alcohol-induced neurodegeneration. PMID:22829701

  17. Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease.

    PubMed Central

    Puchta, H; Dujon, B; Hohn, B

    1993-01-01

    Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination. Images PMID:8255757

  18. DNA damage in cells exhibiting radiation-induced genomic instability

    DOE PAGESBeta

    Keszenman, Deborah J.; Kolodiuk, Lucia; Baulch, Janet E.

    2015-02-22

    Cells exhibiting radiation induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during normal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesismore » that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated cells. The measurement of oxypurine, oxypyrimidine and abasic site endogenous DNA damage showed differences in non-double-strand breaks (DSB) clusters among the three of the four unstable clones evaluated as compared to genomically stable clones and the parental cell line. These three unstable clones also had increased levels of DSB clusters. The results of this study demonstrate that each unstable cell line has a unique spectrum of persistent damage and lead us to speculate that alterations in DNA damage signaling and repair may be related to the perpetuation of genomic instability.« less

  19. DNA damage in cells exhibiting radiation-induced genomic instability

    SciTech Connect

    Keszenman, Deborah J.; Kolodiuk, Lucia; Baulch, Janet E.

    2015-02-22

    Cells exhibiting radiation induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during normal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesis that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated cells. The measurement of oxypurine, oxypyrimidine and abasic site endogenous DNA damage showed differences in non-double-strand breaks (DSB) clusters among the three of the four unstable clones evaluated as compared to genomically stable clones and the parental cell line. These three unstable clones also had increased levels of DSB clusters. The results of this study demonstrate that each unstable cell line has a unique spectrum of persistent damage and lead us to speculate that alterations in DNA damage signaling and repair may be related to the perpetuation of genomic instability.

  20. Metabolic activation of carcinogenic ethylbenzene leads to oxidative DNA damage.

    PubMed

    Midorikawa, Kaoru; Uchida, Takafumi; Okamoto, Yoshinori; Toda, Chitose; Sakai, Yoshie; Ueda, Koji; Hiraku, Yusuke; Murata, Mariko; Kawanishi, Shosuke; Kojima, Nakao

    2004-12-01

    Ethylbenzene is carcinogenic to rats and mice, while it has no mutagenic activity. We have investigated whether ethylbenzene undergoes metabolic activation, leading to DNA damage. Ethylbenzene was metabolized to 1-phenylethanol, acetophenone, 2-ethylphenol and 4-ethylphenol by rat liver microsomes. Furthermore, 2-ethylphenol and 4-ethylphenol were metabolically transformed to ring-dihydroxylated metabolites such as ethylhydroquinone and 4-ethylcatechol, respectively. Experiment with 32P-labeled DNA fragment revealed that both ethylhydroquinone and 4-ethylcatechol caused DNA damage in the presence of Cu(II). These dihydroxylated compounds also induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). Catalase, methional and Cu(I)-specific chelator, bathocuproine, significantly (P<0.05) inhibited oxidative DNA damage, whereas free hydroxyl radical scavenger and superoxide dismutase did not. These results suggest that Cu(I) and H2O2 produced via oxidation of ethylhydroquinone and 4-ethylcatechol are involved in oxidative DNA damage. Addition of an endogenous reductant NADH dramatically enhanced 4-ethylcatechol-induced oxidative DNA damage, whereas ethylhydroquinone-induced DNA damage was slightly enhanced. Enhancing effect of NADH on oxidative DNA damage by 4-ethylcatechol may be explained by assuming that reactive species are generated from the redox cycle. In conclusion, these active dihydroxylated metabolites would be involved in the mechanism of carcinogenesis by ethylbenzene. PMID:15560893

  1. Ultraviolet induced DNA damage and hereditary skin cancer

    SciTech Connect

    Regan, J.D.; Carrier, W.L.; Francis, A.A.

    1984-01-01

    Clearly, cells from normal individuals possess the ability to repair a variety of damage to DNA. Numerous studies indicate that defects in DNA repair may increase an individual's susceptibility to cancer. It is hoped that continued studies of the exact structural changes produced in the DNA by environmental insults, and the correlation of specific DNA changes with particulr cellular events, such as DNA repair, will lead to a better understanding of cell-killing, mutagenesis and carbinogenesis. 1 figure, 2 tables.

  2. DNA Damage among Wood Workers Assessed with the Comet Assay

    PubMed Central

    Bruschweiler, Evin Danisman; Wild, Pascal; Huynh, Cong Khanh; Savova-Bianchi, Dessislava; Danuser, Brigitta; Hopf, Nancy B.

    2016-01-01

    Exposure to wood dust, a human carcinogen, is common in wood-related industries, and millions of workers are occupationally exposed to wood dust worldwide. The comet assay is a rapid, simple, and sensitive method for determining DNA damage. The objective of this study was to investigate the DNA damage associated with occupational exposure to wood dust using the comet assay (peripheral blood samples) among nonsmoking wood workers (n = 31, furniture and construction workers) and controls (n = 19). DNA damage was greater in the group exposed to composite wood products compared to the group exposed to natural woods and controls (P < 0.001). No difference in DNA damage was observed between workers exposed to natural woods and controls (P = 0.13). Duration of exposure and current dust concentrations had no effect on DNA damage. In future studies, workers’ exposures should include cumulative dust concentrations and exposures originating from the binders used in composite wood products. PMID:27398027

  3. DNA Damage among Wood Workers Assessed with the Comet Assay.

    PubMed

    Bruschweiler, Evin Danisman; Wild, Pascal; Huynh, Cong Khanh; Savova-Bianchi, Dessislava; Danuser, Brigitta; Hopf, Nancy B

    2016-01-01

    Exposure to wood dust, a human carcinogen, is common in wood-related industries, and millions of workers are occupationally exposed to wood dust worldwide. The comet assay is a rapid, simple, and sensitive method for determining DNA damage. The objective of this study was to investigate the DNA damage associated with occupational exposure to wood dust using the comet assay (peripheral blood samples) among nonsmoking wood workers (n = 31, furniture and construction workers) and controls (n = 19). DNA damage was greater in the group exposed to composite wood products compared to the group exposed to natural woods and controls (P < 0.001). No difference in DNA damage was observed between workers exposed to natural woods and controls (P = 0.13). Duration of exposure and current dust concentrations had no effect on DNA damage. In future studies, workers' exposures should include cumulative dust concentrations and exposures originating from the binders used in composite wood products. PMID:27398027

  4. Pseudo-DNA damage response in senescent cells

    PubMed Central

    Pospelova, Tatyana V.; Demidenko, Zoya N.; Bukreeva, Elena I.; Pospelov, Valery A.; Gudkov, Andrei V.; Blagosklonny, Mikhail V.

    2016-01-01

    Cellular senescence is currently viewed as a response to DNA damage. In this report, we showed that non-damaging agents such as sodium butyrate-induced p21 and ectopic expression of either p21 or p16 cause cellular senescence without detectable DNA breaks. Nevertheless, senescent cells displayed components of DNA damage response (DDR) such as γH2AX foci and uniform nuclear staining for p-ATM. Importantly, there was no accumulation of 53BP1 in γH2AX foci of senescent cells. Consistently, comet assay failed to detect DNA damage. Rapamycin, an inhibitor of mTOR, which was shown to suppress cellular senescence, decreased γH2AX foci formation. Thus, cellular senescence leads to activation of atypical DDR without detectable DNA damage. Pseudo-DDR may be a marker of general over-activation of senescent cells. PMID:19946210

  5. Synthetic lethal approaches exploiting DNA damage in aggressive myeloma

    PubMed Central

    Cottini, Francesca; Hideshima, Teru; Suzuki, Rikio; Tai, Yu-Tzu; Bianchini, Giampaolo; Richardson, Paul G.; Anderson, Kenneth C.; Tonon, Giovanni

    2015-01-01

    Ongoing DNA damage is a common feature of epithelial cancers. Here we show that tumor cells derived from multiple myeloma (MM), a disease of clonal plasma cells, demonstrate DNA replicative stress leading to DNA damage. We identified a poor prognosis subset of MM with extensive chromosomal instability and replicative stress which rely on ATR to compensate for DNA replicative stress; conversely, silencing of ATR or treatment with a specific ATR inhibitor triggers MM cell apoptosis. We show that oncogenes such as MYC induce DNA damage in MM cells not only by increased replicative stress, but also via increased oxidative stress, and that ROS-inducer piperlongumine triggers further DNA damage and apoptosis. Importantly, ATR inhibition combined with piperlongumine triggers synergistic MM cytotoxicity. This synthetic lethal approach, enhancing oxidative stress while concomitantly blocking replicative stress response, provides a novel combination targeted therapy to address an unmet medical need in this subset of MM. PMID:26080835

  6. Stress-induced DNA damage biomarkers: applications and limitations.

    PubMed

    Nikitaki, Zacharenia; Hellweg, Christine E; Georgakilas, Alexandros G; Ravanat, Jean-Luc

    2015-01-01

    A variety of environmental stresses like chemicals, UV and ionizing radiation and organism's endogenous processes such as replication stress and metabolism can lead to the generation of reactive oxygen and nitrogen species (ROS/RNS) that can attack cellular vital components like DNA, proteins and lipid membranes. Among them, much attention has been focused on DNA since DNA damage plays a role in several biological disorders and aging processes. Thus, DNA damage can be used as a biomarker in a reliable and accurate way to quantify for example radiation exposure and can indicate its possible long term effects and cancer risk. Based on the type of DNA lesions detected one can hypothesize on the most probable mechanisms involved in the formation of these lesions for example in the case of UV and ionizing radiation (e.g., X- or α-, γ-rays, energetic ions, neutrons). In this review we describe the most accepted chemical pathways for DNA damage induction and the different types of DNA lesions, i.e., single, complex DNA lesions etc. that can be used as DNA damage biomarkers. We critically compare DNA damage detection methods and their limitations. In addition, we suggest the use of DNA repair gene products as biomarkes for identification of different types of stresses i.e., radiation, oxidative, or replication stress, based on bioinformatic approaches and meta-analysis of literature data. PMID:26082923

  7. Stress-induced DNA damage biomarkers: applications and limitations

    PubMed Central

    Nikitaki, Zacharenia; Hellweg, Christine E.; Georgakilas, Alexandros G.; Ravanat, Jean-Luc

    2015-01-01

    A variety of environmental stresses like chemicals, UV and ionizing radiation and organism's endogenous processes such as replication stress and metabolism can lead to the generation of reactive oxygen and nitrogen species (ROS/RNS) that can attack cellular vital components like DNA, proteins and lipid membranes. Among them, much attention has been focused on DNA since DNA damage plays a role in several biological disorders and aging processes. Thus, DNA damage can be used as a biomarker in a reliable and accurate way to quantify for example radiation exposure and can indicate its possible long term effects and cancer risk. Based on the type of DNA lesions detected one can hypothesize on the most probable mechanisms involved in the formation of these lesions for example in the case of UV and ionizing radiation (e.g., X- or α-, γ-rays, energetic ions, neutrons). In this review we describe the most accepted chemical pathways for DNA damage induction and the different types of DNA lesions, i.e., single, complex DNA lesions etc. that can be used as DNA damage biomarkers. We critically compare DNA damage detection methods and their limitations. In addition, we suggest the use of DNA repair gene products as biomarkes for identification of different types of stresses i.e., radiation, oxidative, or replication stress, based on bioinformatic approaches and meta-analysis of literature data. PMID:26082923

  8. Delayed chromosomal instability induced by DNA damage.

    PubMed Central

    Marder, B A; Morgan, W F

    1993-01-01

    DNA damage induced by ionizing radiation can result in gene mutation, gene amplification, chromosome rearrangements, cellular transformation, and cell death. Although many of these changes may be induced directly by the radiation, there is accumulating evidence for delayed genomic instability following X-ray exposure. We have investigated this phenomenon by studying delayed chromosomal instability in a hamster-human hybrid cell line by means of fluorescence in situ hybridization. We examined populations of metaphase cells several generations after expanding single-cell colonies that had survived 5 or 10 Gy of X rays. Delayed chromosomal instability, manifested as multiple rearrangements of human chromosome 4 in a background of hamster chromosomes, was observed in 29% of colonies surviving 5 Gy and in 62% of colonies surviving 10 Gy. A correlation of delayed chromosomal instability with delayed reproductive cell death, manifested as reduced plating efficiency in surviving clones, suggests a role for chromosome rearrangements in cytotoxicity. There were small differences in chromosome destabilization and plating efficiencies between cells irradiated with 5 or 10 Gy of X rays after a previous exposure to 10 Gy and cells irradiated only once. Cell clones showing delayed chromosomal instability had normal frequencies of sister chromatid exchange formation, indicating that at this cytogenetic endpoint the chromosomal instability was not apparent. The types of chromosomal rearrangements observed suggest that chromosome fusion, followed by bridge breakage and refusion, contributes to the observed delayed chromosomal instability. Images PMID:8413263

  9. Direct visualization of a DNA glycosylase searching for damage.

    PubMed

    Chen, Liwei; Haushalter, Karl A; Lieber, Charles M; Verdine, Gregory L

    2002-03-01

    DNA glycosylases preserve the integrity of genetic information by recognizing damaged bases in the genome and catalyzing their excision. It is unknown how DNA glycosylases locate covalently modified bases hidden in the DNA helix amongst vast numbers of normal bases. Here we employ atomic-force microscopy (AFM) with carbon nanotube probes to image search intermediates of human 8-oxoguanine DNA glycosylase (hOGG1) scanning DNA. We show that hOGG1 interrogates DNA at undamaged sites by inducing drastic kinks. The sharp DNA bending angle of these non-lesion-specific search intermediates closely matches that observed in the specific complex of 8-oxoguanine-containing DNA bound to hOGG1. These findings indicate that hOGG1 actively distorts DNA while searching for damaged bases. PMID:11927259

  10. Plasmid DNA damage induced by helium atmospheric pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Han, Xu; Cantrell, William A.; Escobar, Erika E.; Ptasinska, Sylwia

    2014-03-01

    A helium atmospheric pressure plasma jet (APPJ) is applied to induce damage to aqueous plasmid DNA. The resulting fractions of the DNA conformers, which indicate intact molecules or DNA with single- or double-strand breaks, are determined using agarose gel electrophoresis. The DNA strand breaks increase with a decrease in the distance between the APPJ and DNA samples under two working conditions of the plasma source with different parameters of applied electric pulses. The damage level induced in the plasmid DNA is also enhanced with increased plasma irradiation time. The reactive species generated in the APPJ are characterized by optical emission spectra, and their roles in possible DNA damage processes occurring in an aqueous environment are also discussed.

  11. Chimeric Proteins to Detect DNA Damage and Mismatches

    SciTech Connect

    McCutchen-Maloney, S; Malfatti, M; Robbins, K M

    2002-01-14

    The goal of this project was to develop chimeric proteins composed of a DNA mismatch or damage binding protein and a nuclease, as well as methods to detect DNA mismatches and damage. We accomplished this through protein engineering based on using polymerase chain reactions (PCRs) to create chimeras with novel functions for damage and mismatch detection. This project addressed fundamental questions relating to disease susceptibility and radiation-induced damage in cells. It also supported and enhanced LLNL's competency in the emerging field of proteomics. In nature, DNA is constantly being subjected to damaging agents such as exposure to ultraviolet (UV) radiation and various environmental and dietary carcinogens. If DNA damage is not repaired however, mutations in DNA result that can eventually manifest in cancer and other diseases. In addition to damage-induced DNA mutations, single nucleotide polymorphisms (SNPs), which are variations in the genetic sequence between individuals, may predispose some to disease. As a result of the Human Genome Project, the integrity of a person's DNA can now be monitored. Therefore, methods to detect DNA damage, mutations, and SNPs are useful not only in basic research but also in the health and biotechnology industries. Current methods of detection often use radioactive labeling and rely on expensive instrumentation that is not readily available in many research settings. Our methods to detect DNA damage and mismatches employ simple gel electrophoresis and flow cytometry, thereby alleviating the need for radioactive labeling and expensive equipment. In FY2001, we explored SNP detection by developing methods based on the ability of the chimeric proteins to detect mismatches. Using multiplex assays with flow cytometry and fluorescent beads to which the DNA substrates where attached, we showed that several of the chimeras possess greater affinity for damaged and mismatched DNA than for native DNA. This affinity was demonstrated in

  12. DETECTION OF DNA DAMAGE USING MELTING ANALYSIS TECHNIQUES

    EPA Science Inventory

    A rapid and simple fluorescence screening assay for UV radiation-, chemical-, and enzyme-induced DNA damage is reported. This assay is based on a melting/annealing analysis technique and has been used with both calf thymus DNA and plasmid DNA (puc 19 plasmid from E. coli). DN...

  13. Chromatin perturbations during the DNA damage response in higher eukaryotes.

    PubMed

    Bakkenist, Christopher J; Kastan, Michael B

    2015-12-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response

  14. Chromatin perturbations during the DNA damage response in higher eukaryotes

    PubMed Central

    Bakkenist, Christopher J.; Kastan, Michael B.

    2016-01-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response

  15. Stress-induced DNA Damage biomarkers: Applications and limitations

    NASA Astrophysics Data System (ADS)

    Nikitaki, Zacharenia; Hellweg, Christine; Georgakilas, Alexandros; Ravanat, Jean-Luc

    2015-06-01

    A variety of environmental stresses like chemicals, UV and ionizing radiation and organism’s endogenous processes like replication stress and metabolism can lead to the generation of reactive oxygen and nitrogen species (ROS/RNS) that can attack cellular vital components like DNA, proteins and lipid membranes. Among them, much attention has been focused on DNA since DNA damages play a role in several biological disorders and aging processes. Thus, DNA damage can be used as a biomarker in a reliable and accurate way to quantify for example radiation exposure and can indicate its possible long term effects and cancer risk. Based on the type of DNA lesions detected one can hypothesize on the most probable mechanisms involved in the formation of these lesions for example in the case of UV and ionizing radiation (e.g. X- or α-, γ-rays, energetic ions, neutrons). In this review we describe the most accepted chemical pathways for DNA damage induction and the different types of DNA lesions, i.e. single, complex DNA lesions etc. that can be used as biomarkers. We critically compare DNA damage detection methods and their limitations. In addition to such DNA damage products, we suggest possible gene inductions that can be used to characterize responses to different types of stresses i.e. radiation, oxidative and replication stress, based on bioinformatic approaches and stringent meta-analysis of literature data.

  16. Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

    DOE PAGESBeta

    Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; Park, Beomseok; Shim, Yoonjung; Kim, Youngchang; Liu, Lili; Van Houten, Bennett; He, Chuan; Ansari, Anjum; et al

    2015-01-06

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

  17. Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

    PubMed Central

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

    2015-01-01

    The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites. PMID:25562780

  18. Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  19. Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

    SciTech Connect

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

    2015-01-06

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

  20. At a crossroads: human DNA tumor viruses and the host DNA damage response.

    PubMed

    Nikitin, Pavel A; Luftig, Micah A

    2011-07-01

    Human DNA tumor viruses induce host cell proliferation in order to establish the necessary cellular milieu to replicate viral DNA. The consequence of such viral-programmed induction of proliferation coupled with the introduction of foreign replicating DNA structures makes these viruses particularly sensitive to the host DNA damage response machinery. In fact, sensors of DNA damage are often activated and modulated by DNA tumor viruses in both latent and lytic infection. This article focuses on the role of the DNA damage response during the life cycle of human DNA tumor viruses, with a particular emphasis on recent advances in our understanding of the role of the DNA damage response in EBV, Kaposi's sarcoma-associated herpesvirus and human papillomavirus infection. PMID:21927617

  1. Metallothionein blocks oxidative DNA damage in vitro

    PubMed Central

    Qu, Wei; Pi, Jingbo; Waalkes, Michael P.

    2012-01-01

    The role of metallothionein (MT) in mitigation of oxidative DNA damage (ODD) induced either by cadmium (Cd) or the direct oxidant hydrogen peroxide (H2O2) was systematically examined by using MT-I/II double knockout (MT-null) or MT-competent wild-type (WT) cells. Both toxicants were much more lethal to MT-null cells (Cd LC50 = 6.6 μM; H2O2 LC50 = 550 μM) than WT cells (Cd LC50 = 16.5 μM; H2O2 LC50 = 930 μM). Cd induced concentration-related MT increases in WT cells, while the basal levels were undetectable and not increased by Cd in MT-null cells. ODD, measured by the immuno-spin trapping method, was minimally induced by sub-toxic Cd levels (1 or 5 μM; 24 h) in WT cells, but markedly increased in MT-null cells (> 430%). Similarly, ODD was induced to higher levels by lower concentrations of H2O2 in MT-null cells than WT cells. Transfection of MT-I into MT-null cells reduced both Cd- and H2O2-induced cytolethality and ODD. Cd increased expression of the oxidant defense genes, HO-1 and GSTa2 to a much greater extent in MT-null cells than WT. Cd or H2O2 exposure increased expression of key transport genes, Mrp1 and Mrp2, in WT cells but not in MT-null cells. MT protects against Cd- and H2O2-induced ODD in MT competent cells possibly by multiple mechanisms, potentially including direct metal ion sequestration and sequestration of oxidant radicals by MT. MT-deficient cells appear to adapt to Cd primarily by turning on oxidant response systems, while MT-competent cells activate MT and transport systems. PMID:22914987

  2. Inflammation-induced DNA damage and damage-induced inflammation: a vicious cycle.

    PubMed

    Pálmai-Pallag, Timea; Bachrati, Csanád Z

    2014-10-01

    Inflammation is the ultimate response to the constant challenges of the immune system by microbes, irritants or injury. The inflammatory cascade initiates with the recognition of microorganism-derived pathogen associated molecular patterns (PAMPs) and host cell-derived damage associated molecular patterns (DAMPs) by the pattern recognition receptors (PRRs). DNA as a molecular PAMP or DAMP is sensed directly or via specific binding proteins to instigate pro-inflammatory response. Some of these DNA binding proteins also participate in canonical DNA repair pathways and recognise damaged DNA to initiate DNA damage response. In this review we aim to capture the essence of the complex interplay between DNA damage response and the pro-inflammatory signalling through representative examples. PMID:25449753

  3. DNA damage and repair after high LET radiation

    NASA Astrophysics Data System (ADS)

    O'Neill, Peter; Cucinotta, Francis; Anderson, Jennifer

    Predictions from biophysical models of interactions of radiation tracks with cellular DNA indicate that clustered DNA damage sites, defined as two or more lesions formed within one or two helical turns of the DNA by passage of a single radiation track, are formed in mammalian cells. These complex DNA damage sites are regarded as a signature of ionizing radiation exposure particularly as the likelihood of clustered damage sites arising endogenously is low. For instance, it was predicted from biophysical modelling that 30-40% of low LET-induced double strand breaks (DSB), a form of clustered damage, are complex with the yield increasing to >90% for high LET radiation, consistent with the reduced reparability of DSB with increasing ionization density of the radiation. The question arises whether the increased biological effects such as mutagenesis, carcinogenesis and lethality is in part related to DNA damage complexity and/or spatial distribution of the damage sites, which may lead to small DNA fragments. With particle radiation it is also important to consider not only delta-rays which may cause clustered damaged sites and may be highly mutagenic but the non-random spatial distribution of DSB which may lead to deletions. In this overview I will concentrate on the molecular aspects of the variation of the complexity of DNA damage on radiation quality and the challenges this complexity presents the DNA damage repair pathways. I will draw on data from micro-irradiations which indicate that the repair of DSBs by non-homologous end joining is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB. In summary the aim is to emphasis the link between the spatial distribution of energy deposition events related to the track, the molecular products formed and the consequence of damage complexity contributing to biological effects and to present some of the outstanding molecular challenges with particle radiation.

  4. Pneumococcal Pneumolysin Induces DNA Damage and Cell Cycle Arrest.

    PubMed

    Rai, Prashant; He, Fang; Kwang, Jimmy; Engelward, Bevin P; Chow, Vincent T K

    2016-01-01

    Streptococcus pneumoniae produces pneumolysin toxin as a key virulence factor against host cells. Pneumolysin is a cholesterol-dependent cytolysin (CDC) toxin that forms lytic pores in host membranes and mediates pneumococcal disease pathogenesis by modulating inflammatory responses. Here, we show that pneumolysin, which is released during bacterial lysis, induces DNA double strand breaks (DSBs), as indicated by ataxia telangiectasia mutated (ATM)-mediated H2AX phosphorylation (γH2AX). Pneumolysin-induced γH2AX foci recruit mediator of DNA damage checkpoint 1 (MDC1) and p53 binding protein 1 (53BP1), to sites of DSBs. Importantly, results show that toxin-induced DNA damage precedes cell cycle arrest and causes apoptosis when DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end joining is inhibited. Further, we observe that cells that were undergoing DNA replication harbored DSBs in greater frequency during pneumolysin treatment. This observation raises the possibility that DSBs might be arising as a result of replication fork breakdown. Additionally, neutralizing the oligomerization domain of pneumolysin with monoclonal antibody suppresses DNA damage and also cell cycle arrest, indicating that pneumolysin oligomerization is important for causing DNA damage. Taken together, this study reveals a previously unidentified ability of pneumolysin to induce cytotoxicity via DNA damage, with implications in the pathophysiology of S. pneumoniae infection. PMID:27026501

  5. Analysis of chromatin integrity and DNA damage of buffalo spermatozoa.

    PubMed

    Mahmoud, K Gh M; El-Sokary, A A E; Abdel-Ghaffar, A E; Abou El-Roos, M E A; Ahmed, Y F

    2015-01-01

    This study was conducted to determine chromatin integrity and DNA damage by DNA electrophoresis and comet assays of buffalo fresh and frozen semen. Semen samples were collected from four buffalo bulls and evaluated after freezing for semen motility, viability, sperm abnormalities, chromatin integrity and DNA damage. A significant variation was found in semen parameters after thawing. Highly significant differences (P<0.001) in chromatin integrity were observed between fresh and frozen semen. For the fresh semen, there was no significant difference between the bulls for chromatin integrity; however, a significant variation (P<0.05) was detected in their frozen semen. No DNA fragmentation was observed by agarose gel electrophoresis. The percentage of sperm with damaged DNA detected by comet assay differed significantly between fresh and frozen semen. A significant negative correlation was recorded between motility and DNA damage (r=-0.68, P<0.05). Sperm abnormalities and DNA fragmentation were significantly positively correlated (r=0.59, P<0.05). In conclusion, DNA damage evaluation can provide reassurance about genomic normalcy and guide the development of improved methods of selecting spermatozoa with intact DNA to be used in artificial insemination. PMID:27175169

  6. Analysis of chromatin integrity and DNA damage of buffalo spermatozoa

    PubMed Central

    Mahmoud, K. Gh. M.; El-Sokary, A. A. E.; Abdel-Ghaffar, A. E.; Abou El-Roos, M. E. A.; Ahmed, Y. F.

    2015-01-01

    This study was conducted to determine chromatin integrity and DNA damage by DNA electrophoresis and comet assays of buffalo fresh and frozen semen. Semen samples were collected from four buffalo bulls and evaluated after freezing for semen motility, viability, sperm abnormalities, chromatin integrity and DNA damage. A significant variation was found in semen parameters after thawing. Highly significant differences (P<0.001) in chromatin integrity were observed between fresh and frozen semen. For the fresh semen, there was no significant difference between the bulls for chromatin integrity; however, a significant variation (P<0.05) was detected in their frozen semen. No DNA fragmentation was observed by agarose gel electrophoresis. The percentage of sperm with damaged DNA detected by comet assay differed significantly between fresh and frozen semen. A significant negative correlation was recorded between motility and DNA damage (r=-0.68, P<0.05). Sperm abnormalities and DNA fragmentation were significantly positively correlated (r=0.59, P<0.05). In conclusion, DNA damage evaluation can provide reassurance about genomic normalcy and guide the development of improved methods of selecting spermatozoa with intact DNA to be used in artificial insemination. PMID:27175169

  7. Pneumococcal Pneumolysin Induces DNA Damage and Cell Cycle Arrest

    PubMed Central

    Rai, Prashant; He, Fang; Kwang, Jimmy; Engelward, Bevin P.; Chow, Vincent T.K.

    2016-01-01

    Streptococcus pneumoniae produces pneumolysin toxin as a key virulence factor against host cells. Pneumolysin is a cholesterol-dependent cytolysin (CDC) toxin that forms lytic pores in host membranes and mediates pneumococcal disease pathogenesis by modulating inflammatory responses. Here, we show that pneumolysin, which is released during bacterial lysis, induces DNA double strand breaks (DSBs), as indicated by ataxia telangiectasia mutated (ATM)-mediated H2AX phosphorylation (γH2AX). Pneumolysin-induced γH2AX foci recruit mediator of DNA damage checkpoint 1 (MDC1) and p53 binding protein 1 (53BP1), to sites of DSBs. Importantly, results show that toxin-induced DNA damage precedes cell cycle arrest and causes apoptosis when DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end joining is inhibited. Further, we observe that cells that were undergoing DNA replication harbored DSBs in greater frequency during pneumolysin treatment. This observation raises the possibility that DSBs might be arising as a result of replication fork breakdown. Additionally, neutralizing the oligomerization domain of pneumolysin with monoclonal antibody suppresses DNA damage and also cell cycle arrest, indicating that pneumolysin oligomerization is important for causing DNA damage. Taken together, this study reveals a previously unidentified ability of pneumolysin to induce cytotoxicity via DNA damage, with implications in the pathophysiology of S. pneumoniae infection. PMID:27026501

  8. Radiation-induced DNA damage and chromatin structure

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2001-01-01

    DNA lesions induced by ionizing radiation in cells are clustered and not randomly distributed. For low linear energy transfer (LET) radiation this clustering occurs mainly on the small scales of DNA molecules and nucleosomes. For example, experimental evidence suggests that both strands of DNA on the nucleosomal surface can be damaged in single events and that this damage occurs with a 10-bp modulation because of protection by histones. For high LET radiation, clustering also occurs on a larger scale and depends on chromatin organization. A particularly significant clustering occurs when an ionizing particle traverses the 30 nm chromatin fiber with generation of heavily damaged DNA regions with an average size of about 2 kbp. On an even larger scale, high LET radiation can produce several DNA double-strand breaks in closer proximity than expected from randomness. It is suggested that this increases the probability of misrejoining of DNA ends and generation of lethal chromosome aberrations.

  9. Superoxide and the production of oxidative DNA damage.

    PubMed Central

    Keyer, K; Gort, A S; Imlay, J A

    1995-01-01

    The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2. The hydroxyl radical then attacks DNA. Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells. Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive. However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2. Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis. That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2. Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- increases the amount of free iron that is available to catalyze hydroxyl radical production. Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation. Thus, O2- may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron. PMID:7592468

  10. p53 and Cell Cycle Effects After DNA Damage

    PubMed Central

    Senturk, Emir; Manfredi, James J.

    2016-01-01

    Flow cytometry, a valuable technique that employs the principles of light scattering, light excitation, and emission of fluorochrome molecules, can be used to assess the cell cycle position of individual cells based on DNA content. After the permeabilization of cells, the DNA can be stained with a fluorescent dye. Cells which have a 2N amount of DNA can be distinguished from cells with a 4N amount of DNA, making flow cytometry a very useful tool for the analysis of cell cycle checkpoints following DNA damage. A critical feature of the cellular response to DNA damage is the ability to pause and repair the damage so that consequential mutations are not passed along to daughter generations of cells. If cells arrest prior to DNA replication, they will contain a 2N amount of DNA, whereas arrest after replication but before mitosis will result in a 4N amount of DNA. Using this technique, the role that p53 plays in cell cycle checkpoints following DNA damage can be evaluated based on changes in the profile of the G1, S, and G2/M phases of the cell cycle. PMID:23150436

  11. Mass Spectrometry-Based Proteomics for Investigating DNA Damage-Associated Protein Ubiquitylation

    PubMed Central

    Heidelberger, Jan B.; Wagner, Sebastian A.; Beli, Petra

    2016-01-01

    Modification of proteins with the 76 amino acid protein ubiquitin plays essential roles in cellular signaling. Development of methods for specific enrichment of ubiquitin remnant peptides and advances in high-resolution mass spectrometry have enabled proteome-wide identification of endogenous ubiquitylation sites. Moreover, ubiquitin remnant profiling has emerged as a powerful approach for investigating changes in protein ubiquitylation in response to cellular perturbations, such as DNA damage, as well as for identification of substrates of ubiquitin-modifying enzymes. Despite these advances, interrogation of ubiquitin chain topologies on substrate proteins remains a challenging task. Here, we describe mass spectrometry-based approaches for quantitative analyses of site-specific protein ubiquitylation and highlight recent studies that employed these methods for investigation of ubiquitylation in the context of the cellular DNA damage response. Furthermore, we provide an overview of experimental strategies for probing ubiquitin chain topologies on proteins and discuss how these methods can be applied to analyze functions of ubiquitylation in the DNA damage response. PMID:27379159

  12. Calculation of complex DNA damage induced by ions

    NASA Astrophysics Data System (ADS)

    Surdutovich, Eugene; Gallagher, David C.; Solov'yov, Andrey V.

    2011-11-01

    This paper is devoted to the analysis of the complex damage of DNA irradiated by ions. The assessment of complex damage is important because cells in which it occurs are less likely to survive because the DNA repair mechanisms may not be sufficiently effective. We study the flux of secondary electrons through the surface of nucleosomes and calculate the radial dose and the distribution of clustered damage around the ion's path. The calculated radial dose distribution is compared to simulations. The radial distribution of the complex damage is found to be different from that of the dose. A comparison with experiments may solve the question of what is more lethal for the cell, damage complexity or absorbed energy. We suggest a way to calculate the probability of cell death based on the complexity of the damage. This work is done within the framework of the phenomenon-based multiscale approach to radiation damage by ions.

  13. Calculation of complex DNA damage induced by ions

    SciTech Connect

    Surdutovich, Eugene; Gallagher, David C.; Solov'yov, Andrey V.

    2011-11-15

    This paper is devoted to the analysis of the complex damage of DNA irradiated by ions. The assessment of complex damage is important because cells in which it occurs are less likely to survive because the DNA repair mechanisms may not be sufficiently effective. We study the flux of secondary electrons through the surface of nucleosomes and calculate the radial dose and the distribution of clustered damage around the ion's path. The calculated radial dose distribution is compared to simulations. The radial distribution of the complex damage is found to be different from that of the dose. A comparison with experiments may solve the question of what is more lethal for the cell, damage complexity or absorbed energy. We suggest a way to calculate the probability of cell death based on the complexity of the damage. This work is done within the framework of the phenomenon-based multiscale approach to radiation damage by ions.

  14. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

    Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. Black-Right-Pointing-Pointer DNA repair foci are found in soluble chromatin. Black-Right-Pointing-Pointer Biophysical analysis reveals global chromatin fibre compaction after DNA damage. Black-Right-Pointing-Pointer DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation ({gamma}H2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and {gamma}H2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by

  15. DNA damage in cancer therapeutics: a boon or a curse?

    PubMed

    Khanna, Anchit

    2015-06-01

    Millions of DNA-damaging lesions occur every day in each cell of our bodies due to various stresses. The failure to detect and accurately repair these lesions can give rise to cells with high levels of endogenous DNA damage, deleterious mutations, or genomic aberrations. Such genomic instability can lead to the activation of specific signaling pathways, including the DNA damage response (DDR) pathway. Constitutive activation of DDR proteins has been observed in human tumor specimens from different cancer stages, including precancerous and metastatic cancers, although not in normal tissues. The tumor-suppressive role of DDR activity during the premalignant stage has been studied, and strong evidence is emerging for an oncogenic role for DDR proteins such as DNA-PK and CHK1 during the later stages of tumor development. However, the majority of current cancer therapies induce DNA damage, potentially exacerbating protumorigenic genomic instability and enabling the development of resistance. Therefore, elucidating the molecular basis of DNA damage-mediated genomic instability and its role in tumorigenesis is critical. Finally, I discuss the potential existence of distinct DNA damage thresholds at various stages of tumorigenesis and what the ramifications of such thresholds would be, including the ambiguous role of the DDR pathway in human cancers, therapy-induced malignancies, and enhanced therapies. PMID:25931285

  16. The DNA damage response: the omics era and its impact

    PubMed Central

    Derks, Kasper W.J.; Hoeijmakers, Jan H.J.; Pothof, Joris

    2014-01-01

    The emergence of high density technologies monitoring the genome, transcriptome and proteome in relation to genotoxic stress have tremendously enhanced our knowledge on global responses and dynamics in the DNA damage response, including its relation with cancer and aging. Moreover, ‘-omics’ technologies identified many novel factors, their post-translational modifications, pathways and global responses in the cellular response to DNA damage. Based on omics, it is currently estimated that thousands of gene(product)s participate in the DNA damage response, recognizing complex networks that determine cell fate after damage to the most precious cellular molecule, DNA. The development of next generation sequencing technology and associated specialized protocols can quantitatively monitor RNA and DNA at unprecedented single nucleotide resolution. In this review we will discuss the contribution of omics technologies and in particular next generation sequencing to our understanding of the DNA damage response and the future prospective of next generation sequencing, its single cell application and omics dataset integration in unraveling intricate DNA damage signaling networks. PMID:24794401

  17. Homologous recombination maintenance of genome integrity during DNA damage tolerance

    PubMed Central

    Prado, Félix

    2014-01-01

    The DNA strand exchange protein Rad51 provides a safe mechanism for the repair of DNA breaks using the information of a homologous DNA template. Homologous recombination (HR) also plays a key role in the response to DNA damage that impairs the advance of the replication forks by providing mechanisms to circumvent the lesion and fill in the tracks of single-stranded DNA that are generated during the process of lesion bypass. These activities postpone repair of the blocking lesion to ensure that DNA replication is completed in a timely manner. Experimental evidence generated over the last few years indicates that HR participates in this DNA damage tolerance response together with additional error-free (template switch) and error-prone (translesion synthesis) mechanisms through intricate connections, which are presented here. The choice between repair and tolerance, and the mechanism of tolerance, is critical to avoid increased mutagenesis and/or genome rearrangements, which are both hallmarks of cancer. PMID:27308329

  18. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA. PMID:25486562

  19. Spatially localized generation of nucleotide sequence-specific DNA damage.

    PubMed

    Oh, D H; King, B A; Boxer, S G; Hanawalt, P C

    2001-09-25

    Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry resulting in psoralen-DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quantum energy required for conventional one-photon excitation, as used in psoralen + UV A radiation (320-400 nm) therapy. Target DNA acquired strand-specific psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA-psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targeting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4- to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen-TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues. PMID:11572980

  20. Effect of oxidative DNA damage in promoter elements on transcription factor binding.

    PubMed Central

    Ghosh, R; Mitchell, D L

    1999-01-01

    Reactive oxygen species produced by endogenous metabolic activity and exposure to a multitude of exogenous agents impact cells in a variety of ways. The DNA base damage 8-oxodeoxyguanosine (8-oxodG) is a prominent indicator of oxidative stress and has been well-characterized as a premutagenic lesion in mammalian cells and putative initiator of the carcinogenic process. Commensurate with the recent interest in epigenetic pathways of cancer causation we investigated how 8-oxodG alters the interaction between cis elements located on gene promoters and sequence-specific DNA binding proteins associated with these promoters. Consensus binding sequences for the transcription factors AP-1, NF-kappaB and Sp1 were modified site-specifically at guanine residues and electrophoretic mobility shift assays were performed to assess DNA-protein interactions. Our results indicate that whereas a single 8-oxodG was sufficient to inhibit transcription factor binding to AP-1 and Sp1 sequences it had no effect on binding to NF-kappaB, regardless of its position. We conclude from these data that minor alterations in base composition at a crucial position within some, but not all, promoter elements have the ability to disrupt transcription factor binding. The lack of inhibition by damaged NF-kappaB sequences suggests that DNA-protein contact sites may not be as determinative for stable p50 binding to this promoter as other, as yet undefined, structural parameters. PMID:10454620

  1. Topoisomerase I-mediated DNA damage.

    PubMed

    Pourquier, P; Pommier, Y

    2001-01-01

    Topoisomerase I is a ubiquitous and essential enzyme in multicellular organisms. It is involved in multiple DNA transactions including DNA replication, transcription, chromosome condensation and decondensation, and probably DNA recombination. Besides its activity of DNA relaxation necessary to eliminate torsional stresses associated with these processes, topoisomerase I may have other functions related to its interaction with other cellular proteins. Topoisomerase I is the target of the novel anticancer drugs, the camptothecins. Recently a broad range of physiological and environmentally-induced DNA modifications have also been shown to poison topoisomerases. This review summarizes the various factors that enhance or suppress top1 cleavage complexes and discusses the significance of such effects. We also review the different mechanisms that have been proposed for the repair of topoisomerase I-mediated DNA lesions. PMID:11034544

  2. Is DNA Damage Response Ready for Action Anywhere?

    PubMed Central

    Terradas, Mariona; Martín, Marta; Hernández, Laia; Tusell, Laura; Genescà, Anna

    2012-01-01

    Organisms are continuously exposed to DNA damaging agents, consequently, cells have developed an intricate system known as the DNA damage response (DDR) in order to detect and repair DNA lesions. This response has to be rapid and accurate in order to keep genome integrity. It has been observed that the condensation state of chromatin hinders a proper DDR. However, the condensation state of chromatin is not the only barrier to DDR. In this review, we have collected data regarding the presence of DDR factors on micronuclear DNA lesions that indicate that micronuclei are almost incapable of generating an effective DDR because of defects in their nuclear envelope. Finally, considering the recent observations about the reincorporation of micronuclei to the main bulk of chromosomes, we suggest that, under certain circumstances, micronuclei carrying DNA damage might be a source of chromosome instability. PMID:23109871

  3. Roles of RNA-Binding Proteins in DNA Damage Response

    PubMed Central

    Kai, Mihoko

    2016-01-01

    Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, “sensor” proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)’s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as

  4. Roles of RNA-Binding Proteins in DNA Damage Response.

    PubMed

    Kai, Mihoko

    2016-01-01

    Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, "sensor" proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)'s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as "liquid-demixing"). Among the

  5. Spatially localized generation of nucleotide sequence-specific DNA damage

    PubMed Central

    Oh, Dennis H.; King, Brett A.; Boxer, Steven G.; Hanawalt, Philip C.

    2001-01-01

    Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry resulting in psoralen–DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quantum energy required for conventional one-photon excitation, as used in psoralen + UV A radiation (320–400 nm) therapy. Target DNA acquired strand-specific psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA–psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targeting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4- to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen–TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues. PMID:11572980

  6. Regulation of the DNA damage response by ubiquitin conjugation

    PubMed Central

    Brinkmann, Kerstin; Schell, Michael; Hoppe, Thorsten; Kashkar, Hamid

    2015-01-01

    In response to DNA damage, cells activate a highly conserved and complex kinase-based signaling network, commonly referred to as the DNA damage response (DDR), to safeguard genomic integrity. The DDR consists of a set of tightly regulated events, including detection of DNA damage, accumulation of DNA repair factors at the site of damage, and finally physical repair of the lesion. Upon overwhelming damage the DDR provokes detrimental cellular actions by involving the apoptotic machinery and inducing a coordinated demise of the damaged cells (DNA damage-induced apoptosis, DDIA). These diverse actions involve transcriptional activation of several genes that govern the DDR. Moreover, recent observations highlighted the role of ubiquitylation in orchestrating the DDR, providing a dynamic cellular regulatory circuit helping to guarantee genomic stability and cellular homeostasis (Popovic et al., 2014). One of the hallmarks of human cancer is genomic instability (Hanahan and Weinberg, 2011). Not surprisingly, deregulation of the DDR can lead to human diseases, including cancer, and can induce resistance to genotoxic anti-cancer therapy (Lord and Ashworth, 2012). Here, we summarize the role of ubiquitin-signaling in the DDR with special emphasis on its role in cancer and highlight the therapeutic value of the ubiquitin-conjugation machinery as a target in anti-cancer treatment strategy. PMID:25806049

  7. Novel Ser/Thr Protein Phosphatase 5 (PP5) Regulated Targets during DNA Damage Identified by Proteomics Analysis

    SciTech Connect

    Ham, Bryan M.; Jayachandran, Hemalatha; Yang, Feng; Jaitly, Navdeep; Polpitiya, Ashoka D.; Monroe, Matthew E.; Wang, Ling; Zhao, Rui; Purvine, Samuel O.; Livesay, Eric A.; Camp, David G.; Rossie, Sandra S.; Smith, Richard D.

    2010-02-05

    The DNA damage response is a global phosphorylation signaling cascade process involved in sensing the damaged DNA condition and coordinating responses to cope with and repair the perturbed cellular state. We utilized a label-free liquid chromatography-mass spectrometry approach to evaluate changes in protein phosphorylation associated with PP5 activity during the DNA damage response. Biological replicate analyses of bleomycin-treated HeLa cells expressing either WT-PP5 or mutant inactive PP5 lead to the identification of six potential target proteins of PP5 action. Four of these putative targets are known to be involved in DNA damage responses. Using phospho-site specific antibodies, we confirmed that phosphorylation of one target, ribosomal protein S6, was selectively decreased in cells overexpressing catalytically inactive PP5. Our findings also suggest that PP5 may play a role in controlling translation and in regulating substrates for proline-directed kinases, such as MAP kinases and cyclin-dependent protein kinases that are involved in response to DNA damage.

  8. Circadian Modulation of 8-Oxoguanine DNA Damage Repair

    PubMed Central

    Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G.; Santarelli, Lory

    2015-01-01

    The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

  9. Leishmania major and Trypanosoma cruzi present distinct DNA damage responses.

    PubMed

    Garcia, Juliana B F; Rocha, João P Vieira da; Costa-Silva, Héllida M; Alves, Ceres L; Machado, Carlos R; Cruz, Angela K

    2016-05-01

    Leishmania major and Trypanosoma cruzi are medically relevant parasites and interesting model organisms, as they present unique biological processes. Despite increasing data regarding the mechanisms of gene expression regulation, there is little information on how the DNA damage response (DDR) occurs in trypanosomatids. We found that L. major presented a higher radiosensitivity than T. cruzi. L. major showed G1 arrest and displayed high mortality in response to ionizing radiation as a result of the inefficient repair of double-strand breaks (DSBs). Conversely, T. cruzi exhibited arrest in the S/G2 cell cycle phase, was able to efficiently repair DSBs and did not display high rates of cell death after exposure to gamma irradiation. L. major showed higher resistance to alkylating DNA damage, and only L. major was able to promote DNA repair and growth recovery in the presence of MMS. ASF1c overexpression did not interfere with the efficiency of DNA repair in either of the parasites but did accentuate the DNA damage checkpoint response, thereby delaying cell fate after damage. The observed differences in the DNA damage responses of T. cruzi and L. major may originate from the distinct preferred routes of genetic plasticity of the two parasites, i.e., DNA recombination versus amplification. PMID:27188657

  10. Circadian Modulation of 8-Oxoguanine DNA Damage Repair.

    PubMed

    Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G; Santarelli, Lory

    2015-01-01

    The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

  11. DNA damage as an intermediate biomarker in intervention studies.

    PubMed

    Santella, R M

    1997-11-01

    The development of sensitive assays for measurement of DNA damage in humans has great potential for enhancing intervention studies. Methods for DNA adduct measurement include immunoassays, [32p] postlabeling, high-performance liquid chromatography with fluorescence or electrochemical detection, and gas chromatography/mass spectroscopy. It is now well established that DNA adducts are a marker of exposure to various environmental, lifestyle, or occupational chemical carcinogens. Our own studies concentrate on immunologic detection of adducts by enzyme-linked immunosorbent assay (ELISA) of isolated DNA or quantitative immunohistochemical analysis of intact cells. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts are elevated in blood cells of foundry and coke oven workers, individuals with high levels of exposure to environmental air pollution, and smokers. The study in smokers also found an inverse relationship between serum antioxidants and PAH-DNA, and is the basis for an ongoing antioxidant intervention. DNA adducts of PAH and 4-aminobiphenyl and oxidative DNA damage (8-oxo-deoxyguanosine) are being measured in blood mononuclear cells and exfoliated oral and bladder cells from subjects on antioxidants or placebo. Data on published intervention studies investigating oxidative damage and general aromatic DNA adducts measured by postlabeling are also summarized. These studies have already demonstrated that DNA adducts can be modulated by interventions and suggest that they can provide important mechanistic information in support of larger scale studies. PMID:9349685

  12. Curcumin Triggers DNA Damage and Inhibits Expression of DNA Repair Proteins in Human Lung Cancer Cells.

    PubMed

    Ting, Chien-Yi; Wang, Hsin-Ell; Yu, Chien-Chih; Liu, Hsin-Chung; Liu, Yu-Chang; Chiang, I-Tsang

    2015-07-01

    The study goal was to evaluate the effects of curcumin on DNA damage and expression of DNA-repair proteins in human lung cancer. Thus, NCI-H460 cells were used to study the effects of curcumin on DNA damage and repair in vitro. We investigated curcumin induces DNA damage by comet the assay and 4',6-diamidino-2-phenylindole (DAPI) staining. The DNA damage/repair-related protein levels were examined and monitored by western blotting and confocal microscopy. Curcumin significantly increased the length of comet tails and DNA condensation in NCI-H460 cells. Curcumin reduced expression of DNA-repair proteins such as 14-3-3 protein sigma (14-3-3σ), O6-methylguanine-DNA methyltransferase (MGMT), breast cancer susceptibility gene 1 (BRCA1), and mediator of DNA damage checkpoint 1 (MDC1). Curcumin also increased phosphorylation of p53 and Histone H2A.X (S140) in the nuclei of NCI-H460 cells. Taken together, our findings indicated that curcumin triggered DNA damage and inhibited expression of DNA-repair-associated proteins in NCI-H460 cells. PMID:26124332

  13. Comparison of DNA damage by methylmelamines and formaldehyde

    SciTech Connect

    Ross, W.E.; McMillan, D.R.; Ross, C.F.

    1981-07-01

    The cytotoxicity and DNA damaging activity of S9-activated hexamethylmelamine (HMM) and pentamethylmelamine (PMM) were compared with suspected active metabolites in mouse leukemia L1210 cells. Following treatment of L1210 cells with high concentrations of activated HMM and PMM, there were no DNA single-strand breaks or interstrand cross-links observed by DNA alkaline elution and only a low frequency of DNA-protein cross-links. Formaldehyde (FA) at nonlethal concentrations caused far greater DNA-protein cross-linking. The cytotoxicities of HMM and PMM were found unlikely to be related to extracellular or intracellular release of FA.

  14. Antioxidant Nutrients and Oxidative DNA Damage in Humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oxidative stress has been implicated in the pathogenesis of chronic diseases related to aging, such as cancer and cardiovascular disease. When the excessive amount of reactive oxygen species accumulates in vivo, it can cause oxidative damage to lipids, proteins and DNA. In particular DNA is one of...

  15. DNA damage induced by the direct effect of radiation

    NASA Astrophysics Data System (ADS)

    Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

    2008-10-01

    We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

  16. Inhibition of transcription by oxidative DNA damage products

    SciTech Connect

    Byrd, S.; Reines, D.; Doetsch, P.W. )

    1991-03-11

    Thymine glycol is a major oxidative DNA base damage product that can be produced spontaneously in normal cells or by certain chemicals and ionizing radiation. This lesion as well as other oxidatively damaged bases are recognized and removed in eukaryotic cells by the DNA repair enzyme redoxyendonuclease which the authors have identified in a variety of cell types. Transcriptional regulation is a key element in the control of gene expression. Deficiencies in the various steps of transcription of an essential gene may have catastrophic effects for a cell. In terminally differentiated cells, the removal of RNA-polymerase blocking lesions could be viewed as a critical function for DNA repair systems in such cells. Very little information exists on the effects of oxidative base damage products on the process of transcription. The authors show here that thymine glycol containing DNA templates can inhibit transcriptional elongation when these lesions are chemically introduced into a DNA template. A DNA segment containing a region of the human H3.3 histone gene was utilized to determine the effects of oxidative DNA base damage on transcription by pure E. coli core RNA polymerase and rat liver RNA polymerase II. Both eukaryotic and prokaryotic RNA polymerases are blocked by the presence of thymine glycols appearing in certain clusters of thymines in the oxidatively damaged transcription template. To obtain quantitative efficiencies of transcriptional arrest, the authors are engineering a DNA template containing a single defined oxidatively damaged residue. The authors' results support the idea that an important function of DNA repair systems in terminally differentiated cells is to ensure the efficient transcription of genes necessary for normal cellular function.

  17. Biomarkers of oxidative damage to DNA and repair.

    PubMed

    Loft, Steffen; Høgh Danielsen, Pernille; Mikkelsen, Lone; Risom, Lotte; Forchhammer, Lykke; Møller, Peter

    2008-10-01

    Oxidative-stress-induced damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. Many lesions have been characterized by MS-based methods after extraction and digestion of DNA. These preparation steps may cause spurious base oxidation, which is less likely to occur with methods such as the comet assay, which are based on nicking of the DNA strand at modified bases, but offer less specificity. The European Standards Committee on Oxidative DNA Damage has concluded that the true levels of the most widely studied lesion, 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine), in cellular DNA is between 0.5 and 5 lesions per 10(6) dG bases. Base excision repair of oxidative damage to DNA can be assessed by nicking assays based on oligonucleotides with lesions or the comet assay, by mRNA expression levels or, in the case of, e.g., OGG1 (8-oxoguanine DNA glycosylase 1), responsible for repair of 8-oxodG, by genotyping. Products of repair in DNA or the nucleotide pool, such as 8-oxodG, excreted into the urine can be assessed by MS-based methods and generally reflects the rate of damage. Experimental and population-based studies indicate that many environmental factors, including particulate air pollution, cause oxidative damage to DNA, whereas diets rich in fruit and vegetables or antioxidant supplements may reduce the levels and enhance repair. Urinary excretion of 8-oxodG, genotype and expression of OGG1 have been associated with risk of cancer in cohort settings, whereas altered levels of damage, repair or urinary excretion in case-control settings may be a consequence rather than the cause of the disease. PMID:18793191

  18. T7 replisome directly overcomes DNA damage

    PubMed Central

    Sun, Bo; Pandey, Manjula; Inman, James T.; Yang, Yi; Kashlev, Mikhail; Patel, Smita S.; Wang, Michelle D.

    2015-01-01

    Cells and viruses possess several known ‘restart' pathways to overcome lesions during DNA replication. However, these ‘bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase–DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly. PMID:26675048

  19. T7 replisome directly overcomes DNA damage

    NASA Astrophysics Data System (ADS)

    Sun, Bo; Pandey, Manjula; Inman, James T.; Yang, Yi; Kashlev, Mikhail; Patel, Smita S.; Wang, Michelle D.

    2015-12-01

    Cells and viruses possess several known `restart' pathways to overcome lesions during DNA replication. However, these `bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase-DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.

  20. GOLPH3 Links the Golgi, DNA Damage, and Cancer

    PubMed Central

    Buschman, Matthew D.; Rahajeng, Juliati; Field, Seth J.

    2014-01-01

    GOLPH3 is the first example of an oncogene that functions in secretory trafficking at the Golgi. The discovery of GOLPH3’s roles in both cancer and Golgi trafficking raises questions about how GOLPH3 and the Golgi contribute to cancer. Our recent investigation of the regulation of GOLPH3 revealed a surprising response by the Golgi upon DNA damage that is mediated by DNA-PK and GOLPH3. These results provide new insight into the DNA damage response with important implications for understanding the cellular response to standard cancer therapeutic agents. PMID:25634214

  1. DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors

    PubMed Central

    Francia, Sofia; Cabrini, Matteo; Matti, Valentina; Oldani, Amanda; d'Adda di Fagagna, Fabrizio

    2016-01-01

    ABSTRACT The DNA damage response (DDR) plays a central role in preserving genome integrity. Recently, we reported that the endoribonucleases DICER and DROSHA contribute to DDR activation by generating small non-coding RNAs, termed DNA damage response RNA (DDRNA), carrying the sequence of the damaged locus. It is presently unclear whether DDRNAs act by promoting the primary recognition of DNA lesions or the secondary recruitment of DDR factors into cytologically detectable foci and consequent signal amplification. Here, we demonstrate that DICER and DROSHA are dispensable for primary recruitment of the DDR sensor NBS1 to DNA damage sites. Instead, the accumulation of the DDR mediators MDC1 and 53BP1 (also known as TP53BP1), markers of secondary recruitment, is reduced in DICER- or DROSHA-inactivated cells. In addition, NBS1 (also known as NBN) primary recruitment is resistant to RNA degradation, consistent with the notion that RNA is dispensable for primary recognition of DNA lesions. We propose that DICER, DROSHA and DDRNAs act in the response to DNA damage after primary recognition of DNA lesions and, together with γH2AX, are essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signaling. PMID:26906421

  2. DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors.

    PubMed

    Francia, Sofia; Cabrini, Matteo; Matti, Valentina; Oldani, Amanda; d'Adda di Fagagna, Fabrizio

    2016-04-01

    The DNA damage response (DDR) plays a central role in preserving genome integrity. Recently, we reported that the endoribonucleases DICER and DROSHA contribute to DDR activation by generating small non-coding RNAs, termed DNA damage response RNA (DDRNA), carrying the sequence of the damaged locus. It is presently unclear whether DDRNAs act by promoting the primary recognition of DNA lesions or the secondary recruitment of DDR factors into cytologically detectable foci and consequent signal amplification. Here, we demonstrate that DICER and DROSHA are dispensable for primary recruitment of the DDR sensor NBS1 to DNA damage sites. Instead, the accumulation of the DDR mediators MDC1 and 53BP1 (also known as TP53BP1), markers of secondary recruitment, is reduced in DICER- or DROSHA-inactivated cells. In addition, NBS1 (also known as NBN) primary recruitment is resistant to RNA degradation, consistent with the notion that RNA is dispensable for primary recognition of DNA lesions. We propose that DICER, DROSHA and DDRNAs act in the response to DNA damage after primary recognition of DNA lesions and, together with γH2AX, are essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signaling. PMID:26906421

  3. DNA damage in dihydroartemisinin-resistant Molt-4 cells.

    PubMed

    Park, Jungsoo; Lai, Henry C; Sasaki, Tomikazu; Singh, Narendra P

    2015-03-01

    Artemisinin generates carbon-based free radicals when it reacts with iron, and induces molecular damage and apoptosis. Its toxicity is more selective toward cancer cells because cancer cells contain a higher level of intracellular free iron. Dihydroartemisinin (DHA), an analog of artemisinin, has selective cytotoxicity toward Molt-4 human lymphoblastoid cells. A major concern is whether cancer cells could develop resistance to DHA, thus limiting its therapeutic efficacy. We have developed a DHA-resistant Molt-4 cell line (RTN) and found out that these cells exhibited resistance to DHA but no significant cross- resistance to artemisinin-tagged holotransferrin (ART-TF), a synthetic artemisinin compound. In the present study, we investigated DNA damage induced by DHA and ART-TF in both Molt-4 and RTN cells using the comet assay. RTN cells exhibited a significantly lower level of basal and X-ray-induced DNA damage compared to Molt-4 cells. Both DHA and ART-TF induced DNA damage in Molt-4 cells, whereas DNA damage was induced in RTN cells by ART-TF, and not DHA. The result of this study shows that by the cell selection method, it is possible to generate a Molt-4 cell line which is not sensitive to DHA, but sensitive to ART-TF, as measured by DNA damage. PMID:25750283

  4. MERIT40 facilitates BRCA1 localization and DNA damage repair

    PubMed Central

    Feng, Lin; Huang, Jun; Chen, Junjie

    2009-01-01

    The product of breast cancer susceptibility gene 1, BRCA1, plays pivotal roles in the maintenance of genomic integrity. Mounting evidence indicates that BRCA1 associates with many proteins or protein complexes to regulate diverse processes important for the cellular response to DNA damage. One of these complexes, which mediates the accumulation of BRCA1 at sites of DNA breaks, involves the ubiquitin-binding motif (UIM)-containing protein RAP80, a coiled-coil domain protein CCDC98/Abraxas, and a deubiquitinating enzyme BRCC36. Here we describe the characterization of a novel component of this complex, MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kd), which together with an adaptor protein BRE/BRCC45, enforces the BRCA1-dependent DNA damage response. MERIT40 is assembled into this RAP80/CCDC98-containing complex via its direct interaction with BRE/BRCC45. Importantly, MERIT40 regulates BRCA1 retention at DNA breaks and checkpoint function primarily via a role in maintaining the stability of BRE and this five-subunit protein complex at sites of DNA damage. Together, our study reveals that a stable complex containing MERIT40 acts early in DNA damage response and regulates damage-dependent BRCA1 localization. PMID:19261748

  5. Quality control mechanisms in cellular and systemic DNA damage responses

    PubMed Central

    Ermolaeva, Maria A.; Dakhovnik, Alexander; Schumacher, Björn

    2016-01-01

    The maintenance of the genome is of pivotal importance for the functional integrity of cells and tissues. The gradual accumulation of DNA damage is thought to contribute to the functional decline of tissues and organs with ageing. Defects in multiple genome maintenance systems cause human disorders characterized by cancer susceptibility, developmental failure, and premature ageing. The complex pathological consequences of genome instability are insufficiently explained by cell-autonomous DNA damage responses (DDR) alone. Quality control pathways play an important role in DNA repair and cellular DDR pathways. Recent years have revealed non-cell autonomous effects of DNA damage that impact the physiological adaptations during ageing. We will discuss the role of quality assurance pathways in cell-autonomous and systemic responses to genome instability. PMID:25560147

  6. Quantitative PCR for detection of DNA damage in mitochondrial DNA of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Senoo, Takanori; Yamanaka, Mayumi; Nakamura, Atori; Terashita, Tomoki; Kawano, Shinji; Ikeda, Shogo

    2016-08-01

    Quantitative polymerase chain reaction (QPCR) has been employed to detect DNA damage and repair in mitochondrial DNA (mtDNA) of human and several model organisms. The assay also permits the quantitation of relative mtDNA copy number in cells. Here, we developed the QPCR assay primers and reaction conditions for the fission yeast Schizosaccharomyces pombe, an important model of eukaryote biology, not previously described. Under these conditions, long targets (approximately 10kb) in mtDNA were quantitatively amplified using 0.1ng of crude DNA templates without isolation of mitochondria and mtDNA. Quantitative detection of oxidative DNA damage in mtDNA was illustrated by using a DNA template irradiated with UVA in the presence of riboflavin. The damage to mtDNA in S. pombe cells treated with hydrogen peroxide and paraquat was also quantitatively measured. Finally, we found that mtDNA copy number in S. pombe cells increased after transition into a stationary phase and that the damage to mtDNA due to endogenous cellular processes accumulated during chronological aging. PMID:27236021

  7. Mouse zygotes respond to severe sperm DNA damage by delaying paternal DNA replication and embryonic development.

    PubMed

    Gawecka, Joanna E; Marh, Joel; Ortega, Michael; Yamauchi, Yasuhiro; Ward, Monika A; Ward, W Steven

    2013-01-01

    Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development. PMID:23431372

  8. Novel apparatus to measure hyperthermal heavy ion damage to DNA: Strand breaks, base loss, and fragmentation

    SciTech Connect

    Sellami, L.; Lacombe, S.; Hunting, D.; Wagner, R. J.; Huels, M. A.

    2007-08-15

    We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100 {mu}g range) spread out over a large surface area (42 cm{sup 2}) with low energy (kT-100 eV) heavy ions and to quantitatively analyze the film substance via standard biochemical techniques afterwards. Here we discuss the details of the apparatus and method and show that it allows us to measure substantial damage to double stranded DNA molecules (plasmids) and its fundamental subunits induced by heavy ions with unprecedented low energies, i.e., 2.5 eV/amu; these energies correspond to track end energies of stopping ions or secondary ions created along primary ion tracks. We find that hyperthermal Ar{sup +} ions interacting with plasmid DNA will lead to the formation of single and double strand breaks, as well as fragmentation of nucleosides, which also involve chemical modifications and site specific rupture along the N1-C1 glycosidic bond, resulting in base release. In cells, such localized clustered damage will enhance the severity of DNA strand lesions, thus making them harder to repair.

  9. Both genetic and dietary factors underlie individual differences in DNA damage levels and DNA repair capacity.

    PubMed

    Slyskova, Jana; Lorenzo, Yolanda; Karlsen, Anette; Carlsen, Monica H; Novosadova, Vendula; Blomhoff, Rune; Vodicka, Pavel; Collins, Andrew R

    2014-04-01

    The interplay between dietary habits and individual genetic make-up is assumed to influence risk of cancer, via modulation of DNA integrity. Our aim was to characterize internal and external factors that underlie inter-individual variability in DNA damage and repair and to identify dietary habits beneficial for maintaining DNA integrity. Habitual diet was estimated in 340 healthy individuals using a food frequency questionnaire and biomarkers of antioxidant status were quantified in fasting blood samples. Markers of DNA integrity were represented by DNA strand breaks, oxidized purines, oxidized pyrimidines and a sum of all three as total DNA damage. DNA repair was characterized by genetic variants and functional activities of base and nucleotide excision repair pathways. Sex, fruit-based food consumption and XPG genotype were factors significantly associated with the level of DNA damage. DNA damage was higher in women (p=0.035). Fruit consumption was negatively associated with the number of all measured DNA lesions, and this effect was mediated mostly by β-cryptoxanthin and β-tocopherol (p<0.05). XPG 1104His homozygotes appeared more vulnerable to DNA damage accumulation (p=0.001). Sex and individual antioxidants were also associated with DNA repair capacity; both the base and nucleotide excision repairs were lower in women and the latter increased with higher plasma levels of ascorbic acid and α-carotene (p<0.05). We have determined genetic and dietary factors that modulate DNA integrity. We propose that the positive health effect of fruit intake is partially mediated via DNA damage suppression and a simultaneous increase in DNA repair capacity. PMID:24674629

  10. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis

    PubMed Central

    Mukherjee, Anirban; Vasquez, Karen M.

    2012-01-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

  11. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

    PubMed

    Mukherjee, Anirban; Vasquez, Karen M

    2011-08-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

  12. DNA Damage in Chronic Kidney Disease: Evaluation of Clinical Biomarkers

    PubMed Central

    Schupp, Nicole; Stopper, Helga; Heidland, August

    2016-01-01

    Patients with chronic kidney disease (CKD) exhibit an increased cancer risk compared to a healthy control population. To be able to estimate the cancer risk of the patients and to assess the impact of interventional therapies thereon, it is of particular interest to measure the patients' burden of genomic damage. Chromosomal abnormalities, reduced DNA repair, and DNA lesions were found indeed in cells of patients with CKD. Biomarkers for DNA damage measurable in easily accessible cells like peripheral blood lymphocytes are chromosomal aberrations, structural DNA lesions, and oxidatively modified DNA bases. In this review the most common methods quantifying the three parameters mentioned above, the cytokinesis-block micronucleus assay, the comet assay, and the quantification of 8-oxo-7,8-dihydro-2′-deoxyguanosine, are evaluated concerning the feasibility of the analysis and regarding the marker's potential to predict clinical outcomes. PMID:27313827

  13. NBS1 and multiple regulations of DNA damage response

    PubMed Central

    Komatsu, Kenshi

    2016-01-01

    DNA damage response is finely tuned, with several pathways including those for DNA repair, chromatin remodeling and cell cycle checkpoint, although most studies to date have focused on single pathways. Genetic diseases characterized by genome instability have provided novel insights into the underlying mechanisms of DNA damage response. NBS1, a protein responsible for the radiation-sensitive autosomal recessive disorder Nijmegen breakage syndrome, is one of the first factors to accumulate at sites of DNA double-strand breaks (DSBs). NBS1 binds to at least five key proteins, including ATM, RPA, MRE11, RAD18 and RNF20, in the conserved regions within a limited span of the C terminus, functioning in the regulation of chromatin remodeling, cell cycle checkpoint and DNA repair in response to DSBs. In this article, we reviewed the functions of these binding proteins and their comprehensive association with NBS1. PMID:27068998

  14. Connecting the Dots: From DNA Damage and Repair to Aging.

    PubMed

    Pan, Mei-Ren; Li, Kaiyi; Lin, Shiaw-Yih; Hung, Wen-Chun

    2016-01-01

    Mammalian cells evolve a delicate system, the DNA damage response (DDR) pathway, to monitor genomic integrity and to prevent the damage from both endogenous end exogenous insults. Emerging evidence suggests that aberrant DDR and deficient DNA repair are strongly associated with cancer and aging. Our understanding of the core program of DDR has made tremendous progress in the past two decades. However, the long list of the molecules involved in the DDR and DNA repair continues to grow and the roles of the new "dots" are under intensive investigation. Here, we review the connection between DDR and DNA repair and aging and discuss the potential mechanisms by which deficient DNA repair triggers systemic effects to promote physiological or pathological aging. PMID:27164092

  15. Connecting the Dots: From DNA Damage and Repair to Aging

    PubMed Central

    Pan, Mei-Ren; Li, Kaiyi; Lin, Shiaw-Yih; Hung, Wen-Chun

    2016-01-01

    Mammalian cells evolve a delicate system, the DNA damage response (DDR) pathway, to monitor genomic integrity and to prevent the damage from both endogenous end exogenous insults. Emerging evidence suggests that aberrant DDR and deficient DNA repair are strongly associated with cancer and aging. Our understanding of the core program of DDR has made tremendous progress in the past two decades. However, the long list of the molecules involved in the DDR and DNA repair continues to grow and the roles of the new “dots” are under intensive investigation. Here, we review the connection between DDR and DNA repair and aging and discuss the potential mechanisms by which deficient DNA repair triggers systemic effects to promote physiological or pathological aging. PMID:27164092

  16. Modulation of irinotecan-induced genomic DNA damage by theanine.

    PubMed

    Attia, Sabry

    2012-05-01

    The possible chemoprotective activity of theanine against irinotecan-induced genomic DNA damage towards mouse bone marrow cells was investigated. Chromosomal aberrations, DNA damage, micronuclei formation and mitotic activity were studied in the current study as markers of genomic damage. Oxidative DNA stress markers such as 8-hydroxydeoxyguanosine, lipid peroxidation, reduced and oxidized glutathione levels were assessed as a possible mechanism underlying this amelioration. Theanine was neither genotoxic nor cytotoxic in mice at doses equivalent to 30 or 60 mg/kg for 12 days. Pretreatment of mice with theanine significantly reduced irinotecan-induced genomic damage in the bone marrow cells and these effects were dose dependent. Irinotecan induced marked biochemical alterations characteristic of oxidative DNA stress, including increased 8-hydroxydeoxyguanosine, enhanced lipid peroxidation and reduction in the reduced/oxidized glutathione ratio. Prior administration of theanine ahead of irinotecan challenge ameliorated these oxidative DNA stress markers. Overall, this study provides for the first time that theanine has a protective role in the abatement of irinotecan-induced genomic damage in the bone marrow cells of mice that resides, at least in part, on its ability to modulate the cellular antioxidant levels and consequently protect bone marrow from irinotecan genotoxicity. PMID:22414655

  17. Involvement of DNA Damage Response Pathways in Hepatocellular Carcinoma

    PubMed Central

    Yang, Sheau-Fang; Wei, Ren-Jie; Shiue, Yow-Ling; Wang, Shen-Nien

    2014-01-01

    Hepatocellular carcinoma (HCC) has been known as one of the most lethal human malignancies, due to the difficulty of early detection, chemoresistance, and radioresistance, and is characterized by active angiogenesis and metastasis, which account for rapid recurrence and poor survival. Its development has been closely associated with multiple risk factors, including hepatitis B and C virus infection, alcohol consumption, obesity, and diet contamination. Genetic alterations and genomic instability, probably resulted from unrepaired DNA lesions, are increasingly recognized as a common feature of human HCC. Dysregulation of DNA damage repair and signaling to cell cycle checkpoints, known as the DNA damage response (DDR), is associated with a predisposition to cancer and affects responses to DNA-damaging anticancer therapy. It has been demonstrated that various HCC-associated risk factors are able to promote DNA damages, formation of DNA adducts, and chromosomal aberrations. Hence, alterations in the DDR pathways may accumulate these lesions to trigger hepatocarcinogenesis and also to facilitate advanced HCC progression. This review collects some of the most known information about the link between HCC-associated risk factors and DDR pathways in HCC. Hopefully, the review will remind the researchers and clinicians of further characterizing and validating the roles of these DDR pathways in HCC. PMID:24877058

  18. Copper oxide nanoparticle mediated DNA damage in terrestrial plant models.

    PubMed

    Atha, Donald H; Wang, Huanhua; Petersen, Elijah J; Cleveland, Danielle; Holbrook, R David; Jaruga, Pawel; Dizdaroglu, Miral; Xing, Baoshan; Nelson, Bryant C

    2012-02-01

    Engineered nanoparticles, due to their unique electrical, mechanical, and catalytic properties, are presently found in many commercial products and will be intentionally or inadvertently released at increasing concentrations into the natural environment. Metal- and metal oxide-based nanomaterials have been shown to act as mediators of DNA damage in mammalian cells, organisms, and even in bacteria, but the molecular mechanisms through which this occurs are poorly understood. For the first time, we report that copper oxide nanoparticles induce DNA damage in agricultural and grassland plants. Significant accumulation of oxidatively modified, mutagenic DNA lesions (7,8-dihydro-8-oxoguanine; 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 4,6-diamino-5-formamidopyrimidine) and strong plant growth inhibition were observed for radish (Raphanus sativus), perennial ryegrass (Lolium perenne), and annual ryegrass (Lolium rigidum) under controlled laboratory conditions. Lesion accumulation levels mediated by copper ions and macroscale copper particles were measured in tandem to clarify the mechanisms of DNA damage. To our knowledge, this is the first evidence of multiple DNA lesion formation and accumulation in plants. These findings provide impetus for future investigations on nanoparticle-mediated DNA damage and repair mechanisms in plants. PMID:22201446

  19. Activation of the DNA Damage Response by RNA Viruses.

    PubMed

    Ryan, Ellis L; Hollingworth, Robert; Grand, Roger J

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  20. Activation of the DNA Damage Response by RNA Viruses

    PubMed Central

    Ryan, Ellis L.; Hollingworth, Robert; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  1. Parvovirus Diversity and DNA Damage Responses

    PubMed Central

    Cotmore, Susan F.; Tattersall, Peter

    2013-01-01

    Parvoviruses have a linear single-stranded DNA genome, around 5 kb in length, with short imperfect terminal palindromes that fold back on themselves to form duplex hairpin telomeres. These contain most of the cis-acting information required for viral “rolling hairpin” DNA replication, an evolutionary adaptation of rolling-circle synthesis in which the hairpins create duplex replication origins, prime complementary strand synthesis, and act as hinges to reverse the direction of the unidirectional cellular fork. Genomes are packaged vectorially into small, rugged protein capsids ∼260 Å in diameter, which mediate their delivery directly into the cell nucleus, where they await their host cell’s entry into S phase under its own cell cycle control. Here we focus on genus-specific variations in genome structure and replication, and review host cell responses that modulate the nuclear environment. PMID:23293137

  2. DNA damage and L1 retrotransposition.

    PubMed

    Farkash, Evan A; Luning Prak, Eline T

    2006-01-01

    Barbara McClintock was the first to suggest that transposons are a source of genome instability and that genotoxic stress assisted in their mobilization. The generation of double-stranded DNA breaks (DSBs) is a severe form of genotoxic stress that threatens the integrity of the genome, activates cell cycle checkpoints, and, in some cases, causes cell death. Applying McClintock's stress hypothesis to humans, are L1 retrotransposons, the most active autonomous mobile elements in the modern day human genome, mobilized by DSBs? Here, evidence that transposable elements, particularly retrotransposons, are mobilized by genotoxic stress is reviewed. In the setting of DSB formation, L1 mobility may be affected by changes in the substrate for L1 integration, the DNA repair machinery, or the L1 element itself. The review concludes with a discussion of the potential consequences of L1 mobilization in the setting of genotoxic stress. PMID:16877815

  3. Parvovirus diversity and DNA damage responses.

    PubMed

    Cotmore, Susan F; Tattersall, Peter

    2013-02-01

    Parvoviruses have a linear single-stranded DNA genome, around 5 kb in length, with short imperfect terminal palindromes that fold back on themselves to form duplex hairpin telomeres. These contain most of the cis-acting information required for viral "rolling hairpin" DNA replication, an evolutionary adaptation of rolling-circle synthesis in which the hairpins create duplex replication origins, prime complementary strand synthesis, and act as hinges to reverse the direction of the unidirectional cellular fork. Genomes are packaged vectorially into small, rugged protein capsids ~260 Å in diameter, which mediate their delivery directly into the cell nucleus, where they await their host cell's entry into S phase under its own cell cycle control. Here we focus on genus-specific variations in genome structure and replication, and review host cell responses that modulate the nuclear environment. PMID:23293137

  4. Ubiquitin-family modifications in the replication of DNA damage.

    PubMed

    Lehmann, Alan R

    2011-09-16

    The cell uses specialised Y-family DNA polymerases or damage avoidance mechanisms to replicate past damaged sites in DNA. These processes are under complex regulatory systems, which employ different types of post-translational modification. All the Y-family polymerases have ubiquitin binding domains that bind to mono-ubiquitinated PCNA to effect the switching from replicative to Y-family polymerase. Ubiquitination and de-ubiquitination of PCNA are tightly regulated. There is also evidence for another as yet unidentified ubiquitinated protein being involved in recruitment of Y-family polymerases to chromatin. Poly-ubiquitination of PCNA stimulates damage avoidance, and, at least in yeast, PCNA is SUMOylated to prevent unwanted recombination events at the replication fork. The Y-family polymerases themselves can be ubiquitinated and, in the case of DNA polymerase η, this results in the polymerase being excluded from chromatin. PMID:21704031

  5. SITE-SPECIFIC RECOMBINATION FOR PLANT GENETIC ENGINEERING: STRATEGY FOR AGRO-MEDIATED GENE STACKING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The precise rearrangement of DNA in planta can be achieved through site-specific recombination. For the past decade and a half, laboratory experiments have shown that site-specific recombination can delete genomic DNA, regulate gene expression, recombine chromosomes, and target new DNA into designat...

  6. DNA Damage to a Single Chromosome End Delays Anaphase Onset*

    PubMed Central

    Silva, Bárbara Alcaraz; Stambaugh, Jessica R.; Yokomori, Kyoko; Shah, Jagesh V.; Berns, Michael W.

    2014-01-01

    Chromosome ends contain nucleoprotein structures known as telomeres. Damage to chromosome ends during interphase elicits a DNA damage response (DDR) resulting in cell cycle arrest. However, little is known regarding the signaling from damaged chromosome ends (designated here as “TIPs”) during mitosis. In the present study, we investigated the consequences of DNA damage induced at a single TIP in mitosis. We used laser microirradiation to damage mitotic TIPs or chromosome arms (non-TIPs) in PtK2 kidney epithelial cells. We found that damage to a single TIP, but not a non-TIP, delays anaphase onset. This TIP-specific checkpoint response is accompanied by differential recruitment of DDR proteins. Although phosphorylation of H2AX and the recruitment of several repair factors, such as Ku70-Ku80, occur in a comparable manner at both TIP and non-TIP damage sites, DDR factors such as ataxia telangiectasia mutated (ATM), MDC1, WRN, and FANCD2 are specifically recruited to TIPs but not to non-TIPs. In addition, Nbs1, BRCA1, and ubiquitin accumulate at damaged TIPs more rapidly than at damaged non-TIPs. ATR and 53BP1 are not detected at either TIPs or non-TIPs in mitosis. The observed delay in anaphase onset is dependent on the activity of DDR kinases ATM and Chk1, and the spindle assembly checkpoint kinase Mps1. Cells damaged at a single TIP or non-TIP eventually exit mitosis with unrepaired lesions. Damaged TIPs are segregated into micronuclei at a significantly higher frequency than damaged non-TIPs. Together, these findings reveal a mitosis-specific DDR uniquely associated with chromosome ends. PMID:24982423

  7. Can graphene quantum dots cause DNA damage in cells?

    NASA Astrophysics Data System (ADS)

    Wang, Dan; Zhu, Lin; Chen, Jian-Feng; Dai, Liming

    2015-05-01

    Graphene quantum dots (GQDs) have attracted tremendous attention for biological applications. We report the first study on cytotoxicity and genotoxicity of GQDs to fibroblast cell lines (NIH-3T3 cells). The NIH-3T3 cells treated with GQDs at dosages over 50 μg mL-1 showed no significant cytotoxicity. However, the GQD-treated NIH-3T3 cells exhibited an increased expression of proteins (p53, Rad 51, and OGG1) related to DNA damage compared with untreated cells, indicating the DNA damage caused by GQDs. The GQD-induced release of reactive oxygen species (ROS) was demonstrated to be responsible for the observed DNA damage. These findings should have important implications for future applications of GQDs in biological systems.Graphene quantum dots (GQDs) have attracted tremendous attention for biological applications. We report the first study on cytotoxicity and genotoxicity of GQDs to fibroblast cell lines (NIH-3T3 cells). The NIH-3T3 cells treated with GQDs at dosages over 50 μg mL-1 showed no significant cytotoxicity. However, the GQD-treated NIH-3T3 cells exhibited an increased expression of proteins (p53, Rad 51, and OGG1) related to DNA damage compared with untreated cells, indicating the DNA damage caused by GQDs. The GQD-induced release of reactive oxygen species (ROS) was demonstrated to be responsible for the observed DNA damage. These findings should have important implications for future applications of GQDs in biological systems. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01734c

  8. Spectrum of complex DNA damages depends on the incident radiation

    NASA Astrophysics Data System (ADS)

    Hada, M.; Sutherland, B.

    Ionizing radiation induces clustered DNA damages in DNA-two or more abasic sites oxidized bases and strand breaks on opposite DNA strands within a few helical turns Clustered damages are considered to be difficult to repair and therefore potentially lethal and mutagenic damages Although induction of single strand breaks and isolated lesions has been studied extensively little is known of factors affecting induction of clusters other than double strand breaks DSB The aim of the present study was to determine whether the type of incident radiation could affect yield or spectra of specific clusters Genomic T7 DNA a simple 40 kbp linear blunt-ended molecule was irradiated in non-scavenging buffer conditions with Fe 970 MeV n Ti 980 MeV n C 293 MeV n Si 586 MeV n ions or protons 1 GeV n at the NASA Space Radiation Laboratory or with 100 kVp X-rays Irradiated DNA was treated with homogeneous Fpg or Nfo proteins or without enzyme treatment for DSB quantitation then electrophoresed in neutral agarose gels DSB Fpg-OxyPurine clusters and Nfo-Abasic clusters were quantified by number average length analysis The results show that the yields of all these complex damages depend on the incident radiation Although LETs are similar protons induced twice as many DSBs than did X-rays Further the spectrum of damage also depends on the radiation The yield damage Mbp Gy of all damages decreased with increasing linear energy transfer LET of the radiation The relative frequencies of DSBs to Abasic- and OxyBase clusters were higher

  9. DNA damage and cell killing. Cause and effect

    SciTech Connect

    Elkind, M.M.

    1985-11-15

    The evidence supporting a cause and effect relationship between DNA damage and cell killing is examined in the light of what is currently known about the organization and replication of genomic DNA in eukaryotic cells and the radio-energetics of DNA breakage. A large disparity is identified between characteristic doses for cell killing and for the production of DNA lesions (i.e., single- or double-strand breaks). In contrast, the sensitive phase of the inhibition of DNA synthesis has a dependence on dose quantitatively similar to that of cell killing. A model is developed in which single- and double-strand breaks are associated with the inhibition of replicon initiation, whereas only double-strand breaks are primarily responsible for strand elongation. Furthermore, the model points to the replisome and the region of replicated DNA just downstream from the fork as the locus of radiation action.

  10. Specificity of damage recognition and catalysis of DNA repair.

    PubMed

    Osman, R; Fuxreiter, M; Luo, N

    2000-05-01

    A common feature of DNA repair enzymes is their ability to recognize the damage independently of sequence in which they are found. The presence of a flipped out base inserted into the protein in several DNA-enzyme complexes suggests a contribution to enzyme specificity. Molecular simulations of damaged DNA indicate that the damage produces changes in DNA structure and changes the dynamics of DNA bending. The reduced bending force constant can be used by the enzyme to induce DNA bending and facilitate base flipping. We show that a thymine dimer (TD) containing DNA requires less energy to bend, lowering the barrier for base flipping. On the other hand, bending in DNA with U-G mismatch is affected only by a small amount and flipping is not enhanced significantly. T4 endonuclease V (endoV), which recognizes TD, utilizes the reduced barrier for flipping as a specific recognition element. In uracil DNA glycosylase (UDG), which recognizes U-G mismatches, base flipping is not enhanced and recognition is encoded in a highly specific binding pocket for the flipped base. Simulations of UDG and endoV in complex with damaged DNA provide insight into the essential elements of the catalytic mechanism. Calculations of pKas of active site residues in endoV and endoV-DNA complex show that the pKa, of the N-terminus is reduced from 8.01 to 6.52 while that of Glu-23 increases from 1.52 to 7.82. Thus, the key catalytic residues are in their neutral form. The simulations also show that Glu-23 is also H-bonded to O4' of the 5'-TD enhancing the nucleophilic attack on Cl and that Arg-26 enhances the hydrolysis by electrostatic stabilization but does not participate in proton transfer. In the enzyme-substrate complex of UDG, the role of electrostatic stabilization is played by His-268, whose pKa increases to 7.1 from 4.9 in the free enzyme. The pKa of Asp-145, the other important catalytic residue, remains around 4.2 in the free enzyme and in the complex. Thus, it can not act as a proton